US20050287165A1

US20050287165A1 - Meningococcal antigens

Info

Publication number: US20050287165A1
Application number: US11/212,443
Authority: US
Inventors: Vincenzo Scarlato; Vega Masignani; Rino Rappuoli; Mariagrazia Pizza; Guido Grandi
Original assignee: Chiron Corp
Current assignee: Novartis AG; Novartis Vaccines and Diagnostics Inc
Priority date: 1998-01-14
Filing date: 2005-08-24
Publication date: 2005-12-29
Also published as: ES2333071T5; JP4399112B2; EP2210945A3; JP2002508966A; CA2317815A1; DK1047784T3; EP1047784B2; HK1030435A1; WO1999036544A2; EP2278011A2; EP1047784B1; CN1292820A; DK1047784T4; EP2278011A3; CN1597694A; EP2210945A2; US6709660B1; ATE446368T1; CN1224708C; AU1979599A

Abstract

The invention provides proteins from Neisseria meningitidis (strains A & B), including amino acid sequences, the corresponding nucleotide sequences, expression data, and serological data. The proteins are useful antigens for vaccines, immunogenic compositions, and/or diagnostics.

Description

This application is a continuation-in-part of international patent application PCT/IB99/00103, filed Jan. 14, 1999, from which priority is claimed under 35 U.S.C. § 119.
This invention relates to antigens from the bacterium Neisseria meningitidis.

BACKGROUND

Neisseria meningitidis is a non-motile, gram negative diplococcus human pathogen. It colonises the pharynx, causing meningitis and, occasionally, septicaemia in the absence of meningitis. It is closely related to N. gonorrhoeae, although one feature that clearly differentiates meningococcus from gonococcus is the presence of a polysaccharide capsule that is present in all pathogenic meningococci.
N. meningitidis causes both endemic and epidemic disease. In the United States the attack rate is 0.6-1 per 100,000 persons per year, and it can be much greater during outbreaks (see Lieberman el al. (1996) Safety and Immunogenicity of a Serogroups A/C Neisseria meningitidis Oligosaccharide-Protein Conjugate Vaccine in Young Children. JAMA 275(19):1499-1503; Schuchat et al (1997) Bacterial Meningitis in the United States in 1995. N Engl J Med 337(14):970-976). In developing countries, endemic disease rates are much higher and during epidemics incidence rates can reach 500 cases per 100,000 persons per year. Mortality is extremely high, at 10-20% in the United States, and much higher in developing countries. Following the introduction of the conjugate vaccine against Haemophilus influenzae, N. meningitidis is the major cause of bacterial meningitis at all ages in the United States (Schuchat et al (1997) supra).
Based on the organism's capsular polysaccharide, 12 serogroups of N. meningitidis have been identified. Group A is the pathogen most often implicated in epidemic disease in sub-Saharan Africa. Serogroups B and C are responsible for the vast majority of cases in the United States and in most developed countries. Serogroups W135 and Y are responsible for the rest of the cases in the United States and developed countries. The meningococcal vaccine currently in use is a tetravalent polysaccharide vaccine composed of serogroups A, C, Y and W135. Although efficacious in adolescents and adults, it induces a poor immune response and short duration of protection, and cannot be used in infants [eg. Morbidity and Mortality weekly report, Vol. 46, No. RR-5 (1997)]. This is because polysaccharides are T-cell independent antigens that induce a weak immune response that cannot be boosted by repeated immunization. Following the success of the vaccination against H. influenzae, conjugate vaccines against serogroups A and C have been developed and are at the final stage of clinical testing (Zollinger W D “New and Improved Vaccines Against Meningococcal Disease” in: New Generation Vaccines, supra, pp. 469-488; Lieberman et al (1996) supra; Costantino et al (1992) Development and phase I clinical testing of a conjugate vaccine against meningococcus A and C. Vaccine 10:691-698).
Meningococcus B remains a problem, however. This serotype currently is responsible for approximately 50% of total meningitis in the United States, Europe, and South America. The polysaccharide approach cannot be used because the menB capsular polysaccharide is a polymer of α(2-8)-linked N-acetyl neuraminic acid that is also present in mammalian tissue. This results in tolerance to the antigen; indeed, if an immune response were elicited, it would be anti-self, and therefore undesirable. In order to avoid induction of autoimmunity and to induce a protective immune response, the capsular polysaccharide has, for instance, been chemically modified substituting the N-acetyl groups with N-propionyl groups, leaving the specific antigenicity unaltered (Romero & Outschoorn (1994) Current status of Meningococcal group B vaccine candidates: capsular or non-capsular? Clin Microbiol Rev 7(4):559-575).
Alternative approaches to menB vaccines have used complex mixtures of outer membrane proteins (OMPs), containing either the OMPs alone, or OMPs enriched in porins, or deleted of the class 4 OMPs that are believed to induce antibodies that block bactericidal activity. This approach produces vaccines that are not well characterized. They are able to protect against the homologous strain, but are not effective at large where there are many antigenic variants of the outer membrane proteins. To overcome the antigenic variability, multivalent vaccines containing up to nine different porins have been constructed (eg. Poolman J T (1992) Development of a meningococcal vaccine. Infect. Agents Dis. 4:13-28). Additional proteins to be used in outer membrane vaccines have been the opa and opc proteins, but none of these approaches have been able to overcome the antigenic variability (eg. Ala'Aldeen & Borriello (1996) The meningococcal transferrin-binding proteins 1 and 2 are both surface exposed and generate bactericidal antibodies capable of killing homologous and heterologous strains. Vaccine 14(1):49-53).
A certain amount of sequence data is available for meningococcal and gonococcal genes and proteins (eg. EP-A-0467714, WO96/29412), but this is by no means complete. The provision of further sequences could provide an opportunity to identify secreted or surface-exposed proteins that are presumed targets for the immune system and which are not antigenically variable. For instance, some of the identified proteins could be components of efficacious vaccines against meningococcus B, some could be components of vaccines against all meningococcal serotypes, and others could be components of vaccines against all pathogenic Neisseriae.
The Invention
The invention provides proteins comprising the N. meningitidis amino acid sequences disclosed in the examples.
It also provides proteins comprising sequences homologous (ie. having sequence identity) to the N. meningitidis amino acid sequences disclosed in the examples. Depending on the particular sequence, the degree of sequence identity is preferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%, 99% or more). These homologous proteins include mutants and allelic variants of the sequences disclosed in the examples. Typically, 50% identity or more between two proteins is considered to be an indication of functional equivalence. Identity between the proteins is preferably determined by the Smith-Waterman homology search algorithm as implemented in the MPSRCH program (Oxford Molecular), using an affine gap search with parameters gap open penalty=12 and gap extension penalty=1.
The invention further provides proteins comprising fragments of the N. meningitidis amino acid sequences disclosed in the examples. The fragments should comprise at least n consecutive amino acids from the sequences and, depending on the particular sequence, n is 7 or more (eg. 8, 10, 12, 14, 16, 18, 20 or more). Preferably the fragments comprise an epitope from the sequence.
The proteins of the invention can, of course, be prepared by various means (eg. recombinant expression, purification from cell culture, chemical synthesis etc.) and in various forms (eg. native, fusions etc.). They are preferably prepared in substantially pure form (ie. substantially free from other N. meningitidis or host cell proteins).
According to a further aspect, the invention provides antibodies which bind to these proteins. These may be polyclonal or monoclonal and may be produced by any suitable means.
According to a further aspect, the invention provides nucleic acid comprising the N. meningitidis nucleotide sequences disclosed in the examples. In addition, the invention provides nucleic acid comprising sequences homologous (ie. having sequence identity) to the N. meningitidis nucleotide sequences disclosed in the examples.
Furthermore, the invention provides nucleic acid which can hybridise to the N. meningitidis nucleic acid disclosed in the examples, preferably under “high stringency” conditions (eg. 65° C. in a 0.1×SSC, 0.5% SDS solution).
Nucleic acid comprising fragments of these sequences are also provided. These should comprise at least n consecutive nucleotides from the N. meningitidis sequences and, depending on the particular sequence, n is 10 or more ( eg 12, 14, 15, 18, 20, 25, 30, 35, 40 or more).
According to a further aspect, the invention provides nucleic acid encoding the proteins and protein fragments of the invention.
It should also be appreciated that the invention provides nucleic acid comprising sequences complementary to those described above (eg. for antisense or probing purposes).
Nucleic acid according to the invention can, of course, be prepared in many ways (eg. by chemical synthesis, from genomic or cDNA libraries, from the organism itself etc.) and can take various forms (eg. single stranded, double stranded, vectors, probes etc.).
In addition, the term “nucleic acid” includes DNA and RNA, and also their analogues, such as those containing modified backbones, and also peptide nucleic acids (PNA) etc.
According to a further aspect, the invention provides vectors comprising nucleotide sequences of the invention (eg. expression vectors) and host cells transformed with such vectors.
According to a further aspect, the invention provides compositions comprising protein, antibody, and/or nucleic acid according to the invention. These compositions may be suitable as vaccines, for instance, or as diagnostic reagents, or as immunogenic compositions.
The invention also provides nucleic acid, protein, or antibody according to the invention for use as medicaments (eg. as vaccines) or as diagnostic reagents. It also provides the use of nucleic acid, protein, or antibody according to the invention in the manufacture of: (i) a medicament for treating or preventing infection due to Neisserial bacteria; (ii) a diagnostic reagent for detecting the presence of Neisserial bacteria or of antibodies raised against Neisserial bacteria; and/or (iii) a reagent which can raise antibodies against Neisserial bacteria Said Neisserial bacteria may be any species or strain (such as N. gonorrhoeae) but are preferably N. meningitidis, especially strain A, strain B or strain C.
The invention also provides a method of treating a patient, comprising administering to the patient a therapeutically effective amount of nucleic acid, protein, and/or antibody according to the invention.
According to further aspects, the invention provides various processes.
A process for producing proteins of the invention is provided, comprising the step of culturing a host cell according to the invention under conditions which induce protein expression.
A process for producing protein or nucleic acid of the invention is provided, wherein the protein or nucleic acid is synthesised in part or in whole using chemical means.
A process for detecting polynucleotides of the invention is provided, comprising the steps of: (a) contacting a nucleic probe according to the invention with a biological sample under hybridizing conditions to form duplexes; and (b) detecting said duplexes.
A process for detecting proteins of the invention is provided, comprising the steps of: (a) contacting an antibody according to the invention with a biological sample under conditions suitable for the formation of an antibody-antigen complexes; and (b) detecting said complexes.
Unlike the sequences disclosed in PCT/IB98/01665, the sequences disclosed in the present application are believed not to have any significant homologs in N. gonorrhoeae. Accordingly, the sequences of the present invention also find use in the preparation of reagents for distinguishing between N. meningitidis and N. gonorrhoeae.
A summary of standard techniques and procedures which may be employed in order to perform the invention (eg. to utilise the disclosed sequences for vaccination or diagnostic purposes) follows. This summary is not a limitation on the invention but, rather, gives examples that may be used, but are not required.
General
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature eg. Sambrook Molecular Cloning; A Laboratory Manual, Second Edition (1989); DNA Cloning, Volumes I and ii (D. N Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed, 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription and Translation (B. D. Hames & S. J. Higgins eds. 1984); Animal Cell Culture (R. I. Freshney ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide to Molecular Cloning (1984); the Methods in Enzymology series (Academic Press, Inc.), especially volumes 154 & 155; Gene Transfer Vectors for Mammalian Cells (J. H. Miller and M. P. Calos eds. 1987, Cold Spring Harbor Laboratory); Mayer and Walker, eds. (1987), Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes, (1987) Protein Purification: Principles and Practice, Second Edition (Springer-Verlag, N.Y.), and Handbook of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell eds 1986).
Standard abbreviations for nucleotides and amino acids are used in this specification.
All publications, patents, and patent applications cited herein are incorporated in full by reference. In particular, the contents of UK patent applications 9800760.2, 9819015.0 and 9822143.5 are incorporated herein.
Definitions
A composition containing X is “substantially free of” Y when at least 85% by weight of the total X+Y in the composition is X. Preferably, X comprises at least about 90% by weight of the total of X+Y in the composition, more preferably at least about 95% or even 99% by weight.
The term “comprising” means “including” as well as “consisting” eg. a composition “comprising” X may consist exclusively of X or may include something additional to X, such as X+Y.
A “conserved” Neisseria amino acid fragment or protein is one that is present in a particular Neisserial protein in at least x % of Neisseria. The value of x may be 50% or more, e.g., 66%, 75%, 80%, 90%, 95% or even 100% (i.e. the amino acid is found in the protein in question in all Neisseria). In order to determine whether an animo acid is “conserved” in a particular Neisserial protein, it is necessary to compare that amino acid residue in the sequences of the protein in question from a plurality of different Neisseria (a reference population). The reference population may include a number of different Neisseria species or may include a single species. The reference population may include a number of different serogroups of a particular species or a single serogroup. A preferred reference population consists of the 5 most common Neisseria.
The term “heterologous” refers to two biological components that are not found together in nature. The components may be host cells, genes, or regulatory regions, such as promoters. Although the heterologous components are not found together in nature, they can function together, as when a promoter heterologous to a gene is operably linked to the gene. Another example is where a Neisserial sequence is heterologous to a mouse host cell. A further examples would be two epitopes from the same or different proteins which have been assembled in a single protein in an arrangement not found in nature.
An “origin of replication” is a polynucleotide sequence that initiates and regulates replication of polynucleotides, such as an expression vector. The origin of replication behaves as an autonomous unit of polynucleotide replication within a cell, capable of replication under its own control. An origin of replication may be needed for a vector to replicate in a particular host cell. With certain origins of replication, an expression vector can be reproduced at a high copy number in the presence of the appropriate proteins within the cell. Examples of origins are the autonomously replicating sequences, which are effective in yeast; and the viral T-antigen, effective in COS-7 cells.
A “mutant” sequence is defined as DNA, RNA or amino acid sequence differing from but having sequence identity with the native or disclosed sequence. Depending on the particular sequence, the degree of sequence identity between the native or disclosed sequence and the mutant sequence is preferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%, 99% or more, calculated using the Smith-Waterman algorithm as described above). As used herein, an “allelic variant” of a nucleic acid molecule, or region, for which nucleic acid sequence is provided herein is a nucleic acid molecule, or region, that occurs essentially at the same locus in the genome of another or second isolate, and that, due to natural variation caused by, for example, mutation or recombination, has a similar but not identical nucleic acid sequence. A coding region allelic variant typically encodes a protein having similar activity to that of the protein encoded by the gene to which it is being compared. An allelic variant can also comprise an alteration in the 5′ or 3′ untranslated regions of the gene, such as in regulatory control regions (eg. see U.S. Pat. No. 5,753,235).
Expression Systems
The Neisserial nucleotide sequences can be expressed in a variety of different expression systems; for example those used with mammalian cells, baculoviruses, plants, bacteria, and yeast.
i. Mammalian Systems
Mammalian expression systems are known in the art. A mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiating region, which is usually placed proximal to the 5′ end of the coding sequence, and a TATA box, usually located 25-30 base pairs (bp) upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site. A mammalian promoter will also contain an upstream promoter element, usually located within 100 to 200 bp upstream of the TATA box. An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation [Sambrook et al. (1989) “Expression of Cloned Genes in Mammalian Cells.” In Molecular Cloning: A Laboratory Manual, 2nd ed.]
Mammalian viral genes are often highly expressed and have a broad host range; therefore sequences encoding mammalian viral genes provide particularly useful promoter sequences. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), and herpes simplex virus promoter. In addition, sequences derived from non-viral genes, such as the murine metallotheionein gene, also provide useful promoter sequences. Expression may be either constitutive or regulated (inducible), depending on the promoter can be induced with glucocorticoid in hormone-responsive cells.
The presence of an enhancer element (enhancer), combined with the promoter elements described above, will usually increase expression levels. An enhancer is a regulatory DNA sequence that can stimulate transcription up to 1000-fold when linked to homologous or heterologous promoters, with synthesis beginning at the normal RNA start site. Enhancers are also active when they are placed upstream or downstream from the transcription initiation site, in either normal or flipped orientation, or at a distance of more than 1000 nucleotides from the promoter [Maniatis et al. (1987) Science 236:1237; Alberts et al. (1989) Molecular Biology of the Cell, 2nd ed.]. Enhancer elements derived from viruses may be particularly useful, because they usually have a broader host range. Examples include the SV40 early gene enhancer [Dijkema et al (1985) EMBO J. 4:761] and the enhancer/promoters derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus [Gorman et al. (1982b) Proc. Natl. Acad. Sci. 79:6777] and from human cytomegalovirus [Boshart et al. (1985) Cell 41:521]. Additionally, some enhancers are regulatable and become active only in the presence of an inducer, such as a hormone or metal ion [Sassone-Corsi and Borelli (1986) Trends Genet. 2:215; Maniatis et al. (1987) Science 236:1237].
A DNA molecule may be expressed intracellularly in mammalian cells. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.
Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in mammalian cells. Preferably, there are processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vivo or in vitro. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. The adenovirus triparite leader is an example of a leader sequence that provides for secretion of a foreign protein in mammalian cells.
Usually, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3′ to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3′ terminus of the mature mRNA is formed by site-specific post-transcriptional cleavage and polyadenylation [Birnstiel et al. (1985) Cell 41:349; Proudfoot and Whitelaw (1988) “Termination and 3′ end processing of eukaryotic RNA. In Transcription and splicing (ed. B. D. Hames and D. M. Glover); Proudfoot (1989) Trends Biochem. Sci. 14:105]. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Examples of transcription terminater/polyadenylation signals include those derived from SV40 [Sambrook et al (1989) “Expression of cloned genes in cultured mammalian cells.” In Molecular Cloning: A Laboratory Manual].
Usually, the above described components, comprising a promoter, polyadenylation signal, and transcription termination sequence are put together into expression constructs. Enhancers; introns with functional splice donor and acceptor sites, and leader sequences may also be included in an expression construct, if desired. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg. plasmids) capable of stable maintenance in a host, such as mammalian cells or bacteria. Mammalian replication systems include those derived from animal viruses, which require trans-acting factors to replicate. For example, plasmids containing the replication systems of papovaviruses, such as SV40 [Gluzman (1981) Cell 23:175] or polyomavirus, replicate to extremely high copy number in the presence of the appropriate viral T antigen. Additional examples of mammalian replicons include those derived from bovine papillomavirus and Epstein-Barr virus. Additionally, the replicon may have two replicaton systems, thus allowing it to be maintained, for example, in mammalian cells for expression and in a prokaryotic host for cloning and amplification. Examples of such mammalian-bacteria shuttle vectors include pMT2 [Kaufman et al. (1989) Mol. Cell. Biol. 9:946] and pHEBO [Shimizu et al. (1986) Mol. Cell. Biol. 6:1074].
The transformation procedure used depends upon the host to be transformed. Methods for introduction of heterologous polynucleotides into mammalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg. Hep G2), and a number of other cell lines.
ii. Baculovirus Systems
The polynucleotide encoding the protein can also be inserted into a suitable insect expression vector, and is operably linked to the control elements within that vector. Vector construction employs techniques which are known in the art. Generally, the components of the expression system include a transfer vector, usually a bacterial plasmid, which contains both a fragment of the baculovirus genome, and a convenient restriction site for insertion of the heterologous gene or genes to be expressed; a wild type baculovirus with a sequence homologous to the baculovirus-specific fragment in the transfer vector (this allows for the homologous recombination of the heterologous gene in to the baculovirus genome); and appropriate insect host cells and growth media.
After inserting the DNA sequence encoding the protein into the transfer vector, the vector and the wild type viral genome are transfected into an insect host cell where the vector and viral genome are allowed to recombine. The packaged recombinant-virus is expressed and recombinant plaques are identified and purified. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego Calif. (“MaxBac” kit). These techniques are generally known to those skilled in the art and fully described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987) (hereinafter “Summers and Smith”).
Prior to inserting the DNA sequence encoding the protein into the baculovirus genome, the above described components, comprising a promoter, leader (if desired), coding sequence of interest, and transcription termination sequence, are usually assembled into an intermediate transplacement construct (transfer vector). This construct may contain a single gene and operably linked regulatory elements; multiple genes, each with its owned set of operably linked regulatory elements; or multiple genes, regulated by the same set of regulatory elements. Intermediate transplacement constructs are often maintained in a replicon, such as an extrachromosomal element (eg. plasmids) capable of stable maintenance in a host, such as a bacterium. The replicon will have a replication system, thus allowing it to be maintained in a suitable host for cloning and amplification.
Currently, the most commonly used transfer vector for introducing foreign genes into AcNPV is pAc373. Many other vectors, known to those of skill in the art, have also been designed. These include, for example, pVL985 (which alters the polyhedrin start codon from ATG to ATT, and which introduces a BamHI cloning site 32 basepairs downstream from the ATT; see Luckow and Summers, Virology (1989) 17:31.
The plasmid usually also contains the polyhedrin polyadenylation signal (Miller et al. (1988) Ann. Rev. Microbiol., 42:177) and a prokaryotic ampicillin-resistance (amp) gene and origin of replication for selection and propagation in E. coli.
Baculovirus transfer vectors usually contain a baculovirus promoter. A baculovirus promoter is any DNA sequence capable of binding a baculovirus RNA polymerase and initiating the downstream (5′ to 3) transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site. A baculovirus transfer vector may also have a second domain called an enhancer, which, if present, is usually distal to the structural gene. Expression may be either regulated or constitutive.
Structural genes, abundantly transcribed at late times in a viral infection cycle, provide particularly useful promoter sequences. Examples include sequences derived from the gene encoding the viral polyhedron protein, Friesen et al., (1986) “The Regulation of Baculovirus Gene Expression,” in: The Molecular Biology of Baculoviruses (ed. Walter Doerfler); EPO Publ. Nos. 127 839 and 155 476; and the gene encoding the p10 protein, Vlak et al., (1988), J. Gen. Virol. 69:765.
DNA encoding suitable signal sequences can be derived from genes for secreted insect or baculovirus proteins, such as the baculovirus polyhedrin gene (Carbonell et al. (1988) Gene, 73:409). Alternatively, since the signals for mammalian cell posttranslational modifications (such as signal peptide cleavage, proteolytic cleavage, and phosphorylation) appear to be recognized by insect cells, and the signals required for secretion and nuclear accumulation also appear to be conserved between the invertebrate cells and vertebrate cells, leaders of non-insect origin, such as those derived from genes encoding human α-interferon, Maeda et al., (1985), Nature 315:592; human gastrin-releasing peptide, Lebacq-Verheyden et al., (1988), Molec. Cell. Biol. 8:3129; human IL-2, Smith et al., (1985) Proc. Nat'l Acad. Sci. USA, 82:8404; mouse IL-3, (Miyajima et al., (1987) Gene 58:273; and human glucocerebrosidase, Martin et al. (1988) DNA, 7:99, can also be used to provide for secretion in insects.
A recombinant polypeptide or polyprotein may be expressed intracellularly or, if it is expressed with the proper regulatory sequences, it can be secreted. Good intracellular expression of nonfused foreign proteins usually requires heterologous genes that ideally have a short leader sequence containing suitable translation initiation signals preceding an ATG start signal. If desired, methionine at the N-terminus may be cleaved from the mature protein by in vitro incubation with cyanogen bromide.
Alternatively, recombinant polyproteins or proteins which are not naturally secreted can be secreted from the insect cell by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in insects. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the translocation of the protein into the endoplasmic reticulum.
After insertion of the DNA sequence and/or the gene encoding the expression product precursor of the protein, an insect cell host is co-transformed with the heterologous DNA of the transfer vector and the genomic DNA of wild type baculovirus—usually by co-transfection. The promoter and transcription termination sequence of the construct will usually comprise a 2-5 kb section of the baculovirus genome. Methods for introducing heterologous DNA into the desired site in the baculovirus virus are known in the art (See Summers and Smith supra; Ju et al. (1987); Smith et al., Mol. Cell. Biol. (1983) 3:2156; and Luckow and Summers (1989)). For example, the insertion can be into a gene such as the polyhedrin gene, by homologous double crossover recombination; insertion can also be into a restriction enzyme site engineered into the desired baculovirus gene. Miller et al., (1989), Bioessays 4:91 The DNA sequence, when cloned in place of the polyhedrin gene in the expression vector, is flanked both 5′ and 3′ by polyhedrin-specific sequences and is positioned downstream of the polyhedrin promoter.
The newly formed baculovirus expression vector is subsequently packaged into an infectious recombinant baculovirus. Homologous recombination occurs at low frequency (between about 1% and about 5%); thus, the majority of the virus produced after cotransfection is still wild-type virus. Therefore, a method is necessary to identify recombinant viruses. An advantage of the expression system is a visual screen allowing recombinant viruses to be distinguished. The polyhedrin protein, which is produced by the native virus, is produced at very high levels in the nuclei of infected cells at late times after viral infection. Accumulated polyhedrin protein forms occlusion bodies that also contain embedded particles. These occlusion bodies, up to 15 μm in size, are highly refractile, giving them a bright shiny appearance that is readily visualized under the light microscope. Cells infected with recombinant viruses lack occlusion bodies. To distinguish recombinant virus from wild-type virus, the transfection supernatant is plaqued onto a monolayer of insect cells by techniques known to those skilled in the art. Namely, the plaques are screened under the light microscope for the presence (indicative of wild-type virus) or absence (indicative of recombinant virus) of occlusion bodies. “Current Protocols in Microbiology” Vol. 2 (Ausubel et al. eds) at 16.8 (Supp. 10, 1990); Summers and Smith, supra; Miller et al. (1989).
Recombinant baculovirus expression vectors have been developed for infection into several insect cells. For example, recombinant baculoviruses have been developed for, inter alia: Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni (WO 89/046699; Carbonell et al., (1985) J. Virol. 56:153; Wright (1986) Nature 321:718; Smith et al., (1983) Mol. Cell. Biol. 3:2156; and see generally, Fraser, et al. (1989) In Vitro Cell. Dev. Biol. 25:225).
Cells and cell culture media are commercially available for both direct and fusion expression of heterologous polypeptides in a baculovirus/expression system; cell culture technology is generally known to those skilled in the art. See, eg. Summers and Smith supra.
The modified insect cells may then be grown in an appropriate nutrient medium, which allows for stable maintenance of the plasmid(s) present in the modified insect host. Where the expression product gene is under inducible control, the host may be grown to high density, and expression induced. Alternatively, where expression is constitutive, the product will be continuously expressed into the medium and the nutrient medium must be continuously circulated, while removing the product of interest and augmenting depleted nutrients. The product may be purified by such techniques as chromatography, eg. HPLC, affinity chromatography, ion exchange chromatography, etc.; electrophoresis; density gradient centrifugation; solvent extraction, or the like. As appropriate, the product may be further purified, as required, so as to remove substantially any insect proteins which are also secreted in the medium or result from lysis of insect cells, so as to provide a product which is at least substantially free of host debris, eg. proteins, lipids and polysaccharides.
In order to obtain protein expression, recombinant host cells derived from the transformants are incubated under conditions which allow expression of the recombinant protein encoding sequence. These conditions will vary, dependent upon the host cell selected. However, the conditions are readily ascertainable to those of ordinary skill in the art, based upon what is known in the art.
iii. Plant Systems
There are many plant cell culture and whole plant genetic expression systems known in the art. Exemplary plant cellular genetic expression systems include those described in patents, such as: U.S. Pat. No. 5,693,506; U.S. Pat. No. 5,659,122; and U.S. Pat. No. 5,608,143. Additional examples of genetic expression in plant cell culture has been described by Zenk, Phytochemistry 30:3861-3863 (1991). Descriptions of plant protein signal peptides may be found in addition to the references described above in Vaulcombe et al., Mol. Gen. Genet. 209:33-40 (1987); Chandler et al., Plant Molecular Biology 3:407-418 (1984); Rogers, J. Biol. Chem. 260:3731-3738 (1985); Rothstein et al., Gene 55:353-356 (1987); Whittier et al., Nucleic Acids Research 15:2515-2535 (1987); Wirsel et al., Molecular Microbiology 3:3-14 (1989); Yu et al., Gene 122:247-253 (1992). A description of the regulation of plant gene expression by the phytohormone, gibberellic acid and secreted enzymes induced by gibberellic acid can be found in R. L. Jones and J. MacMillin, Gibberellins: in: Advanced Plant Physiology, Malcolm B. Wilkins, ed., 1984 Pitman Publishing Limited, London, pp. 21-52. References that describe other metabolically-regulated genes: Sheen, Plant Cell, 2:1027-1038 (1990); Maas et al., EMBO J. 9:3447-3452 (1990); Benkel and Hickey, Proc. Natl. Acad. Sci 84:1337-1339 (1987)
Typically, using techniques known in the art, a desired polynucleotide sequence is inserted into an expression cassette comprising genetic regulatory elements designed for operation in plants. The expression cassette is inserted into a desired expression vector with companion sequences upstream and downstream from the expression cassette suitable for expression in a plant host. The companion sequences will be of plasmid or viral origin and provide necessary characteristics to the vector to permit the vectors to move DNA from an original cloning host, such as bacteria, to the desired plant host. The basic bacterial/plant vector construct will preferably provide a broad host range prokaryote replication origin; a prokaryote selectable marker, and, for Agrobacterium transformations, T DNA sequences for Agrobacterium-mediated transfer to plant chromosomes. Where the heterologous gene is not readily amenable to detection, the construct will preferably also have a selectable marker gene suitable for determining if a plant cell has been transformed. A general review of suitable markers, for example for the members of the grass family, is found in Wilmink and Dons, 1993, Plant Mol. Biol. Reptr, 11(2):165-185.
Sequences suitable for permitting integration of the heterologous sequence into the plant genome are also recommended. These might include transposon sequences and the like for homologous recombination as well as Ti sequences which permit random insertion of a heterologous expression cassette into a plant genome. Suitable prokaryote selectable markers include resistance toward antibiotics such as ampicillin or tetracycline. Other DNA sequences encoding additional functions may also be present in the vector, as is known in the art.
The nucleic acid molecules of the subject invention may be included into an expression cassette for expression of the protein(s) of interest. Usually, there will be only one expression cassette, although two or more are feasible. The recombinant expression cassette will contain in addition to the heterologous protein encoding sequence the following elements, a promoter region, plant 5′ untranslated sequences, initiation codon depending upon whether or not the structural gene comes equipped with one, and a transcription and translation termination sequence. Unique restriction enzyme sites at the 5′ and 3′ ends of the cassette allow for easy insertion into a pre-existing vector.
A heterologous coding sequence may be for any protein relating to the present invention. The sequence encoding the protein of interest will encode a signal peptide which allows processing and translocation of the protein, as appropriate, and will usually lack any sequence which might result in the binding of the desired protein of the invention to a membrane. Since, for the most part, the transcriptional initiation region will be for a gene which is expressed and translocated during germination, by employing the signal peptide which provides for translocation, one may also provide for translocation of the protein of interest. In this way, the protein(s) of interest will be translocated from the cells in which they are expressed and may be efficiently harvested. Typically secretion in seeds are across the aleurone or scutellar epithelium layer into the endosperm of the seed. While it is not required that the protein be secreted from the cells in which the protein is produced, this facilitates the isolation and purification of the recombinant protein.
Since the ultimate expression of the desired gene product will be in a eucaryotic cell it is desirable to determine whether any portion of the cloned gene contains sequences which will be processed out as introns by the host's splicosome machinery. If so, site-directed mutagenesis of the “intron” region may be conducted to prevent losing a portion of the genetic message as a false intron code, Reed and Maniatis, Cell 41:95-105, 1985.
The vector can be microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA. Crossway, Mol. Gen. Genet, 202:179-185, 1985. The genetic material may also be transferred into the plant cell by using polyethylene glycol, Krens, et al., Nature, 296, 72-74, 1982. Another method of introduction of nucleic acid segments is high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface, Klein, et al., Nature, 327, 70-73, 1987 and Knudsen and Muller, 1991, Planta, 185:330-336 teaching particle bombardment of barley endosperm to create transgenic barley. Yet another method of introduction would be fusion of protoplasts with other entities, either minicells, cells, lysosomes or other fusible lipid-surfaced bodies, Fraley, et al., Proc. Natl. Acad. Sci. USA, 79, 1859-1863, 1982.
The vector may also be introduced into the plant cells by electroporation. (Fromm et al., Proc. Natl Acad. Sci. USA 82:5824, 1985). In this technique, plant protoplasts are electroporated in the presence of plasmids containing the gene construct. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and form plant callus.
All plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be transformed by the present invention so that whole plants are recovered which contain the transferred gene. It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugarcane, sugar beet, cotton, fruit and other trees, legumes and vegetables. Some suitable plants include, for example, species from the genera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersion, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hererocallis, Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Lolium, Zea, Triticum, Sorghum, and Datura.
Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts containing copies of the heterologous gene is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced from the protoplast suspension. These embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Shoots and roots normally develop simultaneously. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is fully reproducible and repeatable.
In some plant cell culture systems, the desired protein of the invention may be excreted or alternatively, the protein may be extracted from the whole plant. Where the desired protein of the invention is secreted into the medium, it may be collected. Alternatively, the embryos and embryoless-half seeds or other plant tissue may be mechanically disrupted to release any secreted protein between cells and tissues. The mixture may be suspended in a buffer solution to retrieve soluble proteins. Conventional protein isolation and purification methods will be then used to purify the recombinant protein. Parameters of time, temperature pH, oxygen, and volumes will be adjusted through routine methods to optimize expression and recovery of heterologous protein.
iv. Bacterial Systems
Bacterial expression techniques are known in the art. A bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site. A bacterial promoter may also have a second domain called an operator, that may overlap an adjacent RNA polymerase binding site at which RNA synthesis begins. The operator permits negative regulated (inducible) transcription, as a gene repressor protein may bind the operator and thereby inhibit transcription of a specific gene. Constitutive expression may occur in the absence of negative regulatory elements, such as the operator. In addition, positive regulation may be achieved by a gene activator protein binding sequence, which, if present is usually proximal (5′) to the RNA polymerase binding sequence. An example of a gene activator protein is the catabolite activator protein (CAP), which helps initiate transcription of the lac operon in Escherichia coli (E. coli) [Raibaud et al. (1984) Annu. Rev. Genet. 18:173]. Regulated expression may therefore be either positive or negative, thereby either enhancing or reducing transcription.
Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) [Chang et al. (1977) Nature 198:1056], and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp) [Goeddel et al. (1980) Nuc. Acids Res. 8:4057; Yelverton et al. (1981) Nucl. Acids Res. 9:731; U.S. Pat. No. 4,738,921; EP-A-0036776 and EP-A-0121775]. The g-laotamase (bla) promoter system [Weissmann (1981) “The cloning of interferon and other mistates.” In Interferon 3 (ed I. Gresser)], bacteriophage lambda PL [Shimatake et al. (1981) Nature 292:128] and T5 [U.S. Pat. No. 4,689,406] promoter systems also provide useful promoter sequences.
In addition, synthetic promoters which do not occur in nature also function as bacterial promoters. For example, transcription activation sequences of one bacterial or bacteriophage promoter may be joined with the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter [U.S. Pat. No. 4,551,433]. For example, the tac promoter is a hybrid trp-lac promoter comprised of both trp promoter and lac operon sequences that is regulated by the lac repressor [Amann et al. (1983) Gene 25:167; de Boer et al. (1983) Proc. Natl. Acad. Sci. 80:21]. Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. A naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes. The bacteriophage T7 RNA polymerase/promoter system is an example of a coupled promoter system [Studier et al. (1986) J. Mol. Biol. 189:113; Tabor et al. (1985) Proc Natl. Acad. Sci. 82:1074]. In addition, a hybrid promoter can also be comprised of a bacteriophage promoter and an E. coli operator region (EPO-A-0 267 851).
In addition to a functioning promoter sequence, an efficient ribosome binding site is also useful for the expression of foreign genes in prokaryotes. In E. coli, the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes an initiation codon (ATG) and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon [Shine et al. (1975) Nature 254:34). The SD sequence is thought to promote binding of mRNA to the ribosome by the pairing of bases between the SD sequence and the 3′ and of E. coli 16S rRNA [Steitz et al. (1979) “Genetic signals and nucleotide sequences in messenger RNA.” In Biological Regulation and Development: Gene Expression (ed. R. F. Goldberger)]. To express eukaryotic genes and prokaryotic genes with weak ribosome-binding site [Sambrook et al. (1989) “Expression of cloned genes in Escherichia coli.” In Molecular Cloning: A Laboratory Manual].
A DNA molecule may be expressed intracellularly. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide or by either in vivo on in vitro incubation with a bacterial methionine N-terminal peptidase (EPO-A-0 219 237).
Fusion proteins provide an alternative to direct expression. Usually, a DNA sequence encoding the N-terminal portion of an endogenous bacterial protein, or other stable protein, is fused to the 5′ end of heterologous coding sequences. Upon expression, this construct will provide a fusion of the two amino acid sequences. For example, the bacteriophage lambda cell gene can be linked at the 5′ terminus of a foreign gene and expressed in bacteria The resulting fusion protein preferably retains a site for a processing enzyme (factor Xa) to cleave the bacteriophage protein from the foreign gene [Nagai et al. (1984) Nature 309:810]. Fusion proteins can also be made with sequences from the lacZ [Jia et al. (1987) Gene 60:197], trpE (Allen et al. (1987) J. Biotechnol. 5:93; Makoff et al. (1989) J. Gen. Microbiol. 135:11], and Chey [EP-A-0 324 647] genes. The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site. Another example is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (eg. ubiquitin specific processing-protease) to cleave the ubiquitin from the foreign protein. Through this method, native foreign protein can be isolated [Miller et al. (1989) Bio/Technology 7:698].
Alternatively, foreign proteins can also be secreted from the cell by creating chimeric DNA molecules that encode a fusion protein comprised of a signal peptide sequence fragment that provides for secretion of the foreign protein in bacteria [U.S. Pat. No. 4,336,336]. The signal sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria). Preferably there are processing sites, which can be cleaved either in vivo or in vitro encoded between the signal peptide fragment and the foreign gene.
DNA encoding suitable signal sequences can be derived from genes for secreted bacterial proteins, such as the E. coli outer membrane protein gene (ompA) [Masui et al. (1983), in: Experimental Manipulation of Gene Expression; Ghrayeb et al. (1984) EMBO J. 3:2437] and the E. coli alkaline phosphatase signal sequence (phoA) [Oka et al. (1985) Proc. Natl. Acad. Sci. 82:7212]. As an additional example, the signal sequence of the alpha-amylase gene from various Bacillus strains can be used to secrete heterologous proteins from B. subtilis [Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 244 042].
Usually, transcription termination sequences recognized by bacteria are regulatory regions located 3′ to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Transcription termination sequences frequently include DNA sequences of about 50 nucleotides capable of forming stem loop structures that aid in terminating transcription. Examples include transcription termination sequences derived from genes with strong promoters, such as the trip gene in E. coli as well as other biosynthetic genes.
Usually, the above described components, comprising a promoter, signal sequence (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg. plasmids) capable of stable maintenance in a host, such as bacteria The replicon will have a replication system, thus allowing it to be maintained in a prokaryotic host either for expression or for cloning and amplification. In addition, a replicon may be either a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about 10 to about 150. A host containing a high copy number plasmid will preferably contain at least about 10, and more preferably at least about 20 plasmids. Either a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host.
Alternatively, the expression constructs can be integrated into the bacterial genome with an integrating vector. Integrating vectors usually contain at least one sequence homologous to the bacterial chromosome that allows the vector to integrate. Integrations appear to result from recombinations between homologous DNA in the vector and the bacterial chromosome. For example, integrating vectors constructed with DNA from various Bacillus strains integrate into the Bacillus chromosome (EP-A-0 127 328). Integrating vectors may also be comprised of bacteriophage or transposon sequences.
Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of bacterial strains that have been transformed. Selectable markers can be expressed in the bacterial host and may include genes which render bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), and tetracycline [Davies et al. (1978) Annu. Rev. Microbiol. 32:469]. Selectable markers may also include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways.
Alternatively, some of the above described components can be put together in transformation vectors. Transformation vectors are usually comprised of a selectable market that is either maintained in a replicon or developed into an integrating vector, as described above.
Expression and transformation vectors, either extra-chromosomal replicons or integrating vectors, have been developed for transformation into many bacteria For example, expression vectors have been developed for, inter alia, the following bacteria: Bacillus subtilis [Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541], Escherichia coli [Shimatake et al. (1981) Nature 292:128; Amann et al. (1985) Gene 40:183; Studier et al. (1986) J. Mol. Biol. 189:113; EP-A-0 036 776, EP-A-0 136 829 and EP-A-0 136 907], Streptococcus cremoris [Powell et al. (1988) Appl. Environ. Microbiol. 54:655]; Streptococcus lividans [Powell et al. (1988) Appl. Environ. Microbiol. 54:655], Streptomyces lividans [U.S. Pat. No. 4,745,056].
Methods of introducing exogenous DNA into bacterial hosts are well-known in the art, and usually include either the transformation of bacteria treated with CaCl₂or other agents, such as divalent cations and DMSO. DNA can also be introduced into bacterial cells by electroporation. Transformation procedures usually vary with the bacterial species to be transformed. See eg. [Masson et al. (1989) FEMS Microbiol. Lett. 60:273; Palva et al. (1982) Proc. Natl. Acad. Sci USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541, Bacillus], [Miller et al. (1988) Proc. Natl. Acad. Sci. 85:856; Wang et al. (1990) J. Bacteriol. 172:949, Campylobacter], [Cohen et al. (1973) Proc. Natl. Acad. Sci. 69:2110; Dower et al. (1988) Nucleic Acids Res. 16:6127; Kushner (1978) “An improved method for transformation of Escherichia coli with ColE1-derived plasmids. In Genetic Engineering: Proceedings of the International Symposium on Genetic Engineering (eds. H. W. Boyer and S. Nicosia); Mandel et al. (1970) J. Mol. Biol. 53:159; Taketo (1988) Biochim Biophys. Acta 949:318; Escherichia], [Chassy et al. (1987) FEMS Microbiol. Lett. 44:173 Lactobacillus]; [Fiedler et al. (1988) Anal. Biochem 170:38, Pseudomonas]; [Augustin et al. (1990) FEMS Microbiol. Lett. 66:203, Staphylococcus], [Barany et al. (1980) J. Bacteriol. 144:698; Harlander (1987) “Transformation of Streptococcus lactis by electroporation, in: Streptococcal Genetics (ed. J. Ferretti and R. Curtiss III); Perry et al. (1981) Infect Immun. 32:1295; Powell et al. (1988) Appl. Environ. Microbiol. 54:655; Somkuti et al. (1987) Proc. 4th Evr. Cong. Biotechnology 1:412, Streptococcus].
v. Yeast Expression
Yeast expression systems are also known to one of ordinary skill in the art. A yeast promoter is any DNA sequence capable of binding yeast RNA polymerase and initiating the downstream (3′) transcription of a coding sequence (eg. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5′ end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site (the “TATA Box”) and a transcription initiation site. A yeast promoter may also have a second domain called an upstream activator sequence (UAS), which, if present, is usually distal to the structural gene. The UAS permits regulated (inducible) expression. Constitutive expression occurs in the absence of a UAS. Regulated expression may be either positive or negative, thereby either enhancing or reducing transcription.
Yeast is a fermenting organism with an active metabolic pathway, therefore sequences encoding enzymes in the metabolic pathway provide particularly useful promoter sequences. Examples include alcohol dehydrogenase (ADH) (EP-A-0 284 044), enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, and pyruvate kinase (PyK) (EPO-A-0 329 203). The yeast PHO5 gene, encoding acid phosphatase, also provides useful promoter sequences [Myanohara et al. (1983) Proc. Natl. Acad. Sci. USA 80:1].
In addition, synthetic promoters which do not occur in nature also function as yeast promoters. For example, UAS sequences of one yeast promoter may be joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter. Examples of such hybrid promoters include the ADH regulatory sequence linked to the GAP transcription activation region (U.S. Pat. Nos. 4,876,197 and 4,880,734). Other examples of hybrid promoters include promoters which consist of the regulatory sequences of either the ADH2, GAL4, GAL10, OR PHO5 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK (EP-A-0 164 556). Furthermore, a yeast promoter can include naturally occurring promoters of non-yeast origin that have the ability to bind yeast RNA polymerase and initiate transcription. Examples of such promoters include, inter alia, [Cohen et al. (1980) Proc. Natl. Acad. Sci. USA 77:1078; Henikoff et al. (1981) Nature 283:835; Hollenberg et al. (1981) Curr. Topics Microbiol. Immunol. 96:119; Hollenberg et al. (1979) “The Expression of Bacterial Antibiotic Resistance Genes in the Yeast Saccharomyces cerevisiae,” in: Plasmids of Medical, Environmental and Commercial Importance (eds. K. N. Timmis and A. Puhler); Mercerau-Puigalon et al. (1980) Gene 11:163; Panthier et al. (1980) Curr. Genet. 2:109;].
A DNA molecule may be expressed intracellularly in yeast. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by in vitro incubation with cyanogen bromide.
Fusion proteins provide an alternative for yeast expression systems, as well as in mammalian, baculovirus, and bacterial expression systems. Usually, a DNA sequence encoding the N-terminal portion of an endogenous yeast protein, or other stable protein, is fused to the 5′ end of heterologous coding sequences. Upon expression, this construct will provide a fusion of the two amino acid sequences. For example, the yeast or human superoxide dismutase (SOD) gene, can be linked at the 5′ terminus of a foreign gene and expressed in yeast. The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site. See eg. EP-A-0 196 056. Another example is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (eg. ubiquitin-specific processing protease) to cleave the ubiquitin from the foreign protein. Through this method, therefore, native foreign protein can be isolated (eg. WO88/024066).
Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provide for secretion in yeast of the foreign protein. Preferably, there are processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vivo or in vitro. The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.
DNA encoding suitable signal sequences can be derived from genes for secreted yeast proteins, such as the yeast invertase gene (EP-A-0 012 873; JPO. 62,096,086) and the A-factor gene (U.S. Pat. No. 4,588,684). Alternatively, leaders of non-yeast origin, such as an interferon leader, exist that also provide for secretion in yeast (EP-A-0 060 057).
A preferred class of secretion leaders are those that employ a fragment of the yeast alpha-factor gene, which contains both a “pre” signal sequence, and a “pro” region. The types of alpha-factor fragments that can be employed include the full-length pre-pro alpha factor leader (about 83 amino acid residues) as well as truncated alpha-factor leaders (usually about 25 to about 50 amino acid residues) (U.S. Pat. Nos. 4,546,083 and 4,870,008; EP-A-0 324 274). Additional leaders employing an alpha-factor leader fragment that provides for secretion include hybrid alpha-factor leaders made with a presequence of a first yeast, but a pro-region from a second yeast alphafactor. (eg. see WO 89/02463.)
Usually, transcription termination sequences recognized by yeast are regulatory regions located 3′ to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Examples of transcription terminator sequence and other yeast-recognized termination sequences, such as those coding for glycolytic enzymes.
Usually, the above described components, comprising a promoter, leader (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (eg. plasmids) capable of stable maintenance in a host, such as yeast or bacteria. The replicon may have two replication systems, thus allowing it to be maintained, for example, in yeast for expression and in a prokaryotic host for cloning and amplification. Examples of such yeast-bacteria shuttle vectors include YEp24 [Botstein et al. (1979) Gene 8:17-24], pCl/1 [Brake et al. (1984) Proc. Natl. Acad. Sci USA 81:4642-4646], and YRp17 [Stinchcomb et al. (1982) J. Mol. Biol. 158:157]. In addition, a replicon may be either a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about 10 to about 150. A host containing a high copy number plasmid will preferably have at least about 10, and more preferably at least about 20. Enter a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host. See eg. Brake et al., supra.
Alternatively, the expression constructs can be integrated into the yeast genome with an integrating vector. Integrating vectors usually contain at least one sequence homologous to a yeast chromosome that allows the vector to integrate, and preferably contain two homologous sequences flanking the expression construct. Integrations appear to result from recombinations between homologous DNA in the vector and the yeast chromosome [Orr-Weaver et al. (1983) Methods in Enzymol. 101:228-245]. An integrating vector may be directed to a specific locus in yeast by selecting the appropriate homologous sequence for inclusion in the vector. See Orr-Weaver et al., supra. One or more expression construct may integrate, possibly affecting levels of recombinant protein produced [Rine et al. (1983) Proc. Natl. Acad. Sci. USA 80:6750]. The chromosomal sequences included in the vector can occur either as a single segment in the vector, which results in the integration of the entire vector, or two segments homologous to adjacent segments in the chromosome and flanking the expression construct in the vector, which can result in the stable integration of only the expression construct.
Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of yeast strains that have been transformed. Selectable markers may include biosynthetic genes that can be expressed in the yeast host, such as ADE2, HIS4, LEU2, TRP1, and ALG7, and the G418 resistance gene, which confer resistance in yeast cells to tunicamycin and G418, respectively. In addition, a suitable selectable marker may also provide yeast with the ability to grow in the presence of toxic compounds, such as metal. For example, the presence of CUP1 allows yeast to grow in the presence of copper ions [Butt et al. (1987) Microbiol, Rev. 51:351].
Alternatively, some of the above described components can be put together into transformation vectors. Transformation vectors are usually comprised of a selectable marker that is either maintained in a replicon or developed into an integrating vector, as described above.
Expression and transformation vectors, either extrachromosomal replicons or integrating vectors, have been developed for transformation into many yeasts. For example, expression vectors have been developed for, inter alia, the following yeasts: Candida albicans [Kurtz, et al. (1986) Mol. Cell. Biol. 6:142], Candida maltosa [Kunze, et al. (1985) J. Basic Microbiol. 25:141]. Hansenula polymorpha [Gleeson, et al. (1986) J. Gen. Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302], Kluyveromyces fragilis [Das, et al. (1984) J. Bacteriol. 158:1165], Kluyveromyces lactis [De Louvencourt et al. (1983) J. Bacteriol. 154:737; Van den Berg et al. (1990) Bio/Technology 8:135], Pichia guillerimondii [Kunze et al. (1985) J. Basic Microbiol. 25:141], Pichia pastoris [Cregg, et al. (1985) Mol. Cell. Biol. 5:3376; U.S. Pat. Nos. 4,837,148 and 4,929,555], Saccharomyces cerevisiae [Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75:1929; Ito et al. (1983) J. Bacteriol. 153:163], Schizosaccharomyces pombe [Beach and Nurse (1981) Nature 300:706], and Yarrowia lipolytica [Davidow, et al. (1985) Curr. Genet. 10:380471 Gaillardin, et al (1985) Curr. Genet. 10:49].
Methods of introducing exogenous DNA into yeast hosts are well-known in the art, and usually include either the transformation of spheroplasts or of intact yeast cells treated with alkali cations. Transformation procedures usually vary with the yeast species to be transformed. See eg. [Kurtz et al. (1986) Mol. Cell. Biol. 6:142; Kunze et al. (1985) J. Basic Microbiol. 25:141; Candida]; [Gleeson et al. (1986) J. Gen. Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302; Hansenula]; [Das et al. (1984) J. Bacteriol. 158:1165; De Louvencourt et al. (1983) J. Bacteriol. 154:1165; Van den Berg et al. (1990) Bio/Technology 8:135; Kluyveromyces]; [Cregg et al. (1985) Mol. Cell. Biol. 5:3376; Kunze et al. (1985) J. Basic Microbiol. 25:141; U.S. Pat. Nos. 4,837,148 and 4,929,555; Pichia]; [Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75;1929; Ito et al. (1983) J. Bacteriol. 153:163 Saccharomyces]; [Beach and Nurse (1981) Nature 300:706; Schizosaccharomyces]; [Davidow et al. (1985) Curr. Genet. 10:39; Gaillardin et al. (1985) Curr. Genet. 10:49; Yarrowia].
Antibodies
As used herein, the term “antibody” refers to a polypeptide or group of polypeptides composed of at least one antibody combining site. An “antibody combining site” is the three-dimensional binding space with an internal surface shape and charge distribution complementary to the features of an epitope of an antigen, which allows a binding of the antibody with the antigen. “Antibody” includes, for example, vertebrate antibodies, hybrid antibodies, chimeric antibodies, humanised antibodies, altered antibodies, univalent antibodies, Fab proteins, and single domain antibodies. Antibodies against the proteins of the invention are useful for affinity chromatography, immunoassays, and distinguishing/identifying Neisserial proteins.
Antibodies to the proteins of the invention, both polyclonal and monoclonal, may be prepared by conventional methods. In general, the protein is first used to immunize a suitable animal, preferably a mouse, rat, rabbit or goat. Rabbits and goats are preferred for the preparation of polyclonal sera due to the volume of serum obtainable, and the availability of labeled anti-rabbit and anti-goat antibodies. Immunization is generally performed by mixing or emulsifying the protein in saline, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally (generally subcutaneously or intramuscularly). A dose of 50-200 μg/injection is typically sufficient. Immunization is generally boosted 2-6 weeks later with one or more injections of the protein in saline, preferably using Freund's incomplete adjuvant. One may alternatively generate antibodies by in vitro immunization using methods known in the adt, which for the purposes of this invention is considered equivalent to in vivo immunization. Polyclonal antisera is obtained by bleeding the immunized animal into a glass or plastic container, incubating the blood at 25° C. for one hour, followed by incubating at 4° C. for 2-18 hours. The serum is recovered by centrifugation (eg. 1,000 g for 10 minutes). About 20-50 ml per bleed may be obtained from rabbits.
Monoclonal antibodies are prepared using the standard method of Kohler & Milstein [Nature (1975) 256:495-96], or a modification thereof. Typically, a mouse or rat is immunized as described above. However, rather than bleeding the animal to extract serum, the spleen (and optionally several large lymph nodes) is removed and dissociated into single cells. If desired, the spleen cells may be screened (after removal of nonspecifically adherent cells) by applying a cell suspension to a plate or well coated with the protein antigen. B-cells expressing membrane-bound immunoglobulin specific for the antigen bind to the plate, and are not rinsed away with the rest of the suspension. Resulting B-cells, or all dissociated spleen cells, are then induced to fuse with myeloma cells to form hybridomas, and are cultured in a selective medium (eg. hypoxanthine, aminopterin, thymidine medium, “HAT”). The resulting hybridomas are plated by limiting dilution, and are assayed for the production of antibodies which bind specifically to the immunizing antigen (and which do not bind to unrelated antigens). The selected MAb-secreting hybridomas are then cultured either in vitro (eg. in tissue culture bottles or hollow fiber reactors), or in vivo (as ascites in mice).
If desired, the antibodies (whether polyclonal or monoclonal) may be labeled using conventional techniques. Suitable labels include fluorophores, chromophores, radioactive atoms (particularly ³²P and ¹²⁵I), electron-dense reagents, enzymes, and ligands having specific binding partners. Enzymes are typically detected by their activity. For example, horseradish peroxidase is usually detected by its ability to convert 3,3′,5,5′-tetramethylbenzidine (TMB) to a blue pigment, quantifiable with a spectrophotometer. “Specific binding partner” refers to a protein capable of binding a ligand molecule with high specificity, as for example in the case of an antigen and a monoclonal antibody specific therefor. Other specific binding partners include biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand couples known in the art. It should be understood that the above description is not meant to categorize the various labels into distinct classes, as the same label may serve in several different modes. For example, ¹²⁵I may serve as a radioactive label or as an electron-dense reagent. HRP may serve as enzyme or as antigen for a MAb. Further, one may combine various labels for desired effect. For example, MAbs and avidin also require labels in the practice of this invention: thus, one might label a MAb with biotin, and detect its presence with avidin labeled with ¹²⁵I, or with an anti-biotin MAb labeled with HRP. Other permutations and possibilities will be readily apparent to those of ordinary skill in the art, and are considered as equivalents within the scope of the instant invention.
Pharmaceutical Compositions
Pharmaceutical compositions can comprise either polypeptides, antibodies, or nucleic acid of the invention. The pharmaceutical compositions will comprise a therapeutically effective amount of either polypeptides, antibodies, or polynucleotides of the claimed invention.
The term “therapeutically effective amount” as used herein refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect. The effect can be detected by, for example, chemical markers or antigen levels. Therapeutic effects also include reduction in physical symptoms, such as decreased body temperature. The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by routine experimentation and is within the judgement of the clinician.
For purposes of the present invention, an effective dose will be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the individual to which it is administered.
A pharmaceutical composition can also contain a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.
Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. Liposomes are included within the definition of a pharmaceutically acceptable carrier.
Delivery Methods
Once formulated, the compositions of the invention can be administered directly to the subject. The subjects to be treated can be animals; in particular, human subjects can be treated.
Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications (eg. see WO98/20734), needles, and gene guns or hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule.
Vaccines
Vaccines according to the invention may either be prophylactic (ie. to prevent infection) or therapeutic (ie. to treat disease after infection).
Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid, usually in combination with “pharmaceutically acceptable carriers,” which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. Additionally, these carriers may function as immunostimulating agents (“adjuvants”). Furthermore, the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, etc. pathogens.
Preferred adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc; (2) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59™ (WO 90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds. Powell & Newman, Plenum Press 1995), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP (see below) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) Ribi™ adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); (3) saponin adjuvants, such as Stimulon™ (Cambridge Bioscience, Worcester, Mass.) may be used or particles generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (5) cytokines, such as interleukins (eg. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (eg. gamma interferon), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc; and (6) other substances that act as immunostimulating agents to enhance the effectiveness of the composition. Alum and MF59™ are preferred.
As mentioned above, muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)ethylamine (MTP-PE), etc.
The immunogenic compositions (eg. the immunising antigen/immunogen/polypeptide/protein/nucleic acid, pharmaceutically acceptable carrier, and adjuvant) typically will contain diluents, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
Typically, the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above under pharmaceutically acceptable carriers.
Immunogenic compositions used as vaccines comprise an immunologically effective amount of the antigenic or immunogenic polypeptides, as well as any other of the above-mentioned components, as needed. By “immunologically effective amount”, it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated (eg. nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctors assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
The immunogenic compositions are conventionally administered parenterally, eg. by injection, either subcutaneously, intramuscularly, or transdermally/transcutaneously (eg. WO98/20734). Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories, and transdermal applications. Dosage treatment may be a single dose schedule or a multiple dose schedule. The vaccine may be administered in conjunction with other immunoregulatory agents.
As an alternative to protein-based vaccines, DNA vaccination may be employed [eg. Robinson & Torres (1997) Seminars in Immunology 9:271-283; Donnelly et al. (1997) Annu Rev Immunol 15:617-648; see later herein].
Gene Delivery Vehicles
Gene therapy vehicles for delivery of constructs including a coding sequence of a therapeutic of the invention, to be delivered to the mammal for expression in the mammal, can be administered either locally or systemically. These constructs can utilize viral or non-viral vector approaches in in vivo or ex vivo modality. Expression of such coding sequence can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence in vivo can be either constitutive or regulated.
The invention includes gene delivery vehicles capable of expressing the contemplated nucleic acid sequences. The gene delivery vehicle is preferably a viral vector and, more preferably, a retroviral, adenoviral, adeno-associated viral (AAV), herpes viral, or alphavirus vector. The viral vector can also be an astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picomavirus, poxvirus, or togavirus viral vector. See generally, Jolly (1994) Cancer Gene Therapy 1:51-64; Kimura (1994) Human Gene Therapy 5:845-852; Connelly (1995) Human Gene Therapy 6:185-193; and Kaplitt (1994) Nature Genetics 6:148-153.
Retroviral vectors are well known in the art and we contemplate that any retroviral gene therapy vector is employable in the invention, including B, C and D type retroviruses, xenotropic retroviruses (for example, NZB-X1, NZB-X2 and NZB9-1 (see O'Neill (1985) J. Virol. 53:160) polytropic retrovirus eg. MCF and MCF-MLV (see Kelly (1983) J. Virol. 45:291), spumaviruses and lentiviruses. See RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985.
Portions of the retroviral gene therapy vector may be derived from different retroviruses. For example, retrovector LTRs may be derived from a Murine Sarcoma Virus, a tRNA binding site from a Rous Sarcoma Virus, a packaging signal from a Murine Leukemia Virus, and an origin of second strand synthesis from an Avian Leukosis Virus.
These recombinant retroviral vectors may be used to generate transduction competent retroviral vector particles by introducing them into appropriate packaging cell lines (see U.S. Pat. No. 5,591,624). Retrovirus vectors can be constructed for site-specific integration into host cell DNA by incorporation of a chimeric integrase enzyme into the retroviral particle (see WO96/37626). It is preferable that the recombinant viral vector is a replication defective recombinant virus.
Packaging cell lines suitable for use with the above-described retrovirus vectors are well known in the art, are readily prepared (see WO95/30763 and WO92/05266), and can be used to create producer cell lines (also termed vector cell lines or “VCLs”) for the production of recombinant vector particles. Preferably, the packaging cell lines are made from human parent cells (eg. HT1080 cells) or mink parent cell lines, which eliminates inactivation in human serum.
Preferred retroviruses for the construction of retroviral gene therapy vectors include Avian Leukosis Virus, Bovine Leukemia, Virus, Murine Leukemia Virus, Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis Virus and Rous Sarcoma Virus. Particularly preferred Murine Leukemia Viruses include 4070A and 1504A (Hartley and Rowe (1976) J Virol 19:19-25), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245), Graffi, Gross (ATCC Nol VR-590), Kirsten, Harvey Sarcoma Virus and Rauscher (ATCC No. VR-998) and Moloney Murine Leukemia Virus (ATCC No. VR-190). Such retroviruses may be obtained from depositories or collections such as the American Type Culture Collection (“ATCC”) in Rockville, Md. or isolated from known sources using commonly available techniques.
Exemplary known retroviral gene therapy vectors employable in this invention include those described in patent applications GB2200651, EP0415731, EP0345242, EP0334301, WO89/02468; WO89/05349, WO89/09271, WO90/02806, WO90/07936, WO94/03622, WO93/25698, WO93/25234, WO93/11230, WO93/10218, WO91/02805, WO91/02825, WO95/07994, U.S. Pat. No. 5,219,740, U.S. Pat. No. 4,405,712, U.S. Pat. No. 4,861,719, U.S. Pat. No. 4,980,289, U.S. Pat. No. 4,777,127, U.S. Pat. No. 5,591,624. See also Vile (1993) Cancer Res 53:3860-3864; Vile (1993) Cancer Res 53:962-967; Rain (1993) Cancer Res 53 (1993) 83-88; Takamiya (1992) J Neurosci Res 33:493-503; Baba (1993) J Neurosurg 79:729-735; Mann (1983) Cell 33:153; Cane (1984) Proc Natl Acad Sci 81:6349; and Miller (1990) Human Gene Therapy 1.
Human adenoviral gene therapy vectors are also known in the art and employable in this invention. See, for example, Berkner (1988) Biotechniques 6:616 and Rosenfeld (1991) Science 252:431, and WO93/07283, WO93/06223, and WO93/07282. Exemplary known adenoviral gene therapy vectors employable in this invention include those described in the above referenced documents and in WO94/12649, WO93/03769, WO93/19191, WO94/28938, WO95/11984, WO95/00655, WO95/27071, WO95/29993, WO95/34671, WO96/05320, WO94/08026, WO94/11506, WO93/06223, WO94/24299, WO95/14102, WO95/24297, WO95/02697, WO94/28152, WO94/24299, WO95/09241, WO95/25807, WO95/05835, WO94/18922 and WO95/09654. Alternatively, administration of DNA linked to killed adenovirus as described in Curiel (1992) Hum. Gene Ther. 3:147-154 may be employed. The gene delivery vehicles of the invention also include adenovirus associated virus (AAV) vectors. Leading and preferred examples of such vectors for use in this invention are the AAV-2 based vectors disclosed in Srivastava, WO93/09239. Most preferred AAV vectors comprise the two AAV inverted terminal repeats in which the native D-sequences are modified by substitution of nucleotides, such that at least 5 native nucleotides and up to 18 native nucleotides, preferably at least 10 native nucleotides up to 18 native nucleotides, most preferably 10 native nucleotides are retained and the remaining nucleotides of the D-sequence are deleted or replaced with non-native nucleotides. The native D-sequences of the AAV inverted terminal repeats are sequences of 20 consecutive nucleotides in each AAV inverted terminal repeat (ie. there is one sequence at each end) which are not involved in HP formation. The non-native replacement nucleotide may be any nucleotide other than the nucleotide found in the native D-sequence in the same position. Other employable exemplary AAV vectors are pWP-19, pWN-1, both of which are disclosed in Nahreini (1993) Gene 124:257-262. Another example of such an AAV vector is psub201 (see Samulski (1987) J. Virol. 61:3096). Another exemplary AAV vector is the Double-D ITR vector. Construction of the Double-D ITR vector is disclosed in U.S. Pat. No. 5,478,745. Still other vectors are those disclosed in Carter U.S. Pat. No. 4,797,368 and Muzyczka U.S. Pat. No. 5,139,941, Chartejee U.S. Pat. No. 5,474,935, and Kotin WO94/288157. Yet a further example of an AAV vector employable in this invention is SSV9AFABTKneo, which contains the AFP enhancer and albumin promoter and directs expression predominantly in the liver. Its structure and construction are disclosed in Su (1996) Human Gene Therapy 7:463-470. Additional AAV gene therapy vectors are described in U.S. Pat. No. 5,354,678, U.S. Pat. No. 5,173,414, U.S. Pat. No. 5,139,941, and U.S. Pat. No. 5,252,479.
The gene therapy vectors of the invention also include herpes vectors. Leading and preferred examples are herpes simplex virus vectors containing a sequence encoding a thymidine kinase polypeptide such as those disclosed in U.S. Pat. No. 5,288,641 and EP0176170 (Roizman). Additional exemplary herpes simplex virus vectors include HFEM/ICP6-LacZ disclosed in WO95/04139 (Wistar Institute), pHSVlac described in Geller (1988) Science 241:1667-1669 and in WO90/09441 and WO92/07945, HSV Us3::pgC-lacZ described in Fink (1992) Human Gene Therapy 3:11-19 and HSV 7134, 2 RH 105 and GAL4 described in EP 0453242 (Breakefield), and those deposited with the ATCC as accession numbers ATCC VR-977 and ATCC VR-260.
Also contemplated are alpha virus gene therapy vectors that can be employed in this invention. Preferred alpha virus vectors are Sindbis viruses vectors. Togaviruses, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Middleberg virus (ATCC VR-370), Ross River virus (ATCC VR-373; ATCC VR-1246), Venezuelan equine encephalitis virus (ATCC VR923; ATCC VR-1250; ATCC VR-1249; ATCC VR-532), and those described in U.S. Pat. Nos. 5,091,309, 5,217,879, and WO92/10578. More particularly, those alpha virus vectors described in U.S. Ser. No. 08/405,627, filed Mar. 15, 1995, WO94/21792, WO92/10578, WO95/07994, U.S. Pat. No. 5,091,309 and U.S. Pat. No. 5,217,879 are employable. Such alpha viruses may be obtained from depositories or collections such as the ATCC in Rockville, Md. or isolated from known sources using commonly available techniques. Preferably, alphavirus vectors with reduced cytotoxicity are used (see U.S. Ser. No. 08/679,640).
DNA vector systems such as eukaryotic layered expression systems are also useful for expressing the nucleic acids of the invention. See WO95/07994 for a detailed description of eukaryotic layered expression systems. Preferably, the eukaryotic layered expression systems of the invention are derived from alphavirus vectors and most preferably from Sindbis viral vectors.
Other viral vectors suitable for use in the present invention include those derived from poliovirus, for example ATCC VR-58 and those described in Evans, Nature 339 (1989) 385 and Sabin (1973) J. Biol. Standardization 1:115; rhinovirus, for example ATCC VR-1110 and those described in Arnold (1990) J Cell Biochem L401; pox viruses such as canary pox virus or vaccinia virus, for example ATCC VR-111 and ATCC VR-2010 and those described in Fisher-Hoch (1989) Proc Natl Acad Sci 86:317; Flexner (1989) Ann NY Acad Sci 569:86, Flexner (1990) Vaccine 8:17; in U.S. Pat. No. 4,603,112 and U.S. Pat. No. 4,769,330 and WO89/01973; SV40 virus, for example ATCC VR-305 and those described in Mulligan (1979) Nature 277:108 and Madzak (1992) J Gen Virol 73:1533; influenza virus, for example ATCC VR-797 and recombinant influenza viruses made employing reverse genetics techniques as described in U.S. Pat. No. 5,166,057 and in Enami (1990) Proc Nail Acad Sci 87:3802-3805; Enami & Palese (1991) J Virol 65:2711-2713 and Luytjes (1989) Cell 59:110, (see also McMichael (1983) NEJ Med 309:13, and Yap (1978) Nature 273:238 and Nature (1979) 277:108); human immunodeficiency virus as described in EP-0386882 and in Buchschacher (1992) J. Virol. 66:2731; measles virus, for example ATCC VR-67 and VR-1247 and those described in EP-0440219; Aura virus, for example ATCC VR-368; Bebaru virus, for example ATCC VR-600 and ATCC VR-1240; Cabassou virus, for example ATCC VR-922; Chikungunya virus, for example ATCC VR-64 and ATCC VR-1241; Fort Morgan Virus, for example ATCC VR-924; Getah virus, for example ATCC VR-369 and ATCC VR-1243; Kyzylagach virus, for example ATCC VR-927; Mayaro virus, for example ATCC VR-66; Mucambo virus, for example ATCC VR-580 and ATCC VR-1244; Ndumu virus, for example ATCC VR-371; Pixuna virus, for example ATCC VR-372 and ATCC VR-1245; Tonate virus, for example ATCC VR-925; Triniti virus, for example ATCC VR-469; Una virus, for example ATCC VR-374; Whataroa virus, for example ATCC VR-926; Y-62-33 virus, for example ATCC VR-375; O'Nyong virus, Eastern encephalitis virus, for example ATCC VR-65 and ATCC VR-1242; Western encephalitis virus, for example ATCC VR-70, ATCC VR-1251, ATCC VR-622 and ATCC VR-1252; and coronavirus, for example ATCC VR-740 and those described in Hamre (1966) Proc Soc Exp Biol Med 121:190.
Delivery of the compositions of this invention into cells is not limited to the above mentioned viral vectors. Other delivery methods and media may be employed such as, for example, nucleic acid expression vectors, polycationic condensed DNA linked or unlinked to killed adenovirus alone, for example see U.S. Ser. No. 08/366,787, filed Dec. 30, 1994 and Curiel (1992) Hum Gene Ther 3:147-154 ligand linked DNA, for example see Wu (1989) J Biol Chem 264:16985-16987, eucaryotic cell delivery vehicles cells, for example see U.S. Ser. No. 08/240,030, filed May 9, 1994, and U.S. Ser. No. 08/404,796, deposition of photopolymerized hydrogel materials, hand-held gene transfer particle gun, as described in U.S. Pat. No. 5,149,655, ionizing radiation as described in U.S. Pat. No. 5,206,152 and in WO92/11033, nucleic charge neutralization or fusion with cell membranes. Additional approaches are described in Philip (1994) Mol Cell Biol 14:2411-2418 and in Woffendin (1994) Proc Natl Acad Sci 91:1581-1585.
Particle mediated gene transfer may be employed, for example see U.S. Ser. No. 60/023,867. Briefly, the sequence can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, as described in Wu & Wu (1987) J. Biol. Chem. 262:4429-4432, insulin as described in Hucked (1990) Biochem Pharmacol 40:253-263, galactose as described in Plank (1992) Bioconjugate Chem 3:533-539, lactose or transferrin.
Naked DNA may also be employed. Exemplary naked DNA introduction methods are described in WO 90/11092 and U.S. Pat. No. 5,580,859. Uptake efficiency may be improved using biodegradable latex beads. DNA coated latex beads are efficiently transported into cells after endocytosis initiation by the beads. The method may be improved further by treatment of the beads to increase hydrophobicity and thereby facilitate disruption of the endosome and release of the DNA into the cytoplasm.
Liposomes that can act as gene delivery vehicles are described in U.S. Pat. No. 5,422,120, WO95/13796, WO94/23697, WO91/14445 and EP-524,968. As described in U.S. S No. 60/023,867, on non-viral delivery, the nucleic acid sequences encoding a polypeptide can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then be incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, insulin, galactose, lactose, or transferrin. Other delivery systems include the use of liposomes to encapsulate DNA comprising the gene under the control of a variety of tissue-specific or ubiquitously-active promoters. Further non-viral delivery suitable for use includes mechanical delivery systems such as the approach described in Woffendin et al (1994) Proc. Natl. Acad. Sci. USA 91(24):11581-11585. Moreover, the coding sequence and the product of expression of such can be delivered through deposition of photopolymerized hydrogel materials. Other conventional methods for gene delivery that can be used for delivery of the coding sequence include, for example, use of hand-held gene transfer particle gun, as described in U.S. Pat. No. 5,149,655; use of ionizing radiation for activating transferred gene, as described in U.S. Pat. No. 5,206,152 and WO92/11033
Exemplary liposome and polycationic gene delivery vehicles are those described in U.S. Pat. Nos. 5,422,120 and 4,762,915; in WO 95/13796; WO94/23697, and WO91/14445; in EP-0524968; and in Stryer, Biochemistry, pages 236-240 (1975) W.H. Freeman, San Francisco; Szoka (1980) Biochem Biophys Acta 600:1; Bayer (1979) Biochem Biophys Acta 550:464; Rivnay (1987) Meth Enzymol 149:119; Wang (1987) Proc Natl Acad Sci 84:7851; Plant (1989) Anal Biochem 176:420.
A polynucleotide composition can comprises therapeutically effective amount of a gene therapy vehicle, as the term is defined above. For purposes of the present invention, an effective dose will be from about 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNA constructs in the individual to which it is administered.
Delivery Methods
Once formulated, the polynucleotide compositions of the invention can be administered (1) directly to the subject; (2) delivered ex vivo, to cells derived from the subject; or (3) in vitro for expression of recombinant proteins. The subjects to be treated can be mammals or birds. Also, human subjects can be treated.
Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications (eg. see WO98/20734), needles, and gene guns or hyposprays. Dosage treatment may be a single dose schedule or a multiple dose schedule.
Methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and described in eg. WO93/14778. Examples of cells useful in ex vivo applications include, for example, stem cells, particularly hematopoetic, lymph cells, macrophages, dendritic cells, or tumor cells.
Generally, delivery of nucleic acids for both ex vivo and in vitro applications can be accomplished by the following procedures, for example, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei, all well known in the art.
Polynucleotide and Polypeptide Pharmaceutical Compositions
In addition to the pharmaceutically acceptable carriers and salts described above, the following additional agents can be used with polynucleotide and/or polypeptide compositions.
A. Polypeptides
One example are polypeptides which include, without limitation: asioloorosomucoid (ASOR); transferrin; asialoglycoproteins; antibodies; antibody fragments; ferritin; interleukins; interferons, granulocyte, macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), stem cell factor and erythropoietin. Viral antigens, such as envelope proteins, can also be used. Also, proteins from other invasive organisms, such as the 17 amino acid peptide from the circumsporozoite protein of plasmodium falciparum known as RII.
B. Hormones, Vitamins, etc.
Other groups that can be included are, for example: hormones, steroids, androgens, estrogens, thyroid hormone, or vitamins, folic acid.
C. Polyalkylenes, Polysaccharides, etc.
Also, polyalkylene glycol can be included with the desired polynucleotides/polypeptides. In a preferred embodiment, the polyalkylene glycol is polyethlylene glycol. In addition, mono-, di-, or polysaccharides can be included. In a preferred embodiment of this aspect, the polysaccharide is dextran or DEAE-dextran. Also, chitosan and poly(lactide-co-glycolide)
D. Lipids, and Liposomes
The desired polynucleotide/polypeptide can also be encapsulated in lipids or packaged in liposomes prior to delivery to the subject or to cells derived therefrom.
Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid. The ratio of condensed polynucleotide to lipid preparation can vary but will generally be around 1:1 (mg DNA:micromoles lipid), or more of lipid. For a review of the use of liposomes as carriers for delivery of nucleic acids, see, Hug and Sleight (1991) Biochim. Biophys. Acta. 1097:1-17; Straubinger (1983) Meth. Enzymol. 101:512-527.
Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Felgner (1987) Proc. Natl. Acad. Sci. USA 84:7413-7416); mRNA (Malone (1989) Proc. Natl. Acad. Sci. USA 86:6077-6081); and purified transcription factors (Debs (1990) J. Biol. Chem. 265:10189-10192), in functional form.
Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner supra). Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, eg. Szoka (1978) Proc. Nail. Acad. Sci. USA 75:41944198; WO90/11092 for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.
Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, Ala.), or can be easily prepared using readily available materials. Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.
The liposomes can comprise multilammelar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). The various liposome-nucleic acid complexes are prepared using methods known in the art See eg. Straubinger (1983) Meth. Immunol. 101:512-527; Szoka (1978) Proc. Natl. Acad. Sci. USA 75:4194-4198; Papahadjopoulos (1975) Biochim. Biophys. Acta 394:483; Wilson (1979) Cell 17:77); Deamer & Bangham (1976) Biochim. Biophys. Acia 443:629; Ostro (1977) Biochem. Biophys. Res. Commun. 76:836; Fraley (1979) Proc. Natl. Acad. Sci. USA 76:3348); Enoch & Strittmatter (1979) Proc. Natl. Acad. Sci. USA 76:145; Fraley (1980) J. Biol. Chem. (1980) 255:10431; Szoka & Papahadjopoulos (1978) Proc. Natl. Acad. Sci. USA 75:145; and Schaefer-Ridder (1982) Science 215:166.
E. Lipoproteins
In addition, lipoproteins can be included with the polynucleotide/polypeptide to be delivered. Examples of lipoproteins to be utilized include: chylomicrons, HDL, DL, LDL, and VLDL. Mutants, fragments, or fusions of these proteins can also be used. Also, modifications of naturally occurring lipoproteins can be used, such as acetylated LDL. These lipoproteins can target the delivery of polynucleotides to cells expressing lipoprotein receptors. Preferably, if lipoproteins are including with the polynucleotide to be delivered, no other targeting ligand is included in the composition.
Naturally occurring lipoproteins comprise a lipid and a protein portion. The protein portion are known as apoproteins. At the present, apoproteins A, B, C, D, and E have been isolated and identified. At least two of these contain several proteins, designated by Roman numerals, AI, AII, AIV; CI, CII, CIII.
A lipoprotein can comprise more than one apoprotein. For example, naturally occurring chylomicrons comprises of A, B, C, and E, over time these lipoproteins lose A and acquire C and E apoproteins. VLDL comprises A, B, C, and E apoproteins, LDL comprises apoprotein B; and HDL comprises apoproteins A, C, and E.
The amino acid of these apoproteins are known and are described in, for example, Breslow (1985) Annu Rev. Biochem 54:699; Law (1986) Adv. Exp Med. Biol. 151:162; Chen (1986) J Biol Chem 261:12918; Kane (1980) Proc Natl Acad Sci USA 77:2465; and Utermann (1984) Hum Genet 65:232.
Lipoproteins contain a variety of lipids including, triglycerides, cholesterol (free and esters), and phospholipids. The composition of the lipids varies in naturally occurring lipoproteins. For example, chylomicrons comprise mainly triglycerides. A more detailed description of the lipid content of naturally occurring lipoproteins can be found, for example, in Meth. Enzymol. 128 (1986). The composition of the lipids are chosen to aid in conformation of the apoprotein for receptor binding activity. The composition of lipids can also be chosen to facilitate hydrophobic interaction and association with the polynucleotide binding molecule.
Naturally occurring lipoproteins can be isolated from serum by ultracentrifugation, for instance. Such methods are described in Meth. Enzymol. (supra); Pitas (1980) J. Biochem. 255:5454-5460 and Mahey (1979) J. Clin. Invest 64:743-750. Lipoproteins can also be produced by in vitro or recombinant methods by expression of the apoprotein genes in a desired host cell. See, for example, Atkinson (1986) Annu Rev Biophys Chem 15:403 and Radding (1958) Biochim Biophys Acia 30: 443. Lipoproteins can also be purchased from commercial suppliers, such as Biomedical Techniologies, Inc., Stoughton, Mass., USA. Further description of lipoproteins can be found in Zuckermann et al. PCT/US97/14465.
F. Polycationic Agents
Polycationic agents can be included, with or without lipoprotein, in a composition with the desired polynucleotide/polypeptide to be delivered.
Polycationic agents, typically, exhibit a net positive charge at physiological relevant pH and are capable of neutralizing the electrical charge of nucleic acids to facilitate delivery to a desired location. These agents have both in vitro, ex vivo, and in vivo applications. Polycationic agents can be used to deliver nucleic acids to a living subject either intramuscularly, subcutaneously, etc.
The following are examples of useful polypeptides as polycationic agents: polylysine, polyarginine, polyornithine, and protamine. Other examples include histones, protamines, human serum albumin, DNA binding proteins, non-histone chromosomal proteins, coat proteins from DNA viruses, such as (X174, transcriptional factors also contain domains that bind DNA and therefore may be useful as nucleic aid condensing agents. Briefly, transcriptional factors such as C/CEBP, c-jun, c-fos, AP-1, AP-2, AP-3, CPF, Prot-1, Sp-1, Oct-1, Oct-2, CREP, and TFIID contain basic domains that bind DNA sequences.
Organic polycationic agents include: spermine, spermidine, and purtrescine.
The dimensions and of the physical properties of a polycationic agent can be extrapolated from the list above, to construct other polypeptide polycationic agents or to produce synthetic polycationic agents.
Synthetic polycationic agents which are useful include, for example, DEAE-dextran, polybrene. Lipofectin™, and lipofectAMINE™ are monomers that form polycationic complexes when combined with polynucleotides/polypeptides.
Immunodiagnostic Assays
Neisserial antigens of the invention can be used in immunoassays to detect antibody levels (or, conversely, anti-Neisserial antibodies can be used to detect antigen levels). Immunoassays based on well defined, recombinant antigens can be developed to replace invasive diagnostics methods. Antibodies to Neisserial proteins within biological samples, including for example, blood or serum samples, can be detected. Design of the immunoassays is subject to a great deal of variation, and a variety of these are known in the art. Protocols for the immunoassay may be based, for example, upon competition, or direct reaction, or sandwich type assays. Protocols may also, for example, use solid supports, or may be by immunoprecipitation. Most assays involve the use of labeled antibody or polypeptide; the labels may be, for example, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays which amplify the signals from the probe are also known; examples of which are assays which utilize biotin and avidin, and enzyme-labeled and mediated immunoassays, such as ELISA assays.
Kits suitable for immunodiagnosis and containing the appropriate labeled reagents are constructed by packaging the appropriate materials, including the compositions of the invention, in suitable containers, along with the remaining reagents and materials (for example, suitable buffers, salt solutions, etc.) required for the conduct of the assay, as well as suitable set of assay instructions.
Nucleic Acid Hybridisation
“Hybridization” refers to the association of two nucleic acid sequences to one another by hydrogen bonding. Typically, one sequence will be fixed to a solid support and the other will be free in solution. Then, the two sequences will be placed in contact with one another under conditions that favor hydrogen bonding. Factors that affect this bonding include: the type and volume of solvent; reaction temperature; time of hybridization; agitation; agents to block the non-specific attachment of the liquid phase sequence to the solid support (Denhardt's reagent or BLOTTO); concentration of the sequences; use of compounds to increase the rate of association of sequences (dextran sulfate or polyethylene glycol); and the stringency of the washing conditions following hybridization. See Sambrook et al. [supra] Volume 2, chapter 9, pages 9.47 to 9.57.
“Stringency” refers to conditions in a hybridization reaction that favor association of very similar sequences over sequences that differ. For example, the combination of temperature and salt concentration should be chosen that is approximately 120 to 200° C. below the calculated Tm of the hybrid under study. The temperature and salt conditions can often be determined empirically in preliminary experiments in which samples of genomic DNA immobilized on filters are hybridized to the sequence of interest and then washed under conditions of different stringencies. See Sambrook et al. at page 9.50.
Variables to consider when performing, for example, a Southern blot are (1) the complexity of the DNA being blotted and (2) the homology between the probe and the sequences being detected. The total amount of the fragment(s) to be studied can vary a magnitude of 10, from 0.1 to 1 g for a plasmid or phage digest to 10⁻⁹to 10⁻⁸g for a single copy gene in a highly complex eukaryotic genome. For lower complexity polynucleotides, substantially shorter blotting, hybridization, and exposure times, a smaller amount of starting polynucleotides, and lower specific activity of probes can be used. For example, a single-copy yeast gene can be detected with an exposure time of only 1 hour starting with 1 μg of yeast DNA, blotting for two hours, and hybridizing for 4-8 hours with a probe of 10⁸cpm/μg. For a single-copy mammalian gene a conservative approach would start with 10 μg of DNA, blot overnight, and hybridize overnight in the presence of 10% dextran sulfate using a probe of greater than 10⁸cpm/μg, resulting in an exposure time of 24 hours.
Several factors can affect the melting temperature (Tm) of a DNA-DNA hybrid between the probe and the fragment of interest, and consequently, the appropriate conditions for hybridization and washing. In many cases the probe is not 100% homologous to the fragment. Other commonly encountered variables include the length and total G+C content of the hybridizing sequences and the ionic strength and formamide content of the hybridization buffer. The effects of all of these factors can be approximated by a single equation:
Tm—81+16.6(log₁₀ Ci)+0.4[% (G+C)]−0.6(% formamide)-600/n−1.5(% mismatch).
where Ci is the salt concentration (monovalent ions) and n is the length of the hybrid in base pairs (slightly modified from Meinkoth & Wahl (1984) Anal. Biochem. 138: 267-284).
In designing a hybridization experiment, some factors affecting nucleic acid hybridization can be conveniently altered. The temperature of the hybridization and washes and the salt concentration during the washes are the simplest to adjust. As the temperature of the hybridization increases (ie. stringency), it becomes less likely for hybridization to occur between strands that are nonhomologous, and as a result, background decreases. If the radiolabeled probe is not completely homologous with the immobilized fragment (as is frequently the case in gene family and interspecies hybridization experiments), the hybridization temperature must be reduced, and background will increase. The temperature of the washes affects the intensity of the hybridizing band and the degree of background in a similar manner. The stringency of the washes is also increased with decreasing salt concentrations.
In general, convenient hybridization temperatures in the presence of 50% formamide are 42° C. for a probe with is 95% to 100% homologous to the target fragment, 37° C. for 90% to 95% homology, and 32° C. for 85% to 90% homology. For lower homologies, formamide content should be lowered and temperature adjusted accordingly, using the equation above. If the homology between the probe and the target fragment are not known, the simplest approach is to start with both hybridization and wash conditions which are nonstringent. If non-specific bands or high background are observed after autoradiography, the filter can be washed at high stringency and reexposed. If the time required for exposure makes this approach impractical, several hybridization and/or washing stringencies should be tested in parallel.
Nucleic Acid Probe Assays
Methods such as PCR, branched DNA probe assays, or blotting techniques utilizing nucleic acid probes according to the invention can determine the presence of cDNA or mRNA. A probe is said to “hybridize” with a sequence of the invention if it can form a duplex or double stranded complex, which is stable enough to be detected.
The nucleic acid probes will hybridize to the Neisserial nucleotide sequences of the invention (including both sense and antisense strands). Though many different nucleotide sequences will encode the amino acid sequence, the native Neisserial sequence is preferred because it is the actual sequence present in cells. mRNA represents a coding sequence and so a probe should be complementary to the coding sequence; single-stranded cDNA is complementary to mRNA, and so a cDNA probe should be complementary to the non-coding sequence.
The probe sequence need not be identical to the Neisserial sequence (or its complement)—some variation in the sequence and length can lead to increased assay sensitivity if the nucleic acid probe can form a duplex with target nucleotides, which can be detected. Also, the nucleic acid probe can include additional nucleotides to stabilize the formed duplex. Additional Neisserial sequence may also be helpful as a label to detect the formed duplex. For example, a non-complementary nucleotide sequence may be attached to the 5′ end of the probe, with the remainder of the probe sequence being complementary to a Neisserial sequence. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the a Neisserial sequence in order to hybridize therewith and thereby form a duplex which can be detected.
The exact length and sequence of the probe will depend on the hybridization conditions, such as temperature, salt condition and the like. For example, for diagnostic applications, depending on the complexity of the analyte sequence, the nucleic acid probe typically contains at least 10-20 nucleotides, preferably 15-25, and more preferably at least 30 nucleotides, although it may be shorter than this. Short primers generally require cooler temperatures to form sufficiently stable hybrid complexes with the template.
Probes may be produced by synthetic procedures, such as the triester method of Matteucci et al. [J. Am. Chem. Soc. (1981) 103:3185], or according to Urdea et al. [Proc. Natl. Acad. Sci. USA (1983) 80: 7461], or using commercially available automated oligonucleotide synthesizers.
The chemical nature of the probe can be selected according to preference. For certain applications, DNA or RNA are appropriate. For other applications, modifications may be incorporated eg. backbone modifications, such as phosphorothioates or methylphosphonates, can be used to increase in vivo half-life, alter RNA affinity, increase nuclease resistance etc. [eg. see Agrawal & Iyer (1995) Curr Opin Biotechnol 6:12-19; Agrawal (1996) TIBTECH 14:376-387]; analogues such as peptide nucleic acids may also be used [eg. see Corey (1997) TIBTECH 15:224-229; Buchardt et al. (1993) TIBTECH 11:384-386].
Alternatively, the polymerase chain reaction (PCR) is another well-known means for detecting small amounts of target nucleic acids. The assay is described in: Mullis et al. [Meth. Enzymol. (1987) 155: 335-350]; U.S. Pat. Nos. 4,683,195 and 4,683,202. Two “primer” nucleotides hybridize with the target nucleic acids and are used to prime the reaction. The primers can comprise sequence that does not hybridize to the sequence of the amplification target (or its complement) to aid with duplex stability or, for example, to incorporate a convenient restriction site. Typically, such sequence will flank the desired Neisserial sequence.
A thermostable polymerase creases copies of target nucleic acids from the primers using the original target nucleic acids as a template. After a threshold amount of target nucleic acids are generated by the polymerase, they can be detected by more traditional methods, such as Southern blots. When using the Southern blot method, the labelled probe will hybridize to the Neisserial sequence (or its complement).
Also, mRNA or cDNA can be detected by traditional blotting techniques described in Sambrook et al [supra]. mRNA, or cDNA generated from mRNA using a polymerase enzyme, can be purified and separated using gel electrophoresis. The nucleic acids on the gel are then blotted onto a solid support, such as nitrocellulose. The solid support is exposed to a labelled probe and then washed to remove any unhybridized probe. Next, the duplexes containing the labeled probe are detected. Typically, the probe is labelled with a radioactive moiety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-7 show biochemical data and sequence analysis pertaining to Examples 1, 2, 3, 7, 13, 16 and 19, respectively, with ORFs 40, 38, 44, 52, 114, 41 and 124. M1 and M2 are molecular weight markers. Arrows indicate the position of the main recombinant product or, in Western blots, the position of the main N. meningitidis immunoreactive band. TP indicates N. meningitidis total protein extract; OMV indicates N. meningitidis outer membrane vesicle preparation. In bactericidal assay results: a diamond (♦) shows preimmune data; a triangle (▴) shows GST control data; a circle (●) shows data with recombinant N. meningitidis protein. Computer analyses show a hydrophilicity plot (upper), an antigenic index plot (middle), and an AMPHI analysis (lower). The AMPHI program has been used to predict T-cell epitopes [Gao et al. (1989) J. Immunol. 143:3007; Roberts et al. (1996) AIDS Res Hum Retrovir 12:593; Quakyi et al. (1992) Scand J Immunol suppl. 11:9) and is available in the Protean package of DNASTAR, Inc. (1228 South Park Street, Madison, Wis. 53715 USA).
FIG. 8 shows an alignment comparison of amino acid sequences for ORF 40 for several strains of Neisseria. Dark shading indicates regions of homology, and gray shading indicates the conservation of amino acids with similar characteristics. The Figure demonstrates a high degree of conservation among the various strains, further confirming its utility as an antigen for both vaccines and diagnostics.

EXAMPLES

The examples describe nucleic acid sequences which have been identified in N. meningitidis, along with their putative translation products. Not all of the nucleic acid sequences are complete ie. they encode less than the full-length wild-type protein. It is believed at present that none of the DNA sequences described herein have significant homologs in N. gonorrhoeae.
The examples are generally in the following format:

- a nucleotide sequence which has been identified in N. meningitidis (strain B)
- the putative translation product of this sequence
- a computer analysis of the translation product based on database comparisons
- a corresponding gene and protein sequence identified in N. meningitidis (strain A)
- a description of the characteristics of the proteins which indicates that they might be suitably antigenic
- results of biochemical analysis (expression, purification, ELISA, FACS etc.)

The examples typically include details of sequence homology between species and strains. Proteins that are similar in sequence are generally similar in both structure and function, and the homology often indicates a common evolutionary origin. Comparison with sequences of proteins of known function is widely used as a guide for the assignment of putative protein function to a new sequence and has proved particularly useful in whole-genome analyses.
Sequence comparisons were performed at NCBI (http://www.ncbi.nlm.nih.gov) using the algorithms BLAST, BLAST2, BLASTn, BLASTp, tBLASTn, BLASTx, & tBLASTx [eg. see also Altschul et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research 25:2289-3402]. Searches were performed against the following databases: non-redundant GenBank+EMBL+DDBJ+PDB sequences and non-redundant GenBank CDS translations+PDB+SwissProt+SPupdate+PIR sequences.
Dots within nucleotide sequences (eg. position 288 in Example 12) represent nucleotides which have been arbitrarily introduced in order to maintain a reading frame. In the same way, double-underlined nucleotides were removed. Lower case letters (eg. position 589 in Example 12) represent ambiguities which arose during alignment of independent sequencing reactions (some of the nucleotide sequences in the examples are derived from combining the results of two or more experiments).
Nucleotide sequences were scanned in all six reading frames to predict the presence of hydrophobic domains using an algorithm based on the statistical studies of Esposti et al. [Critical evaluation of the hydropathy of membrane proteins (1990) Eur J Biochem 190:207-219]. These domains represent potential transmembrane regions or hydrophobic leader sequences.
Open reading frames were predicted from fragmented nucleotide sequences using the program ORFFINDER (NCBI).
Underlined amino acid sequences indicate possible transmembrane domains or leader sequences in the ORFs, as predicted by the PSORT algorithm (http://www.psort.nibb.ac.jp). Functional domains were also predicted using the MOTIFS program (GCG Wisconsin & PROSITE).
Various tests can be used to assess the in vivo immunogenicity of the proteins identified in the examples. For example, the proteins can be expressed recombinantly and used to screen patient sera by immunoblot A positive reaction between the protein and patient serum indicates that the patient has previously mounted an immune response to the protein in question ie. the protein is an immunogen. This method can also be used to identify immunodominant proteins.
The recombinant protein can also be conveniently used to prepare antibodies eg. in a mouse. These can be used for direct confirmation that a protein is located on the cell-surface. Labelled antibody (eg. fluorescent labelling for FACS) can be incubated with intact bacteria and the presence of label on the bacterial surface confirms the location of the protein.
In particular, the following methods (A) to (S) were used to express, purify and biochemically characterise the proteins of the invention:
A) Chromosomal DNA Preparation
N. meningitidis strain 2996 was grown to exponential phase in 100 ml of GC medium, harvested by centrifugation, and resuspended in 5 ml buffer (20% Sucrose, 50 mM Tris-HCl, 50 mM EDTA, pH8). After 10 minutes incubation on ice, the bacteria were lysed by adding 10 ml lysis solution (50 mM NaCl, 1% Na-Sarkosyl, 50 μg/ml Proteinase K), and the suspension was incubated at 37° C. for 2 hours. Two phenol extractions (equilibrated to pH 8) and one ChCl₃/isoamylalcohol (24:1) extraction were performed. DNA was precipitated by addition of 0.3M sodium acetate and 2 volumes ethanol, and was collected by centrifugation. The pellet was washed once with 70% ethanol and redissolved in 4 ml buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). The DNA concentration was measured by reading the OD at 260 nm.
B) Oligonucleotide Design
Synthetic oligonucleotide primers were designed on the basis of the coding sequence of each ORF, using (a) the meningococcus B sequence when available, or (b) the gonococcus/meningococcus A sequence, adapted to the codon preference usage of meningococcus as necessary. Any predicted signal peptides were omitted, by deducing the 5′-end amplification primer sequence immediately downstream from the predicted leader sequence.
The 5′ primers included two restriction enzyme recognition sites (BamHI-NdeI, BamHI-NheI, or EcoRI-NheI, depending on the gene's own restriction pattern); the 3′ primers included a XhoI restriction site. This procedure was established in order to direct the cloning of each amplification product (corresponding to each ORF) into two different expression systems: pGEX-KG (using either BamHI-XhoI or EcoRI-XhoI), and pET21b+ (using either NdeI-XhoI or NheI-XhoI).

5′end primer tail:

CGCGGATCCCATATG (BamHI-NdeI)

CGCGGATCCGCTAGC (BamHI-NheI)

CCGGAATTCTAGCTAGC (EcoRI-NheI)

3′-end primer tail:

CCCGCTCGAG (XhoI)
As well as containing the restriction enzyme recognition sequences, the primers included nucleotides which hybridised to the sequence to be amplified. The number of hybridizing nucleotides depended on the melting temperature of the whole primer, and was determined for each primer using the formulae:
T _m=4 (G+C)+2 (A+T) (tail excluded)
T _m=64.9+0.41 (% GC)−600/N (whole primer)
The average melting temperature of the selected oligos were 65-70° C. for the whole oligo and 50-55° C. for the hybridising region alone.

Table I shows the forward and reverse primers used for each amplification. Oligos were synthesized by a Perkin Elmer 394 DNA/RNA Synthesizer, eluted from the columns in 2 ml NH₄OH, and deprotected by 5 hours incubation at 56° C. The oligos were precipitated by addition of 0.3M Na-Acetate and 2 volumes ethanol. The samples were then centrifuged and the pellets resuspended in either 100 μl or 1 ml of water. OD₂₆₀was determined using a Perkin Elmer Lambda Bio spectrophotometer and the concentration was determined and adjusted to 2-10 pmol/μl.

TABLE I


PCR primers

ORF	Primer	Sequence	Restriction sites

ORF 38	Forward	CGCGGATCCCATATG-TCGCCGCAAAATTCCGA	BamHI-NdeI
		<SEQ ID 112>
	Reverse	CCCGCTCGAG-TTTTGCCGCGTTAAAAGC	XhoI
		<SEQ ID 113>

ORF 40	Forward	CGCGGATCCCATATG-ACCGTGAAGACCGCC	BamHI-NdeI
		<SEQ ID 114>
	Reverse	CCCGCTCGAG-CCACTGATAACCGACAGA	XhoI
		<SEQ ID 115>

ORF 41	Forward	CGCGGATCCCATATG-TATTTGAAACAGCTCCAAG	BamHI-NdeI
		<SEQ ID 116>
	Reverse	CCCGCTCGAG-TTCTGGGTGAATGTTA	XhoI
		<SEQ ID 117>

ORF 44	Forward	GCGGATCCCATATG-GGCACGGACAACCCC	BamHI-NdeI
		<SEQ ID 118>
	Reverse	CCCGCTCGAG-ACGTGGGGAACAGTCT	XhoI
		<SEQ ID 119>

ORF 51	Forward	GCGGATCCCATATG-AAAAATATTCAAGTAGTTGC	BamHI-NdeI
		<SEQ ID 120>
	Reverse	CCCGCTCGAG-AAGTTTGATTAAACCCG	XhoI
		<SEQ ID 121>

ORF 52	Forward	CGCGGATCCCATATG-TGCCAACCGCAATCCG	BamHI-NdeI
		<SEQ ID 122>
	Reverse	CCCGCTCGAG-TTTTTCCAGCTCCGGCA	XhoI
		<SEQ ID 123>

ORF 56	Forward	GCGGATCCCATATG-GTTATCGGAATATTACTCG	BamHI-NdeI
		<SEQ ID 124>
	Reverse	CCCGCTCGAG-GGCTGCAGAAGCTGG	XhoI
		<SEQ ID 125>

ORF 69	Forward	CGCGGATCCCATATG-CGGACGTGGTTGGTTTT	BamHI-NdeI
		<SEQ ID 126>
	Reverse	CCCGCTCGAG-ATATCTTCCGTTTTTTTCAC	XhoI
		<SEQ ID 127>

ORF 82	Forward	CGCGGATCCGCTAGC-GTAAATTTATTATTTTTAGAA	BamHI-NheI
		<SEQ ID 128>
	Reverse	CCCGCTCGAG-TCCAACTCATTGAAGTA	XhoI
		<SEQ ID 129>

ORF 114	Forward	CGCGGATCCCATATG-AATAAAGGTTTACATCGCAT	BamHI-NheI
		<SEQ ID 130>
	Reverse	CCCGCTCGAG-AATCGCTGCACCGGCT	XhoI
		<SEQ ID 131>

ORF 124	Forward	CGCGGATCCCATATG-ACTGCCTTTTCGACA	BamHI-NheI
		<SEQ ID 132>
	Reverse	CCCGCTCGAG-GCGTGAAGCGTCAGGA	XhoI
		<SEQ ID 133>

C) Amplification

The standard PCR protocol was as follows: 50-200 ng of genomic DNA were used as a template in the presence of 20-40 μM of each oligo, 400-8004M dNTs solution, 1×PCR buffer (including 1.5 mM MgCl₂), 2.5 units TaqI DNA polymerase (using Perkin-Elmer AmpliTaQ, GIBCO Platinum, Pwo DNA polymerase, or Tahara Shuzo Taq polymerase).
In some cases, PCR was optimised by the addition of 10 μl DMSO or 50 μl 2M betaine.
After a hot start (adding the polymerase during a preliminary 3 minute incubation of the whole mix at 95° C.), each sample underwent a double-step amplification: the first 5 cycles were performed using as the hybridization temperature the one of the oligos excluding the restriction enzymes tail, followed by 30 cycles performed according to the hybridization temperature of the whole length oligos. The cycles were followed by a final 10 minute extension step at 72° C.
The standard cycles were as follows:

Denaturation Hybridisation Elongation

First

5 cycles 30 seconds 30 seconds 30-60 seconds

95° C. 50-55° C. 72° C.

Last 30 cycles 30 seconds 30 seconds 30-60 seconds

95° C. 65-70° C. 72° C.
The elongation time varied according to the length of the ORF to be amplified.
The amplifications were performed using either a 9600 or a 2400 Perkin Elmer GeneAmp PCR System. To check the results, 1/10 of the amplification volume was loaded onto a 1-1.5% agarose gel and the size of each amplified fragment compared with a DNA molecular weight marker.
The amplified DNA was either loaded directly on a 1% agarose gel or first precipitated with ethanol and resuspended in a suitable volume to be loaded on a 1% agarose gel. The DNA fragment corresponding to the right size band was then eluted and purified from gel, using the Qiagen Gel Extraction Kit, following the instructions of the manufacturer. The final volume of the DNA fragment was 30 μl or 500 of either water or 10 mM Tris, pH 8.5.
D) Digestion of PCR Fragments
The purified DNA corresponding to the amplified fragment was split into 2 aliquots and double-digested with:

- NdeI/XhoI or NheI/XhoI for cloning into pET-21b+ and further expression of the protein as a C-terminus His-tag fusion
- BamHI/XhoI or EcoRI/XhoI for cloning into pGEX-KG and further expression of the protein as N-terminus GST fusion.
- EcoRI/PstI, EcoRI/SalI, SalI/PstI for cloning into pGex-His and further expression of the protein as N-terminus His-tag fusion

Each purified DNA fragment was incubated (37° C. for 3 hours to overnight) with 20 units of each restriction enzyme (New England Biolabs) in a either 30 or 40 μl final volume in the presence of the appropriate buffer. The digestion product was then purified using the QIAquick PCR purification kit, following the manufacturer's instructions, and eluted in a final volume of 30 or 50 μl of either water or 10 mM Tris-HCl, pH 8.5. The final DNA concentration was determined by 1% agarose gel electrophoresis in the presence of titrated molecular weight marker.
E) Digestion of the Cloning Vectors (pET22B, pGEX-KG, pTRC-His A, and pGex-His)
10 μg plasmid was double-digested with 50 units of each restriction enzyme in 200 μl reaction volume in the presence of appropriate buffer by overnight incubation at 37° C. After loading the whole digestion on a 1% agarose gel, the band corresponding to the digested vector was purified from the gel using the Qiagen QIAquick-Gel Extraction Kit and the DNA was eluted in 50 μl of 10 mM Tris-HCl, pH 8.5. The DNA concentration was evaluated by measuring OD₂₆₀of the sample, and adjusted to 50 μg/μl. 1 μl of plasmid was used for each cloning procedure.
The vector pGEX-His is a modified pGEX-2T vector carrying a region encoding six histidine residues upstream to the thrombin cleavage site and containing the multiple cloning site of the vector pTRC99 (Pharmacia).
F) Cloning
The fragments corresponding to each ORF, previously digested and purified, were ligated in both pET22b and pGEX-KG. In a final volume of 20 μl, a molar ratio of 3:1 fragment/vector was ligated using 0.5 μl of NEB T4 DNA ligase (400 units/μl), in the presence of the buffer supplied by the manufacturer. The reaction was incubated at room temperature for 3 hours. In some experiments, ligation was performed using the Boehringer “Rapid Ligation Kit”, following the manufacturer's instructions.
In order to introduce the recombinant plasmid in a suitable strain, 100 μl E. coli DH5 competent cells were incubated with the ligase reaction solution for 40 minutes on ice, then at 37° C. for 3 minutes, then, after adding 800 μl LB broth, again at 37° C. for 20 minutes. The cells were then centrifuged at maximum speed in an Eppendorf microfuge and resuspended in approximately 200 μl of the supernatant. The suspension was then plated on LB ampicillin (100 mg/ml).
The screening of the recombinant clones was performed by growing 5 randomly-chosen colonies overnight at 37° C. in either 2 ml (pGEX or pTC clones) or 5 ml (pET clones) LB broth+100 μg/ml ampicillin. The cells were then pelletted and the DNA extracted using the Qiagen QIAprep Spin Miniprep Kit, following the manufacturer's instructions, to a final volume of 30 μl. 5 μl of each individual miniprep (approximately 1 g) were digested with either NdeI/XhoI or BamHI/XhoI and the whole digestion loaded onto a 1-1.5% agarose gel (depending on the expected insert size), in parallel with the molecular weight marker (1 Kb DNA Ladder, GIBCO). The screening of the positive clones was made on the base of the correct insert size.
G) Expression
Each ORF cloned into the expression vector was transformed into the strain suitable for expression of the recombinant protein product. 1 μl of each construct was used to transform 30 μl of E. coli BL21 (pGEX vector), E. coli TOP 10 (pTRC vector) or E. coli BL21-DE3 (pET vector), as described above. In the case of the pGEX-His vector, the same E. coli strain (W3110) was used for initial cloning and expression. Single recombinant colonies were inoculated into 2 ml LB+Amp (100 μg/ml), incubated at 37° C. overnight, then diluted 1:30 in 20 ml of LB+Amp (100 μg/ml) in 100 ml flasks, making sure that the OD₆₀₀ranged between 0.1 and 0.15. The flasks were incubated at 30° C. into gyratory water bath shakers until OD indicated exponential growth suitable for induction of expression (0.4-0.8 OD for pET and pTRC vectors; 0.8-1 OD for pGEX and pGEX-His vectors). For the pET, pTRC and pGEX-His vectors, the protein expression was induced by addition of 1 mM IPTG, whereas in the case of pGEX system the final concentration of IPTG was 0.2 mM. After 3 hours incubation at 30° C., the final concentration of the sample was checked by OD. In order to check expression, 1 ml of each sample was removed, centrifuged in a microfuge, the pellet resuspended in PBS, and analysed by 12% SDS-PAGE with Coomassie Blue staining. The whole sample was centrifuged at 6000 g and the pellet resuspended in PBS for further use.
H) GST-Fusion Proteins Large-Scale Purification.
A single colony was grown overnight at 37° C. on LB+Amp agar plate. The bacteria were inoculated into 20 ml of LB+Amp liquid culture in a water bath shaker and grown overnight. Bacteria were diluted 1:30 into 600 ml of fresh medium and allowed to grow at the optimal temperature (20-37° C.) to OD₅₅₀0.8-1. Protein expression was induced with 0.2 mM IPTG followed by three hours incubation. The culture was centrifuged at 800 rpm at 4° C. The supernatant was discarded and the bacterial pellet was resuspended in 7.5 ml cold PBS. The cells were disrupted by sonication on ice for 30 sec at 40 W using a Branson sonifier B-15, frozen and thawed twice and centrifuged again. The supernatant was collected and mixed with 150 μl Glutatione-Sepharose 4B resin (Pharmacia) (previously washed with PBS) and incubated at room temperature for 30 minutes. The sample was centrifuged at 700 g for 5 minutes at 4° C. The resin was washed twice with 10 ml cold PBS for 10 minutes, resuspended in 1 ml cold PBS, and loaded on a disposable column. The resin was washed twice with 2 ml cold PBS until the flow-through reached OD₂₈₀of 0.02-0.06. The GST-fusion protein was eluted by addition of 70011 cold Glutathione elution buffer (10 mM reduced glutathione, 50 mM Tris-HCl) and fractions collected until the OD₂₈₀was 0.1. 21 μl of each fraction were loaded on a 12% SDS gel using either Biorad SDS-PAGE Molecular weight standard broad range (M1) (200, 116.25, 97.4, 66.2, 45, 31, 21.5, 14.4, 6.5 kDa) or Amersham Rainbow Marker (M2) (220, 66, 46, 30, 21.5, 14.3 kDa) as standards. As the MW of GST is 26 kDa, this value must be added to the MW of each GST-fusion protein.
I) His-Fusion Solubility Analysis
To analyse the solubility of the His-fusion expression products, pellets of 3 ml cultures were resuspended in buffer M1 [500 μl PBS pH 7.2]. 25 μl lysozyme (10 mg/ml) was added and the bacteria were incubated for 15 min at 4° C. The pellets were sonicated for 30 sec at 40 W using a Branson sonifier B-15, frozen and thawed twice and then separated again into pellet and supernatant by a centrifugation step. The supernatant was collected and the pellet was resuspended in buffer M2 [8M urea, 0.5M NaCl, 20 mM imidazole and 0.1M NaH₂PO₄] and incubated for 3 to 4 hours at 4° C. After centrifugation, the supernatant was collected and the pellet was resuspended in buffer M3 [6M guanidinium-HCl, 0.5M NaCl, 20 mM imidazole and 0.1M NaH₂PO₄] overnight at 4° C. The supernatants from all steps were analysed by SDS-PAGE.
J) His-Fusion Large-Scale Purification.
A single colony was grown overnight at 37° C. on a LB+Amp agar plate. The bacteria were inoculated into 20 ml of LB+Amp liquid culture and incubated overnight in a water bath shaker. Bacteria were diluted 1:30 into 600 ml fresh medium and allowed to grow at the optimal temperature (20-37° C.) to OD₅₅₀0.6-0.8. Protein expression was induced by addition of 1 mM IPTG and the culture further incubated for three hours. The culture was centrifuged at 8000 rpm at 4° C., the supernatant was discarded and the bacterial pellet was resuspended in 7.5 ml of either (i) cold buffer A (300 mM NaCl, 50 mM phosphate buffer, 10 mM imidazole, pH 8) for soluble proteins or (ii) buffer B (urea 8M, 10 mM Tris-HCl, 100 mM phosphate buffer, pH 8.8) for insoluble proteins.
The cells were disrupted by sonication on ice for 30 sec at 40 W using a Branson sonifier B-15, frozen and thawed two times and centrifuged again.
For insoluble proteins, the supernatant was stored at −20° C., while the pellets were resuspended in 2 ml buffer C (6M guanidine hydrochloride, 100 mM phosphate buffer, 10 mM Tris-HCl, pH 7.5) and treated in a homogenizer for 10 cycles. The product was centrifuged at 13000 rpm for 40 minutes.
Supernatants were collected and mixed with 150 μl Ni²⁺-resin (Pharmacia) (previously washed with either buffer A or buffer B, as appropriate) and incubated at room temperature with gentle agitation for 30 minutes. The sample was centrifuged at 700 g for 5 minutes at 4° C. The resin was washed twice with 10 ml buffer A or B for 10 minutes, resuspended in 1 ml buffer A or B and loaded on a disposable column. The resin was washed at either (i) 4° C. with 2 ml cold buffer A or (ii) room temperature with 2 ml buffer B, until the flow-through reached OD₂₈₀of 0.02-0.06.
The resin was washed with either (i) 2 ml cold 20 mM imidazole buffer (300 mM NaCl, 50 mM phosphate buffer, 20 mM imidazole, pH 8) or (ii) buffer D (urea 8M, 10 mM Tris-HCl, 100 mM phosphate buffer, pH 6.3) until the flow-through reached the O.D₂₈₀of 0.02-0.06. The His-fusion protein was eluted by addition of 700 μl of either (i) cold elution buffer A (300 mM NaCl, 50 mM phosphate buffer, 250 mM imidazole, pH 8) or (ii) elution buffer B (urea 8M, 10 mM Tris-HCl, 100 mM phosphate buffer, pH 4.5) and fractions collected until the O.D₂₈₀was 0.1. 21 μl of each fraction were loaded on a 12% SDS gel.
K) His-Fusion Proteins Renaturation
10% glycerol was added to the denatured proteins. The proteins were then diluted to 20 μg/ml using dialysis buffer I (10% glycerol, 0.5M arginine, 50 mM phosphate buffer, 5 mM reduced glutathione, 0.5 mM oxidised glutathione, 2M urea, pH 8.8) and dialysed against the same buffer at 4° C. for 12-14 hours. The protein was further dialysed against dialysis buffer II (10% glycerol, 0.5M arginine, 50 mM phosphate buffer, 5 mM reduced glutathione, 0.5 mM oxidised glutathione, pH 8.8) for 12-14 hours at 4° C. Protein concentration was evaluated using the formula:
Protein (mg/ml)=(1.55×OD ₂₈₀)−(0.76×OD ₂₆₀)
L) His-Fusion Large-Scale Purification
500 ml of bacterial cultures were induced and the fusion proteins were obtained soluble in buffer M1, M2 or M3 using the procedure described above. The crude extract of the bacteria was loaded onto a Ni-NTA superflow column (Qiagen) equilibrated with buffer M1, M2 or M3 depending on the solubilization buffer of the fusion proteins. Unbound material was eluted by washing the column with the same buffer. The specific protein was eluted with the corresponding buffer containing 500 mM imidazole and dialysed against the corresponding buffer without imidazole. After each run the columns were sanitized by washing with at least two column volumes of 0.5 M sodium hydroxide and reequilibrated before the next use.
M) Mice Immunisations
20 μg of each purified protein were used to immunise mice intraperitoneally. In the case of ORF 44, CD1 mice were immunised with Al(OH)₃as adjuvant on days 1, 21 and 42, and immune response was monitored in samples taken on day 56. For ORF 40, CD1 mice were immunised using Freund's adjuvant, rather than Al(OH)₃, and the same immunisation protocol was used, except that the immune response was measured on day 42, rather than 56. Similarly, for ORF 38, CD1 mice were immunised with Freund's adjuvant, but the immune response was measured on day 49.
N) ELISA Assay (Sera Analysis)
The acapsulated MenB M7 strain was plated on chocolate agar plates and incubated overnight at 37° C. Bacterial colonies were collected from the agar plates using a sterile dracon swab and inoculated into 7 ml of Mueller-Hinton Broth (Difco) containing 0.25% Glucose. Bacterial growth was monitored every 30 minutes by following OD₆₂₀. The bacteria were let to grow until the OD reached the value of 0.3-0.4. The culture was centrifuged for 10 minutes at 10000 rpm. The supernatant was discarded and bacteria were washed once with PBS, resuspended in PBS containing 0.025% formaldehyde, and incubated for 2 hours at room temperature and then overnight at 4° C. with stirring. 100 μl bacterial cells were added to each well of a 96 well Greiner plate and incubated overnight at 4° C. The wells were then washed three times with PBT washing buffer (0.1% Tween-20 in PBS). 200 μl of saturation buffer (2.7% Polyvinylpyrrolidone 10 in water) was added to each well and the plates incubated for 2 hours at 37° C. Wells were washed three times with PBT. 200 μl of diluted sera (Dilution buffer: 1% BSA, 0.1% Tween-20, 0.1% NaN₃in PBS) were added to each well and the plates incubated for 90 minutes at 37° C. Wells were washed three times with PBT. 100 μl of HRP-conjugated rabbit anti-mouse (Dako) serum diluted 1:2000 in dilution buffer were added to each well and the plates were incubated for 90 minutes at 37° C. Wells were washed three times with PBT buffer. 100 μl of substrate buffer for HRP (25 ml of citrate buffer pH5, 10 mg of O-phenildiamine and 10 μl of H₂O) were added to each well and the plates were left at room temperature for 20 minutes. 100 μl H₂SO₄was added to each well and OD₄₉₀was followed. The ELISA was considered positive when OD₄₉₀was 2.5 times the respective pre-immune sera.
O) FACScan Bacteria Binding Assay Procedure.
The acapsulated MenB M7 strain was plated on chocolate agar plates and incubated overnight at 37° C. Bacterial colonies were collected from the agar plates using a sterile dracon swab and inoculated into 4 tubes containing 8 ml each Mueller-Hinton Broth (Difco) containing 0.25% glucose. Bacterial growth was monitored every 30 minutes by following OD₆₂₀. The bacteria were let to grow until the OD reached the value of 0.35-0.5. The culture was centrifuged for 10 minutes at 4000 rpm. The supernatant was discarded and the pellet was resuspended in blocking buffer (1% BSA, 0.4% NaN₃) and centrifuged for 5 minutes at 4000 rpm. Cells were resuspended in blocking buffer to reach OD₆₂₀of 0.07. 100 μl bacterial cells were added to each well of a Costar 96 well plate. 100 μl of diluted (1:200) sera (in blocking buffer) were added to each well and plates incubated for 2 hours at 4° C. Cells were centrifuged for 5 minutes at 4000 rpm, the supernatant aspirated and cells washed by addition of 200 μl/well of blocking buffer in each well. 100 μl of R-Phicoerytin conjugated F(ab)₂goat anti-mouse, diluted 1:100, was added to each well and plates incubated for 1 hour at 4° C. Cells were spun down by centrifugation at 4000 rpm for 5 minutes and washed by addition of 200 μl/well of blocking buffer. The supernatant was aspirated and cells resuspended in 200 μl/well of PBS, 0.25% formaldehyde. Samples were transferred to FACScan tubes and read. The condition for FACScan setting were: FL1 on, FL2 and FL3 off; FSC-H threshold: 92; FSC PMT Voltage: E 02; SSC PMT: 474; Amp. Gains 7.1; FL-2 PMT: 539; compensation values: 0.
P) OMV Preparations
Bacteria were grown overnight on 5 GC plates, harvested with a loop and resuspended in 10 ml 20 mM Tris-HCl. Heat inactivation was performed at 56° C. for 30 minutes and the bacteria disrupted by sonication for 10 minutes on ice (50% duty cycle, 50% output). Unbroken cells were removed by centrifugation at 5000 g for 10 minutes and the total cell envelope fraction recovered by centrifugation at 50000 g at 4° C. for 75 minutes. To extract cytoplasmic membrane proteins from the crude outer membranes, the whole fraction was resuspended in 2% sarkosyl (Sigma) and incubated at room temperature for 20 minutes. The suspension was centrifuged at 10000 g for 10 minutes to remove aggregates, and the supernatant further ultracentrifuged at 50000 g for 75 minutes to pellet the outer membranes. The outer membranes were resuspended in 10 mM Tris-HCl, pH8 and the protein concentration measured by the Bio-Rad Protein assay, using BSA as a standard.
Q) Whole Extracts Preparation
Bacteria were grown overnight on a GC plate, harvested with a loop and resuspended in 1 ml of 20 mM Tris-HCl. Heat inactivation was performed at 56° C. for 30 minutes.
R) Western Blotting
Purified proteins (500 ng/lane), outer membrane vesicles (5 μg) and total cell extracts (25 μg) derived from MenB strain 2996 were loaded on 15% SDS-PAGE and transferred to a nitrocellulose membrane. The transfer was performed for 2 hours at 150 mA at 4° C., in transferring buffer (0.3% Tris base, 1.44% glycine, 20% methanol). The membrane was saturated by overnight incubation at 4° C. in saturation buffer (10% skimmed milk, 0.1% Triton X100 in PBS). The membrane was washed twice with washing buffer (3% skimmed milk, 0.1% Triton X100 in PBS) and incubated for 2 hours at 37° C. with mice sera diluted 1:200 in washing buffer. The membrane was washed twice and incubated for 90 minutes with a 1:2000 dilution of horseradish peroxidase labelled anti-mouse Ig. The membrane was washed twice with 0.1% Triton X100 in PBS and developed with the Opti-4CN Substrate Kit (Bio-Rad). The reaction was stopped by adding water.
S) Bactericidal Assay
MC58 strain was grown overnight at 37° C. on chocolate agar plates. 5-7 colonies were collected and used to inoculate 7 ml Mueller-Hinton broth. The suspension was incubated at 37° C. on a nutator and let to grow until OD₆₂₀was 0.5-0.8. The culture was aliquoted into sterile 1.5 ml Eppendorf tubes and centrifuged for 20 minutes at maximum speed in a microfuge. The pellet was washed once in Gey's buffer (Gibco) and resuspended in the same buffer to an OD₆₂₀of 0.5, diluted 1:20000 in Gey's buffer and stored at 25° C.
50 μl of Gey's buffer/1% BSA was added to each well of a 96-well tissue culture plate. 25 μl of diluted mice sera (1:100 in Gey's buffer/0.2% BSA) were added to each well and the plate incubated at 4° C. 25 μl of the previously described bacterial suspension were added to each well. 25 μl of either heat-inactivated (56° C. waterbath for 30 minutes) or normal baby rabbit complement were added to each well. Immediately after the addition of the baby rabbit complement, 22 μl of each sample/well were plated on Mueller-Hinton agar plates (time 0). The 96-well plate was incubated for 1 hour at 37° C. with rotation and then 22 μl of each sample/well were plated on Mueller-Hinton agar plates (time 1). After overnight incubation the colonies corresponding to time 0 and time 1 hour were counted.

Table II gives a summary of the cloning, expression and purification results.

TABLE II


Cloning, expression and purification

		His-fusion	GST-fusion
ORF	PCR/cloning	expression	expression	Purification

orf 38	+	+	+	His-fusion
orf
40	+	+	+	His-fusion
orf 41	+	n.d.	n.d.
orf 44	+	+	+	His-fusion
orf 51	+	n.d.	n.d.
orf 52	+	n.d.	+	GST-fusion
orf 56	+	n.d.	n.d.
orf 69	+	n.d.	n.d.
orf 82	+	n.d.	n.d.
orf 114	+	n.d.	+	GST-fusion
orf 124	+	n.d.	n.d.

Example 1

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 1>:


1	ACACTGTTGT TTGCAACGGT TCAGGCAAGT GCTAACCAAT GAAGAGCAAG

51	AAGAAGATTT ATATTTAGAC CCCGTACAAC GCACTGTTGC CGTGTTGATA

101	GTCAATTCCG ATAAAGAAGG CACGGGAGAA AAAGAAAAAG TAGAAGAAAA

151	TTCAGATTGG GCAGTATATT TCAACGAGAA AGGAGTACTA ACAGCCAGAG

201	AAATCACCyT CAAAGCCGGC GACAACCTGA AAATCAAACA AAACGGCACA

251	AACTTCACCT ACTCGCTGAA AAALGACCTC ACAGATCTGA CCAGTGTTGG

301	AACTGAAAAA TTATCGTTTA GCGCAAACGG CAATAAAGTC AACATCACAA

351	GCGACACCAA AGGCTTGAAT TTTGCGAAAG AAACGGCTGG sACGAACGgC

401	GACACCACGG TTCATCTGAA CGGTATTGGT TCGACTTTGA CCGATACGCT

451	GCTGAATACC GGAGCGACCA CAAACGTAAC CAACGACAAC GTTACCGATG

501	ACGAGAAAAA ACGTGCGGCA AGCGTTAAAG ACGTATTAAA CGCTGGCTGG

551	AACATTAAAG GCGTTAAACC CGGTACAACA GCTTCCGATA ACGTTGATTT

601	CGTCCGCACT TACGACACAG TCGAGTTCTT GAGCGCAGAT ACGAAAACAA

651	CGACTGTTAA TGTGGAAAGC AAAGACAACG GCAAGAAAAC CGAAGTTAAA

701	ATCGGTGCGA AGACTTCTGT TATTAAAGAA AAAGAC...

This corresponds to the amino acid sequence <SEQ ID 2; ORF40>:


1	..TLLFATVQAS ANQEEQEEDL YLDPVQRTVA VLIVNSDKEG TGEKEKVEEN

51	SDWAVYFNEK GVLTAREITX KAGDNLKIKQ NGTNFTYSLK KDLTDLTSVG

101	TEKLSFSANG NKVNITSDTK GLNFAKETAG TNGDTTVHLN GIGSTLTDTL

151	LNTGATTNVT NDNVTDDEKK RAASVKDVLN AGWNIKGVKP GTTASDNVDF

201	VRTYDTVEFL SADTKTITVN VESKDNGKKT EVKIGAXTSV IKEKD...

Further work revealed the complete DNA sequence <SEQ ID 3>:


1	ATGAACAAAA TATACCGCAT CATTTGGAAT AGTGCCCTCA ATGCCTGGGT

51	CGTCGTATCC GAGCTCACAC GCAACCACAC CAAACGCGCC TCCGCAACCG

101	TGAAGACCGC CGTATTGGCG ACACTGTTGT TTGCAACGGT TCAGGCAAGT

151	GCTAACAATG AAGAGCAAGA AGAAGATTTA TATTTAGACC CCGTACAACG

201	CACTGTTGCC GTGTTGATAG TCAATTCCGA TAAAGAAGGC ACGGGAGAAA

251	AAGAAAAAGT AGAAGAAAAT TCAGATTGGG CAGTATATTT CAACGAGAAA

301	GGAGTACTAA CAGCCAGAGA AATCACCCTC AAAGCCGGCG ACAACCTGAA

351	AATCAAACAA AACGGCACAA ACTTCACCTA CTCGCTGAAA AAAGACCTCA

401	CAGATCTGAC CAGTGTTGGA ACTGAAAAAT TATCGTTTAG CGCAAACGGC

451	AATAAAGTCA ACATCACAAG CGACACCAAA GGCTTGAATT TTGCGAAAGA

501	AACGGCTGGG ACGAACGGCG ACACCACGGT TCATCTGAAC GGTATTGGTT

551	CGACTTTGAC CGATACGCTG CTGAATACCG GAGCGACCAC AAACGTAACC

601	AACGACAACG TTACCGATGA CGAGAAAAAA CGTGCGGCAA GCGTTAAAGA

651	CGTATTAAAC GCTGGCTGGA ACATTAAAGG CGTTAAACCC GGTACAACAG

701	CTTCCGATAA CGTTGATTTC GTCCGCACTT ACGACACAGT CGAGTTCTTG

751	AGCGCAGATA CGAAAACAAC GACTGTTAAT GTGGAAAGCA AAGACAACGG

801	CAAGAAAACC GAAGTTAAAA TCGGTGCGAA GACTTCTGTT ATTAAAGAAA

851	AAGACGGTAA GTTGGTTACT GGTAAAGACA AAGGCGAGAA TGGTTCTTCT

901	ACAGACGAAG GCGAAGGCTT AGTGACTGCA AAAGAAGTGA TTGATGCAGT

951	AAACAAGGCT GGTTGGAGAA TGAAAACAAC AACCGCTAAT GGTCAAACAG

1001	GTCAAGCTGA CAAGTTTGAA ACCGTTACAT CAGGCACAAA TGTAACCTTT

1051	GCTAGTGGTA AAGGTACAAC TGCGACTGTA AGTAAAGATG ATCAAGGCAA

1101	CATCACTGTT ATGTATGATG TAAATGTCGG CGATGCCCTA AACGTCAATC

1151	AGCTGCAAAA CAGCGGTTGG AATTTGGATT CCAAAGCGGT TGCAGGTTCT

1201	TCGGGCAAAG TCATCAGCGG CAATGTTTCG CCGAGCAAGG GAAAGATGGA

1251	TGAAACCGTC AACATTAATG CCGGCAACAA CATCGAGATT ACCCGCAACG

1301	GTAAAAATAT CGACATCGCC ACTTCGATGA CCCCGCAGTT TTCCAGCGTT

1351	TCGCTCGGCG CGGGGGCGGA TGCGCCCACT TTGAGCGTGG ATGGGGACGC

1401	ATTGAATGTC GGCAGCAAGA AGGACAACAA ACCCGTCCGC ATTACCAATG

1451	TCGCCCCGGG CGTTAAAGAG GGGGATGTTA CAAACGTCGC ACAACTTAAA

1501	GGCGTGGCGC AAAACTTGAA CAACCGCATC GACAATGTGG ACGGCAACGC

1551	GCGTGCGGGC ATCGCCCAAG CGATTGCAAC CGCAGGTCTG GTTCAGGCGT

1601	ATTTGCCCGG CAAGAGTATG ATGGCGATCG GCGGCGGCAC TTATCGCGGC

1651	GAAGCCGGTT ACGCCATCGG CTACTCCAGT ATTTCCGACG GCGGAAATTG

1701	GATTATCAAA GGCACGGCTT CCGGCAATTC GCGCGGCCAT TTCGGTGCTT

1751	CCGCATCTGT CGGTTATCAG TGGTAA

This corresponds to the amino acid sequence <SEQ ID 4; ORF40-1>:


1	MNKIYRIIWN SALNAWVVVS ELTRNHTKRA SATVKTAVLA TLLFATVQAS

51	ANNEEQEEDL YLDFVQRTVA VLIVNSDKEG TGEKEKVEEN SDWAVYFNEK

101	GVLTAREITL KAGDNLKIKQ NGTNFTYSLK KDLTDLTSVG TEKLSFSMIG

151	NKVNITSDTK GLNFAKETAG TNGDTTVHLN GIGSTLTDTL LNTGATTNVT

201	NDNVTDDEKK RAASVKDVLN AGWNIKGVKP GTTASDNVDF VRTYDTVEFL

251	SADTKTTTVN VESKDNGKKT EVKIGAKTSV IKEKDGKLVT GKDKGENGSS

301	TDEGEGLVTA KEVIDAYNKA GWRMKTTTAN GQTGQADKFE TVTSGTNVTF

351	ASGKGTTATV SKDDQGNITV NYDVNVGDAL NVNQLQNSGW NLDSKAVAGS

401	SGKVISGNVS PSKGKMDETV NINAGNNIEI TRNGKNIDIA TSHTPQFSSV

451	SLGAGADAPT LSVDGDALNV GSKKDNKPVR ITNVAPGVKE GOVTNVAQLK

501	GVAQNLNNRI DNVDGNARAG ZAQAIATAGL VQAYLPGKSM MAIGGGTYRG

551	EAGYAIGYSS ISDGGNWIIK GTASGNSRGH FGASASVGYQ W*

Further work identified the corresponding gene in strain A of N. meningitidis <SEQ ID 5>:


1	ATGAACAAAA TATACCGCAT CATTTGGAAT AGTGCCCTCA ATGCCTGNGT

51	CGCCGTATCC GAGCTCACAC GCAACCACAC CAAACGCGCC TCCGCAACCG

101	TGAAGACCGC CGTATTGGCG ACACTGTTGT TTGCAACGGT TCAGGCGAAT

151	GCTACCGATG AAGATGAAGA AGAAGAGTTA GAATCCGTAC AACGCTCTGT

201	CGTAGGGAGC ATTCAAGCCA GTATGGAAGG CAGCGGCGAA TTGGAAACGA

251	TATCATTATC AATGACTAAC GACAGCAAGG AATTTGTAGA CCCATACATA

301	GTAGTTACCC TCAAAGCCGG CGACAACCTG AAAATCAAAC AAAACACCAA

351	TGAAAACACC AATGCCAGTA GCTTCACCTA CTCGCTGAAA AAAGACCTCA

401	CAGGCCTGAT CAATGTTGAN ACTGAAAAAT TATCGTTTGG CGCAAACGGC

451	AAGAAAGTCA ACATCATAAG CGACACCAAA GGCTTGAATT TCGCGAAAGA

501	AACGGCTGGG ACGAACGGCG ACACCACGGT TCATCTGAAC GGTATCGGTT

551	CGACTTTGAC CGATACGCTT GCGGGTTCTT CTGCTTCTCA CGTTGATGCG

601	GGTAACCNAA GTACACATTA CACTCGTGCA GCAAGTATTA AGGATGTGTT

651	GAATGCGGGT TGGAATATTA AGGGTGTTAA ANNNGGCTCA ACAACTGGTC

701	AATCAGAAAA TGTCGATTTC GTCCGCACTT ACGACACAGT CGAGTTCTTG

751	AGCGCAGATA CGNAAACAAC GACNGTTAAT GTGGAAAGCA AAGACAACGG

801	CAAGAGAACC GAAGTTAAAA TCGGTGCGAA GACTTCTGTT ATTAAAGAAA

851	AAGACGGTAA GTTGGTTACT GGTAAAGGCA AAGGCGAGAA TGGTTCTTCT

901	ACAGACGAAG GCGAAGGCTT AGTGACTGCA AAAGAAGTGA TTGATGCAGT

951	AAACAAGGCT GGTTGGAGAA TGAAAACAAC AACCGCTAAT GGTCAAACAG

1001	GTCAAGCTGA CAAGTTTGAA ACCGTTACAT CAGGCACAAA TGTAACCTTT

1051	GCTAGTGGTA AAGGTACAAC TGCGACTGTA AGTAAAGATG ATCAAGGCAA

1101	CATCACTGTT ATGTATGATG TAAATGTCGG CGATGCCCTA AACGTCAATC

1151	AGCTGCAAAA CAGCGGTTGG AATTTGGATT CCAAAGCGGT TGCAGGTTCT

1201	TCGGGCAAAG TCATCAGCGG CAATGTTTCG CCGAGCAAGG GAAAGATGGA

1251	TGAAACCGTC AACATTAATG CCGGCAACAA CATCGACATT AGCCGCAACG

1301	GTAAAAATAT CGACATCGCC ACTTCGATGG CGCCGCAGTT TTCCAGCGTT

1351	TCGCTCGGCG CGGGGGCAGA TGCGCCCACT TTAAGCGTGG ATGACGAGGG

1401	CGCGTTGAAT GTCGGCAGCA AGGATGCCAA CAAACCCGTC CGCATTACCA

1451	ATGTCGCCCC GGGCGTTAAA GANGGGGATG TTACAAACGT CNCACAACTT

1501	AAAGGCGTGG CGCAAAACTT GAACAACCGC ATCGACAATG TGGACGGCAA

1551	CGCGCGTGCN GGCATCGCCC AAGCGATTGC AACCGCAGGT CTGGTTCAGG

1601	CGTATCTGCC CGGCAAGAGT ATGATGGCGA TCGGCGGCGG CACTTATCGC

1651	GGCGAAGCCG GTTACGCCAT CGGCTACTCC AGTATTTCCG ACGGCGGAAA

1701	TTGGATTATC AAAGGCACGG CTTCCGGCAA TTCGCGCGGC CATTTCGGTG

1751	CTTCCGCATC TGTCGGTTAT CAGTGGTAA

This encodes a protein having amino acid sequence <SEQ ID 6; ORF40a>:


1	MNKIYRIIWN SALNPXVAVS ELTRNHTKRA SATVKTAVLA TLLFATVQAN

51	ATDEDEKEEL ESVQRSVVGS IQASMEGSGE LETISLSHTN DSKEFVDPYI

101	VVTLKAGDNL KIKONTHENT NASSFTYSLK KDLTGLINVX TEKLSFGANG

151	KKVNIISDTK GLNFAXETAG TNGDTTVHLN GIGSTLTDTL AGSSASHVDA

201	GNXSTHYTRA ASIKDVLNAG WNIKGVKXGS TTGQSENVDF VRTYDTVEFL

251	SADTXTTTVN VESKDNGKRT EVXIGAXTSV IKEKDGKLVT GKGKGENGSS

301	TDEGEGLVTA KEVIDAVNKA GWRMKTTTAN GQTGQADKFE TVTSGTNVTF

351	ASGKGTTATV SXDDQGNITV MYDVNVGDAL NVNQLONSGW NLDSKAVAGS

401	SGKVISGNVS PSKGKMDETV NINAGNNIEI SRNGKNIDIA TSMAPQFSSV

451	SLGAGADAPT LSVDDEGALN VGSKDANKPV RITNVAPGVK XGDVTNVXQL

501	KGVAQNLNNR IDNYOGNARA GIAQAIATAG LVQAYLPGKS NNAIGGGTYR

551	GEAGYAIGYS SISDGGNWII KGTASGNSRG HFGASASVGY QW

The originally-identified partial strain B sequence (ORF40) shows 65.7% identity over a 254 aa overlap with ORF40a:


	10 20 30
orf40.pep	TLLFATVQASANQEEQEEDLYLDPVQRTVA
	\|\|\|\|\|\|\|\|\|:\|::\|::\|\|:\| : \|\|\|:\|
orf40a	SALNAXVAVSELTRNHTKRASATVKTAVLATLLFATVQANATDEDEEEEL--ESVQRSV-
	20 30 40 50 60

	40 50 60 70 80
orf40.pep	VLIVNSDKEGTGEKEKVEEN-SDWAVYFNEKGVLTAREITXKAGDNLKIKQN------GT
	\| :::: \|\|:\|\| \| : : :: : \| : :: :\| \|\|\|\|\|\|\|\|\|\|\| ::
orf40a	VGSIQASMEGSGELETISLSMTNDSKEFVDPYIV----VTLKAGDNLKIKQNTNENTNAS
	70 80 90 100 110 120

	90 100 110 120 130 140
orf40.pep	NFTYSLKKDLTDLTSVGTEKLSFSANGNKVNITSDTKGLNFAKETAGTNGDTTVHLNGIG
	:\|\|\|\|\|\|\|\|\|\| \| :\| \|\|\|\|\|\|:\|\|\|:\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf40a	SFTYSLKKDLTGLINVXTEKLSFGANGKKVNIISDTKGLNFAKETAGTNGDTTVHLNGIG
	130 140 150 160 170 180

	150 160 170 180 190 200
orf40.pep	STLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTA--SDNVDFV
	\|\|\|\|\|\|\| :::\|: :\| \| : : \|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|:\|: \|:\|\|\|\|\|
orf40a	STLTDTLAGSSAS-HVDAGNXST-HYTRAASIKDVLNAGWNIKGVKXGSTTGQSENVDFV
	190 200 210 220 230 240

	210 220 230 240
orf40.pep	RTYDTVEFLSADTKTTTVNVESKDNGKKTEVKIGAKTSVIKEKD
	\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf40a	RTYDTVEFLSADTXTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSST
	250 260 270 280 290 300

The complete strain B sequence (ORF44-1) and ORF40a show 83.7% identity in 601 aa overlap:


	10 20 30 40 50 60
orf40-1.pep	MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVKTAVLATLLFATVQASANNEEQEEDL
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|::\|::\|\|:\|
orf40a	MNKIYRIIWNSALNAXVAVSELTRNHTKRASATVKTAVLATLLFATVQANATDEDEEEEL
	10 20 30 40 50 60

	70 80 90 100 110 119
orf40-1.pep	YLDPVQRTVAVLIVNSDKEGTGEKEKVEEN-SDWAVYFNEKGVLTAREITLKAGDNLKIK
	: \|\|\|:\| \| :::: \|\|:\|\| \| : : :: : \| : :: :\|\|\|\|\|\|\|\|\|\|\|
orf40a	--ESVQRSV-VGSIQASMEGSGELETISLSMTNDSKEFVDPYIV----VTLKAGDNLKIK
	70 80 90 100 110

	120 130 140 150 160 170
orf40-1.pep	QN------GTNFTYSLKKDLTDLTSVGTEKLSFSANGNKVNITSDTKGLNFAKETAGTNG
	\|\| :::\|\|\|\|\|\|\|\|\| \| :\| \|\|\|\|\|\|:\|\|\|:\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf40a	QNTNENTNASSFTYSLKKDLTGLINVXTEKLSFGANGKKVNIISDTKGLNFAKETAGTNG
	120 130 140 150 160 170

	180 190 200 210 220 230
orf40-1.pep	DTTVHLNGIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTT
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| :::\|: :\| \| : : \|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|:\|
orf40a	DTTVHLNGIGSTLTDTLAGSSAS-HVDAGNXST-HYTRAASIKDVLNAGWNIKGVKXGST
	180 190 200 210 220 230

	240 250 260 270 280 290
orf40-1.pep	A--SDNVDFVRTYDTVEFLSADTKTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTG
	: \|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf40a	TGQSENVDFVRTYDTVEFLSADTXTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTG
	240 250 260 270 280 290

	300 310 320 330 340 350
orf40-1.pep	KDKGENGSSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFA
	\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf40a	KGKGENGSSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFA
	300 310 320 330 340 350

	360 370 360 390 400 410
orf40-1.pep	SGKGTTATVSKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSP
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf40a	SGKGTTATVSKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSP
	360 370 360 390 400 410

	420 430 440 450 460 470
orf40-1.pep	SKGKMDETVNINAGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPTLSVDGD-ALNV
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| : \|\|\|\|
orf40a	SKGKMDETVNINAGNNIEISRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNV
	420 430 440 450 460 470

	480 490 500 510 520 530
orf40-1.pep	GSKKDNKPVRITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGL
	\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf40a	GSKDANKPVRITNVAPGVKXGDVTNVXQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGL
	480 490 500 510 520 530

	540 550 560 570 580 590
orf40-1.pep	VQAYLPGKSMMAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQ
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf40a	VQAYLPGKSMMAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQ
	540 550 560 570 580 590

orf40-1.pep	WX
	\|\|
orf40a	WX

Computer analysis of these amino acid sequences gave the following results:
Homology with Hsf Protein Encoded by the Type b Surface Fibrils Locus of H. influenzae (Accession Number U41852)

ORF40 and Hsf protein show 54% aa identity in 251 aa overlap:


Orf40	1 TLLFATVQASANQEEQEEDLYLDPVQRTVAVLVINSDXXXXXXXXXXXXNSDWAVYFNEK 60
	TLLFATVQA+A E++E LDPV RT VL +SD NS+W +YF+ K
Hsf	41 TLLFATVQANATDEDEE----LDPVVRTAPVLSFHSDKEGTGEKEVTE-NSNWGIYFDNK 95

Orf40	61 GVLTAREITXKAGDNLKIKQN------GTNFTYSLKKDLTDLTSVGTEKLSFSANGNKVN 114
	GVL A IT KAGDNLKIKQN ++FTYSLKKDLTDLTSV TEKLSF ANG+KV+
Hsf	96 GVLKAGAITLKAGDNLKIKQNTDESTNASSFTYSLKKDLTDLTSVATEKLSFGANGDKVD 155

Orf40	115 ITSDTKGLNFAKETAGTNGDTTVhLNGIGSTLTDTLLNTGAXXXXXXXXXXXXEKKRAAS 174
	ITSD GL AK G+ VHLNG+ STL D + NTG EK RAA+
Hsf	156 ITSDANGLKLAK-----TGNGNVHLNGLDSTLPDAVTNTGVLSSSSFTPNDV-EKTRAAT 209

Orf40	175 VKDVLNAGWNIKGVKPGTTASDNVDFVRTYDTVEFLSADTKTTTVNVESKDNGKKTEVKI 234
	VKDVLNAGWNIKG K ++VD V Y+ VEF++ D T V ++K+NGK TEVK
Hsf	210 VKDVLNAGWNIKGAKTAGGNVESVDLVSAYNNVEFITGDKNTLDVVLTAKENGKTTEVKF 269

Orf40	235 GAKTSVIKEKD 245
	KTSVIKEKD
Hsf	270 TPKTSVIKEKD 280

ORF40a also shows homology to Hsf:


gi\|1666683 (U41852) hsf gene product [Haemophilus influenzae] Length = 2353
Score = 153 (67.7 bits), Expect = 1.5−116, Sum P(11) = 1.5e−116
Identities = 33/36 (91%), Positives = 34/36 (94%)
Query: 16 VAVSELTRNHTKRASATVKTAVLATLLFATVQANAT 51
V VSELTR HTKRASATV+TAVLATLLFATVQNAT
Sbjct: 17 VVVSELTRTHTKRASATVETAVLATLLFATVQANAT 52

Score = 161 (71.2 bits), Expect = 1.5e−116, Sum P(11) 1.5e−116
Identities = 32/38 (84%), Positives = 36/38 (94%)
Query: 101 VTLKAGDNLKIKQNTNENTNASSFTYSLKKDLTGLINV 138
+TLAGDNLKIKQNT+E+TNASSFTYSLKKDLT L +V
Sbjct: 103 ITLKAGDNLKIKQNTDESTNASSFFYSLKKDLTDLTSV 140

Score = 110 (48.7 bits), Expect = 1.5e−116, Sum P(11) = 1.5e−116
Identities = 21/29 (72%), Positives = 25/29 (86%)
Query: 138 VTEKLSFGANGKKVNIISDTKGLNFAKET 166
V++KLS G NG KVNI SDTKGLNFAK++
Sbjct: 1439 VSDKLSLGTNGNKVNITSDTXGLNFAKDS 1467

Score = 85 (37.6 bits), Expect = 1.5e−116, Sum P(11) = 1.5e−116
Identities = 18/32 (56%), Positives = 20/32 (62%)
Query: 169 TNGDTTVHLNGIGSTLTDTLAGSSASHVDAGN 200
T D +HLNGI STLTDTL S A+ GN
Sbjct: 1469 TGDDANIHLNGIASTLTDTLLNSGATTNLGGN 1500

Score = 92 (40.7 bits), Expect = 1.5e−116, Sum P(11) = 1.5e−116
Identities = 16/19 (84%), Positives = 19/19 (100%)
Query: 206 RAASIKOVLNAGWNIKGVK 224
RAAS+KDVLNAGWN++GVK
Sbjct: 1509 RAASVKDVLNAGWNVRGVK 1527

Score = 90 (39.8 bits), Expect = 1.5e−116, Sum P(11) = 1.5e−116
Identities = 17/28 (60%), Positives 20/28 (71%)
Query: 226 STTGQSENVDFVRTYDTVEFLSADTTTT 253
S Q EN+DFV TYDTV+F+S D TT
Sbjct: 1530 SANNQVENIDFVATYDTVDFVSGDKDTT 1557

Based on homology with Hsf, it was predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.
ORF40-1 (61 kDa) was cloned in pET and pGex vectors and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIG. 1A shows the results of affinity purification of the His-fusion protein, and FIG. 1B shows the results of expression of the GST-fusion in E. coli. Purified His-fusion protein was used to immunise mice, whose sera were used for FACS analysis (FIG. 1C), a bactericidal assay (FIG. 1D), and ELISA (positive result). These experiments confirm that ORF40-1 is a surface-exposed protein, and that it is a useful immunogen.
FIG. 1E shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF40-1.

Example 2

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 7>


	1	ATGTFACGTt TGACTGCtTT AGCCGTATGC ACCGCCCTCG CTTTGGGCGC

	51	GTGTT~GCCG CAAAATTCCG ACTCTGCCCC ACAAGCCAAA GaACAGGCGG

	101	TTTCCGCCGC ACAAACCGAA GgCGCGTCCG TTACCGTCAA AACCGCGCGC

	151	GGCGACGTTC AAATACCGCA AAACCCCGAA CGCATCGCCG TTTACGATTT

	201	GGGTATGCTC GACACCTTGA GCAAACTGGG CGTGAAAACC GGTTTGTCCG

	251	TCGATAAAAA CCGCCTGCCG TATTTAGAGG AATATTTCAA AACGACAAAA

	301	CCTGCcGGCA CTTTGTTCGA GCCGGATTAC GAAACGCTCA ACGCTTACAA

	351	ACCGCAGCTC ATCATCATCG GCAGCCGCGC CgCCAAGGCG TTTGACAAAT

	401	TGAAcGAAAT CGCGCCGACC ATCGrmwTGA CCGCCGATAC CGCCAACCTC

	451	AAAGAAAGTG CCAArGAGGC ATCGACGCTG GCGCAAATCT TC..

This corresponds to the amino acid sequence <SEQ ID 8; ORF38>:


1	MLRLTALAVC TALALGACSP QNSDSAPOAK EQAVSAAQTE GASVTVKTAR

51	GDVQIPQNPE RIAVYDLGHL DTLSKLGVKT GLSVDKNRLP YLEEYFKTTK

101	PAGTLFEPDY ETLNAYKPQL IIIGSRAAKA FDKLNEIAPT IXXTADTANL

151	KESAKEASTL AQIF..

Further work revealed the complete nucleotide sequence <SEQ ID 9>:


1	ATGTTACGTT TGACTGCTTT AGCCGTATGC ACCGCCCTCG CTTTGGGCGC

51	GTGTTCGCCG CAAAATTCCG ACTCTGCCCC ACAAGCCAAA GAACAGGCGG

101	TTTCCGCCGC ACAAACCGAA GGCGCGTCCG TTACCGTCAA AACCGCGCGC

151	GGCGACGTTC AAATACCGCA AAACCCCGAA CGCATCGCCG TTTACGATTT

201	GGGTATGCTC GACACCTTGA GCAAACTGGG CGTGAAAACC GGTTTGTCCG

251	TCGATAAAAA CCGCCTGCCG TATTTAGAGG AATATTTCAA AACGACAAAA

301	CCTGCCGGCA CTTTGTTCGA GCCGGATTAC GAAACGCTCA ACGCTTACAA

351	ACCGCAGCTC ATCATCATCG GCAGCCGCGC CGCCAAGGCG TTTGACAAAT

401	TGAACGAAAT CGCGCCGACC ATCGAAATGA CCGCCGATAC CGCCAACCTC

451	AAAGAAAGTG CCAAAGAGCG CATCGACGCG CTGGCGCAAA TCTTCGGCAA

501	ACAGGCGGAA GCCGACAAGC TGAAGGCGGA AATCGACGCG TCTTTTGAAG

551	CCGCGAAAAC TGCCGCACAA GGTAAGGGCA AAGGTTTGGT GATTTTGGTC

601	AACGGCGGCA AGATGTCGGC TTTCGGCCCG TCTTCACGCT TGGGCGGCTG

651	GCTGCACAAA GACATCGGCG TTCCCGCTGT CGATGAATCA ATTAAAGAAG

701	GCAGCCACGG TCAGCCTATC AGCTTTGAAT ACCTGAAAGA GAAAAATCCC

751	GACTGGCTGT TTGTCCTTGA CCGAAGCGCG GCCATCGGCG AAGAGGGTCA

801	GGCGGCGAAA GACGTGTTGG ATAATCCGCT GGTTGCCGAA ACAACCGCTT

851	GGAAAAAAGG ACAGGTCGTG TACCTCGTTC CTGAAACTTA TTTGGCAGCC

901	GGTGGCGCGC AAGAGCTGCT GAATGCAAGC AAACAGGTTG CCGACGCTTT

951	TAACGCGGCA AAATAA

This corresponds to the amino acid sequence <SEQ ID 10; ORF38-1>:


1	MLRLTALAVC TALALGACSP QNSDSAPQAK EQAVSAAQTE GASVTVKTAR

51	GDVQIPQNPE RIAVYDLQIL DTLSXLGVKT GLSVDKNRLP YLEEYFKTTK

101	PAGTLFEPDY ETLNAYKPQL IIIGSRAAKA FDKLNEIAPT IENTADTANL

151	KESAKERIDA LAQIFGKQAE ADKLKAEIDA SFEAAKTAAQ GKGKGLVILV

201	NGGKMSAFGP SSRLGGWLKK DIGVPAVDES IKEGSHGQPI SFEYLKEKNP

251	DWLFVLDRSA AIGEEGQAAK DVLDNPLVAE TTAWKKGQVV YLVPETYLAA

301	GGAQELLNAS KQVADAFNAA K*

Computer analysis of this amino acid sequence reveals a putative prokaryotic membrane lipoprotein lipid attachment site (underlined).

Further work identified the corresponding gene in strain A of N. meningitidis <SEQ ID 11>:


1	ATGTTACGTT TGACTGCTTT AGCCGTATGC ACCGCCCTCG CTTTGGGCGC

51	GTGTTCGCCG CAAAATTCCG ACTCTGCCCC ACAAGCCAAA GAACAGGCGG

101	TTTCCGCCGC ACAATCCGAA GGCGTGTCCG TTACCGTCAA AACGGCGCGC

151	GGCGATGTTC AAATACCGCA AAACCCCGAA CGTATCGCCG TTTACGATTT

201	GGGTATGCTC GACACCTTGA GCAAACTGGG CGTGAAAACC GGTTTGTCCG

251	TCGATAAAAA CCGCCTGCCG TATTTAGAGG AATATTTCAA AACGACAAAA

301	CCTGCCGGAA CTTTGTTCGA GCCGGATTAC GAAACGCTCA ACGCTTACAA

351	ACCGCAGCTC ATCATCATCG GCAGCCGCGC AGCCAAAGCG TTTGACAAAT

401	TGAACGAAAT CGCGCCGACC ATCGAAATGA CCGCCGATAC CGCCAACCTC

451	AAAGAAAGTG CCAAAGAGCG TATCGACGCG CTGGCGCAAA TCTTCGGCAA

501	AAAGGCGGAA GCCGACAAGC TGAAGGCGGA AATCGACGCG TCTTTTGAAG

551	CCGCGAAAAC TGCCGCGCAA GGCAAAGGCA AGGGTTTGGT GATTTTGGTC

601	AAcGGCGGCA AGATGTCCGC CTTCGGCCCG TCTTCACGAC TGGGCGGCTG

651	GCTGCACAAA GACATCGGCG TTCCCGCTGT TGACGAAGCC ATCAAAGAAG

701	GCAGCCACGG TCAGCCTATC AGCTTTGAAT ACCTGAAAGA GAAAAATCCC

751	GACTGGCTGT TTGTCCTTGA CCGCAGCGCG GCCATCGGCG AAAAGGGTCA

601	GGCGGCGAAA GACGTGTTGA ACAATCCGCT GGTTGCCGAA ACAACCGCTT

851	GGAAAAATGG ACAAGTCGTT TACCTTGTTC CTGAAACTTA TTTGGCAGCC

901	GGTGGCGCGC AAGAGCTACT GAATGCAAGC AAACAGGTTG CCGACGCTTT

951	TAACGCGGCA AAATAA

This encodes a protein having amino acid sequence <SEQ ID 12; ORF38a>:


1	MLRLTALAVC TALALGACSP QNSDSAPOAK EQAVSAAQSE GVSVTVKTAR

51	GDVQIPQNPE RIAVYDLGHL DTLSKLGVKT GLSVDKNRLP YLEEYFKTTK

101	PAGTLFEPDY ETLNAYKPQL IIIGSRAAKA FDKLNEIAPT IENTADTANL

151	KESAKERIDA LAOIFGKKAE ADKLKAEIDA SFEAAKTAAQ GKGKGLVILV

201	NGGKMSAFGP SSRLGGWLHK DIGVPAVDEA IKEGSHGQPI SFEYLKEKNP

251	DWLFVLDRSA AIGEEGQAAK DVLNNPLVAE TTAWKKGQVV YLVPETYLAA

301	GGAQELLNAS KQVAOAFWAA K*

The originally-identified partial strain B sequence (ORF38) shows 95.2% identity over a 165 aa overlap with ORF38a:


	10 20 30 40 50 60
orf38.pep	MLRLTALAVCTALALGACSPQNSDSAPQAKEQAVSAAQTEGASVTVKTARGDVQIPQNPE
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf38a	MLRLTALAVCTALALGACSPQNSDSAPQAKEQAVSAAQSEGVSVTVKTARGDVQIPQNPE
	10 20 30 40 50 60

	70 80 90 100 110 120
orf38.pep	RIAVYDLGMLDTLSKLGVKTGLSVDKNRLPYLEEYFKTTKPAGTLFEPDYETLNAYKPQL
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf38a	RIAVYDLGMLDTLSKLGVKTGLSVDKNRLPYLEEYFKTTKPAGTLFEPDYETLNAYKPQL
	70 80 90 100 110 120

	130 140 150 160
orf38.pep	IIIGSRAAKAFDKLNEIAPTIXXTADTANLKESAKE-ASTLAQIF
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\| ::\|\|\|\|\|
orf39a	IIIGSRAAKAFDKLNEIAPTIEMTADTANLKESAKERIDALAQIFGKKAEADKLKAEIDA
	130 140 150 160

orf38a	SFEAAKTAAQGKGKGLVILVNGGKMSAFGPSSRLGGWLHKDIGVPAVDEAIKEGSHGQPI
	190 200 210 220 230 240

The complete strain B sequence (ORF38-1) and ORF38a show 98.4% identity in 321 aa overlap:


orf38a.pep	MLRLTALAVCTALALGACSPQNSDSAPQAKEQAVSAAQSEGVSVTVKTARGDVQIPQNPE
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf38-1	MLRLTALAVCTALALGACSPQNSDSAPQAKEQAVSAAQTEGASVTVKTARGDVQIPQNPE

orf38a.pep	RIAVYDLGMLDTLSKLGVKTGLSVDKNRLPYLEEYFKTTKPAGTLFEPDYETLNAYKPQL
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf38-1	RIAVYDLGMLDTLSKLGVKTGLSVDKNRLPYLEEYFKTTKPAGTLFEPDYETLNAYKPQL

orf38a.pep	IIIGSRAAKAFDKLNEIAPTIEMTADTANLKESAKERIDALAQIFGKKAEADKLKAEIDA
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|
orf38-1	IIIGSRAAKAFDKLNEIAPTIEMTADTANLKESAKERIDALAQIFGKQAEADKLKAEIDA

orf38a.pep	SFEAAKTAAQGKGKGLVILVNGGKMSAFGPSSRLGGWLHKDIGVPAVDEAIKEGSHGQPI
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|
orf38-1	SFEAAKTAAQGKGKGLVILVNGGKMSAFGPSSRLGGWLHKDIGVPAVDESIKEGSHGQPI

orf38a.pep	SFEYLKEKNPDWLFVLDRSAAIGEEGQAAKDVLNNPLVAETTAWKKGQVVYLVPETYLAA
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf38-1	SFEYLKEKNPDWLFVLDRSAAIGEEGQAAKDVLDNPLVAETTAWKKGQVVYLVPETYLAA

orf38a.pep	GGAQELLNASKQVADAFNAAK
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf38-1	GGAQELLNASKQVADAFNAAK

Computer analysis of these sequences revealed the following:
Homology with a Lipoprotein (lipo) of C. jejuni (Accession Number X82427)

ORF38 and lipo show 38% aa identity in 96 aa overlap:


Orf38:	40 EGASVTVKTARGDVQIPQNPERIAVYDLGMLDTLSKLGVKTGLS-VKDNRLPYLEEYFKT 98
	EG S VK + G+ + P+NP ++ + DLG+LDT L + ++ V LP + FK
Lipo:	51 EGDSFLVKDSLGENKTPKNPSKVVILDLGILDTFDALKLNDKVAGVPAKNLPKYLQQFKN 110

Orf38:	99 TKPAGTLFEPDYETLNAYKPQLIIIGSRAAKAFDKL 134
	G + + D+E +NA KP LIII R +K +DKL
Lipo:	111 KPSVGGVQQVDFEAINALKPDLIIISGRQSKFYDKL 146

Based on this analysis, it was predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.
ORF38-1 (32 kDa) was cloned in pET and pGex vectors and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIG. 2A shows the results of affinity purification of the His-fusion protein, and FIG. 2B shows the results of expression of the GST-fusion in E. coli. Purified His-fusion protein was used to immunise mice, whose sera were used for Western blot analysis (FIG. 2C) and FACS analysis (FIG. 2D). These experiments confirm that ORF38-1 is a surface-exposed protein, and that it is a useful immunogen.
FIG. 2E shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF38-1.

Example 3

The following N. meningitidis DNA sequence was identified <SEQ ID 13>:


	1	ATGAAACTTC TGACCACCGC AATCCTGTCT TCCGCAATCG CGCTCAGCAG

	51	TATGGCTGCC GCCGCTGGCA CGGACAACCC CACTGTTGCA AAAAAAACCG

	101	TCAGCTACGT CTGCCAGCAA GGTAAAAAAG TCAAAGTAAC CTACGGCTTC

	151	AACAAACAGG GTCTGACCAC ATACGCTTCC GCCGTCATCA ACGGCAAACG

	201	CGTGCAAATG CCTGTCAATT TGGACAAATC CGACAATGTG GAAACATTCT

	251	ACGGCAAAGA AGGCGGTTAT GTTTTGGGTA CCGGCGTGAT GGATGGCAAA

	301	TCCTACCGCA AACAGCCCAT TATGATTACC GCACCTGACA ACCAAATCGT

	351	CTTCAAAGAC TGTTCCCCAC GTTAA

This corresponds to the amino acid sequence <SEQ ID 14; ORF44>:

1 MKLLTTAILS SAIALSSMAA AAGTDWPTVA KKTVSYVCQQ GKKVKVTYGF

51 NKQGLTTYAS AVINGKRVQH PVNLDKSDNV ETFYGKEGGY VLGTGVMDGK

101 SYRKQPIHIT APDNQIVFKD CSPR*
Computer analysis of this amino acid sequence predicted the leader peptide shown underlined.

Further work identified the corresponding gene in strain A of N. meningitidis <SEQ ID 15>:


1	ATGAAACTTC TGACCACCGC AATCCTGTCT TCCGCAATCG CGCTCAGCAG

51	TATGGCTGCT GCTGCCGGCA CGAACAACCC CACCGTTGCC AAAAAAACCG

101	TCAGCTACGT CTGCCAGCAA GGTAAAAAAG TCAAAGTAAC CTACGGCTTT

151	AACAAACAGG GCCTGACCAC ATACGCTTCC GCCGTCATCA ACGGCAAACG

201	TGTGCAAATG CCTGTCAATT TGGACAAATC CGACAATGTG GAAACATTCT

251	ACGGCAAAGA AGGCGGTTAT GTTTTGGGTA CCGGCGTGAT GGATGGCAAA

301	TCCTATCGCA AACAGCCTAT TATGATTACC GCACCTGACA ACCAAATCGT

351	CTTCAAAGAC TGTTCCCCAC GTTAA

This encodes a protein having amino acid sequence <SEQ ID 16; ORF44a>:

1 MKLLTTAILS SAIALSSMAA AAGTNNPTVA KKTVSYVCQQ GKKVKVTYGF

51 NKQGLTTYAS AVINGKRVQM PVNLDKSDNV ETFYGKEGGY VLGTGVMDGK

101 SYRKQPIMIT APDNQIVFKD CSPR*

The strain B sequence (ORF44) shows 99.2% identity over a 124 aa overlap with ORF44a:


	10 20 30 40 50 60
orf44.pep	MKLLTTAILSSAIALSSMAAAAGTDNPTVAKKTVSYVCQQGKKVKVTYGFNKQGLTTYAS
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf44a	MKLLTTAILSSAIALSSMAAAAGTNNPTVAKKTVSYVCQQGKKVKVTYGFNKQGLTTYAS
	10 20 30 40 50 60

70 80 90 100 110 120
orf44.pep	AVINGKRVQMPVNLDKSDNVETFYGKEGGYVLGTGVMDGKSYRKQPIMITAPDNQIVFKD
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf44a	AVINGKRVQMPVNLDKSDNVETFYGKEGGYVLGTGVMDGKSYRKQPIMITAPDNQIVFKD
	70 80 90 100 110 120

orf44.pep	CSPRX
	\|\|\|\|\|
orf44a	CSPRX

Computer analysis gave the following results:
Homology with the LecA Adhesin of Eikenella corrodens (Accession Number D78153)

ORF44 and LecA protein show 45% aa identity in 91 aa overlap:


Orf44	33 TVSYVCQQGKKVKVTYGFNKQGLTTYASAVINGKRVQMPVNLDKSDNVETFYGKEGGYVL 92
	+V+YVCQQG+++ V Y FN G+ T A +N + +++P NL SDNV+T + GY L
LecA	135 SVAYVCQQGRRLNVNYRFNSAGVPTSAELRVNNRNLRLPYNLSASDNVDTVF-SANGYRL 193

Orf44	93 GTGVHDGKSYRKQPIHITAPDNQIVFKDCSP 123
	T MD +YR Q I+++AP+ Q+++KDCSP
LecA	194 TTNAMDSANYRSQDIIVSAPNGQNLYKDCSP 224

Based on homology with the adhesin, it was predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.
ORF44-1 (11.2 kDa) was cloned in pET and pGex vectors and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIG. 3A shows the results of affinity purification of the His-fusion protein, and FIG. 3B shows the results of expression of the GST-fusion in E-coli. Purified His-fusion protein was used to immunise mice, whose sera were used for ELISA, which gave positive results, and for a bactericidal assay (FIG. 3C). These experiments confirm that ORF44-1 is a surface-exposed protein, and that it is a useful immunogen.
FIG. 3D shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF44-1.

Example 4

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 17>


1	GGCACCGAAT TCAAAACCAC CCTTTCCGGA GCCGACATAC AGGCAGGGGT

51	GGGTGAAAAA GCCCGAGCCG ATGCGAAAAT TATCCTAAAA GGCATCGTTA

101	ACCGCATCCA AACCGAAGAA AAGCTGGAAT CCAACTCGAC CGTATGGCAA

151	AAGCAGGCCG GAAGCGGCAG CACGGTTGAA ACGCTGAAGC TACCGAGCTT

201	TGAAGGGCCG GCACTGCCTA AGCTGACCGC TCCCGGCGGC TATATCGCCG

251	ACATCCCCAA AGGCAACCTC AAAACCGAAA TCGAAAAGCT GGCCAAACAG

301	CCCGAATATG CCTATCTGAA ACAGCTTCAG ACGGTCAAGG ACGTGAACTG

351	GAACCAAGTA CAGCTCGCTT ACGACAAATG GGACTATAAA CAGGAAGGCC

401	TAACCGGAGC CGGAGCCGCA ATTANCGCAC TGGCCGTTAC CGTGGTCACC

451	TCAGGCGCAG GAACCGGAGC CGTATTGGGA TTAANACGNG TGGCCGCCGC

501	CGCAACCGAT GCAGCATTT...

This corresponds to the amino acid sequence <SEQ ID 18; ORF49>:


1	GTEFKTTLSG ADIQAGVGEK ARADPKIILK GIVNRIQTEE KLESNSTVWQ

51	KQAGSGSTVE TLKLPSFEGP ALPKLTAPGG YIADIPKGNL KTEIEKLAKQ

101	PEYAYLKQLQ TVKDVNWNQV QLAYDKWDYK QEGLTCAGAA IXALAVTVVT

151	SGAGTGAVLG LXRVAAAATD AAF..

Further work revealed the complete nucleotide sequence <SEQ ID 19>:


1	ATGCAACTGC TGGCAGCCGA AGGCATTCAC CAACACCAAT TGAATGTTCA

51	GAAAAGTACC CGTTTCATCG GCATCAAAGT GGGTAAAAGC AATTACAGCA

101	AAAACGAGCT GAACGAAACC AAACTGCCCG TACGCGTTAT CGCCCAAACA

151	GCCAAAACCC GTTCCGGCTG GGATACCGTA CTCGAAGGCA CCGAATTCAA

201	AACCACCCTT TCCGGAGCCG ACATACAGGC AGGGGTGGGT GAAAAAGCCC

251	GAGCCGATGC GAAAATTATC CTAAAAGGCA TCGTTAACCG CATCCAAACC

301	GAAGAAAAGC TGGAATCCAA CTCGACCGTA TGGCAAAAGC AGGCCGGAAG

351	CGGCAGCACG GTTGAAACGC TGAAGCTACC GAGCTTTGAA GGGCCGGCAC

401	TGCCTAAGCT GACCGCTCCC GGCGGCTATA TCGCCGACAT CCCCAAAGGC

451	AACCTCAAAA CCGAAATCGA AAAGCTGGCC AAACAGCCCG AATATGCCTA

501	TCTGAAACAG CTTCAGACGG TCAAGGACGT GAACTGGAAC CAAGTACAGC

551	TCGCTTACGA CAAATGGGAC TATAAACAGG AAGGCCTAAC CGGAGCCGGA

601	GCCGCAATTA TCGCACTGGC CGTTACCGTG GTCACCTCAG GCGCAGGAAC

651	CGGAGCCGTA TTGGGATTAA ACGGTGCGGC CGCCGCCGCA ACCGATGCAG

701	CATTTGCCTC TTTGGCCAGC CAGGCTTCCG TATCGTFCAT CAACAACAAA

751	CGCAATATCG GTAACACCCT GAAAGAGCTG GGCAGAAGCA GCACGGTGAA

801	AAATCTGATG GTTGCCGTCG CTACCGCAGG CGTAGCCGAC AAAATCGGTG

851	CTTCGGCACT GAACAATGTC AGCGATAAGC AGTGGATCAA CAACCTGACC

901	GTCAACCTGG CCAATGCGGG CAGTGCCGCA CTGATTAATA CCGCTGTCAA

951	CGGCGGCAGC CTGAAAGACA ATCTGGAAGC GAATATCCTT GCGGCTTTGG

1001	TGAATACTGC GCATGGAGAG GCAGCAAGTA AAATCAAACA GTTGGATCAG

1051	CACTACATTG CCCATAAGAT TGCCCATGCC ATAGCGGGCT GTGCGGCAGC

1101	GGCGGCGAAT AAGGGCAAGT GTCAAGATGG TGCGATCGGT GCGGCGGTCG

1151	GTGAAATCCT TGGCGAAACC CTACTGGACG GCAGAGACCC TGGCAGCCTG

1201	AATGTGAAGG ACAGGGCAAA AATCATTGCT AAGGCGAAGC TGGCAGCAGG

1251	GGCGGTTGCG GCGTTGAGTA AGGGGGATGT GAGTACGGCG GCGAATGCGG

1301	CTGCTGTGGC GGTAGAGAAT AATTCTTTAA ATOATATACA GGATCGTTTG

1351	TTGAGTGGAA ATTATGCTTT ATGTATGAGT GCAGGAGGAG CAGAAAGCTT

1401	TTGTGAGTCT TATCGACCAC TGGGCTTGCC ACACTTTGTA AGTGTTTCAG

1451	GAGAAATGAA ATTACCTAAT AAATTCGGGA ATCGTATGGT TAATGGAAAA

1531	TTAATTATTA ACACTAGAAA TGGCAATGTA TATTTCTCTG TAGGTAAAAT

1551	ATGGAGTACT GTAAAATCAA CAAAATCAAA TATAAGTGGG GTATCTGTCG

1601	GTTGGGTTTT AAATGTTTCC CCTAATGATT ATTTAAAAGA AGCATTTATG

1651	AATGATTTCA GAAATAGTAA TCAAAATAAA GCCTATGCAG AAATGATTTC

1701	CCAGACTTTG GTAGGTGAGA GTGTTGGTGG TAGTCTTTGT CTGACAAGAG

1751	CCTGCTTTTC GGTAAGTTCA ACAATATCTA AATCTAAATC TCCTTTTAAA

1801	GATTCAAAAA TTATTGGGGA AATCGGTTTG GGAAGTGGTG TTGCTGCAGG

1851	AGTAGAAAAA ACAATATACA TAGGTAACAT AAAAGATATT GATAAATTTA

1901	TTAGTGCAAA CATAAAAAAA TAG

This corresponds to the amino acid sequence <SEQ ID 20; ORF49-1>:


1	MQLLAAEGIH QHQLNVQKST RFIGIKVGKS NYSKNELNET KLPVRVIAQT

51	AKTRSGWDTV LEGTEFKTTL SGADIQAGVG EKARADAKII LKGIVNRIQT

101	EEKLESNSTV WQKQAGSGST VETLKLPSFE GPALPKLTAP GGYIADIPKG

151	NLKTEIEKLA KQPEYAYLKQ LQTVKDVNWN QVQLAYDKWD YKQEGLTGAG

201	AAIIALAVTV VTSGAGTGAV LGLNGAAAAA TDAAFASLAS QASVSFINNK

251	GNIGNTLKEL GRSSTVKNLM VAVATAGVAD KIGASALNNV SDKQWINNLT

301	VNLANAGSAA LINTAVNGGS LKDNLEANIL AALVNTAHGE AASKIKQLDQ

351	HYIAHKIAHA IAGCAAAAAN KGKCQDGAIG AAVGEILGET LLDGRDPGSL

401	NVKDRAKIIA KAKLAAGAVA ALSKGDVSTA ANAAAVAVEN NSLNDIQDRL

451	LSGNYALCNS AGGAESFCES YRPLGLPHFV SVSGENKLPN KFGNRNVNGK

501	LIINTRNGNV YFSVGKIWST VKSTKSNISG VSVGWVLNVS PNDYLKEASM

551	NDFRNSNQNK AYAEMISQTL VGESVGGSLC LTRACFSVSS TISKSKSPFK

601	DSKIIGEIGL GSGVAAGVEK TIYIGNIKDI DKFISANIKK *

Computer analysis predicts a transmembrane domain and also indicates that ORF49 has no significant amino acid homology with known proteins. A corresponding ORF from N. meningitidis strain A was, however, identified:

ORF49 shows 86.1% identity over a 173 aa overlap with an ORF (ORF49a) from strain A of N. meningitidis:


	10 20 30
orf49.pep	GTEFKTTLSGADIQAGVGEKARADAKIILK
	\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\| \|\|\|\|:\|\|\|\|\|\|\|
orf49a	SKNELNETKLPVRVVAQXAATRSGWDTVLEGTEFKTTLAGADIQAGVXEKARVDAKIILK
	40 50 60 70 80 90

	40 50 60 70 80 90
orf49.pep	GIVNRIQTEEKLESNSTVWQKQAGSGSTVETLKLPSFEGPALPKLTAPGGYIADIPKGNL
	\|\|\|\|\|\|\|:\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\| \|\|\|:\|\|\|\|\|\|\|\|\|:\|: \|\|\|:\|\|\|\|\|\|:\|\|\|\|\|\|\|
orf49a	GIVNRIQSEEKLETNSTVWQKQAGRGSTIETLKLPSFESPTPPKLSAPGGYIVDIPKGNL
	100 110 120 130 140 150

	100 110 120 130 140 150
orf49.pep	KTEIEKLAKQPEYAYLKQLQTVKDVNWNQVQLAYDKWDYKQEGLTGAGAAIXALAVTVVT
	\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|::\|::\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\| \|\|\|\|\| \|\|\|\|\|\|\|\|
orf49a	KTEIEKLSKQPEYAYLKQLQVAKNINWNQVQLAYDRWDYKQEGLTEAGAAIIALAVTVVT
	160 170 180 190 200 210

	160 170
orf49.pep	SGAGTGAVLGLXRVAAAATDAAF
	\|\|\|\|\|\|\|\|\|\|\| : \|\|\|\|\|\|\|\|
orf49a	SGAGTGAVLGLNGAXAAATDAAFASLASQASVSFINNKGDVGKTLKELGRSSTVKNLVVA
	220 230 240 250 260 270

ORF49-1 and ORF49a show 83.2% identity in 457 aa overlap:


orf49a.pep	XQLLAEEGIHKHELDVQKSRRFIGIKVGXSNYSKNELNETKLPVRVVAQXAATRSGWDTV
	\|\|\|\| \|\|\|\|:\|:\|:\|\|\|\| \|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|:\| \|\|\|\|\|\|\|\|
orf49-1	MQLLAAEGIHQHQLNVQKSTRFIGIKVGKSNYSKNELNETKLPVRVIAQTAKTRSGWDTV

orf49a.pep	LEGTEFKTTLAGADIQAGVXEKARVDAKIILKGIVNRIQSEEKLETNSTVWQKQAGRGST
	\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\| \|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\| \|\|\|
orf49-1	LEGTEFKTTLSGADIQAGVGEKARADAXIILKGIVNRIQTEEKLESNSTVWQKQAGSGST

orf49a.pep	IETLKLPSFESPTPPKLSAPGGYIVDIPKGNLKTEIEKLSKQPEYAYLKQLQVAKNINWN
	:\|\|\|\|\|\|\|\|\|:\|: \|\|\|:\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|::\|::\|\|\|
orf49-1	VETLKLPSFEGPALPKLTAPGGYIADIPKGNLKTEIEKLAKQPEYAYLKQLQTVKDVNWN

orf49a.pep	QVQLAYDRWDYKQEGLTEAGAAIIALAVTVVTSGAGTGAVLGLNGAXAAATDAAFASLAS
	\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|
orf49-1	QVQLAYDKWDYKQEGLTGAGAAIIALAVTVVTSGAGTGAVLGLNGAAAAATDAAFASLAS

orf49a.pep	QASVSFINNKGDVGKTLKELGRSSTVKNLVVAAATAGVADKIGASALXNVSDKQWINNLT
	\|\|\|\|\|\|\|\|\|\|\|::\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|
orf49-1	QASVSFINNKGNIGNTLKELGRSSTVKNLMVAVATAGVADKIGASALNNVSDKQWINNLT

orf49a.pep	VNLANAGSAALINTAVNGGSLKDXLEANILAALVNTAHGEAASKIKQLDQHYIVHKIAHA
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|
orf49-1	VNLANAGSAALINTAVNGGSLKDNLEANILAALVNTAHGEAASKIKQLDQHYIAHKIAHA

orf49a.pep	IAGCAAAAANKGKCQDGAIGAAVGEIVGEALTNGKNPDTLTAKEREQILAYSKLVAGTVS
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|:\| :\|::\| :\|::\|:\| :\|:\| :\|\|:\|\|:\|:
orf49-1	IAGCAAAAANKGKCQDGAIGAAVGEILGETLLDGRDPGSLNVKDRAKIIAKAKLAAGAVA

orf49a.pep	GVVGGDVNAAANAAEVAVKNNQLSDXEGREFDNEMTACAKQNXPQLCRKNTVKKYQNVAD
	:: \|\|\|::\|\|\|\|\| \|\|\|:\|\|:\|:\| : \| :::::: \|
orf49-1	ALSKGDVSTAANAAAVAVENNSLNDIQDRLLSGNYALCMSAGGAESFCESYRPLGLPHFV

orf49a.pep	KRLAASIAICTDISRSTECRTIRKQHLIDSRSLHSSWEAGLIGKDDEWYKLFSKSYTQAD

orf49-1	SVSGEMKLPNKFGNRMVNGKLIINTRNGNVYFSVGKIWSTVKSTKSNISGVSVGWVLNVS

The complete length ORF49a nucleotide sequence <SEQ ID 21> is:


1	NTGCAACTGC TGGCAGAAGA AGGCATCCAC AAGCACGAGT TGGATGTCCA

51	AAAAAGCCGC CGCTTTATCG GCATCAAGGT AGGTNAGAGC AATTACAGTA

101	AAAACGAACT GAACGAAACC AAATTGCCTG TCCGCGTCGT CGCCCAAANT

151	GCAGCCACCC GTTCAGGCTG GGATACCGTG CTCGAAGGTA CCGAATTCAA

201	AACCACGCTG GCCGGTGCCG ACATTCAGGC AGGTGTANGC GAAAAAGCCC

251	GTGTCGATGC GAAAATTATC CTCAAAGGCA TTGTGAACCG TATCCAGTCG

301	GAAGAAAAAT TAGAAACCAA CTCAACCGTA TGGCAGAAAC AGGCCGGACG

351	CGGCAGCACT ATCGAAACGC TAAAACTGCC CAGCTTCGAA AGCCCTACTC

401	CGCCCAAATT GTCCGCACCC GGCGGNTATA TCGTCGACAT TCCGAAAGGC

451	AATCTGAAAA CCGAAATCGA AAAGCTGTCC AAACAGCCCG AGTATGCCTA

501	TCTGAAACAG CTCCAAGTAG CGAAAAACAT CAACTGGAAT CAGGTGCAGC

551	TTGCTTACGA CAGATGGGAC TACAAACAGG AGGGCTTAAC CGAAGCAGGT

601	GCGGCGATTA TCGCACTGGC CGTTACCGTG GTCACCTCAG GCGCAGGAAC

651	CGGAGCCGTA TTGGGATTAA ACGGTGCGNC CGCCGCCGCA ACCGATGCAG

701	CATTCGCCTC TTTGGCCAGC CAGGCTTCCG TATCGTTCAT CAACAACAAA

751	GGCGATGTCG GCAAAACCCT GAAAGAGCTG GGCAGAAGCA GCACGGTGAA

801	AAATCTGGTG GTTGCCGCCG CTACCGCAGG CGTAGCCGAC AAAATCGGCG

851	CTTCGGCACT GANCAATGTC AGCGATAAGC AGTGGATCAA CAACCTGACC

901	GTCAACCTAG CCAATGCGGG CAGTGCCGCA CTGATTAATA CCGCTGTCAA

951	CGGCGGCAGC CTGAAAGACA NTCTGGAAGC GAATATCCTT GCGGCTTTGG

1001	TCAATACCGC GCATGGAGAA GCAGCCAGTA AAATCAAACA GTTGGATCAG

1051	CACTACATAG TCCACAAGAT TGCCCATGCC ATAGCGGGCT GTGCGGCAGC

1101	GGCGGCGAAT AAGGGCAAGT GTCAGGATGG TGCGATAGGT GCGGCTGTGG

1151	GCGAGATAGT CGGGGAGGCT TTGACAAACG GCAAAAATCC TGACACTTTG

1201	ACAGCTAAAG AACGCGAACA GATTTTGGCA TACAGCAAAC TGGTTGCCGG

1251	TACGGTAAGC GGTGTGGTCG GCGGCGATGT AAATGCGGCG GCGAATGCGG

1301	CTGAGGTAGC GGTGAAAAAT AATCAGCTTA GCGACTAAGA GGGTAGAGAA

1351	TTTGATAACG AAATGACTGC ATGCGCCAAA CAGAATANTC CTCAACTGTG

1401	CAGAAAAAAT ACTGTAAAAA AGTATCAAAA TGTTGCTGAT AAAAGACTTG

1451	CTGCTTCGAT TGCAATATGT ACGGATATAT CCCGTAGTAC TGAATGTAGA

1501	ACAATCAGAA AACAACATTT GATCGATAGT AGAAGCCTTC ATTCATCTTG

1551	GGAAGCAGGT CTAATTGGTA AAGATGATGA ATGGTATAAA TTATTCAGCA

1601	AATCTTACAC CCAAGCAGAT TTGGCTTTAC AGTCTTATCA TTTGAATACT

1651	GCTGCTAAAT CTTGGCTTCA ATCGGGCAAT ACAAAGCCTT TATCCGAATG

1701	GATGTCCGAC CAAGGTTATA CACTTATTTC AGGAGTTAAT CCTAGATTCA

1751	TTCCAATACC AAGAGGGITF GTAAAACAAA ATACACCTAT TACTAATGTC

1801	AAATACCCGG AAGGCATCAG TTTCGATACA AACCTANAAA GACATCTGGC

1851	AAATGCTGAT GGTTTTAGTC AAGAACAGGG CATTAAAGGA GCCCATAACC

1901	GCACCAATNT TATGGCAGAA CTAAATTCAC GAGGAGGANG NGTAAAATCT

1951	GAAACCCANA CTGATATTGA AGGCATTACC CGAATTAAAT ATGAGATTCC

2001	TACACTAGAC AGGACAGGTA AACCTGATGG TGGATTTAAG GAAATTTCAA

2051	GTATAAAAAC TGTTTATAAT CCTAAAAANT TTTNNGATGA TAAAATACTT

2101	CAAATGGCTC AANATGCTGN TTCACAAGGA TATTCAAAAG CCTCTAAAAT

2151	TGCTCAAAAT GAAAGAACTA AATCAATATC GGAAAGAAAA AATGTCATTC

2201	AATTCTCAGA AACCTTTGAC GGAATCAAAT TTAGANNNTA TNTNGATGTA

2251	AATACAGGAA GAATTACAAA CATTCACCCA GAATAATTTA A

This encodes a protein having amino acid sequence <SEQ ID 22>:


1	XQLLAEEGIH KHELDVQKSR RFIGIKVGXS NYSKNELNET KLPVRVVAQX

51	AATRSGWDTV LEGTEFKTTL AGADIQAGVX EKARVOAKII LKGIVNRIQS

101	EEKLETNSTV WQKQAGRGST IETLKLPSFE SPTPPKLSAP GGYIVDIPKG

151	NLKTEIEKLS KQPEYAYLKQ LQVAKNINWN QVQLAYDRWD YKQEGLTEAG

201	AAIIALAVTV VTSGAGTGAV LGLNGAXAAA TORAFASLAS QASVSFINNK

251	GDVGKTLKEL GRSSTVKNLV VAAATAGVAD KIGASALXNV SDKQNINNLT

301	VNLANAGSAA LINTAVNGGS LKDXLEANIL AALVNTAHGE AASKIKQLDQ

351	HYIVHKIAHA IAGCAAAAAN KGKCQDGAIG AAVGEIVGEA LTNGKNPDTL

401	TAKEREQILA YSKLVAGTVS GVVGGDVNAA ANAAEVAVKN NQLSDXEGRE

451	FDWEHTACAK QNXPQLCRXN TVKKYQNVAD KRLAASIAIC TDISRSTECR

501	TIRKQHLIDS RSLHSSWEAG LIGKDDEWYK LFSKSYTQAD LALOSYHLNT

551	AAKSWLQSGN TKPLSEWNSD QGYTLISGVN PRFIPIPRGF VKQNTPITNV

601	KYPEGISFDT NLXRHLATAD GFSQEQGIKG AHNRTNXMAE LNSRGGXVKS

651	ETXTDIEGIT RIKYEIPTLD RTGKPDGGFK EISSIKTVYN FKXFKDDKIL

701	QMAQXAXSQG YSKASKIAQN ERTKSISERK NVIQFSETFD GIKFRXYXDV

751	NTGRITNIHP E

Based on the presence of a putative transmembrane domain, it is predicted that these proteins from N. meningitidis, and their epitopes, could be useful antigens for vaccines or diagnostics.

Example 5

The following partial DNA sequence was identified in N. meningitidis SEQ ID 23>


1	..CGGATCGTTG TAGGTTTGCG GATTTCTTGC GCCGTAGTCA CCGTAGTCCC

51	AAGTATAACC CAAGGCTTTG TCTTCGCCTT TCATTCCGAT AAGGGATATG

101	ACGCTTTGGT CGGTATAGCC GTCTTGGGAA CCTTTGTCCA CCCAACGCAT

151	ATCTGCCTGC GGATTCTCAT TGCCGCTTCT TGGCTGCTGA TTTTTCTGCC

201	TTCGCGTTTT TCAACTTCGC GCTTGAGGGC TTCGGCATAT TTGTCGGCCA

251	ACGCCATTTC TTTCGGATGC AGCTGCCTAT TGTTCCAATC TACATTCGCA

301	CCCACCACAG CACCACCACT ACCACCAGTT GCATAG

This corresponds to the amino acid sequence <SEQ ID 24; ORF50>:

1 ..RIVVGLRISC AVVTVVPSIT QGFVFAFHSD KGYDALVGIA VLGTFVHPTH

51 ICLRILIAAS WLLIFLPSRF STSRLRASAY LSANAISFGC SCLLFQSTFA

101 PTTAPPLPPV A*
Computer analysis predicts two transmembrane domains and also indicates that ORF50 has no significant amino acid homology with known proteins.
Based on the presence of a putative transmembrane domain, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 6

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 25>


1	..AAGTTTGACT TTACCTGGTT TATTCCGGCG GTAATCAAAT ACCGCCGGTT

51	GTTTTTTGAA GTATTGGTGG TGTCGGTGGT GTTGCAGCTG TTTGCGCTGA

101	TTACGCCTCT GTTTTTCCAA GTGGTGATGG ACAAGGTGCT GGTACATCGG

151	GGATTCTCTA CTTTGGATGT GGTGTCGGTG GCTTTGTTGG TGGTGTCGCT

201	GTTTGAGATT GTGTTGGGCG GTTTGCGGAC GTATCTGTTT GCACATACGA

251	CTTCACGTAT TGATGTGGAA TTGGGCGCGC GTTTGTTCCG GCATCTGCTT

301	TCCCTGCCTT TATCCTATTT CGAGCACAGA CGAGTGGGTG ATACGGTGGC

351	TCGGGTGCGG GAATTGGAGC AGATTCGCAA TTTCTTGACC GGTCAGGCGC

401	TGACTTCGGT GTTGGATTTG GCGTTTTCGT TTATCTTTCT GGCGGTGATG

451	TGGTATTACA GCTCCACTCT GACTTGGGTG GTATTGGCTT CGTTG.....

	//

1451	.......... .......... .......... .......... ..........

1501	.......... .......... .......... .......... ..ATTTGCGC

1551	CAACCGGACG GTGCTGATTA TCGCCCACCG TCTGTCCACT GTTAAAACGG

1601	CACACCGGAT CATTGCCATG GATAAAGGCA GGATTGTGGA AGCGGGAACA

1651	CAGCAGGAAT TGCTGGCGAA CG..AACGGA TATTACCGCT ATCTGTATGA

1701	TTTACAGAAC GGGTAG

This corresponds to the amino acid sequence <SEQ ID 26; ORF39>:


1	..KFDFTWFIPA VIKYRRLFFE VLVVSVVLQL FALITPLFFQ VVMDKVLVHR

51	GFSTLDVVSV ALLVVSLFEI VLGGLRTYLF AHTTSRIDVE LGARLFRHLL

101	SLPLSYFEHP RVGDTVARVR ELEQIRNFLT GQALTSVLDL AFSFIFLAVM

151	WYYSSTLTWV VLASL..... .......... .......... ..........

	//

501	.......... ....ICANRT VLIIAHRLST VKTAHRIIAH DKGRIVEAGT

551	QQELLANXNG YYRYLYDLQN G*

Further work revealed the complete nucleotide sequence <SEQ ID 27>:


1	ATGTCTATCG TATCCGCACC GCTCCCCGCC CTTTCCGCCC TCATCATCCT

51	CGCCCATTAC CACGGCATTG CCGCCAATCC TGCCGATATA CAGCATGAAT

101	TTTGTACTTC CGCACAGAGC GATTTAAATG AAACGCAATG GCTGTTAGCC

151	GCCAAATCTT TGGGATTGAA GGCAAAGGTA GTCCGCCAGC CTATTAAACG

201	TTTGGCTATG GCGACTTTAC CCGCATTGGT ATGGTGTGAT GACGGCAACC

251	ATTTCATTTT GGCCAAAACA GACGGTGAGG GTGAGCATGC CCAATTTTFG

301	ATACAGGATT TGGTTACGAA TAAGTCTGCG GTATTGTCTT TTGCCGAATT

351	TTCTAACAGA TATTCGGGCA AACTGATATT GGTTGCTTCC CGCGCTTCGG

401	TATTGGGCAG TTTGGCAAAG TTTGACTTTA CCTGGTTTAT TCCGGCGGTA

451	ATCAAATACC GCCGGTTGTT TTTTGAAGTA TTGGTGGTGT CGGTGGTGTT

501	GCAGCTGTTT GCGCTGATTA CGCCTCTGTT TTTCCAAGTG GTGATGGACA

551	AGGTGCTGGT ACATCGGGGA TTCTCTACTT TGGATGTGGT GTCGGTGGCT

601	TTGTTGGTGG TGTCGCTGTT TGAGATTGTG TTGGGCGGTT TGCGGACGTA

651	TCTGTTTGCA CATACGACTT CACGTATTGA TGTGGAATTG GGCGCGCGTT

701	TGTTCCGGCA TCTGCTTTCC CTGCCTTTAT CCTATTTCGA GCACAGACGA

751	GTGGGTGATA CGGTGGCTCG GGTGCGGGAA TTGGAGCAGA TTCGCAATTT

801	CTTGACCGGT CAGGCGCTGA CTTCGGTGTT GGATTTGGCG TTTTCGTTTA

951	TCTTTCTGGC GGTGATGTGG TATTACAGCT CCACTCTGAC TTGGGTGGTA

901	TTGGCTTCGT TGCCTGCCTA TGCGTTTTGG TCGGCATTTA TCAGTCCGAT

951	ACTGCGGACG CGTCTGAACG ATAAGTTCGC GCGCAATGCA GACAACCAGT

1001	CGTTTTTAGT AGAAAGCATC ACTGCGGTGG GTACGGTAAA GGCGATGGCG

1051	GTGGAGCCGC AGATGACGCA GCGTTGGGAC AATCAGTTGG CGGCTTATGT

1101	GGCTTCGGGA TTTCGGGTAA CGAAGTTGGC GGTGGTCGGC CAGCAGGGGG

1151	TGCAGCTGAT TCAGAAGCTG GTGACGGTGG CGACGTTGTG GATTGGCGCA

1201	CGGCTGGTAA TTGAGAGCAA GCTGACGGTG GGGCAGCTGA TTGCGTTTAA

1251	TATGCTCTCG GGACACGTGG CGGCGCCTGT TATCCGTTTG GCGCAGTTGT

1301	GGCAGGATTT CCAGCAGGTG GGGATTTCGG TGGCGCGTTT GGGGGATATT

1351	CTGAATGCGC CGACCGAGAA TGCGTCTTCG CATTTGGCTT TGCCCGATAT

1401	CCGGGGGGAG ATTACGTTCG AACATGTCGA TTTCCGCTAT AAGGCGGACG

1451	GCAGGCTGAT TTTGCAGGAT TTGAACCTGC GGATTCCGGC GGGGGAAGTG

1501	CTGGGGATTG TGGGACGTTC GGGGTCGGGC AAATCCACAC TCACCAAATT

1551	GGTGCAGCGT CTGTATGTAC CGGAGCAGGG ACGGGTGTTG GTGGACGGCA

1601	ACGATTTGGC TTTGGCCGCT CCTGCCTGGC TGCGGCGGCA GGTCGGCGTG

1651	GTCTTGCAGG AGAATGTGCT GCTCAACCGC AGCATACGCG ACAATATCGC

1701	GCTGACGGAT ACGGGTATGC CGCTGGAACG CATTATCGAA GCAGCCAAAC

1751	TGGCGGGCGC ACACGAGTTT ATTATGGAGC TGCCGGAAGG CTACGGCACC

1801	GTGGTGGGCG AACAAGGGGC CGGCTTGTCG GGCGGACAGC GGCAGCGTAT

1951	TGCGATTGCC CGCGCGTTAA TCACCAATCC GCGCATTCTG ATTTTTGATG

1901	AAGCCACCAG CGCGCTGGAT TATGAAAGTG AACGAGCGAT TATGCAGAAC

1951	ATGCAGGCCA TTTGCGCCAA CCGGACGGTG CTGATTATCG CCCACCGTCT

2001	GTCCACTGTT AAAACGGCAC ACCGGATCAT TGCCATGGAT AAAGGCAGGA

2051	TTGTGGAAGC GGGAACACAG CAGGAATTGC TGGCGAAGCC GAACGGATAT

2101	TACCGCTATC TGTATGATTT ACAGAACGGG TAG

This corresponds to the amino acid sequence <SEQ ID 28; ORF39-1>:


1	MSIVSAPLPA LSALIILAMY HGIAANPADI QHEFCTSAQS DLNETQWLLA

51	AKSLGLKAKV VRQPIKRLAM ATLPALVWCD DGNHFILAKT DGEGEHAQFL

101	IQDLVTNKSA VLSFAEFSNR YSGKLILVAS RASVLGSLAK FDFTWFIPAV

151	IKYRRLFFEV LVVSVVLQLF ALITPLFFQV VMDKVLVHRG FSTLDVVSVA

201	LLVVSLFEIV LGGLRTYLFA HTTSRIDVEL GARLFRHLLS LPLSYFEHPA

251	VGDTVARVRE LEQIRNFLTG QALTSVLDLA FSFIFLAVMW YYSSTLTWVV

301	LASLPAYAFW SAFISPILRT RLNDKFAPNA DNQSFLVESI TAVGTVKAMA

351	VEPQHTQRWD NQLAAYVASG FRVTKLAVVG QQGVQLIQKL VTVATLWIGA

401	RLVIESRLTV GQLIAFNMLS GQVAAPVIRL AQLWQDFQQV GISVARLGDI

451	VVAPTENASS HLALPDIRGE ITFEHVDFRY KADGRLILQD LNLRIRAGEV

501	LGIVGRSGSG KSTLTKLVQR LYVPEQGRVL VDGNDLALAA PAWLRRQVGV

551	VLOENVLLNR SIRDNIALTD TGNPLERIIE AAKLAGAHEF IMELPEGYGT

601	VVGEQGAGLS GGQRQRIAIA RALITNPRIL IFDEATSALD YESERAINQN

651	MQAICANRTV LIIAHRLSTV KTAHRIIAND KGRIVEAGTQ QELLAKPNGY

701	YRYLYDLQNG *

Computer analysis of this amino acid sequence gave the following results:
Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF39 shows 100% identity over a 165 aa overlap with an ORF (ORF39a) from strain A of N. meningitidis:


	10 20 30
orf39.pep	KFDFTWFIPAVIKYRRLFFEVLVVSVVLQL
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf39a	AVLSFAEFSNRYSGKLILVASRASVLGSLAKFDFTWFIPAVIKYRRLFFEVLVVSVVLQL
	110 120 130 140 150 160

	40 50 60 70 80 90
orf39.pep	FALITPLFFQVVMDKVLVHRGFSTLDVVSVALLVVSLFEIVLGGLRTYLFAHTTSRIDVE
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf39a	FALITPLFFQVVMDKVLVHRGFSTLDVVSVALLVVSLFEIVLGGLRTYLFAHTTSRIDVE
	170 180 190 200 210 220

	100 110 120 130 140 150
orf39.pep	LGARLFRHLLSLPLSYFEHRRVGDTVARVRELEQIRNFLTGQALTSVLDLAFSFIFLAVM
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf39a	LGARLFRHLLSLPLSYFEHRRVGDTVARVRELEQIRNFLTGQALTSVLDLAFSFIFLAVM
	230 240 250 260 270 280

	160 170 180 190 200 210
orf39.pep	WYYSSTLTWVVLASLXXXXXXXXXXXXXXXXXXXXXXXXXXXXICANRTVLIIAHRLSTV
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf39a	WYYSSTLTWVVLASLPAYAFWSAFISPILRTRLNDKFARNADNQSFLVESITAVGTVKAM
	290 300 310 320 330 340

ORF39-1 and ORF39a show 99.4% identity in 710 aa overlap:


orf39-1.pep	MSIVSAPLPALSALIILAHYHGIAANPADIQHEFCTSAQSDLNETQWLLAAKSLGLKAKV
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf39a	MSIVSAPLPALSALIILAHYHGIAANPADIQHEFCTSAQSDLNETQWLLAAKSLGLKAKV

orf39-1.pep	VRQPIKRLAMATLPALVWCDDGNHFILAKTDGEGEHAQFLIQDLVTNKSAVLSFAEFSNR
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|:\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf39a	VRQPIKRLAMATLPALVWCDDGNHFILAKTDGGGEHAQYLIQDLTTNKSAVLSFAEFSNR

orf39-1.pep	YSGKLILVASRASVLGSLAKFDFTWFIPAVIKYRRLFFEVLVVSVVLQLFALITPLFFQV
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf39a	YSGKLILVASRASVLGSLAKFDFTWFIPAVIKYRRLFFEVLVVSVVLQLFALITPLFFQV

orf39-1.pep	VMDKVLVHRGFSTLDVVSVALLVVSLFEIVLGGLRTYLFAHTTSRIDVELGARLFRHLLS
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf39a	VMDKVLVHRGFSTLDVVSVALLVVSLFEIVLGGLRTYLFAHTTSRIDVELGARLFRHLLS

orf39-1.pep	LPLSYFEHRRVGDTVARVRELEQIRNFLTGQALTSVLDLAFSFIFLAVMWYYSSTLTWVV
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf39a	LPLSYFEHRRVGDTVARVRELEQIRNFLTGQALTSVLDLAFSFIFLAVMWYYSSTLTWVV

orf39-1.pep	LASLPAYAFWSAFISPILRTRLNDKFARNADNQSFLVESITAVGTVKAMAVEPQMTQRWD
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf39a	LASLPAYAFWSAFISPILRTRLNDKFARNADNQSFLVESITAVGTVKAMAVEPQMTQRWD

orf39-1.pep	NQLAAQVASGFRVTKLAVVGQQGVQLIQKLVTVATLWIGARLVIESKLTVGQLIAFNMLS
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf39a	NQLAAQVASGFRVTKLAVVGQQGVQLIQKLVTVATLWIGARLVIESKLTVGQLIAFNMLS

orf39-1.pep	GQVAAPVIRLAQLWQDFQQVGISVARLGDILNAPTENASSHLALPDIRGEITFEHVDFRY
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf39a	GQVAAPVIRLAQLWQDFQQVGISVARLGDILNAPTENASSHLALPDIRGEITFEHVDFRY

orf39-1.pep	KADGRLILQDLNLRIRAGEVLGIVGRSGSGKSTLTKLVQRLYVPEQGRVLVDGNDLALAA
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf39a	KADGRLILQDLNLRIRAGEVLGIVGRSGSGKSTLTKLVQRLYVPEQGRVLVDGNDLALAA

orf39-1.pep	PAWLRRQVGVVLQENVLLNRSIRDNIALTDTGMPLERIIEAAKLAGAHEFIHELPEGYGT
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf39a	PAWLRRQVGVVLQENVLLNRSIRDNIALTDTGMPLERIIEAAKLAGAHEFIHELPEGYGT

orf39-1.pep	VVGEQGAGLSGGQRQRIAIARALITNPRILIFDEATSALDYESERAIMQNMQAICANRTV
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf39a	VVGEQGAGLSGGQRQRIAIARALITNPRILIFDEATSALDYESERAIMQNMQAICANRTV

orf39-1.pep	LIIAHRLSTVKTAHRIIAMDKGRIVEAGTQQELLAKPNGYYRYLYDLQNGX
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf39a	LIIAHRLSTVKTAHRIIAMDKGRIVEAGTQQELLAKPNGYYRYLYDLQNGX

The complete length ORF39a nucleotide sequence <SEQ ID 29> is:


1	ATGTCTATCG TATCCGCACC GCTCCCCGCC CTTTCCGCCC TCATCATCCT

51	CGCCCATYAC CACGGCATTG CCGCCAATCC TGCCGATATA CAGCATGAAT

101	TTTGTACTTC CGCACAGAGC GATTTAAATG AAACGCAATG GCTGTTAGCC

151	GCCAAATCTT TGGGATTGAA GGCAAAGGTA GTCCGCCAGC CTATTAAACG

201	TTTGGCTATG GCGACTTTAC CCGCATTGGT ATGGTGTGAT GACGGCAACC

251	ATTTTATTTT GGCTAAAACA GACGGTGGGG GTGAGCATGC CCAATATCTA

301	ATACAGGATT TAACTACGAA TAAGTCTGCG GTATTGTCTT TTGCCGAATT

351	TTCTAACAGA TATTCGGGCA AACTGATATT GGTTGCTTCC CGCGCTTCGG

401	TATTGGGCAG TTTGGCAAAG TTTGACTTTA CCTGGTTTAT TCCGGCGGTA

451	ATCAAATACC GCCGGTTGTT TTTTGAAGTA TTGGTGGTGT CGGTGGTGTT

501	GCAGCTGTTT GCGCTGATTA CGCCTCTGTT TTTCCAAGTG GTGATGGACA

551	AGGTGCTGGT ACATCGGGGA TTCTCTATTT TGGATGTGGT GTCGGTGGCT

601	TTGTTGGTGG TGTCGCTGTT TGAGATTGTG TTGGGCGGTT TGCGGACGTA

651	TCTGTTTGCA CATACGACTT CACGTATTGA TGTGGAATTG GGCGCGCGTT

701	TGTTCCGGCA TCTGCTTTCC CTGCCTTTAT CCTATTTCGA GCACAGACGA

751	GTGGGTGATA CGGTGGCTCG GGTGCGGGAA TTGGAGCAGA TTCGCAATTT

801	CTTGACCGGT CAGGCGCTGA CTTCGGTGTT GGATTTGGCG TTTTCGTTTA

951	TCTTTCTGGC GGTGATGTGG TATTACAGCT CCACTCTGAC TTGGGTGGTA

901	TTGGCTTCGT TGCCTGCCTA TGCGTTTTGG TCGGCATTTA TCAGTCCGAT

951	ACTGCGGACG CGTCTGAACG ATAAGTTCGC GCGCAATGCA GACAACCAGT

1001	CGTTTTTAGT AGAAAGCATC ACTGCGGTGG GTACGGTAAA GGCGATGGCG

1051	GTGGAGCCGC AGATGACGCA GCGTTGGGAC AATCAGTTGG CGGCTTATGT

1101	GGCTTCGGGA TTTCGGGTAA CGAAGTTGGC GGTGGTCGGC CAGCAGGGGG

1151	TGCAGCTGAT TCAGAAGCTG GTGACGGTGG CGACGTTGTG GATTGGCGCA

1201	CGGCTGGTAA TTGAGAGCAA GCTGACGGTG GGGCAGCTGA TTGCGTTTAA

1251	TATGCTCTCG GGACAGGTGG CGGCGCCTGT TATCCGTTTG GCGCAGTTGT

1301	GGCAGGATTT CCAGCAGGTG GGGATTTCGG TGGCGCGTTT CGGGGATATT

1351	CTGAATGCGC CGACCGAGAA TGCGTCTTCG CATTTGGCTT TGCCCGATAT

1401	CCGGGGGGAG ATTACGTTCG AACATGTCGA TTTCCGCTAT AAGGCGGACG

1451	GCAGGCTGAT TTTGCAGGAT TTGAACCTGC GGATTCGGGC GGGGGAAGTG

1501	CTGGGGATTG TGGGACGTTC GGGGTCGGGC AAATCCACAC TCACCAAATT

1551	GGTGCAGCGT CTGTATGTAC CGGCGCAGGG ACGGGTGTTG GTGGACGGCA

1601	ACGATTTGGC TTTGGCCGCT CCTGCTTGGC TGCGGCGGCA GGTCGGCGTG

1651	GTCTTGCAGG AGAATGTGCT GCTCAACCGC AGCATACGCG ACAATATCGC

1701	GCTGACGGAT ACGGGTATGC CGCTGGAACG CATTATCGAA GCAGCCAAAC

1751	TGGCGGGCGC ACACGAGTTT ATTATGGAGC TGCCGGAAGG CTACGGCACC

1801	GTGGTGGGCG AACAAGGGGC CGGCTTGTCG GGCGGACAGC GGCAGCGTAT

1851	TGCGATTGCC CGCGCGTTAA TCACCAATCC GCGCATTCTG ATTTTTGATG

1901	AAGCCACCAG CGCGCTGGAT TATGAAAGTG AACGAGCGAT TATGCAGAAC

1951	ATGCAGGCCA TTTGCGCCAA CCGGACGGTG CTGATTATCG CCCACCGTCT

2001	GTCCACTGTT AAAACGGCAC ACCGGATCAT TGCCATGGAT AAAGGCAGGA

2051	TTGTGGAAGC GGGAACACAG CAGGAATTGC TGGCGAAGCC GAACGGATAT

2101	TACCGCTATC TGTATGATTT ACAGAACGGG TAG

This encodes a protein having amino acid sequence <SEQ ID 30>:


1	MSIVSAPLPA LSALIILAHY HGIAANPADI QHEFCTSAQS DLNETQWLLA

51	AKSLGLKAKV VRQPIKRLAN ATLPALVWCD DGNHFILAKT DGGGEHAQYL

101	IQDLTTNKSA VLSFAEFSNR YSGKLILVAS RASVIASLAX FDFTWFIPAV

151	IKYRRLFFEV LVVSVVLQLF ALITPLFFQV VNDKVLVHRG FSTLDVVSVA

201	LLVVSLFEIV LGGLRTYLFA HTTSRIDVEL GARLFRHLLS LPLSYFEHRR

251	VGDTVARVRE LEQIRNFLTG QALTSVLDLA FSFIFLAVMW YYSSTLTWVV

301	LASLPAYAFW SAFISPILRT RLNDKFARNA ONQSFLVESI TAVGTVKAMA

351	VEPQNTQRWD NQLAAYVASG FRVTKLAVVG QQGVQLIQKL VTVATLWIGA

401	RLVIESKLTV GQLIAFNHLS GQVAAPVIRL AQLWQOFQQV GISVARLGDI

451	LNAPTENASS HLALPDIRGE ITFEHVDFRY KADGRLILQD LNLRIRAGEV

501	LGIVGRSGSG KSTLTKLVQR LYVPAQGRVL VDGNDLALAA PAWLREQVGV

551	VLQENVLLNR SIRDNIALTD TGMPLERIIE AAKLAGAHEF IHPLPEGYGT

601	VVGEQGAGLS GGQRQRIAIA RALITNPRIL IFDEATSALD YESERAIMQN

651	NQAICANRTV LIIAHRLSTV KTAMRIIAMD KGRIVEAGTQ QELLAKPNGY

701	YRYLYOLQNG *

ORF39a is homologous to a cytolysin from A. pleuropneumoniae:


sp\|P26760\|RT1B_ACTPL RTX-I TOXIN DETERMINANT B (TOXIN RTX-I SECRETION ATP-
BINDING PROTEIN) (APX-IB) (HLY-IB) (CYTOLYSIN IB) (CLY-IB)
>gi\|97137\|pir\|\|D43599 cytolysin IB - Actinobacillus pleuropneumoniae
(serotype 9) >gi\|36944 (X61112) ClyI-B protein [Actinobacillus
pleuropneumoniae] Length = 707 Score = 931 bits (2379), Expect =0.0
Identities = 472/690 (68%), Positives = 540/690 (77%),
Gaps = 3/690 (0%)
Query: 20 YHGIAANPADIQHEFCTSAQSDLNETQWXXXXXXXXXXXXVVRQPIKRLAMATLPALVWC 79
YH IA NP +++H+F + L+ T W V++ I RLA LPALVW
Sbjct: 20 YHNIAVNPEELKHKFDLEGKG-LDLTAWLLAAKSLELKAKQVKKAIDRLAFIALPALVWR 78

Query: 80 DDGNHFILAKTDGGGEHAQYLIQDLTTNKSAVLSFAEFSNRYSGKLILVASRASVLGSLA 139
+DG HFIL K D E +YLI DL T+ +L AEF + Y GKLILVASRAS++G LA
Sbjct: 79 EDGKHFILTKIDN--EAKKYLIFDLETHNPRILEQAEFESLYQGKLILVASRASIVGKLA 136

Query: 140 KFDFTWFIPAVIKYRRXXXXXXXXXXXXXXXXXITPLFFQVVMDKVLVHRGFXXXXXXXX 199
KFDFTWFIPAVIKYR+ ITPLFFQVVMDKVLVHRGF
Sbjct: 137 KFDFTWFIPAVIKYRKIFIETLIVSIFLQIFALITPLFFQVVMDKVLVHRGFSTLNVITV 196

Query: 200 XXXXXXXFEIVLGGLRTYLFAHTTSRIDVELGARLFRHLLSLPLSYFEHRRVGDTVARVR 259
FEIVL GLRTY+FAH+TSRIDVELGARLFRHLL+LP+SYFE+RRVGDTVARVR
Sbjct: 197 ALAIVVLFEIVLNGLRTYIFAHSTSRIDVELGARLFRHLLALPISYFENRRVGDTVARVR 256

Query: 260 ELEQIRNFLTGQALTSVLDLAFSFIFLAVMWYYSSTLTWVVLASLPAYAFWSAFISPILR 319
EL+QIRNFLTGQALTSVLDL FSFIF AVMWYYS LT V+L SLP Y WS FISPILR
Sbjct: 257 ELDQIRNFLTGQALTSVLDIMFSFIFFAVMWYYSPKLTLVILGSLPFYNGWSIFISPILR 316

Query: 320 TRLNDKFARNADNQSFLVESITAVGTVKAMAVEPQNTQRWDNQLAAYVASGFRVTKLAVV 379
RL++KFAR ADNQSFLVES+TA+ T+KA+AV PQMT WD QLA+YV++GFRVT LA +
Sbjct: 317 RRLDEKFARGADNQSFLVESVTAINTIKALAVTPQMTNTWDKQLASYVSAGFRVTTLATI 376

Query: 380 GQQGVQLIQKLVTVATLWIGARLVIESKLTVGOLIAFNNLSGQVAAPVIRLAQLWQDFQQ 439
GQQGVQ IQK+V V TLW+GA LVI L++GQLIAFNNLSGQV APVIRLAQLWQDFQQ
Sbjct: 377 GQQGVQFIQKVVNVITLWLGAMLVISGDLSIGQLIAFNNLSGQVIAPVIRLAQLWQDFQQ 436

Query: 440 VGISVAPLGDILNAPTENASSHLALPDIRGEITFEHVDFRYKADGRLILQDLNLRIRAGE 499
VGISV RLGD+LN+PTE+ LALP+I+G+ITF ++ FRYX D +IL D+NL I+ GE
Sbjct: 437 VGISVTRLGDVLNSPTESYQGKLALPEIKGDITFRNIRFRYXPDAPVILNDVNLSIQQGE 496

Query: 500 VLGIVGRSGSGKSTLTKLVQRLYVPAOGRVLVDGNDLALAAPAWLRRQVGVVLQENVLLN 559
V+GIVGRSGSGKSTLTKL+QR Y+P G+VL+DG+DLALA P WLRRQVGVVLQ+NVLLN
Sbjct: 497 VIGIVGRSGSGKSTLTKLIQRFYIPENGQVLIDGHDLALADPNWLRRQVGVVLQDNVLLN 556

Query: 560 RSIRDNIALTDTGMPLERIIEAAKLAGAIIEFINELPEGYGTVVGEQGNLSGGQRQRIAI 619
RSIRDNIAL D GMP+E+I+ AAKLAGAHEFI EL EGY T+VGEQGAGLSGGQRQRIAI
Sbjct: 557 RSIRDNIALADPGMPMEKIVHAAKLAGAHEFISELREGYNTIVGEOGAGLSGGQRQRIAI 616

Query: 620 ARALITNPRILIFDEATSALDYESERAIMQNMQAICANRTVLIIAHRLSTVKTAHRIIAM 679
ARAL+ NP+ILIFDEATSALDYESE IM+NM IC RTV+IIAHRLSTVK A RII M
Sbjct: 617 ARALVNNPKILIFDEATSALDYESEHIIMRNMHQICKGRTVIIIAHRLSTVKNADRIIVM 676

Query: 680 DKGRIVEAGTQQELLAKPNGYYRYLYDLQN 709
+KG+IVE G +ELLA PNG Y YL+ LQ+
Sbjct: 677 EKGQIVEQGKHKELLADPNGLYHYLHQLQS 706

Homology with the HlyB Leucotoxin Secretion ATP-Binding Protein of Haemophilus actinomycetemcomitans (Accession Number X53955)

ORF39 and HlyB protein show 71% and 69% amino acid identity in 167 and 55 overlap at the N- and C-terminal regions, respectively:


Orf39	1 KFDFTWFIPAVIKYRRXXXXXXXXXXXXXXXXXITPLFFQVVMDKVLVHRGFXXXXXXXX 60
	KFDFTWFIPAVIKYR+ ITPLFFQVVMDKVLVHRGF
HlyB	137 KFDFTWFIPAVIKYRKIFIETLIVSIFLQIFALITPLFFQVVMDKVLVHRGFSTLNVITV 196

Orf39	61 XXXXXXXFEIVLGGLRTYLFAHTTSRIDVELGARLFRHLLSLPLSYFEHRRVGDTVARVR 120
	FEI+LGGLRTY+FAH+TSRIDVELGARLFMLL+LP+SYFE RRVGDTVARVR
HlyB	197 ALAIVVLFEIILGGLRTYVFAHSTSRIDVELGARLFRHLLALPISYFEARRVGDTVARVR 256

Orf39	121 ELEQIRNFLTGQALTSVLDLAFSFIFLAVMWYYSSTLTWVVLASLIC 167
	EL+QIRNFLTGQALTS+LDL FSFIF AVMWYYS LT VVL SL C
HlyB	257 ELDQIRNFLTGQALTSILDLLFSFIFFAVMWYYSPKLTLVVLGSLPC 303
	//

Orf39	166 ICANRTVLIIAHRLSTVKTAHRIIAMDKGRIVEAGTQQELLANXNGYYRYLYDLQ	220
IC NRTVLIIAHRLSTVK A RII MDKG I+E G QELL + G Y YL+ LQ
HlyB	651 ICQNRTVLIIAHRLSTVKNADRIIVMDKGEIIEQGKHQELLKDEKGLYSYLHQLQ 705

Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 7

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 31>


1	ATGAAATACT TGATCCGCAC CGCCTTACTC GCAGTCGCAG CCGCCGGCAT

51	CTACGCCTGC CAACCGCAAT CCGAAGCCGC AGTGCAAGTC AAGGCTGAAA

101	ACAGCCTGAC CGCTATGCGC TTAGCCGTCG CCGACAAACA GGCAGAGATT

151	GACGGGTTGA ACGCCCAAAk sGACGCCGAA ATCAGA...

This corresponds to the amino acid sequence SEQ ID 32; ORF52>:

1 MKYLIRTALL AVAAAGIYAC QPQSEAAVQV KAZNSLTANR LAVADKQAEI

51 DGLNAQXDAE IR..

Further work revealed the complete nucleotide sequence <SEQ ID 33>:


1	ATGAAATACT TGATCCGCAC CGCCTTACTC GCAGTCGCAG CCGCCGGCAT

51	CTACGCCTGC CAACCGCAAT CCGAAGCCGC AGTGCAAGTC AAGGCTGAAA

101	ACAGCCTGAC CGCTATGCGC TTAGCCGTCG CCGACAAACA GGCAGAGATT

151	GACGGGTTGA ACGCCCAAAT CGACGCCGAA ATCAGACAAC GCGAAGCCGA

201	AGAATTGAAA GACTACCGAT GGATACACGG CGACGCGGAA GTGCCGGAGC

251	TGGAAAAATG A

This corresponds to the amino acid sequence <SEQ ID 34; ORF52-1>:

1 MKYLIRTALL AVAAAGIYAC QPQSEAAVQV KAENSLTAMR LAVADKQAEI

51 DGLNAQIDAE IRQREAEELK DThWIHGDAE VPELEK
Computer analysis of this amino acid sequence predicts a prokaryotic membrane lipoprotein lipid attachment site (underlined).
ORF52-1 (7 kDa) was cloned in the pGex vectors and expressed in E. coli, as described above. The products of protein expression and purification were analyzed by SDS-PAGE. FIG. 4A shows the results of affinity purification of the GST-fusion. FIG. 4B shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF52-1.
Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 8

The following DNA sequence was identified in N. meningitidis <SEQ ID 35>


1	ATGGTTATCG GAATATTACT CGCATCAAGC AAGCATGCTC TTGTCATTAC

51	TCTATTGTTA AATCCCGTCT TCCATGCATC CAGTTGCGTA TCGCGTTSGG

101	CAATACGGAA TAAAATCTGC TGTTCTGCTT TGGCTAAATT TGCCAAATTG

151	TTTATTGTTT CTTTAGGAGC AGCTTGCTTA GCCGCCTTCG CTTTCGACAA

201	CGCCCCCACA GGCGCTTCCC AAGCGTTGCC TTCCGTTACC GCACCCGTGG

251	CGATTCCCGC GCCCGCTTCG GCAGCCTGA

This corresponds to the amino acid sequence <SEQ ID 36; ORF56>:

1 MVIGILLASS KHALVITLLL NPVFHASSCV SRXAIRNKIC CSALAKFAKL

51 FIVSLGAACL AAFAFDNAPT GASQALPTVT APVAIPAPAS AA*

Further work revealed the complete nucleotide sequence <SEQ ID 37>:


	1	ATGGCTTGTA CAGGTTTGAT GGTTTTTCCG TTAATGGTYA TCGGAATATT

	51	ACTTGCATCA AGCAAGCCTG CTCCTTTCCT TACTCTATTG TTAAATCCCG

	101	TCTTCCATGC ATCCAGTTGC GTATCGCGTT GGGCAATACG GAATAAAATC

	151	TGCTGTTCTG CTTTGGCTAA ATTTGCCAAA TTGTTTATTG TTTCTTTAGG

	201	AGCAGCTTGC TTAGCCGCCT TCGCTTTCGA CAACGCCCCC ACAGGCGCTT

	251	CCCAAGCGTT GCCTACCGTT ACCGCACCCG TGGCGATTCC CGCGCCCGCT

	301	TCGGCAGCCT GA

This corresponds to the amino acid sequence <SEQ ID 38; ORF56-1>:

1 MACTGLMVFP LNVZGILLAS SKPAPFLTLL LNPVFHASSC VSRWAIRNKI

51 CCSALAKFAK LFIVSLGAAC LAAFAFDNAP TGASQALPTV TAPVAIPAPA

101 SAA*
Computer analysis of this amino acid sequence predicts a leader peptide (underlined) and suggests that ORF56 might be a membrane or periplasmic protein.
Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 9

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 39>


1	ATGTTCAGTA TTTTAAATGT GTTTCTTCAT TGTATTCTGG CTTGTGTAGT

51	CTCTGGTGAG ACGCCTACTA TATTTGGTAT CCTTGCTCTT TTTTACTTAT

101	TGTATCTTTC TTATCTTGCT GTTTTTAAGA TTTTCTTTTC TTTTTTCTTA

151	GACAGAGTTT CACTCCGGTC TCCCAGGCTG GAGTGCAAAT GGCATGACCC

201	TTTGGCTCAC TGGCTCACGG CCACTTCTGC TATTCTGCCG CCTCAGCCTC

251	CAGGG...

This corresponds to the amino acid sequence <SEQ ID 40; ORF63>:

1 MFSILNVFLR CILACVVSGE TPTIFGILAL FYLLYLSYLA VFKIFFSFFL

51 DRVSLRSPRL ECKWNDPLAH WLTATSAILP PQPPG...
Computer analysis of this amino acid sequence predicts a transmembrane region.
Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 10

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 41>


1	..GTGCGGACGT GGTTGGTTTT TTGGTTGCAG CGTTTGAAAT ACCCGTTGTT

51	GCTTTGGATT GCGGATATGT TGCTGTACCG GTTGTTGGGC GGCGCGGAAA

101	TCGAATGCGG CCGTTGCCCT GTGCCGCCGA TGACGGATTG GCAGCATTTT

151	TTGCCGGCGA TGGGAACGGT GTCGGCTTGG GTGGCGGTGA TTTGGGCATA

201	CCTGATGATT GAAAGTGAAA AAAACGGAAG ATATTGA

This corresponds to the amino acid sequence <SEQ ID 42; ORF69>:

1 ..VRTWLVFWLQ RLKYPLLLWI ADNLLYRLLG GAE1ECGRCP VPPMTDWQHF

51 LPANGTVSAW VAVIWAYLMI ESEKNGRY*
Computer analysis of this amino acid sequence predicts a transmembrane region.
A corresponding ORF from strain A of N. meningitidis was also identified:
Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF69 shows 96.2% identity over a 78 aa overlap with an ORF (ORF69a) from strain A of N. meningitidis:


	10 20 30 40 50 60
orf69.pep	VRTWLVFWLQRLKYPLLLWIADMLLYRLLGGAEIECGRCPVPPMTDWQHFLPAMGTVSAW
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|:\|\|
orf69a	VRTWLVFWLQRLKYPLLLCIADMLLYRLLGGAEIECGRCPVPPMTDWQHFLPTMGTVAAW
	10 20 30 40 50 60

	70 79
orf69.pep	VAVIWAYLMIESEKNGRYX
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf69a	VAVIWAYLMIESEKNGRYX
	70 79

The ORF69a nucleotide sequence <SEQ ID 43> is:


1	GTGCGGACGT GGTTGGTTTT TTGGTTGCAG CGTTTGAAAT ACCCGTTGTT

51	GCTTTGTATT GCGGATATGC TGCTGTACCG GTTGTTGGGC GGCGCGGAAA

101	TCGAATGCGG CCGTTGCCCT GTACCGCCGA TGACGGATTG GCAGCATTTT

151	TTGCCGACGA TGGGAACGGT GGCGGCTTGG GTGGCGGTGA TTTGGGCATA

201	CCTGATGATT GAAAGTGAAA AAAACGGAAG ATATTGA

This encodes a protein having amino acid sequence <SEQ ID 44>:

1 VRTWLVFWLQ RLKYPLLLCI ADMLLYRLLG GAEIECGRCP VPPNTDWQHF

51 LPTMGTVAAW VAVIWAYLMI ESEKNGRY*
Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 11

The following DNA sequence was identified in N. meningitidis <SEQ ID 45>


1	ATGTTTCAAA ATTTTGATTT GGGCGTGTTC CTGCTTGCCG TCCTCCCCGT

51	GCTGCCCTCC ATTACCGTCT CGCACGTGGC GCGCGGCTAT ACGGCGCGCT

101	ACTGGGGAGA CAACACTGCC GAACAATACG GCAGGCTGAC ACTGAACCCC

151	CTGCCCCATA TCGATTTGGT CGGCACAATC ATCgTACCGC TGCTTACTTT

201	GATGTTCACG CCCTTCCTGT TCGGCTGGGC GCGTCCGATT CCTATCGATT

251	CGCGCAACTT CCGCAACCCG cGCCTTGCCT GGCGTTGCGT TGCCGCGTCC

301	GGCCCGCTGT CGAATCTAGC GATGGCTGTw CTGTGGGGCG TGGTTTTGGT

351	GCTGACTCCG TATGTCGGCG GGGCGTATCA GATGCCGTTG GCTCAAATGG

401	CAAACTACGG TATTCTGATC AATGCGATTC TGTTCGCGCT CAACATCATC

451	CCCATCCTGC CTTGGGACGG CGGCATTTTC ATCGACACCT TCCTGTCGGC

501	GAAATATTCG CAAGCGTTCC GCAAAATCGA ACCTTATGGG ACGTGGATTA

551	TCCTACTGCT GATGCTGACC SGGGTTTTGG GTGCGTTTAT wGCACCGATT

601	sTGCGGmTGc GTGATTGCrT TTGTGCAGAT GTwCGTCTGA CTGGCTTTCA

651	GACGGCATAA

This corresponds to the amino acid sequence <SEQ ID 46; ORF77>:


1	MFQNFDLGVF LLAVLPVLPS ITVSNVARGY TARYWGDNTA EQYGRLTLNP

51	LPHIDLVGTI IVPLLTLMFT PFLFGWPRPI PIDSRNFRNP RLAWRCVAAS

101	GPLSNLAMAV LWGVVLVLTP YVGGAYQMPL AQMANYGILI NAILFPLNII

151	PILPWDGGIF IDTFLSAKYS QAFRKIEPYG TWIILLLMLT XVLGAFIAPI

201	XRXRDCXCAD VRLTGFQTA*

Further work revealed the complete nucleotide sequence <SEQ ID 47>:


1	ATGTTTCAAA ATTTTGATTT GGGCGTGTTT CTGCTTGCCG TCCTGCCCGT

51	GCTGCTCTCC ATTACCGTCA GGGAGGTGGC GCGCGGCTAT ACGGCGCGCT

101	ACTGGGGAGA CAACACTGCC GAACAATACG GCAGGCTGAC ACTGAACCCC

151	CTGCCCCATA TCGATTTGGT CGGCACAATC ATCGTACCGC TGCTTACTTT

201	GATGTTCACG CCCTTCCTGT TCGGCTGGGC GCGTCCGATT CCTATCGATT

251	CGCGCAACTT CCGCAACCCG CGCCTTGCCT GGCGTTGCGT TGCCGCGTCC

301	GGCCCGCTGT CGAATCTAGC GATGGCTGTT CTGTGGGGCG TGGTTTTGGT

351	GCTGACTCCG TATGTCGGCG GGGCGTATCA GATGCCGTTG GCTCAAATGG

401	CAAACTACGG TATTCTGATC AATGCGATTC TGTTCGCGCT CAACATCATC

451	CCCATCCTGC CTTGGGACGG CGGCATTTTC ATCGACACCT TCCTGTCGGC

501	GAAATATTCG CAAGCGTTCC GCAAAATCGA ACCTTATGGG ACGTGGATTA

551	TCCTACTGCT GATGCTGACC GGGGTTTTGG GTGCGTTTAT TGCACCGATT

601	GTGCGGCTGG TGATTGCGTT TGTGCAGATG TTCGTCTGA

This corresponds to the amino acid sequence <SEQ ID 48; ORF77-1>:


1	MFQNFDLGVF LLAVLPVLLS ITVREVARGY TARYWGDNTA EQYGRLTLNP

51	LPHIDLVGTI IVPLLTLMFT PFLFGWARPI PIDSRNFRNP RLAWRCVAAS

101	GPLSNLAMAV LWGVVLVLTP YVGGAYQMPL AQMANYGILI NAILFALNII

151	PILPWDGGIF IDTFLSAXYS QAYRRIEPYG TWIILLLNLT GVLGAFIAPI

201	VRLVIAFVQH FV*

Computer analysis of this amino acid sequence reveals a putative leader sequence and several transmembrane domains.
A corresponding ORF from strain A of N. meningitidis was also identified:
Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF77 shows 96.5% identity over a 173 aa overlap with an ORF (ORF77a) from strain A of N. meningitidis:


	10 20 30 40 50 60
ort77.pep	MFQNFDLGVFLLAVLPVLPSITVSHVARGYTARYWGDNTAEQYGRLTLNPLPHIDLVGTI
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf77a	RGYTARYWGDNTAEQYGRLTLNPLPHIDLVGTI
	10 20 30

	70 80 90 100 110 120
orf77.pep	IVPLLTLMFTPFLFGWARPIPIDSRNFRNPRLAWRCVAASGPLSNLAMAVLWGVVLVLTP
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf77a	IVPLLTLMFTPFLFGWARPIPIDSRNFRNPRLAWRCVAASGPLSNLAMAVLWGVVLVLTP
	40 50 60 70 80 90

	130 140 150 160 170 180
orf77.pep	YVGGAYQMPLAQMANYGILINAILFALNIIPILPWDGGIFIDTFLSAKYSQAFRKIEPYG
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|
orf77a	YVGGAYQMPLAQMANYXILINAILXALNIIPILPWDGGIFIDTFLSAKXSQAFRKIEPYG
	100 110 120 130 140 150

	190 200 210 220
orf77.pep	TWIILLLMLTGVLGAFIAPIVRLVIAFVQMFVX
	\|\|\|\| \|\|\|\|\|\|\|\|\|\| \|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|
orf77a	TWIIXLLMLTGVLGAXIAPIVQLVIAFVQMFVX
	160 170 180

ORF77-1 and ORF77a show 96.8% identity in 185 aa overlap:


	10 20 30 40 50 60
orf77-1.pep	MFQNFDLGVFLLAVLPVLLSITVREVARGYTARYWGDNTAEQYGRLTLNPLPHIDLVGTI
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf77a	RGYTARYWGDNTAEQYGRLTLNPLPHIDLVGTI
	10 20 30

	70 80 90 100 110 120
orf77-1.pep	IVPLLTLMFTPFLFGWARPIPIDSRNFRNPRLAWRCVAASGPLSNLAMAVLWGVVLVLTP
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf77a	IVPLLTLMFTPFLFGWARPIPIDSRNFRNPRLAWRCVAASGPLSNLAMAVLWGVVLVLTP
	40 50 60 70 80 90

	130 140 150 160 170 180
orf77-1.pep	YVGGAYQMPLAQMANYGILINAILFALNIIPILPWDGGIFIDTFLSAKYSQAFRKIEPYG
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|
orf77a	YVGGAYQMPLAQMANYXILINAILXALNIIPILPWDGGIFIDTFLSAKXSQAFRKIEPYG
	100 110 120 130 140 150

	190 200 210
orf77-1.pep	TWIILLLMLTGVLGAFIAPIVRLVIAFVQMFVX
	\|\|\|\| \|\|\|\|\|\|\|\|\|\| \|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|
orf77a	TWIIXLLMLTGVLGAXIAPIVQLVIAFVQMFVX
	160 170 180

A partial ORF77a nucleotide sequence <SEQ ID 49> was identified:


1	..CGCGGCTATA CAGCGCGCTA CTGGGGTGAC AACACTGCCG AACAATACGG

51	CAGGCTGACA CTGAACCCCC TGCCCCATAT CGATTTGGTC GGCACAATCA

101	TCGTACCGCT GCTTACTTTG ATGTTTACGC CCTTCCTGTT CGGCTGGGCG

151	CGTCCGATTC CTATCGATTC GCGCAACTTC CGCAACCCGC GCCTTGCCTG

201	GCGTTGCGTT GCCGCGTCCG GCCCGCTGTC GAATCTGGCG ATGGCTGTTC

251	TGTGGGGCGT GGTTTTGGTG CTGACTCCGT ATGTCGGTGG GGCGTATCAG

301	ATGCCGTTGG CNCAAATGGC AAACTACNNN ATTCTGATCA ATGCGATTCT

351	GTNCGCGCTC AACATCATCC CCATCCTGCC TTGGGACGGC GGCATTTTCA

401	TCGACACCTT CCTGTCGGCN AAATANTCGC AAGCGTTCCG CAAAATCGAA

451	CCTTATGGGA CGTGGATTAT CCNGCTGCTT ATGCTGACCG GGGTTTTGGG

501	TGCGTNTATT GCACCGATTG TGCAGCTGGT GATTGCGTTT GTGCAGATGT

551	TCGTCTGA

This encodes a protein having amino acid sequence <SEQ ID 50>:


1	..RGYTARYWGD NTAEQYGRLT LNPLPHIDLV GTIIVPLLTL MFTPFLFGWA

51	RPIPIDSRNF RNPRLAWRCV AASGFLSNLA MAVLWGVVLV LTPYVGGAYQ

101	MPLAQNANYX ILINAILXAL NIIPILPWDG GIFIDTFLSA KXSQAFRKIE

151	PYGTWIIXLL MLTGVLGAXI APIVQLVIAF VQNFV*

Example 12

The following partial DNA sequence was identified in N. meningitidis SEQ ID 51>


1	ATGAACCTGA TTTCACGTTA CATCATCCGT CAAATGGCGG TTATGGCGGT

51	TTACGCGCTC CTTGCCTTCC TCGCTTTGTA CAGCTTTTTT GAAATCCTGT

101	ACGAAACCGG CAACCTCGGC AAAGGCAGTT ACGGCATATG GGAAATGCTG

151	GGCTACACCG CCCTCAAAAT GCCCGCCCGC GCCTACGAAC TGATTCCCCT

201	CGCCGTCCTT ATCGGCGGAC TGGTCTCCCT CAGCCAGCTT GCCGCCGGCA

251	GCGAACTGAC CGTCATCAAA GCCAGCGGCA TGAGCACCAA AAAGCTGCTG

301	TTGATTCTGT CGCAGTTCGG TTTTATTTTT GCTATTGCCA CCGTCGCGCT

351	CGGCGAATGG GTTGCGCCCA CACTGAGCCA AAAAGCCGAA AACATCAAAG

401	CCGCCGCCAT CAACGGCAAA ATCAACACCG GCAATACCGG CCTTTGGCTG

451	AAAGAAAAAA ACAGCGTGAT CAATGTGCGC GAAATGTTGC CCGACCAT..

This corresponds to the amino acid sequence SEQ ID 52; ORF112>:


1	HNLISRYIIR QMAVMAVYAL LAFLALYSFF EILYETGNLG KGSYGIWEML

51	GYTALIQPAR AYELIPLAVL IGGLVSLSQL AAGSELTVIK ASGNSTKKLL

101	LILSQFGFIF AIATVPLGEW VAPTLSQKAE NIKAAAINGK ISTGNTGLWL

151	KEKNSVINVR EHLPDH...

Further work revealed further partial nucleotide sequence <SEQ ID 53>:


1	ATGAACCTGA TTTCACGTTA CATCATCCGT CAAATGGCGG TTATGGCGGT

51	TTACGCGCTC CTTGCCTTCC TCGCTTTGTA CAGCTTTTTT GAAATCCTGT

101	ACGAAACCGG CAACCTCGGC AAAGGCAGTT ACGGCATATG GGAAATGCTG

151	GGCTACACCG CCCTCAAAAT GCCCGCCCGC GCCTACGAAC TGATTCCCCT

201	CGCCGTCCTT ATCGGCGGAC TGGTCTCCCT CAGCCAGCTT GCCGCCGGCA

251	GCGAACTGAC CGTCATCAAA GCCAGCGGCA TGAGCACCAA AAAGCTGCTG

301	TTGATTCTGT CGCAGTTCGG TTTTATTTTT GCTATTGCCA CCGTCGCGCT

351	CGGCGAATGG GTTGCGCCCA CACTGAGCCA AAAAGCCGAA AACATCAAAG

401	CCGCCGCCAT CAACGGCAAA ATCAGCACCG GCAATACCGG CCTTGCTCTG

451	AAAGAAAAAA ACAGCTTAAT CAATGTGCGC GAAATGTTGC CCGACCATAC

501	GCTTTTGGGC ATCAAAATTT GGGCGCGCAA CGATAAAAAC GAATTGGCAG

551	AGGCAGTGGA AGCCGATTCC GCCGTTTTGA ACAGCGACGG CAGTTGGCAG

601	TTGAAAAACA TCCGCCGCAG CACGCTTGGC GAAGACAAAG TCGAGGTCTC

651	TATTGCGGCT GAAGAAAACT GGCCGATTTC CGTCAAACGC AACCTGATGG

701	ACGTATTGCT CGTCAAACCC GACCAAATGT CCGTCGGCGA ACTGACCACC

751	TACATCCGCC ACCTCCAAAA CAACAGCCAA AACACCCGAA TCTACGCCAT

601	CGCATGGTGG CGCAAATTGG TTTACCCCGC CGCAGCCTGG GTGATGGCGC

851	TCGTCGCCTT TGCCTTTACC CCGCAAACCA CCCGCCACGG CAATATGGGC

901	TTAAAACTCT TCGGCGGCAT CTGTSTCGGA TTGCTGTTCC ACCTTGCCGG

951	ACGGCTCTTT GGGTTTACCA GCCAACTCGG...

This corresponds to the amino acid sequence <SEQ ID 54; ORF112-1>:


1	MNLISRYIIR QMAVMAVYAL LAFLALYSFF EILYETGNLG KGSYGIWEHL

51	GYTALKMPAR AYELIPLAVL IGGLVSLSQL AAGSELTVIK ASGMSTKKLL

101	LILSQFGFIF AIATVALGEW VAPTLSQKAE NIKAAAINGK ISTGNTGLWL

151	KEKNSXINVR EHLPDHTLLG IKIWARNDKN ELAEAVEADS AVLNSDGSWQ

201	LKNIRRSTLG EDKVEVSIAA EENWPISVKR NLTDVLLVKP DQMSVGELTT

251	YIRHLONNSQ NTRIYAIAWW RKLVYPAAAW VMALVAFAFT PQTTRHGNMG

301	LKLFGGICXG LLFHLAGRLF GFTSQL...

Computer analysis of this amino acid sequence predicts two transmembrane domains.
A corresponding ORF from strain A of N. meningitidis was also identified:
Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF112 shows 96.4% identity over a 166 aa overlap with an ORF (ORF112a) from strain A of N. meningitidis:


	10 20 30 40 50 60
orf112.pep	MNLISRYIIRQMAVMAVYALLAFLALYSFFEILYETGNLGKGSYGIWEMLGYTALKMPAR
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\| \|\|
orf112a	MNLISRYIIRQMAVMAVYALLAFLALYSFFEILYETGNLGKGSYGIWEMXGYTALKMXAR
	10 20 30 40 50 60

	70 80 90 100 110 120
orf112.pep	AYELIPLAVLIGGLVSLSQLAAGSELTVIKASGMSTKKLLLILSQFGFIFAIATVALGEW
	\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf112a	AYELMPLAVLIGGLVSXSQLAAGSELTVIKASGMSXKKLLLILSQFGFIFAIATVALGEW
	70 80 90 100 110 120

	130 140 150 160
orf112.pep	VAPTLSQKAENIKAAAINGKISTGNTGLWLKEKNSVINVREMLPDH
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|
orf112a	VAPTLSQKAENIKAAAINGKISTGNTGLWLKEKNSIINVREMLPDHTLLGIKIWARNDKN
	130 140 150 160

orf112a	ELAEAVEADSAVLNSDGSWQLKNIRRSTLGEDKVEVSIAAEEXWPISVKRNLMDVLLVKP
	190 200 210 220 230 240

A partial ORF112a nucleotide sequence <SEQ ID 55> was identified:


1	ATGAACCTGA TTTCACGTTA CATCATCCGT CAAATGGCGG TTATGGCGGT

51	TTACGCGCTC CTTGCCTTCC TCGCTTTGTA CAGCTTTTTT GAAATCCTGT

101	ACGAAACCGG CAACCTCGGC AAAGGCAGTT ACGGCATATG GGAAATGNTG

151	GGNTACACCG CCCTCAAAAT GNCCGCCCGC GCCTACGAAC TGATGCCCCT

201	CGCCGTCCTT ATCGGCGGAC TGGTCTCTNT CAGCCAGCTT GCCGCCGGCA

251	GCGAACTGAN CGTCATCAAA GCCAGCGGCA TGAGCACCAA AAAGCTGCTG

301	TTGATTCTGT CGCAGTTCGG TTTTATTTTT GCTATTGCCA CCGTCGCGCT

351	CGGCGAATGG GTTGCGCCCA CACTGAGCCA AAAAGCCGAA AACATCAAAG

401	CCGCGGCCAT CAACGGCAAA ATCAGTACCG GCAATACCGG CCTTTGGCTG

451	AAAGAAAAAA ACAGCATTAT CAATGTGCGC GAAATGTTGC CCGACCATAC

501	CCTGCTGGGC ATTAAAATCT GGGCCCGCAA CGATAAAAAC GAACTGGCAG

551	AGGCAGTGGA AGCCGATTCC GCCGTTTTGA ACAGCGACGG CAGTTGGCAG

601	TTGAAAAACA TCCGCCGCAG CACGCTTGGC GAAGACAAAG TCGAGGTCTC

651	TATTGCGGCT GAAGAAAANT GGCCGATTTC CGTCAAACGC AACCTGATGG

701	ACGTATTGCT CGTCAAACCC GACCAAATGT CCGTCGGCGA ACTGACCACC

751	TACATCCGCC ACCTCCAAAN NNACAGCCAA AACACCCGAA TCTACGCCAT

801	CGCATGGTGG CGCAAATTGG TTTACCCCGC CGCAGCCTGG GTGATGGCGC

851	TCGTCGCCTT TGCCTTTACC CCGCAAACCA CCCGCCACGG CAATATGGGC

901	TTAAAANTCT TCGGCGGCAT CTGTCTCGGP TTGCTGTTCC ACCTTGCCGG

951	NCGGCTCTTC NGGTTTACCA GCCAACTCTA CGGCATCCCG CCCTTCCTCG

1001	NCGGCGCACT ACCTACCATA GCCTTCGCCT TGCTCGCCGT TTGGCTGATA

1051	CGCAAACAGG AAAAACGCTA A

This encodes a protein having amino acid sequence <SEQ ID 56>:


1	MNLISRYIIR QMAVMAVYAL LAFLALYSFF EILYETGNLG KGSYGIWEMK

51	GYTALKMXAR AYELMPLAVL IGGLVSXSQ L AAGSELXVIX ASGNSTKKLL

101	LILSQFGFIF AIATVALGEV VAPTLSQKAE NIKAAAINGK ISTGNTGLWL

151	KEKNSIINVR EMLPDHTLLG IKIWAIWDKN ELAEAVFADS AVLNSDGSWQ

201	LKNIRRSTLG EDKVEVSIAA EEXWPISVKR NLMDVLLVKP DQMSVGELTT

251	YIRHLQXXSQ NTRIYAIAWW RKLVYPAAAW VMALVAFAFT PQTTRHGNMG

301	LKXFGGICLG LLFHLAGRLF XFTSQLYGIP PFLXGALPTI AFALLAVWLI

351	RKQEKR*

ORF112a and ORF112-1 show 96.3% identity in 326 aa overlap:


orf112a.pep	MNLISRYIIRQMAVMAVYALLAFLALYSFFEILYETGNLGKGSYGIWEMXGYTALKMXAR
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\| \|\|
orf112-1	MNLISRYIIRQMAVMAVYALLAFLALYSFFEILYETGNLGKGSYGIWEMLGYTALKMXAR

orf112a.pep	AYEIIPLAVLIGGLVSXSQLAAGSELXVIKASGHSTKKLLLILSQFGFIFAIATVALGEW
	\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf112-1	AYEIIPLAVLIGGLVSLSQLAAGSELTVIKASGHSTKKLLLILSQFGFIFAIATVALGEW

orf112a.pep	VAPTLSQKAENIKAAAINGKISTGNTGLWLKEKNSIINVREMLPDHTLLGIKIWARNDKN
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf112-1	VAPTLSQKAENIKAAAINGKISTGNTGLWLKEKNSXINVREMLPDHTLLGIKIWARNDKN

orf112a.pep	ELAEAVEADSAVLNSDGSWQLKNIRRSTLGEDKVEVSIAAEEXWPISVKRNLMDVLLVKP
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf112-1	ELAEAVEADSAVLNSDGSWQLKNIRRSTLGEDKVEVSIAAEENWPISVKRNLMDVLLVKP

orf112a.pep	DQMSVGELTTYIRHLQXXSQNTRIYAIAWWRKLVYPAAAWVMALVAFAFTPQTTRHGNMG
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf112-1	DQMSVGELTTYIRHLQXXSQNTRIYAIAWWRKLVYPAAAWVMALVAFAFTPQTTRHGNMG

orf112a.pep	LKXFGGICLGLLFHLAGRLFXFTSQLYGIPPFLXGALPTIAFALLAVWLIRKQEXRX
	\|\| \|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|
orf112-1	LKLFGGICXGLLFHLAGRLFGFTSQL

Example 13

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 57>


1	..GCAGTAGCCG AAACTGCCAA CAGCCAGGGC AAAGGTAAAC AGGCAGGCAG

51	TTCGGTTTCT GTTTCACTGA AAACTTCAGG CGACCTTTGC GGCAAACTCA

101	AAACCACCCT TAAAACTTTG GTCTGCTCTT TGGTTTCCCT GAGTATGGTA

151	TTGCCTGCCC ATGCCCAAAT TACCACCGAC AAATCACCAC CTAAAAACCA

201	GCAGGTCGTT ATCCTTAAAA CCAACACTGG TGCCCCCTTG GTGAATATCC

251	AAACTCCGAA TGGACGCGGA TTGAGCCACA ACCGCTA.TA CGCATTTGAT

301	GTTGACAACA AAGGGGCAGT GTTAAACAAC GACCGTAACA ATAATCCGTT

351	TGTGGTCAAA GGCAGTGCGC AATTGATTTT GAACGAGGTA CGCGGTACGG

401	CTAGCAAACT CAACGGCATC GTTACCGTAG GCGGTCAAAA GGCCGACGTG

451	ATTATTGCCA ACCCCAACGG CATTACCGTT AATGGCGGCG GCTTTAAAAA

501	TGTCGGTCGG GGCATCTTAA CTACCGGTGC GCCCCAAATC GGCAAAGACG

551	GTGCACTGAC AGGATTTGAT GTGCGTCAAG GCACATTGGA CCGTAGTAGC

601	AGCAGGTTGG AATGATAAAG GCGGAGCmrm yTACACCGGG GTACTTGCTC

651	GTGCAGTTGC TTTGCAGGGG AAATTwmmGG GTAAA.AACT GGCGGTTTCT

701	ACCGGTCCTC AGAAAGTAGA TTACGCCAGC GGCGAAATCA GTGCAGGTAC

751	GGCAGCGGGT ACGAAACCGA cTATTGCCCT TGATACTGCC GCACTGGGCG

901	GTATGTACGC CGACAGCATC ACACTGATTG CCAATGAAAA AGGCGTAGGC

951	GTCTAA

This corresponds to the amino acid sequence <SEQ ID 58; ORF114>:


1	..AVAETANSQG KGKQAGSSVS VSLKTSGDLC GKLKTTLKTL VCSLVSLSHV

51	LPAHAQITTD KSAPKNQQVV ILKTNTGAPL VNIQTPNGRG LSHNRXYAFD

101	VDNKGAVLNN DRNNNPFVVK GSAQLILNEV RGTASKLNGI VTVGGQKADV

151	IIANPNGITV NGGGFXNVGR GILTTGAPQI GKDGALTGFD VVKAHVTVXA

201	AGWNDKGGAX YTGVLAPAVA LQGKXXGLXL AVSTGPQKVD YASGEISAGT

251	AAGTKPTIAL DTAALGGNYA DSITLIANEX GVGV*

Further work revealed the complete nucleotide sequence SEQ ID 59>:


1	ATGAATAAAG GTTTACATCG CATTATCTTT AGTAAAAAGC ACAGCACCAT

51	GGTTGCAGTA GCCGAAACTG CCAACAGCCA GGGCAAAGGT AAACAGGCAG

101	GCAGTTCGGT TTCTGTTTCA CTGAAAACTT CAGGCGACCT TTGCGGCAAA

151	CTCAAAACCA CCCTTAAAAC TTTGGTCTGC TTTTTGGTTT CCCTGAGTAT

201	GGTATTGCCT GCCCATGCCC AAATTACCAC CGACAAATCA GCACCTAAAA

251	ACCAGCAGGT CGTTATCCTT AAAACCAACA CTGGTGCCCC CTTGGTGAAT

301	ATCCAAACTC CGAATGGACG CGGATTGAGC CACAACCGCT ATACGCAGTT

351	TGATGTTGAC AACAAAGGGG CAGTGTTAAA CAACGACCGT AACAATAATC

401	CGTTTGTGGT CAAAGGCAGT GCGCAATTGA TTTTGAACGA GGTACGCGGT

451	ACGGCTAGCA AACTCAACGG CATCGTTACC GTAGGCGGTC AAAAGGCCGA

501	CGTGATTATT GCCAACCCCA ACGGCATTAC CGTTAATGGC GGCGGCTTTA

551	AAAATGTCGG TCGGGGCATC TTAACTACCG GTGCGCCCCA AATCGGCAAA

601	GACGGTGCAC TGACAGGATT TGATGTGCGT CAAGGCACAT TGACCGTAGG

651	AGCAGCAGGT TGGAATGATA AAGGCGGAGC CGACTACACC GGGGTACTTG

701	CTCGTGCAGT TGCTTTGCAG GGGAAATTAC AGGGTAAAAA CCTGGCGGTT

751	TCThCCGGTC CTCAGAAAGT AGATTACGCC AGCGGCGAAA TCAGTGCAGG

801	TACGGCAGCG GGTACGAAAC CGACTATTGC CCTTGATACT GCCGCACTGG

951	GCGGTATGTA CGCCGACACC ATCACACTGA TTGCCAATGA AAAAGGCGTA

901	GGCGTCAAAA ATGCCGGCAC ACTCGAAGCG GCCAAGCAAT TGATTGTGAC

951	TTCGTCAGGC CGCATTGAAA ACAGCGGCCG CATCGCCACC ACTGCCGACG

1001	GCACCGAAGC TTCACCGACT TATCTCTCCA TCGAAACCAC CGAAAAAGGA

1051	GCGGCAGGCA CATTTATCTC CAATGGTGGT CGGATCGAGA GCAAAGGCTT

1101	ATTGGTTATT GAGACGGGAG AAGATATCAG CTTGCGTAAC GGAGCCGTGG

1151	TGCAGAATAA CGGCAGTCTC CCAGCTACCA CGGTATTAAA TGCTGGTCAT

1201	AATTTGGTGA TTGAGAGCAA AACTAATGTG AACAATGCCA AAGGCCCGGC

1251	TACTCTGTCG GCCGACGGCC GTACCGTCAT CAAGGAGGCC AGTATTCAGA

1301	CTGGCACTAC CGTATACAGT TCCAGCAAAG GCAACGCCGA ATTAGGCAAT

1351	AACACACGCA TTACCGGGGC AGATGTTACC GTATTATCCA ACGGCACCAT

1401	CAGCAGTTCC GCCGTAATAG ATGCCAAAGA CACCGCACAC ATCGAAGCAG

1451	GCAAACCGCT TTCTTTGGAA GCTTCAACAG TTACCTCCGA TATCCGCTTA

1501	AACGGAGGCA GTATCAAGGG CGGCAAGCAG CTTGCTTTAC TGGCAGACGA

1551	TAACATTACT GCCAAAACTA CCAATCTGAA TACTCCCGGC AATCTGTATG

1601	TTCATACAGG TAAAGATCTG AATTIGAATG TTGATAAAGA TTTGTCIGCC

1651	GCCAGCATCC ATTTGAAATC GGATAACGCT GCCCATATTA CCGGCACCAG

1701	TAAAACCCTC ACTGCCTCAA AAGACATGGG TGTGGAGGCA GGCTCGCTGA

1751	ATGTTACCAA TACCAATCTG CGTACCAACT CGGGTAATCT GCACATTCAG

1801	GCAGCCAAAG GCAATATTCA GCTTCGCAAT ACCAAGCTGA ACGCAGCCAA

1851	GGCTCTCGAA ACCACCGCAT TGCAGGGCAA TATCGTTTCA GACGGCCTTC

1901	ATGCTGTTTC TGCAGACGGT CATGTATCCT TATCGGCCAA CGGTAATGCC

1951	GACTTTACCG GTCACAATAC CCTGACAGCC AAGGCCGATG TCAATGCAGG

2001	ATCGGTTGGT AAAGGCCGTC TGAAAGCAGA CAATACCAAT ATCACTTCAT

2051	CTTCAGGAGA TATTACGTTG GTTGCCGGCA ACGGTATTCA GCTTGGTGAC

2101	GGAAAACAAC GCAATTCAAT CAACGGAAAA CACATCAGCA TCAAAAACAA

2151	CGGTGGTAAT GCCGACTTAA AAAACCTTAA CGTCCATGCC AAAAGCGGGG

2201	CATTGAACAT TCATTCCGAC CGGGCATTGA GCATAGAAAA TACCAAGCTG

2251	GAGTCTACCC ATAATACGCA TCTTAATGCA CAACACGAAG GGGTAACGCT

2301	CAACCAAGTA GATGCCTACG CACACCGTCA TCTAAGCATT ACCGGCAGCC

2351	AGATTTGGCA AAACGACAAA CTGCCTTCTG CCAACAAGCT GGTGGCTAAC

2401	GGTGTATTGG CACTCAATGC GCGCTATTCC CAAATTGCCG ACAACACCAC

2451	GCTGAGAGCG GGTGCAATCA ACCTTATTGC CGGTACCGCC CTAGTCAAGC

2501	GCGGCAACAT CAATTGGAGT ACCGTTGCGA CCAAAACTTT GGAAGATAAT

2551	GCCGAATTAA AACCATTGGC CGGACGGCTG AATATTGAAG CAGGTAGCGG

2601	CACATTAACC ATCGAACCTG CCAACCGCAT CAGTGCGCAT ACCGACCTGA

2651	GCATCAAAAC AGGCGGAAAA TTGCTGTTGT CTGCAAAAGG AGGAAATGCA

2701	GGTGCGCCTA GTGCTCAAGT TTCCTCATTG GAAGCAAAAG GCAATATCCG

2751	TCTGGTTACA GGAGAAACAG ATTTAAGAGG TTCTAAAATT ACAGCCGGTA

2801	AAAACTTGGT TGTCGCCACC ACCAAAGGCA AGTTGAATAT CGAAGCCGTA

2951	AACAACTCAT TCAGCAATTA TTTTCCTACA CAAAAAGCGG CTGAACTCAA

2901	CCAAAAATCC AAAGAATTGG AACAGCAGAT TGCGCAGTTG AAAAAAAGCT

2951	CGCCTAAAAG CAAGCTGATT CCAACCCTGC AAGAAGAACG CGACCGTCTC

3001	GCTTTCTATA TTCAAGCCAT CAACAAGGAA GTTAAAGGTA AAAAACCCAA

3051	AGGCAAAGAA TACCTGCAAG CCAAGCTTTC TGCACAAAAT ATTGACTTGA

3101	TTTCCGCACA AGGCATCGAA ATCAGCGGTT CCGATATTAC CGCTTCCAAA

3151	AAACTGAACC TTCACGCCGC AGGCGTATTG CCAAAGGCAG CAGATTCAGA

3201	GGCGGCTGCT ATTCTGATTG ACGGCATAAC CGACCAATAT GAAATTGGCA

3251	AGCCCACCTA CAAGAGTCAC TACGACAAAG CTGCTCTGAA CAAGCCTTCA

3301	CGTTTGACCG GACGTACAGG GGTAAGTATT CATGCAGCTG CGGCACTCGA

3351	TGATGCACGT ATTATTATCG GTGCATCCGA AATCAAAGCT CCCTCAGGCA

3401	GCATAGACAT CAAAGCCCAT AGTGATATTG TACTGGAGGC TGGACAAAAC

3451	GATGCCTATA CCTTCTTAAA AACCAAAGGT AAAAGCGGCA AAATCATCAG

3501	AAAAACCAAG TTTACCAGCA CCCGCGACCA CCTGATTATG CCAGCCCCCG

3551	TCGAGCTGAC CGCCAACGGC ATAACGCTTC AGGCAGGCGG CAACATCGAA

3601	GCTAATACCA CCCGCTTCAA TGCCCCTGCA GGTAAAGTTA CCCTGGTTGC

3651	GGGTGAAGAG CTGCAACTGC TGGCAGAASA AGGCATCCAC AAGCACGAGT

3701	TGGATGTCCA AAAAAGCCGC CGCTTTATCG GCATCAAGGT AGGCAAGAGC

3751	AATTACAGTA AAAACGAACT GAACGAAACC AAATTGCCTG TCCGCGTCGT

3801	CGCCCAAACT GCAGCCACCC GTTCAGGCTG GGATACAGTG CTCGAAGGTA

3051	CCGAATTCAA AACCACGCTG GCCGGTGCGG ACATTCAGGC AGGTGTAGGC

3901	GAAAAAGCCC GTGCCGATGC GAAAATTATC CTCAAAGGCA TTGTGAACCG

3951	TATCCAGTCG GAAGAAAAAT TAGAAACCAA CTCAACCGTA TGGCAGAAAC

4001	AGGCCGGACG CGGCAGCACT ATCGAAACGC TGAAACTGCC CAGCTTCGAA

4051	AGCCCTACTC CGCCCAAACT GACCGCCCCC GGTGGCTATA TCGTCGACAT

4101	TCCGAAAGGC AATTTGAAAA CCGAAATCGA AAAGCTGGCC AAACAGCCCG

4151	AGTATGCCTA TCTGAAACAG CTCCAAGTAG CGAAAAACGT CAACTGGAAC

4201	CAGGTGCAAC TGGCTTACGA TAAATGGGAC TATAAGCAGG AAGGCTTAAC

4251	CAGAGCCGGT GCAGCGATTG TTACCATAAT CGTAACCGCA CTGACTTATG

4301	GATACGGCGC AACCGCAGCG GGCGGTGTAG CCGCTTCAGG AAGTAGTACA

4351	GCCGCAGCTG CCGGAACAGC CGCCACAACG ACAGCAGCAG CTACTACCGT

4401	TTCTACAGCG ACTGCCATGC AAACCGCTGC TTTAGCCTCC TTGTATAGCC

4451	AAGCAGCTGT ATCCATCATC AATAATAAAG GTGATGTCGG CAAAGCGTTG

4501	AAAGATCTCG GCACCAGTGA TACGGTCAAG CAGATTGTCA CTTCTGCCCT

4551	GACGGCGGGT GCATTAAATC AGATGGGCGC AGATATIGCC CAATTGAACA

4601	GCAAGGTAAG AACCGAACTG TTCAGCAGTA CGGGCAATCA AACTATTGCC

4651	AACCTTGGAG GCAGATTGGC TACCAATCTC AGTAATGCAG GTATCTCAGC

4701	TGGTATCAAT ACCGCCGTCA ACGGCGGCAG CCTGAAAGAC AACTTAGGCA

4751	ATGCCGCATT AGGAGCATTG GTTAATAGCT TCCAAGGAGA AGCCGCCAGC

4801	AAAATCAAAA CAACCTTCAG CGACGATTAT GTTGCCAAAC AGTTCGCCCA

4851	CGCTTTGGCT GGGTGTGTTA GCGGATTGGT ACAAGGAAAA TGTAAAGACG

4901	GGGCAATTGG CGCAGCAGTT GGGGAAATCG TAGCCGACTC CATGCTTGGC

4951	GGCAGAAACC CTGCTACACT CAGCGATGCG GAAAAGCATA AGGTTATCAG

5001	TTACTCGAAG ATTATTGCCG GCAGCGTGGC GGCACTCAAC GGCGGCGATG

5051	TGAATACTGC GGCGAATGCG GCTGAGGTGG CGGTAGTGAA TAATGCTTTG

5101	AATTTTGACA GTACCCCTAC CAATGCGAAA AAGCATCAAC CGCAGAAGCC

5151	CGACAAAACC GCACTGGAAA AAATTATCCA AGGTATTATG CCTGCACATG

5201	CAGCAGGTGC GATGACTAAT CCGCAGGATA AGGATGCTGC CATTTGGATA

5251	AGCAATATCC GTAATGGCAT CACAGGCCCG ATTGTGATTA CCAGCTATGG

5301	GGTTTATGCT GCAGGTTGGA CAGCTCCGCT GATCGGTACA GCGGGTAAAT

5351	TAGCTATCAG CACCTGCATG GCTAATCCTT CTGGTTGTAC TGTCATGGTC

5401	ACTCAGGCTG CCGAAGCGGG CGCGGGAATC GCCACGGGTG CGGTAACGGT

5451	AGGCAACGCT TGGGAAGCGC CTGTGGGGGC GTTGTCGAAA GCGAAGGCGG

5501	CCAAGCAGGC TATACCAACC CAGACAGTTA AAGAACTTGA TGGCTTACTA

5551	CAAGAATCAA AAAATATAGG TGGTGTAAAT ACACGAATAA ATATAGCGAA

5601	TAGTACTACT CGATATACAC CAATGAGACA AACGGGACAA CCGCTATCTG

5651	CTGGCTTTGA GCATGTFCTT GAGGGGGACT TCCATAGGCC TATTGCGAAT

5701	AACCGTTCAG TTTTTACCAT CTCCCCAAAT GAATTGAAGG TTATACTTCA

5751	AAGTAATAAA GTAGTTTCTT CTCCCGTATC GATGACTCCT GATGGCCAAT

5801	ATATGCGGAC TGTCGATGTA GGAAAAGTTA TTGGTACTAC TTCTATTAAA

5651	GAAGGTGGAC AACCCACAAC TACAATTAAA GTATTTACAG ATAAGTCAGG

5901	AAATTTGATT ACTACATACC CAGTAAAAGG AAACTAA

This corresponds to the amino acid sequence <SEQ ID 60; ORF114-1>:


1	HNKGLHRIIF SXKHSTMVAV AETANSQGKG KQAGSSVSVS LKTSGDLCGK

51	LKTTLKTLVC SLVSLSHVLP AHAQITTDKS APKNQQVVIL KFNTGAPLVN

101	IQTPNGRGLS HNRYTQFDVD NKGAVLNNDR NNNPFVVKGS AQLILNEVRG

151	TASKLNGIVT VGGQKADVII ANPNGITVNG GGFKNVGRGI LTTGAPQIGK

201	DGALTGFDVR QGTLTVGAAG WNDKGGADYT GVLAAAVALQ GKLQGKLLAV

251	STGFQKVDYA SGEISAGTAA GTKPTIALDT AALGGHYADS ITLIANEKGV

301	GVKNAGTLKA AXQLIVTSSG RIENSGRIAT TADGTFASPT YLSIETTEKG

351	AAGTFISNGG RIESKGLLVI ETGEDISLRN GAVVQINGSR PATTVLNAGH

401	HLVIESKTNV NNAKGFATLS ADGRTVIKEA SIQTGTTVYS SSKGTAELGN

451	NTRZTGADVT VLSNGTISSS AVIDAKOTAN IEAGKPLSLT ASTVTSDIRL

501	NGGSIKGGKQ LALLADDNIT AXTTNLNTPG NLYVHTGKDL NIMVDKDLSA

551	ASIHLKSDNA AHITGTSKTL TASKDMGVEA GSLNVTNTNL RTNSGNLHIQ

601	AAKGNIQLRN TKLNAAKALE TTALQGNIVS OGLHAVSADG HVSLLANGNA

651	DFTGHNTLTA KADVNAGSVG KGRLKADNTN ITSSSGDITL VAGNGIQLGD

701	GKQRNSINGK HISIKNNGGN ADLIQLNVHA KSGALNIHSD RALSIENTKL

751	ESTRNTHLNA QHERVTLNQV DAYAHRHLSI TGSQIWQNOK LPSANKLVTN

801	GVLALNARYS QIADNTTLRA GAINLTAGTA LVKRGNINWS TVSTKTLEDN

851	AELKPLAGRL NIEAGSGTLT IEPANRISAH TDLSIKTGGK LLLSAKGGNA

901	GAFSAQVSSL EAKGNIRLVT GETDLRGSKI TAGKNLVVAT TKGKLNIEAV

951	NNSFSNYFPT QKAAELNQKS KELEQQIAQL KKSSPKSKLI PTLQEERDRL

1001	AFYIQAINKE VKGKKPKGKE YLQAKLSAQN IDLISAQGIE ISGSDITASK

1051	KLNLHAAGVL PKAADSEAAA ILIDGITDQY EIGKPTYKSH YOKAALNKPS

1101	RLTGRTGVSI HAAAALDDAR IIIGASEIKA FSGSIDIKAH SDIVLEAGQN

1151	DAYTFLKTKG KSGKIIRXTK FTSTRDHLIM PAPVELTANG ITLQAGGNIE

1201	ANTTRFNAPA GKVTLVAGEE LQLLAEEGIH KHELDVQKSR RFIGIKVGKS

1251	NYSKNELNET KLPVRVVAQT AATRSGWDTV LEGTEFKTTL AGADIQAGVG

1301	EKAPADAKII LKGIVNRIQS EEKLETNSTV WQKQAGRGST IETLKLPSFE

1351	SPTPPKLTAP GGYIVDIPKG NLKTEIEKLA KQPEPEYLKQ LQVAKNVNWN

1401	QVQLAYDKWD YKQEGLTRAG AAIVTIIVTA LTYGYGATAA GGVAASGSST

1451	AAAAGTAATT TAAATTVSTA TANQTAALAS LYSQAAVSII NNKGDVGKAL

1501	KDLGTSDTVK QIVISALTAG ALNQMGADIA QLNSKVRTEL FSSTGVQTIA

1551	NLGGRLATNL SNAGISAGIN TAVNGGSLKD NLGNAALGAL VNSFQGEAAS

1601	KIKTTFSDDY VAKQFAHALA GCVSGLVQGK CKDGAIGAAV GEIVADSNLG

1651	GRNPATLSDA EKHKVISYSK IIAGSVAALN GGDVNTAANA AEVAVVNNAL

1701	NFDSTPTNAK KNQPQKPDKT ALEKIIQGIM PAHAAGAMTN PQDKDAAIWI

1751	SNIRNGITGP IVITSYGVYA AGWTAPLIGT AGKLAISTCM ANPSGCTVNV

1801	TQAAEAGAGI ATGAVTVGNA WEAPVGALSK AKAAKQAIPT QTVKELDGLL

1851	QESKNIGAVN TRINIANSTT RYTPNRQTGQ PVSAGFENVL EGHFHRPIAN

1901	NRSVFTXSPN ELKVILQSNK VVSSPVSMTP DGQYNRTVDV GKVIGTTSIK

1951	EGGQPTTTIK VFTDKSGNLI TTYPVKGN*

Computer analysis of this amino acid sequence predicts a transmembrane region and also gives the following results:
Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF114 shows 91.9% identity over a 284 aa overlap with an ORF (ORF114a) from strain A of N. meningitidis:


	10 20 30 40
orf114.pep	AVAETANSQGKGKQAGSSVSVSLKTSGDLCGKLKTTLKTLVC
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf114a	MNKGLHRIIFSKKHSTMVAVAETANSQGKGKQAGSSVSVSLKTSGDLCGKLKTTLKTLVC
	10 20 30 40 50 60

	50 60 70 80 90 100
orf114.pep	SLVSLSMVLPAHAQITTDKSAPKNQQVVILKTNTGAPLVNIQTPNGRGLSHNRXYAFDVD
	\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|
orf114a	SLVSLSMXXXXXXQITTDKSAPKNXQVVILKTNTGAPLVNIQTPNGRGLSHNRYTQFDVD
	70 80 90 100 110 120

	110 120 130 140 150 160
orf114.pep	NKGAVLNNDRNNNPFVVKGSAQLILNEVRGTALKLNGIVTVGGQKADVIIANPNGITVNG
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf114a	NKGAVLNNDRNNNPFLVKGSAQLILNEVRGTASKLNGIVTVGGQKADVIIANPNGITVNG
	130 140 150 160 170 180

	170 180 190 200 210 220
orf114.pep	GGFKNVGRGILTTGAPQIGKDGALTGFDVVKAHWTVXAAGWNDKGGAXYTGVLARAVALQ
\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| :: \|\| \|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|
orf114a	GGFKNVGRGILTIGAPQIGKDGALTGFDVRQGTLTVAAAGWNDKGGADYTGVLARAVALQ
	190 200 210 220 230 240

	230 240 250 260 270 290
orf114.pep	GKXXGKXLAVSTGPQKVDYASGEISAGTAAGTKPTIALDTAALGGMYADSITLIANEKGV
	\|\| \|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|
orf114a	GKLQGKNLAVSTGPQKVDYASGEISAGTAAGTKPTIALDTAALGGMYADSITLIAXEKGV
	250 260 270 280 290 300

orf114.pep	GVX
	\|\|
orf114a	GVKNAGTLEAAKQLIVTSSGRIENSGRIATTADGTEASPTYLXIETTEKGAXGTFISNGG
	310 320 330 340 350 360

The complete length ORF114a nucleotide sequence <SEQ ID 61> is:


1	ATGAATAAAG GTTTACATCG CATTATCTTT AGTAAAAAGC ACAGCACCAT

51	GGTTGCAGTA GCCGAAACTG CCAACAGCCA GGGCAAAGGT AAACAGGCAG

101	GCAGTTCGGT TTCTGTTTCA CTGAAAACTT CAGGCGACCT TTGCGGCAAA

151	CTCAAAACCA CCCTTAAAAC CTTGGTCTGC TCTTTGGTTT CCCTGAGTAT

201	GGNATTNCNN NNCNNTNCCC AAATTACCAC CGACAAATCA GCACCTAAAA

251	ACCANCAGGT CGTTATCCTT AAAACCAACA CTGGTGCCCC CTTGGTGAAT

301	ATCCAAACTC CGAATGGACG CGGATTGAGC CACAACCGCT ATACGCAGTT

351	TGATGTTGAC AACAAAGGGG CAGTGTTAAA CAACGACCGT AACAATAATC

401	CGTTTCTGGT CAAAGGCAGT GCGCAATTGA TTTTGAACGA GGTACGCGGT

451	ACGGCTAGCA AACTCAACGG CATCGTTACC GTAGGCGGTC AAAAGGCCGA

501	CGTGATTATT GCCAACCCCA ACGGCATTAC TGTTAATGGC GGCGGCTTTA

551	AAAATGTCGG TCGGGGCATC TTAACTATCG GTGCGCCCCA AATCGGCAAA

601	GACGGTGCAC TGACAGGATT TGATGTGCGT CAAGGCACAT TGACCGTAGG

651	AGCAGCAGGT TGGAATGATA AAGGCGGAGC CGACTACACC GGGGTACTTG

701	CTCGTGCAGT TGCTTTGCAG GGGAAATTAC AAGGTAAAAA CCTGGCGGTT

751	TCTACCGGTC CTCAGAAAGT AGATTACGCC AGCGGCGAAA TCAGTGCAGG

801	TACGGCAGCG GGTACGAAAC CGACTATTGC CCTTGATACT GCCGCACTGG

951	GCGGTATGTA CGCCGAGAAA ATCACACTGA TTGCCAATGA AAAAGGCGTA

901	GGcGTCAAAA ATGCCGGCAC ACTCGAAGCG GCCAAGCAAT TGATTGTGAC

951	TTCGTCAGGC CGCATTGAAA ACAGCGGCCG CATCGCCACC ACTGCCGACG

1001	GCACCGAAGC TTCACCGACT TATCTNNCNA TCGAAACCAC CGAAAAAGGA

1051	GCNNCAGGCA CATTTATCTC CAATGGTGGT CGGATCGAGA GCAAAGGCTT

1101	ATTGGTTATT GAGACGGGAG AAGATATCAT CTTGCGTAAC GGAGCCGTGG

1151	TGCAGAATAA CGGCAGTCGC CCAGCTACCA CGGTATTAAA TGCTGGTCAT

1201	AATTTGGTGA TTGAGAGTAA AACTAATGTG AACAATGCCA AAGGCTCGNC

1251	TAATCTGTCG GCCGGCGGTC GTACTACGAT CAATGATGCT ACTATTCAAG

1301	CGGGCAGTTC CGTGTACAGC TCCACCAAAG GCGATACTGA NTTGGGTGAA

1351	AATACCCGTA TTATTGCTGA AAACGTAACC GTATTATCTA ACGGTAGTAT

1401	TGGCAGTGCT GCTGTAATTG AGGCTAAAGA CACTGCACAC ATTGAATCGG

1451	GCAAACCGCT TTCTTTAGAA ACCTCGACCG TTGCCTCCAA CATCCGTTTG

1501	AACAACGGTA ACATTAAAGG CGGAAAGCAG CTTGCTTTAC TGGCAGACGA

1551	TAACATTACT GCCAAAACTA CCAATCTGAA TACTCCCGGC AATCTGTATG

1601	TTCATACAGG TAAAGATCTG AATTTGAATG TTGATAAAGA TTTGTCTGCC

1651	GCCAGCATCC ATTTGAAATC GGATAACGCT GCCCATATTA CCGGCACCAG

1701	TAAAACCCTC ACTGCCTCAA AAGACATGGG TGTGGAGGCA GGCTTGCTGA

1751	ATGTTACCAA TACCAATCTG CGTACCAACT CGGGTAATCT GCACATTCAG

1801	GCAGCCAAAG GCAATATTCA GCTTCGCAAT ACCAAGCTGA ACGCAGCCAA

1851	GGCTCTCGAA ACCACCGCAT TGCAGGGCAA TATCGTTTCA GACGGCCTTC

1901	ATGCTGTTTC TGCAGACGGT CATGTATCCT TATTGGCCAA CGGTAATGCC

1951	GACTTTACCG GTCACAATAC CCTGACAGCC AAGGCCGATG TCNATGCAGG

2001	ATCGGTTGGT AAAGGCCGTC TGAAAGCAGA CAATACCAAT ATCACTTCAT

2051	CTTCAGGAGA TATTACGTTG GTTGCCGNNN NCGGTATTCA GCTTGGTGAC

2101	GGAAAACAAC GCAATTCAAT CAACGGAAAA CACATCAGCA TCAAAAACAA

2151	CGGTGGTAAT GCCGACTTAA AAAACCTTAA CGTCCATGCC AAAAGCGGGG

2201	CATTGAACAT TCATTCCGAC CGGGCATTGA GCATAGAAAA TACNAAGCTG

2251	GAGTCTACCC ATAATACGCA TCTTAATGCA CAACACGAGC GGGTAACGCT

2301	CAACCAAGTA GATGCCTACG CACACCGTCA TCTAAGCATT ANCGGCAGCC

2351	AGATTTGGCA AAACGACAAA CTGCCTTCTG CCAACAAGCT GGTGGCTAAC

2401	GGTGTATTGG CAATCAATGC GCGCTATTCC CAAATTGCCG ACAACACCAC

2451	GCTGAGAGCG GGTGCAATCA ACCTTACTGC CGGTACCGCC CTAGTCAAGC

2501	GCGGCAACAT CAATTGGATT ACCGTTTCGA CCAAGACTTT GGAAGATAAT

2551	GCCGAATTAA AACCATTGGC CGGACGGCTG AATATTGAAG CAGGTAGCGG

2601	CACATTAACC ATCGAACCTG CCAACCGCAT CAGTGCGCAT ACCGACCTGA

2651	GCATCAAAAC AGGCGGAAAA TTGCTGTTGT CTGCAAAAGG AGGAAATGCA

2701	GGTGCGCNTA GTGCTCAAGT TTCCTCATTG GAAGCAAAAG GCAATATCCG

2751	TCTGGTTACA GGAGNAACAG ATTTAAGAGG TTCTAAAATT ACAGCCGGTA

2901	AAAACTTGGT TGTCGCCACC ACCAAAGGCA AGTTGAATAT CGAAGCCGTA

2951	AACAACTCAT TCAGCAATTA TTTTCNTACA CAAAAAGNGN NNGNNCTCAA

2901	CCAAAAATCC AAAGAATTGG AACAACAGAT TGCGCAGTIG AAAAAAAGCT

2951	CGCNTAAAAG CAAGCTGATT CCAACCCTGC AAGAAGAACG CGACCGTCTC

3001	GCTTTCTATA TTCAAGCCAT CAACAAGGAA GTTAAAGGTA AAAAACCCAA

3051	AGGCAAAGAA TACCTGCAAG CCAAGCTTTC TGCACAAAAT ATTGACTTGA

3101	TTTCCGCACA AGGCATCGAA ATCAGCGGTT CCGATATTAC CGCTTCCAAA

3151	AAACTGAACC TTCACGCCGC AGGCGTATTG CCAAAGGCAG CAGATTCAGA

3201	GGCGGCTGCT ATTCTGATTG ACGGCATAAC CGACCAATAT GAAATTGGCA

3251	AGCCCACCTA CAAGAGTCAC TACGACAAAG CTGCTCTGAA CAAGCCTTCA

3301	CGTTTGACCG GACGTACGGG GGTAAGTATT CATGCAGCTG CGGCACTCGA

3351	TGATGCACGT ATTATTATCG GTGCATCCGA AATCAAAGCT CCCTCAGGCA

3401	GCATAGACAT CAAAGCCCAT AGTGATATTG TACTGGAGGC TGGACAAAAC

3451	GATGCCTATA CCTTCTTAAA AACCAAAGGT AAAAGCGGCA NAATNATCAG

3501	AAAAACNAAG TTTACCAGCA CCNGCGANCA CCTGATTATG CCAGCCCCNG

3551	TCGAGCTGAC CGCCAACGGT ATCACGCTTC ACGCAGGCGG CAACATCGAA

3601	GCTAATACCA CCCGCTTCAA TGCCCCTGCA GGTAAAGTIA CCCTGGTTGC

3651	GGGTGAANAG NTGCAACTGC TGGCAGAAGA AGGCATCCAC AAGCACGAGT

3701	TGGATGTCCA AAAAAGCCGC CGCTTTATCG GCATCAAGGT AGGTNAGAGC

3751	AATTACAGTA AAAACGAACT GAACGAAACC AAATTGCCTG TCCGCGTCGT

3801	CGCCCAAAAT GCAGCCACCC GTTCAGGCTG GGAThCCGTG CTCGAAGGTA

3851	CCGAATTCAA ATCCACGCTG GCCGGTGCCG ACATTCAGGC AGGTGTANGC

3901	GAAAAAGCCC GTGTCGATGC GAAAATCATC CTCAAAGGCA TTGTGAACCG

3951	TATCCAGTCG GAAGAAAAAT TAGAAACCAA CTCAACCGTA TGGCAGAAAC

4001	AGGCCGGACG CGGCAGCACT ATCGAAACGC TAAAACTGCC CAGCTTCGAA

4051	AGCCCTACTC CGCCCAAATT GTCCGCACCC GGCGGNTATA TCGTCGACAT

4101	TCCGAAAGGC AATCTGAAAA CCGAAATCGA AAAGCTGTCC AAACAGCCCG

4151	AGTATGCCtA TCTGAAACAG CTCCAAGTAG CGAAAAACAT CAACTGGAAT

4201	CAGGTGCAGC TTGCTTACGA CAGATGGGAC TACAAACAGG AGGGCTTAAC

4251	CGAAGCAGGT GCGGCGATTA TCGCACTGGC CGTTACCGTG GTCACCTCAG

4301	GCGCAGGAAC CGGAGCCGTA TTGGGATTAA ACGGTGCGNC CGCCGCCGCA

4351	ACCGATGCAG CATTCGCCTC TTTGGCCAGC CAGGCTTCCG TATCGTTCAT

4401	CAACAACAAA GGCGATGTCG GCAAAACCCT GAAAGAGCTG GGCAGAAGCA

4451	GCACGGTGAA AAATCTGGTG GTTGCCGCCG CTACCGCAGG CGTAGCCGAC

4501	AAAATCGGCG CTTCGGCACT GANCAATGTC AGCGATAAGC AGTGGATCAA

4551	CAACCTGACC GTCAACCTAG CCAATGNCGG GCAGTGCCGC ACTGAttaa

This encodes a protein having amino acid sequence <SEQ ID 62>:


1	MNKGLHRIIF SKKHSTMVAV AETANSQGKG KQAGSSVSVS LKTSGDLCGK

51	LKTTLKTLVC SLVSLSMXXX XXXQITTDKS APIDXQVVIL KTNTGAPLVN

101	IQTPNGRGLS HNRYTQFDVD NKGAVLNNDR NNNPFLVKGS AQLILNEVRG

151	TASKLNGIVT VGGQKADVII ANPNGITVNG GGFKNVGRGI LTIGAPQIGK

201	DGALTGFDVR QGTLTVGAAG WNDKGGADYT GVLARAVALQ GKLQGKNLAV

251	STGPQKVDYA SGEISAGTAA GTKPTIALDT AALGGMYADS ITLTAXEKGV

301	GVKNAGTLEA AKQLIVTSSG RIENSGRIAT TADGTEASPT YLXIETTEKG

351	AXGTFISNGG RIESKGLLVI ETGEDIXLPA GAVVQNNGSR PATTVLNAGH

401	NLVIESKTNV NNAXGSXNLS AGGRTTINDA TIQAGSSVYS STKGDTXLGE

451	NTRIIAENVT VLSNGSIGSA AVIEAKDTAN IESGKPLSLE TSTVASNIRL

501	NNGNIKGGKQ LALLADDNIT AKTTNLNTPG NLYVHTGKDL NLNVDKDLSA

551	ASIHLKSDNA AHITGTSKTL TASKDNGVEA GLLNVTNTNL RTNSGNLHIQ

601	AAKGNZQLRH TKLNAAKALE TTALQGNIVS DGLHAVSADG HVSLLANGNA

651	DFTGHNTLTA KADVXAGSVG KGRLKADNTN ITSSSGDITL VAXXGIQLGD

701	GKQRNSINGK HISIKNNGGN ADLKNLNVHA KSGALNIHSO RALSIENTKL

751	ESTHNTHLNA QHERVTLNQV DAYAHRHLSI XGSQIWQNDK LPSANKLVAN

801	GVLAXNARYS QIADNTTLRA CAINLTAGTA LVKRGNINWS TVSTKTLEDN

851	AELKPLAGRL NIEAGSGTLT IEFANRISAH TDLSIKTGGK LLLSAXGGNA

901	GAXSAQVSSL EAKGNIRLVT GXTDLRGSKI TAGKNLVVAT TKGKLNIEAV

951	NNSFSNYFXT QKXXXLNQKS KELEOQIAQL KKSSXKSKLI PTLQEERDRL

1001	AFYIQAINKE VKGKKPKGKE YLQAXLSAQN IDLISAQGIE ISGSDITASK

1051	KLNLHAAGVL PKAADSEAAA ILIDGITOQY EIGKPTYKSH YDKAALNKPS

1101	RLTGRTGVSI HAAAALDDAR IIIGASEIKA PSGSIDIKAR SDIVLEAGQN

1151	DAYTFLXTKG KSGXXIRKTK FTSTXXHLIM PAPVELTANG ITLQAGGNIE

1201	ANTTRFHAPA GKVTLVAGEK XQLLAEEGIK KHELDVQKSR RFIGIKVGXS

1251	NYSINELNET KLPVRVVAQX AATRSGWDTV LEGTEFKTTL AGADIQAGVX

1301	EKARVQAXII LKGIVNRIQS EEKLETNSTV WQKQAGRGST IETLKLPSFE

1351	SPTPPKLSAP GGYIVDIPKG NLKTEIEKLS KQPEYAYLKQ LQVAKNINWN

1401	QVQLAYQRWD YKQEGLTEAG AAIIALAVTV VTSGAGTGAV LGLNGAXAAA

1451	TDAAFASLAS QASVSFINNK GDVGKTLKEL GRSSTVKNLV VAAATAGVAD

1501	KIGASALXNV SDKQWINNLT VNLANXGQCR TD*

ORF114-1 and ORF114a show 89.8% identity in 1564 aa overlap


orf114a.pep	MNKGLHRIIFSKKHSTMVAVAETANSQGKGKQAGSSVSVSLKTSGDLCGKLKTTLKTLVC
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf114-1	MNKGLHRIIFSKKHSTMVAVAETANSQGKGKQAGSSVSVSLKTSGDLCGKLKTTLKTLVC

orf114a.pep	SLVSLSMXXXXXXQITTDKSAPKNXQVVILKTNTGAPLVNIQTPNGRGLSHNRYTQFDVD
	\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf114-1	SLVSLSMXXXXXXQITTDKSAPKNQQVVILKTNTGAPLVNIQTPNGRGLSHNRYTQFDVD

orf114a.pep	NKGAVLNNDRNNNPFLVKGSAQLILNEVRGTASKLNGIVTVGGQKADVIIANPNGITVNG
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf114-1	NKGAVLNNDRNNNPFVVKGSAQLILNEVRGTASKLNGIVTVGGQKADVIIANPNGITVNG

orf114a.pep	GGFKNVGRGILTIGAPQIGKDGALTGFDVRQGTLTVGAAGWNDKGGADYTGVLARAVALQ
	\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf114-1	GGFKNVGRGILTTGAPQIGKDGALTGFDVRQGTLTVGAAGWNDKGGADYTGVLARAVALQ

orf114a.pep	GKLQGKNLAVSTGFQKVDYASGEISAGTAAGTKPTIALDTAALGGMYADSITLIAXEKGV
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|
orf114-1	GKLOGKNLAVSTGPQKVDYASGEISAGTAAGTKPTIALDTAALGGMYADSITLIANEKGV

orf114a.pep	GVKNAGTLEAAXQLIVTSSGRIENSGRIATTADGTLASPTYLXIETTEKGAXGTFISNGG
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|
orf114-1	GVKNAGTLEAAXQLIVTSSGRIENSGRIATTADGTLASPTYLSIETTEKGAAGTFISNGG

orf114a.pep	RIESKGLLVIETGEDIXLRNGAVVQNNGSRPATTVLNAGHNLVIESKTNVNNAKGSXNLS
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| :\|\|
orf114-1	RIESKGLLVIETGEDISLRNGAVVQNNGSRPATTVLNAGHNLVIESKTNVNNAKGPANLS

orf114a.pep	AGGRTTINDATIQAGSSVYSSTKGDTXLGENTRIIAENVTVLSNGSIGSAAVIEAKDTAH
	\| \|\|\|:\|::\|:\|\|:\|::\|\|\|\|:\|\|:: \|\|:\|\|\|\| : :\|\|\|\|\|\|\|\|\|:\|:\|\|\|:\|\|\|\|\|\|
orf114-1	ADGRTVIKEASIQTGTTVYSSSKGNAELGNNTRITGADVTVLSNGTISSSAVIDAKDTAH

orf114a.pep	IESGKPLSLETSTVASNIRLNNGNIKGGKQLALLADDNITAKTTNLNTPGNLYVHTGKDL
	\|\|:\|\|\|\|\|\|\|\|\|\|\|:\|\|\|:\|:\|\|\|\|:\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf114-1	IEAGKPLSLEASTVTSDIRLNGGSIKGGKQLALLADDNITAKTTNLNTPGNLYVHTGKDL

orf114a.pep	NLNVDKDLSAASIHLKSDNAAHITGTSKTLTASKDMGVEAGLLNVTNTNLRTNSGNLHIQ
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf114-1	NLNVDKDLSAASIHLKSDNAAHITGTSKTLTASKDMGVEAGSLNVTNTNLRTNSGNLHIQ

orf114a.pep	AAKGNIQLRNTKLNAAKALETTALQGNIVSDGLHAVSADGHVSLLANGNADFTGHNTLTA
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf114-1	AAKGNIQLRNTKLNAAKALETTALQGNIVSDGLHAVSADGHVSLLANGNADFTGHNTLTA

orf114a.pep	KADVXAGSVGKGRLKADNTNITSSSGDITLVAXXGIQLGDGKQRNSINGKHISIKNNGGN
	\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf114-1	KADVNAGSVGKGRLKADNTNITSSSGDITLVAGNGIQLGDGKQRNSINGKHISIKNNGGN

orf114a.pep	ADLKNLNVHAKSGALNIHSDRALSIENTKLESTHNTHLNAQHERVTLNQVDAYAHRHLSI
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf114-1	ADLKNLNVHAKSGALNIHSDRALSIENTKLESTHNTHLNAQHERVTLNQVDAYAHRHLSI

orf114a.pep	XGSQIWQNDKLPSANKLVANGVLAXNARYSQIADNTTLRAGAINLTAGTALVKRGNINWS
	:\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf114-1	TGSQIWQNDKLPSANKLVANGVLALNARYSQIADNTTLRAGAINLTAGTALVKRGNINWS

orf114a.pep	TVSTKTLEDNAELKPLAGRLNIEAGSGTLTIEPANRISAHTDLSIKTGGKLLLSAKGGNA
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf114-1	TVSTKTLEDNAELKPLAGRLNIEAGSGTLTIEPANRISAHTDLSIKTGGKLLLSAKGGNA

orf114a.pep	GAXSAQVSSLEAKGNIRLVTGXTDLRGSKITAGKNLVVATTKGKLNIEAVNNSFSNYFXT
	\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|
orf114-1	GAPSAQVSSLEAKGNIRLVTGETDLRGSKITAGKNLVVATTKGKLNIEAVNNSFSNYFPT

orf114a.pep	QKXXXLNQKSKELEQQIAQLKKSSXKSKLIPTLQEERDRLAFYIQAINKEVKGKKPKGKE
	\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf114-1	QKAAELNQKSKELEQQIAQLKKSSPKSKLIPTLQEERDRLAFYIQAINKEVKGKKPKGKE

orf114a.pep	YLQAKLSAQNIDLISAQGIEISGSDITASKKLNLHAAGVLPKAADSEAAAILIDGITDQY
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf114-1	YLQAKLSAQNIDLISAQGIEISGSDITASKKLNLHAAGVLPKAADSEAAAILIDGITDQY

orf114a.pep	EIGKPTYKSHYDKAALNKPSRLTGRTGVSIHAAAALDDARIIIGASEIKAPSGSIDIKAH
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf114-1	EIGKPTYKSHYDKAALNKPSRLTGRTGVSIHAAAALDDARIIIGASEIKAPSGSIDIKAH

orf114a.pep	SDIVLEAGQNDAYTFLXTKGKSGXXIRKTKFTSTXXHLIMPAPVELTANGITLQAGGNIE
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\| \|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf114-1	SDIVLEAGQNDAYTFLKTKGKSGKIIRKTKFTSTRDHLIMPAPVELTANGITLQAGGNIE

orf114a.pep	ANTTRFNAPAGKVTLVAGEXXQLLAEEGIHKHELDVQKSRRFIGIKVGXSNYSKNELNET
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|
orf114-1	ANTTRFNAPAGKVTLVAGEELQLLAEEGIHKHELDVQKSRRFIGIKVGKSNYSKNELNET

orf114a.pep	KLPVRVVAQXAATRSGWDTVLEGTEFKTTLAGADIQAGVXEKARVDAKIILKGIVNRIQS
	\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf114-1	KLPVRVVAQTAATRSGWDTVLEGTEFKTTLAGADIQAGVGEKARADAKIILKGIVNRIQS

orf114a.pep	EEKLETNSTVWQKQAGRGSTIETLKLPSFESPTPPKLSAPGGYIVDIPKGNLKTEIEKLS
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:
orf114-1	EEKLETNSTVWQKQAGRGSTIETLKLPSFESPTPPKLSTPGGYIVDIPKGNLKTEIEKLA

orf114a.pep	KQPEYAYLKQLQVAKNINWNQVQLAYDRWDYKQEGLTEAGAAIIALAVTVVTSGAGTGAV
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|:\|\|\|\|\|::: \|\|::\| \| \|: \|:
orf114-1	KQPEYAYLKQLQVAKNVNWNQVQLAYDKWDYKQEGLTRAGAAIVTIIVTALTYGYGATAA

orf114a.pep	LGLNGA--------------XAATD----------AAFASLASQASVSFINNKGDVGKTL 1477
	\|: :: :\|\|\|\| \|\|:\|\|\| \|\|\|:\|\|:\|\|\|\|\|\|\|\|\|:\|
orf114-1	GGVAASGSSTAAAAGTAATTTAAATTVSTATAMQTAALASLYSQAAVSIINNKGDVGKAL 1500

orf114a.pep	KELGRSSTVKNLVVAAATAGVADKIGA----------SALXNVSDKQWINNL----TVNL 1523
	\|:\|\| \|:\|\|\|::\|::\| \|\|\|: :::\|\| : \| : : :\| \| \|\| ::\|\|
orf114-1	KDLGTSDTVKQIVTSALTAGALNQMGADIAQLNSKVRTELFSSTGNQTIANLGGRLATNL 1560

orf114a.pep	ANXGQCRTDX
	:\| \|
orf114-1	SNAGISAGINTAVN...

Homology with pspA Putative Secreted Protein of N. meningitidis (Accession Number AF030941)

ORF114 and pspA protein show 36% aa identity in 302 aa overlap:


Orf114:	1	AVAETANSQGKGKQAGSSVSVSL----KTSGDXXXXXXXXXXXXXXXXXXXXXXXPAHAQ 56
		AVAE + GK Q + SV + S PA A
pspA:	19	AVAENVHRDGKSMQDSEAASVRVTGAASVSSARAAFGFRMAAFSVMLALGVAAFSPAPAS 78

Orf114:	57	-ITTDKSAPKNQQVVILKTNTGAPLVNIQTPNGRGLSHNRXYAFDVDNKGAVLNNDRNN- 114
		I DKSAPKNQQ VIL+T G P VNIQTP+ +G+S NR FDVD KG +LNN R+N
pspA:	79	GIIADKSAPKNQQAVILQTANGLPQVNIQTPSSQGVSVNRFKQFDVDEKGVILNNSRSNT 138

Orf114:	115	----------NPFVVKGSAQLILNEV-RGTASKLNGIVTVGGQKADVIIANPNGITVNGG 163
		NP + +G A++I+N++ S LNG + VGG++A+V++ANP+GI VNGG
pspA:	139	QTQLGGWIQGNPHLARGEARVIVNQIDSSNPSLLNGYIEVGGKRAEVVVANPSGIRVNGG 198

Orf114:	164	GFKNVGRGILTTGAPQIGKDGALTGFDVVKAHWTVXAAGWNDKGGAXYTGVLARAVALQG 223
		G N LT+G P + +G LTGFDV + G D A YT +L+RA +
papA:	199	GLINAASVTLTSGVPVL-NNGNLTGFDVSSGKVVIGGKGL-DTSDADYTRILSRAAEINA 256

Orf114:	224	KXXGKXLAVSTGPQKVDYASGEISAGTAAGTK----PTIALDTAALGGMYADSITLIANE 279
		GK + V +G K+D+ +A + PT+A+DTA LGGMYAD ITLI+ +
pspA:	257	GVWGKDVKVVSGKNKLDFDGSLAKTASAPSSSDSVTPTVAIDTATLGGMYAQKITLISTD 316

Orf114:	280	KG 291
		G
papA:	317	NG 318

ORF114a is also homologous to pspA:


gil2623258 (AF030941) putative secreted protein
(Neisseria meningitidis) Length = 2273
Score +32 261 bits (659), Expect +32 3e−69
Identities = 203/663 (30%), Positives 314/663 (46%), Gaps 76/663 (11%)

Query: 1	MNKGLHRIIFSKKHSTMVAVAETANSQGKGKQAGSSVSVSLK-----TSGDXXXXXXXXX 55
	MNK +++IF+KK S M+AVAE + GK Q + SV + +S
Sbjct: 1	MNKRCYKVIFNKKRSCMMAVAENVHRDGKSMQDSEAASVRVTGAASVSSARAAFGFRMAA 60

Query: 56	XXXXXXXXXXXXXXXXXXQITTKDSAPKNXQVVILKTNTGAPLVNIQTPNGRGLSHNRYT 115
	I DKSAPKN Q VIL+T G P VNIQTP+ +G+S NR+
Sbjct: 61	FSVMLALGVAAFSPAPASGIIADKSAPKNQQAVILQTANGLPQVNIQTPSSQGVSVNRFK 120

Query: 116	QFDVDNKGAVLNNDRNN-----------NPFLVKGSAQLILNEV-RGTASKLNGIVTVGG 163
	QFDVD KG +LNN R+N-----------NP L +G A++I+N++ S LNG + VGG
Sbjct: 121	QFDVDEKGVILNNSRSNTQTQLGGWIQGNPHLARGEARVIVNQIDSSNPSLLNGYIEVGG 180

Query: 164	QKADVIIANPNGITVNGGGFKNVGRGILTIGAPQIGKDGALTGFDVRQGTLTVGAAGWND 223
	++A+V++ANP+GI VNGGG N LT G P + +G LTGFDV G + +G G D
Sbjct: 181	KRAEVVVANPSGIRVNGGGLINAASVTLTSGVPVL-NNGNLTGFDVSSGKVVIGGKGL-D 238

Query: 224	KGGADYTGVLARAVALQGKLQGKNLAVSTGPQKVDYASGEISAGTAAGTK----PTIALD 279
	ADYT +L+RA + + GK++ V +G K+D+ +A + PT+A+D
Sbjct: 239	TSDADYTRILSRAAEINAGVWGKDVKVVSGKNKLDFDGSLAKTASAPSSSDSVTPTVAID 298

Query: 280	TAALGGMYADSITLIAXEKGVGVKNAGTLEAAK-QLIVTSSGRIENSGRIATTADGTEAS 338
	TA LGGMYAD ITLI+ + G ++N G + AA + +++ G++ NSG I
Sbjct: 299	TATLGGMYADKITLISTDNGAVIRNKGRIFAATGGVTLSADGKLSNSGSI-------DAA 351

Query: 339	PTYLXIETTEKGAXGTFISNGGRIESKGLLVIETGEDIXLRNGAVVQNNGSRPATTVLNA 398
	+ +T + + G I S V++ + I + G + GS + +
Sbjct: 352	EITISAQTVD--------NRQGFIRSGKGSVLKVSDGINNQAGLI----GSAGLLDIRDT 399

Query: 399	GHNLVIESKTNVNNAKGS----XNLSAGGRTTINDATIQAGSSVYSSTKGDTXLGENTRI 454
	G +S ++NN G+ ++S ++ ND + A V S + D G+
Sbjct: 400	G-----KSSLHINNTDGTIIAGKDVSLQAKSLDNDGILTAARDV-SVSLHDDFAGKRDIE 453

Query: 455	IAENVTVLSNGSIGSAAVIEAKDTAHIESGKPLSLETSTVASNIRLNNGNIKGGKQLALL 514
	+T + G + + +I+A DT + + + + + + S R G L+
Sbjct: 454	AGRTLTFSTQGRLKNTRIIQAGDTVSLTAAQIDNTVSGKIQSGNRTGLNGKNGITNRGLI 513

Query: 515	ADDNIT-----AKTTNLNTPGNLYVHTGKDLNLNVDKDLSAASIHLKSDNAAHITGTSKT 569
	+ IT AK+ N T G +Y G + + D L+ AA
Sbjct: 514	NSNGITLLQTEAKSDNAGT-GRIY---GSRVAVEADTLLNREETVNGETKAA-------V 562

Query: 570	LTASKDMGVEAGXXXXXXXXXXXXSGNLHIQAA---KGNIQLRNTKL-NAAKALETTALQ 625
	+ A + + + A SG+LHI +A +Q NT L N + A+E++
Sbjct: 563	IAARERLDIGAREIENREAALLSSSGDLHIGSALNGSRQVQGANTSLHNRSAAIESS--- 619

Query: 626	GNI 628
	GNI
Sbjct: 620	GNI 622

Score +32 37.5 bits (65), Expect = 0.53
Identities = 87/432 (20%), Positives +32 159/432 (36%), Gaps = 62/432 (14%)

Query: 239	LQGKLQGKNLAVSTGPQKVDYASGEISAGTAAGTKPTIALDTAALGGMYADSITLIAXEK 298
	LQG LQGKN+ + G + +G I A A K A + + S T +
Sbjct: 1023	LQGDLQGKNIFAAAGSDITN--TGSIGAENALLLK--------ASNNIESRSETRSNQNE 1072

Query: 299	GVGVKNAGTLEAAKQLIVTSSGRI--ENSGRIATTADGTEASPTYLXIETTEKGAXG-TF 355
	V+N G + A L +G + + I TA E T + G T
Sbjct: 1073	QGSVRNIGRV-AGIYLTGRQNGSVLLDAGNNIVLTAS-----------ELTNQSEDGQTV 1120

Query: 356	ISNGGRIESKGLLVIETGEDIXLRNGAVVQNNGSRPATTVLNAGHNLVIESK-------T 408
	++ GG I S + I + V++ + +T+ G NL + +K
Sbjct: 1121	LNAGGDIRSDTTGISRNQNTIFDSDNYVIRKEQNEVGSTIRTRG-NLSLNAKGDIRIPAA 1179

Query: 409	NVNNAKGSXNLSAGGRTTINDATIQAGSS--------VYSSTKGDTXLGENTRIIAENVT 460
	V + +G L+AG D ++AG + Y+ G + TR +
Sbjct: 1180	EVGSEQGRLKLAAG-----RDIKVEAGKAHTETEDALKYTGRSGGGIKQKMTRHLKNQNG 1234

Query: 461	VLSNGSIGSAAVIEAKDTAHIESGKPLSLETSTVASWIRLNNGNIKGGKQLALLADDNIT 520
	+G++ +I +G + + T+ S NN +K + + A+ N
Sbjct: 1235	QAVSGTLDGKEIILVSGRDITVTGSNIIADNHTILS--AKNNIVLKAAETRSRSAEMNKK 1292

Query: 521	AKTTNLNTPG-NLYVHTGKDLNLNVDKDLSAASIHLKSDN-------AAHITGTSKTLTA 572
	K+ + + G + KD N + +S + S N H T T T+++
Sbjct: 1293	EKSGLMGSGGIGFTAGSKKDTQTNRSETVSHTESVVGSLNGNTLISAGKHYTQTGSTISS 1352

Query: 573	SK-DMGVEAGXXXXXXXXXXXXSGNLHIQAAKG-----NIQLRNTKLNAAKALETTALQG 626
	+ D+G+ +G + + KG ++ + NT + A A++ G
Sbjct: 1353	PQGDVGISSGKISIDAAQNRYSQESKQVYEQKGVTVAISVPVVNTVMGAVDAVKAVQTVG 1412

Query: 627	NIVSDGLHAVSA 638
	+ ++A++A
Sbjct: 1413	KSKNSRVNAMAA 1424

Amino acids 1-1423 of ORF114-1 were cloned in the pGex vector and expressed in E. coli, as described above. GST-fusion expression was visible using SDS-PAGE, and FIG. 5 shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF114-1.
Based on these results, including the homology with the putative secreted protein of N. meningitidis and on the presence of a transmembrane domain, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 14

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 63>


1	CGCTTCATTC ATGATGAAGC AGTCGGCAGC AACATCGGCG GCGGCAAAAT

51	GATTGTTGCA GCCGGGCAGG ATATCAATGT ACGCGGCAnA AGCCTTATTT

101	CTGATAAGGG CATTGTTTTA AAAGCAGGAC ACGACATCGA TATTTCTACT

151	GCCCATAATC GCTATACCGG CAATGAATAC CACGAGAGCA wAAAwTCAGG

201	CGTCATGGGT ACTGGCGGAT TGGGCTTTAC TATCGGTAAC CGGAAAACTA

251	CCGATGACAC TGATCGTACC AATATTGTsC ATACAGGCAG CATTATAGGC

301	AGCCTGAaTG GAGACACCGT TACAGTTGCA GGAAACCGCT ACCGACAAAC

351	CGGCAGTACC GTCTCCAGCC CCGACGGGCG CAATACCGTC ACAGCCAAAw

401	GCATAGATGT AGAGTTCGCA AACAACCGGT ATGCCACTGA CTACGcCCAT

451	ACCCAGGGAA CAAAAAGGCC TTACCGTCGC CCTCAATGTC CCGGTTGTCC

501	AAGCTGCACA AAACTTCATA CAAGCAGCCC AAAATGTGGG CAAAAGTAAA

551	AATAAACGCG TTAATGCCAT GGCTGCAGCC AATGCTGCAT GGCAGAGTTA

601	TCAAGCAACC CAACAAATGC AACAATTTGC TCCAAGCAGC AGTGCGGGAC

651	AAGGTCAAAA CTACAATCAA AGCCCCAGTA TCAGTGTGTC CATTAC.TAC

701	GGCGAACAGA AAAGTCGTAA CGAGCAAAAA AGACATTACA CCGAAgCGGC

751	AgCAAGTCAA ATTATCGGCA AAGGGCAAAC CACACTTGCG GCAACAGGAA

801	GTGGGGAGCA GTCCAATATC AATATTACAG GTTCCGATGT CATCGGCCAT

951	GCAGGTACTC C.CTCATTGC AAGCAACCAT ATCAGACTCC AATCTGCCAA

901	ACAGGACGGC AGCGAGCAAA GCAAAAACAA AAGCAGTGGT TGGAATGCAG

951	GCGTACGTnn CAAAATAGGC AAcGGCATCA GGTTTGGAAT TACCGCCGGA

1001	GGAAATATCG GTAAAGGTAA AGAGCAAGGG GGAAGTACTA CCCACCGCCA

1051	CACCCATGTC GGCAGCACAA CCGGCAAAAC TACCATCCGA AGCGGCGGGG

1101	GATACCACCC TCAAAGGTGT GCAGCTCATC GGCAAAGGCA TACAGGCAGA

1151	TACGCGCAAC CTGCATATAG AAAGTGTTCA AGATACTGAA ACCTATCAGA

1201	GCAAACAGCA AAACGGCAAT GTCCAAGTTt ACTGTCGGTT ACGGATTCAG

1251	TGCAAGCGGC AGTTACCGCC AAAGCAAAGT CAAAGCAGAC CATGCCTCCG

1301	TAACCGGGCA AAgCGGTATT TATGCCGGAG AAGACGGCTA TCAAATyAAA

1351	GTyAGAGACA ACACAGACCT yAAGGGCGGT ATCATCACGT CTAGCCAAAG

1401	CGCAGAAGAT AAGGGCAAAA ACCTTTTTCA GACGGCCACC CTTACTGCCA

1451	GCGACATTCA AAACCACAGC CGCTACGAAG GCAGAAGCTT CGGCATAGGC

1501	GGCAGTTTCG ACCTGAACGG CGGCTGGGAC GGCACGGTTA CCGACAAACA

1551	AGGCAGGCCT ACCGACAGGA TAAGCCCGGC AGCCGGCTAC GGCAGCGACG

1601	GAGACAGCAA AAACAGCACC ACCCGCAGCG GCGTCAACAC CCACAACATA

1651	CACATCACCG ACGAAGCGGG ACAACTTGCC CGAACAGGCA GGACTGCAAA

1701	AGAAACCGAA GCGCGTATCT ACACCGGCAT CGACACCGAA ACTGCGGATC

1751	AACACTCAGG CCATCTGAAA AACAGCTTCG AC...

This corresponds to the amino acid sequence <SEQ ID 64; ORF116>:


1	..RFIHDEAVGS NIGGGKNIVA AGQDINVRGX SLISDKGIVL KAGADIDIST

51	AHNRYTGNEY HESXXSGVMG TGGLGFTIGN RKTTDDTDRT NIVHTGSIIG

101	SLNGDTVTVA GNRYRQTGST VSSPEGRNTV TAKXIDVEFA NNRYATDYAH

151	TQEQKGLTVA LNVPVVQAAQ NFIQAAQNVG KSKNKRVNAM AAANAAWQSY

201	QATQQMQQFA PSSSAGQGQN YNQSPSISVS IXYGEQKSRN EQKRNYTEAA

251	ASQIIGKGQT TLAATGSGEQ SNINITGSDV IGHAGTXLIA DNHIRLQSAX

301	QDGSEQSKNK SSGWNAGVRX KIGNGIRFGI TAGGNIGKGK EQGGSTTHRH

351	THVGSTTGKT TIRSGGDTTL KGVQLIGXGI QADTRNLHIE SVQDTETYQS

401	KQQNGNVQVT VGYGFSASGS YRQSKVKADH ASVTGQSGIY AGEDGYQIKV

451	RDNTDLKGGI ITSSQSAEDK GKNLFQTATL TASDIQNHSR YEGRSFGIGG

501	SFDLNGGWDG TVTDKQGRPT DRISPAAGYG SDGDSKNSTT RSGVNTHNIH

551	ITDEAGQLAR TGRTAKETEA RIYTGIDTET ADQHSGHLKN SFD...

Computer analysis of this amino acid sequence gave the following results:
Homology with pspA Putative Secreted Protein of N. meningitidis (Accession Number AF030941)

ORF116 and pspA protein show 38% aa identity in 502 aa overlap:


Orf116:	6 EAVGSNIGGGKMIVAAGQDINVRGXSLISDKGIVLKAGHDIDISTAHNRYTGNEYHESXX 65
	+AV + G ++I+ +G+DI V G ++I+D +L A ++I + A R E ++
PspA:	1235 QAVSGTLDGKEIILVSGRDITVTGSNIIADNHTILSAKNNIVLKAAETRSRSAEMNKKEK 1294

Orf116:	66 XXXXXXXXXXXXXXNRKXXXXXXRTNIVHTGSIIGSLNGDTVTVAGNRYRQTGSTVSSPE 125
	++K + HT S++GSLNG+T+ AG Y QTGST+SSP+
PspA:	1295 SGLMGSGGIGFTAGSKKDTQTNRSETVSHTESVVGSLNGNTLISAGKHYTQTGSTISSPQ 1354

Orf116:	126 GRNTVTAKXIDVEFANNRYATDYAHTQEQKGLTVALNVPXXXX---XXXXXXXXXXXGKS 182
	G +++ I ++ A NRY+ + EQKG+TVA++VP GKS
PspA:	1355 GDVGISSGKISIDAAQNRYSQESKQVYEQKGVTVAISVPVVNTVMGAVDAVKAVQTVGKS 1414

Orf116:	183 KNKRVXXXXXXXXXWQSYQATQQMQQFA--PSSSAGQGQNYNQSPSISVSIXYGEQKSRN 240
	KN RV + + + A P +AGQG ISVS+ YGEQK+ +
PspA:	1415 KNSRVNAMAAANALNKGVDSGVALYNAARNPKKAAGQG--------ISVSVTYGEQKNTS 1466

Orf116:	241 EQKRHYTEAAASQIIGKGQTTLAATGSGEQSNINITGSDVIGHAGTXLIADNHIRLQSAK 300
	E + T+ +I G G+ +L A+G+G+ S I ITGSDV G GT L A+N +++++A+
PspA:	1467 ESRIKGTQVQEGKITGGGKVSLTASGAGKDSRITITGSDVYGGKGTRLKAENAVQIEAAR 1526

Orf116:	301 QDGSEQSKNKSSGWNAGVRXKIGNGIRFGITAXXXXXXXXXXXXSTTHRHTHVGSTTGKT 360
	Q E+S+NKS+G+NAGV I GI FG TA T +R++H+GS +T
PspA:	1527 QTHQERSENKSAGFNAGVAIAINKGISFGFTAGANYGKGYGNGDETAYRNSHIGSKDSQT 1586

Orf116:	361 TIRSGGDTTLKGVQLIGKGIQADTRNLHIESVQDTETYQSKQQNGNVQVTVGYGFSASGS 420
	I SGGDT +KG QL GKG+ +LHIES+QDT ++ KQ+N + QVTVGYGFS GS
PspA:	1587 AIESGGDTVIKGGQLKGKGVGVTAESLHIESLQDTAVFKGKQENVSAQVTVGYGFSVGGS 1646

Orf116:	421 YRQSKVKADHASVTGQSGIYAGEDGYQIKVRDNTDLKGGIITSSQSAEDKGKNLFQTATL 480
	Y +SK +D+ASV QSGI+AG DGY+I+V T L G + S DK KNL +T+ +
PspA:	1647 YNRSKSSSDYASVNEQSGIFAGGDGYRIRVNGKTGLVGAAVVSD---ADKSKNLLKTSEI 1703

Orf116:	481 TASDIQNHSRYEGRSFGIGGSF 502
	DIQNH+ + G+ G F
PspA:	1704 WHKDIQNHASAAASALGLSGGF 1725

Based on homology with pspA, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 15

The following partial DNA sequence was identified in N. meningitidis SEQ ID 65>


1	..ACGACCGGCA GCCTCGGCGG CATACTGGCC GGCGGCGGCA CTTCCCTTGC

51	CGCACCGTAT TTGGACAAAG CGGCGGAAAA CCTCGGTCCG GCGGGCAAAG

101	CGGCGGTCAA CGCACTGGGC GGTGCGGCCA TCGGCTATGC AACTGGTGGT

151	AGTGGTGGTG CTGTGGTGGG TGCGAATGTA GATTGGAACA ATAGGCAGCT

201	GCATCCGAAA GAAATGGCGT TGGCCGACAA ATATGCCGAA GCCCTCAAGC

251	GCGAAGTTGA AAAACGCGAA GGCAGAAAAA TCAGCAGCCA AGAAGCGGCA

301	ATGAGAATCC GCAGGCAGAT ATGCGTTGGG TGGACAAAGG TTCCCAAGAC

351	GGCTATACCG ACCAAAGCGT CATATCCCTT ATCGGAATGA

This corresponds to the amino acid sequence <SEQ ID 66; ORF118>:

1 ..TTGSLGGILA GGGTSLAAPY LDKAAENLGP AGKAAVNALG GAAIGYATGG

51 SGGAVVGANV DWNNRQLHPK EMALADKYAE ALKREVEKRE GRKISSQEAA

101 MRIRRQICVG WTKVPKTAIP TKASYPLSE*
Computer analysis of this amino acid sequence reveals two putative transmembrane domains.
Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 16

The following partial DNA sequence was identified in N. meningitidis SEQ ID 67>


1	..CAATGCCGTC TGAAAAGCTC ACAATYTTAC AGACGGCATT TGTTATGCAA

51	GTACATATAC AGATTCCCTA TATACTGCCC AGrkGCGTGC GTgGCTGAAG

101	ACACCCCCTA CGCTTGCTAT TTGrAACAGC TCCAAGTCAC CAAAGACGTC

151	AACTGGAACC AGGTACWACT GGCGTACGAC AAATGGGACT ATAAACAGGA

201	AGGCTTAACC GGAGCCGGAG CAGCGATTAT TGCGCTGGCT GTTACCGTGG

251	TTACTGCGGG CGCGGGAgCC GGAGCCGCAC TGGGcTTAAA CGGCGCGGCc

301	GCAGCGGCAA CCGATGCCGC ATTCGCCTCG CTGGCCAGCC AGGcTTCCGT

351	ATCGCTCATC AaCAACAAAG GCAATATCGG TAaCACCCTG AAAGAGCTGG

401	GCAGAAGCAG CACGGTGAAA AATCTGATGG TTGCCGTCGc tACCGCAgGC

451	GTagCcgaCA AAATCGGTGC TTCGGCACTG AACAATGTCA GCGATAAGCA

501	GTGGATCAAC AACCTGACCG TCAACCTGGC CAATGCGGGC AGTGCCGCAC

551	TGATTAATAC CGCTGTCAAC GGCGGCAGCc tgAAAGACAA TCTGGAAGCG

601	AATATCCTTG CGGCTTTGGT GAATACTGCG CATGGAGAAG CAGCCAGTAA

651	AATCAAACAG TTGGATCAGC ACTACATTAC CCACAAGATT GCCCaTGCCA

701	TAGCGGGCTG TGCGGcTGCG GCGGCGAATA AGGGCAAGTG TCAGGATGGT

751	GCGATAgGTG CGGCTGTGGG CGAGATAGTC GGGGAgGCTT TGACAAACGG

801	CAAAAATCCT GACACTTTGA CAGCTAAAgA ACGCGaACAG ATTTTGGCAT

851	ACAGCAAACT GGTTGCCGGT ACGGTAAGCG GTGTGGTCGG CGGCGATGTA

901	AATGCGGCGG CGAATGCGGC TGAGGTAGCG GTGAAAAATA ATCAGCTTAG

951	CGACAAAtGA

This corresponds to the amino acid sequence <SEQ ID 68; ORF41>:


1	..QCRLKSSQFY RRHLLCKYIY RFPIYCPXAC VAEDTPYACY LXQLQVTKDV

51	HWNQVXLAYD KWDYKQEGLT GAGAAIIALA VTVVT AGAGA GAALGLNGAA

101	AAATDAAFAS LASQASVSLI NNKGNIGNTL KELGRSSTVK NU4VAVATAG

151	VADKIGASAL NNVSDKQWIN NLTVNLANAG SAALINTAVN GGSLKDNLEA

201	NILAALVNTA HGEAASKIKQ LDQHYITHKI AHAIAGCAAA AANKGKCQDG

251	AIGAAVGEIV GEALTNGKNP DTLTAKEREQ ILAYSKLVAG TVSGVVGGDV

301	NAAANAAEVA VKNNQLSDK*

Further work revealed the complete nucleotide sequence <SEQ ID 69>:


1	ATGCAAGTAA ATATTCAGAT TCCCTATATA CTGCCCAGAT GCGTGCGTGC

51	TGAAGACACC CCCTACGCTT GCTATTTGAA ACAGCTCCAA GTCACCAAAG

101	ACGTCAACTG GAACCAGGTA CAACTGGCGT ACGACAAATG GGACTATAAA

151	CAGGAAGGCT TAACCGGAGC CGGAGCAGCG ATTATTGCGC TGGCTGTTAC

201	CGTGGTTACT GCGGGCGCGG GAGCCGGAGC CGCACTGGGC TTAAACGGCG

251	CGGCCGCAGC GGCAACCGAT GCCGCATTCG CCTCGCTGGC CAGCCAGGCT

301	TCCGTATCGC TCATCAACAA CAAAGGCAAT ATCGGTAACA CCCTGAAAGA

351	GCTGGGCAGA AGCAGCACGG TGAAAAATCT GATGGTTGCC GTCGCTACCG

401	CAGGCGTAGC CGACAAAATC GGTGCTTCGG CACTGAACAA TGTCAGCGAT

451	AAGCAGTGGA TCAACAACCT GACCGTCAAC CTGGCCAATG CGGGCAGTGC

501	CGCACTGATT AATACCGCTG TCAACGGCGG CAGCCTGAAA GACAATCTGG

551	AAGCGAATAT CCTTGCGGCT TTGGTGAATA CTGCGCATGG AGAAGCAGCC

601	AGTAAAATCA AACAGTTGGA TCAGCACTAC ATTACCCACA AGATTGCCCA

651	TGCCATAGCG GGCTGTGCGG CTGCGGCGGC GAATAAGGGC AAGTGTCAGG

701	ATGGTGCGAT AGGTGCGGCT GTGGGCGAGA TAGTCGGGGA GGCTTTGACA

751	AACGGCAAAA ATCCTGACAC TTTGACAGCT AAAGAACGCG AACAGATTTT

801	GGCATACAGC AAACTGGTTG CCGGTACGGT AAGCGGTGTG GTCGGCGGCG

851	ATGTAAATGC GGCGGCGAAT GCGGCTGAGG TAGCGGTGAA AAATAATCAG

901	CTTAGCGACA AAGAGGGTAG AGAATTTGAT AACGAAATGA CTGCATGCGC

951	CAAACAGAAT AATCCTCAAC TGTGCAGAAA AAATACTGTA AAAAAGTATC

1001	AAAATGTTGC TGATAAAAGA CTTGCTGCTT CGATTGCAAT ATGTACGGAT

1051	ATATCCCGTA GTACTGAATG TAGAACAATC AGAAAACAAC ATTTGATCGA

1101	TAGTAGAAGC CTTCATTCAT CTTGGGAAGC AGGTCTAATT GGTAAAGATG

1151	ATGAATGGTA TAAATTATTC AGCAAATCTT ACACCCAAGC AGATTTGGCT

1201	TTACAGTCTT ATCATTTGAA TACTGCTGCT AAATCTTGGC TTCAATCGGG

1251	CAATACAAAG CCTTTATCCG AATGGATGTC CGACCAAGGT TATACACTTA

1301	TTTCAGGAGT TAATCCTAGA TTCATTCCAA TACCAAGAGG GTTTGTAAAA

1351	CAAAATACAC CTATTACTAA TGTCAAATAC CCGGAAGGCA TCAGTTTCGA

1401	TACAAACCTA AAAAGACATC TGGCAAATGC TGATGGTTTT AGTCAAAAAC

1451	AGGGCATTAA AGGAGCCCAT AACCGCACCA ATTTTATGGC AGAACTAAAT

1501	TCACGAGGAG GACGCGTAAA ATCTGAAACC CAAACIGATA TTGAAGGCAT

1551	TACCCGAATT AAATATGAGA TTCCTACACT AGACAGGACA GGTAAACCTG

1601	ATGGTGGATT TAAGGAAATT TCAAGTATAA AAACTGTTTA TAATCCTAAA

1651	AAATTTTCTG ATGATAAAAT ACTTCAAATG GCTCAAAATG CTGCTTCACA

1701	AGGATATTCA AAAGCCTCTA AAATTGCTCA AAATGAAAGA ACTAAATCAA

1751	TATCGGAAAG AAAAAATGTC ATTCAATTCT CAGAAACCTT TGACGGAATC

1801	AAATTTAGAT CATATTTTGA TGTAAATACA GGAAGAATTA CAAACATTCA

1851	CCCAGAATAA

This corresponds to the amino acid sequence <SEQ ID 70; ORF41-1>:


1	MQVNIQIPYI LPRCVRAEDT PYACYLKQLQ VTKDVNWNQV QLAYOKWDYK

51	QEGLTGAGAA IIALAVTVVT AGA GAGAALG LNGAAAAATD AAFASLASQA

101	SVSLINNKGN IGNTLKELGR SSTVKNLHVA VATAGVADKI GASALNNVSD

151	KQWINNLTVN LANAGSAALI NTAVNGGSLX DNLEANILAA LVNTAHGEAA

201	SKIKQLDQHY ITHKIAHAIA GCAAAAANKG KCQDGAIGAA VGEIVGEALT

251	NGKNPDTLTA KEREQILAYS KLVAGTVSGV VGGDVNAAAN AAEVAVKNNQ

301	LSDKLGREFD NEMTACAKQN NPQLCRKNTV KKYQNVADKR LAASIAICTD

351	ISRSTECRTI RKQHLIDSRS LHSSWEAGLX GKDDEWYKLF SKSYTQADLA

401	LQSYHLNTAA KSWLOSGNTK PLSEWNSDQG YTLISGVNPR FIPIPRGFVK

451	QHTFITNVKY PEGISFDTNL KRMLANADGF SQKQGIKGAH NRTNFNAELN

501	SRGGRVKSET QTDIEGITRI KYEIPTLDRT GKPDGGFKEI SSIKTVYNPK

551	KFSDDKILQH AQNAASQGYS KASKIAQNER TKSISERKNV IQFSETFDGI

601	KFRSYFDVNT GRITNIHPE*

Computer analysis of this amino acid sequence predicts a transmembrane domain, and homology with an ORF from N. meningitidis (strain A) was also found.

ORF41 shows 92.8% identity over a 279 aa overlap with an ORF (ORF41a) from strain A of N. meningitidis:


	10 20 30 40 50 60 69
orf41.pep	YRRHLLCKYIYRFPIYCPXACVAEDTPYACYLXQLQVTKDVNWNQVXLAYDKWDYKQEGL
	11 1111:1::11111 1111:11111111
orf41a	YLKQLQVAXNINWNQVQLAYDRWDYKQEGL
	10 20 30

	70 80 90 100 110 120 129
orf41.pep	TGAGAAIIALAVTVVTAGAGAGAALGLNGAAAAATDAAFASLASQASVSLINNKGNIGNT
	\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|:\|\|:\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|::\|:\|
orf41a	TEAGAAIIALAVTVVTSGAGTGAVLGLNGAXAAATDAAFASLASQASVSFINNKGDVGKT
	40 50 60 70 80 90

	130 140 150 160 170 180 189
orf41.pep	LKELGRSSTVKNLMVAVATAGVADKIGASALNNVSDKQWINNLTVNLANAGSAALINTAV
	\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf41a	LKELGRSSTVKNLVVAAATAGVADKIGASALXNVSDKQWINNLTVNLANAGSAALINTAV
	100 110 120 130 140 150

	190 200 210 220 230 240 249
orf41.pep	NGGSLKDNLEANILAALVNTAHGEAASKIKQLDQHYITHKIAHAIAGCAAAAANKGKCQD
	\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf41a	NGGSLKDXLEANILAALVNTAHGEAASKIKQLDQHYIVHKIAHAIAGCAAAAANKGKCQD
	160 170 180 190 200 210

	250 260 270 280 290 300 309
orf41.pep	GAIGAAVGEIVGEALTNGKNPDTLTAKEREQILAYSKLVAGTVSGVVGGDVNAAANAAEV
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf41a	GAIGAAVGEIVGEALTNGKNPDTLTAKEREQILAYSKLVAGTVSGVVGGDVNAAANAAEV
	220 230 240 250 260 270

	310 320
orf41.pep	AVKNNQLSDKX
	\|\|\|\|\|\|\|\|\|
orf41a	AVKNNQLSDXEGREFDNENTACAKQNXPQLCRXWIVKKYQNVADKRLAASIAICTDISRS
	280 290 300 310 320 330

A partial ORF41a nucleotide sequence <SEQ ID 71> is:


1	TATCTGAAAC AGCTCCAAGT AGCGAAAAAC ATCAACTGGA ATCAGGTGCA

51	GCTTGCTTAC GACAGATGGG ACTACAAACA GGAGGGCTTA ACCGAAGCAG

101	GTGCGGCGAT TATCGCACTG GCCGTTACCG TGGTCACCTC AGGCGCAGGA

151	ACCGGAGCCG TATTGGGATT AAACGGTGCG NCCGCCGCCG CAACCGATGC

201	AGCATTCGCC TCTTTGGCCA GCCAGGCTTC CGTATCGTTC ATCAACAACA

251	AAGGCGATGT CGGCAAAACC CTGAAAGAGC TGGGCAGAAG CAGCACGGTG

301	AAAAATCTGG TGGTTGCCGC CGCTACCGCA GGCGTAGCCG ACAAAATCGG

351	CGCTTCGGCA CTGANCAATG TCAGCGATAA GCAGTGGATC AACAACCTGA

401	CCGTCAACCT AGCCAATGCG GGCAGTGCCG CACTGATTAA TACCGCTGTC

451	AACGGCGGCA GCCTGAAAGA CANTCTGGAA GCGAATATCC TTGCGGCTTT

501	GGTCAATACC GCGCATGGAG AAGCAGCCAG TAAAATCAAA CAGTTGGATC

551	AGCACTACAT AGTCCACAAG ATTGCCCATG CCATAGCGGG CTGTGCGGCA

601	GCGGCGGCGA ATAAGGGCAA GTGTCAGGAT GGTGCGATAG GTGCGGCTGT

651	GGGCGAGATA GTCGGGGAGG CTTTGACAAA CGGCAAAAAT CCTGACACTT

701	TGACAGCTAA AGAACGCGAA CAGATTTTGG CATACAGCAA ACTGGTTGCC

751	GGTACGGTAA GCGGTGTGGT CGGCGGCGAT GTAAATGCGG CGGCGAATGC

801	GGCTGAGGTA GCGGTGAAAA ATAATCAGCT TAGCGACNAA GAGGGTAGAG

851	AATTTGATAA CGAAATGACT GCATGCGCCA AACAGAATAN TCCTCAACTG

901	TGCAGAAAAA ATACTGTAAA AAAGTATCAA AATGTTGCTG ATAAAAGACT

951	TGCTGCTTCG ATTGCAATAT GTACGGATAT ATCCCGTAGT ACTGAATGTA

1001	GAACAATCAG AAAACAACAT TTGATCGATA GTAGAAGCCT TCATTCATCT

1051	TGGGAAGCAG GTCTAATTGG TAAAGATGAT GAATGGTATA AATTATTCAG

1101	CAAATCTTAC ACCCAAGCAG ATTTGGCTTT ACAGTCTTAT CATTTGAATA

1151	CTGCTGCTAA ATCTTGGCTT CAATCGGGCA ATACAAAGCC TTTATCCGAA

1201	TGGATGTCCG ACCAAGGTTA TACACTTATT TCAGGAGTTA ATCCTAGATT

1251	CATTCCAATA CCAAGAGGGT TTGTAAAACA AAATACACCT ATTACTAATG

1301	TCAAATACCC GGAAGGCATC AGTTTCGATA CAAACCTANA AAGACATCTG

1351	GCAAATGCTG ATGGTTTTAG TCAAGAACAG GGCATTAAAG GAGCCCATAA

1401	CCGCACCAAT NTTATGGCAG AACTAAATTC ACGAGGAGGA NGNGTAAAAT

1451	CTGAAACCCA NACTGATATT GAAGGCATTA CCCGAATTAA ATATGATATT

1501	CCTACACTAG ACAGGACAGG TAAACCTGAT GGTGGATTTA AGGAAATTTC

1551	AAGTATAAAA ACTGTTTATA ATCCTAAAAA NTTTTNNGAT GATAAAATAC

1601	TTCAAATGGC TCAANATGCT GNTTCACAAG GATATTCAAA AGCCTCTAAA

1651	ATTGCTCAAA ATGAAAGAAC TAAATCAATA TCGGAAAGAA AAAATGTCAT

1701	TCAATTCTCA GAAACCTTTG ACGGAATCAA ATTTAGANNN TATNTNGATG

1751	TAAATACAGG AAGAATTACA AACATTCACC CAGAATAA

This encodes a protein having the partial amino acid sequence <SEQ ID 72>:


1	YLKQLQVAKN INWNQVQLAY DRWDYKQEGL TEAGAAIIAL AVTVVTSGAG

51	TGAVLGLNGA XAAATDAAFA SLASQASVSF INNKGDVGKT LKELGRSSTV

101	KNLVVAAATA GVADKIGASA LXNVSDKQWI NNLTVNLANA GSAALINTAV

151	NGGSLKDXLE ANILAALVNT AHGEAASKIK QLDQHYIVRK IAHAIAGCAA

201	AAANKGKCQD GAIGAAVGEI VGEALTNGKN PDTLTAKERE QILAYSKLVA

251	GTVSGVVGGD VNAAANAAEV AVKNNQLSDX EGREFONENT ACAKQNXPQL

301	CRKNTVKKYQ NVADKRLAAS IAICTDISRS TECRTIRKQH LIDSRSLHSS

351	WEAGLIGKDD EWYKLFSKSY TQADLALQSY BLNTAAKSWL QSGNTKPLSE

401	VNSDQGYTLI SGVNPRFIFI PRGFVKQNTP ITNVKYPEGI SFDTNLXRHL

451	ANADGFSQEQ GIKGAHNRTN XMAELNSRGG XVKSETXTDI EGITRIKYEI

501	PTLDRTGKPD GGFKEISSIK TVYNPKXFXD DKILQMAQXA XSQGYSKASK

551	IAQNERTKSI SERKNVIQFS ETFDGIKFRX YXDVNTGRIT NIHPE*

ORF41a and ORF41-1 show 94.8% identity in 595 aa overlap:


	10 20 30
orf41a.pep	YLKQLQVAKNINWNQVQLAYDRWDYKQEGLTEAGAA
	\|\|\|\|\|\|\|:\|::\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\| \|\|\|\|
orf41-1	MQVNIQIPYILPRCVRAEDTPYACYLKQLQVTKDVNWNQVQLAYDKWDYKQEGLTGAGAA
	10 20 30 40 50 60

	40 50 60 70 80 90
orf41a.pep	IIALAVTVVTSGAGTGAVLGLNGAXAAATDAAFASLASQASVSFINNKGDVGKTLKELGR
	\|\|\|\|\|\|\|\|\|\|:\|\|\|:\|\|:\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|::\|:\|\|\|\|\|\|\|
orf41-1	IIALAVTVVTAGAGAGAALGLNGAAAAATDAAFASLASQASVSLINNKGNIGNTLKELGR
	70 80 90 100 110 120

	100 110 120 130 140 150
orf41a.pep	SSTVKNLVVAAATAGVADKIGASALXNVSDKQWINNLTVNLANAGSAALINTAVNGGSLK
	\|\|\|\|\|\|\|:\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf41-1	SSTVKNLMVAVATAGVADKIGASALNNVSDKQWINNLTVNLANAGSAALINTAVNGGSLK
	130 140 150 160 170 180

	160 170 180 190 200 210
orf41a.pep	DXLEANILAALVNTAHGEAASKIKQLDQHYIVHKIAHAIAGCAAAAANKGKCQDGAIGAA
	\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf41-1	DNLEANILAALVNTAHGEAASKIKQLDQHYITHKIAHAIAGCAAAAANKGKCQDGAIGAA
	190 200 210 220 230 240

	220 230 240 250 260 270
orf41a.pep	VGEIVGEALTNGKNPDTLTAKEREQILAYSKLVAGTVSGVVGGDVNAAANAAEVAVKNNQ
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf41-1	VGEIVGEALTNGKNPDTLTAKEREQILAYSKLVAGTVSGVVGGDVNAAANAAEVAVKNNQ
	250 260 270 280 290 300

	280 290 300 310 320 330
orf41a.pep	LSDXEGREFDNEMTACAKQNXPQLCRKNTVKKYQNVADKRLAASIAICTDISRSTECRTI
	\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf41-1	LSDKEGREFDNEMTACAKQNNPQLCRKNTVKKYQNVADKRLAASIAICTDISRSTECRTI
	310 320 330 340 350 360

	340 350 360 370 380 390
orf41a.pep	RKQHLIDSRSLHSSWEAGLIGKODEWYKLFSKSYTQADLALQSYHLNTAMCSWLQSGNTK
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf41-1	RKQHLIDSRSLHSSWEAGLIGKDDEWYKLFSKSYTQADLALQSYHLNTAAXSWLOSGNTK
	370 380 390 400 410 420

	400 410 420 430 440 450
orf41a.pep	PLSEWMSDQGYTLISGVNPRFIPIPRGFVKQNTPITNVKYPEGISFDTNLXRHLANADGF
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|
orf41-1	PLSEWMSDQGYTLISGVNPRFIPIPRGFVKQNTPITNVKYPEGISFDTNLKRHLANADGF
	430 440 450 460 470 480

	460 470 480 490 500 510
orf41a.pep	SQEQGIKGAHNRTNXMAELNSRGGXVKSETXTDIEGITRIKYEIPTLDRTGKPDGGFKEI
	\|\|:\|\|\|\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\| \|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf41-1	SQKQGIKGAHNRTNFMAELNSRGGRVKSETQTDIEGITRIKYEIPTLDRTGKPDGGFKEI
	490 500 510 520 530 540

	520 530 540 550 560 570
orf41a.pep	SSIKTVYNPKXFXDDKILQMAQXAXSQGYSKASKIAQNERTKSISERKNVIQFSETFDGI
	\|\|\|\|\|\|\|\|\|\| \| \|\|\|\|\|\|\|\|\| \| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf41-1	SSIKTVYNPKKFSDDKILQMAQNAASQGYSKASKIAQNERTKSISERKNVIQFSETFDGI
	550 560 570 580 590 600

	580 590
orf41a.pep	KFRXYXDVNTGRITNIHPEX
	\|\|\| \| \|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf41-1	KFRSYFDVNTGRITNIHPEX
	610 620

Amino acids 25-619 of ORF41-1 were amplified as described above. FIG. 6 shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF41-1.
Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 17

The following DNA sequence was identified in N. meningitidis <SEQ ID 73>


1	ATGGCAATCA TTACATTGTA TTATTCTGTC AATGGTATTT TAAATGTATG

51	TGCAAAAGCA AAAAATATTC AAGTAGTTGC CAATAATAAG AATATGGTTC

101	TTTTTGGGTT TTTGGSmrGC ATCATCGGCG GTTCAACCAA TGCCATGTCT

151	CCCATATTGT TAATATTTTT GCTTAGCGAA ACAGAAAATA AAAATcgTAT

201	CGTAAAATCA AGCAATCTAT GCTATCTTTT GGCGAAAATT GTTCAAATAT

251	ATATGCTAAG AGACCAGTAT TGGTTATTAA ATAAGAGTGA ATACGdTTTA

301	ATATTTTTAC TGTCCGTATT GTCTGTTATT GGATTGTATG TTGGAATTCG

351	GTTAAGGACT AAGATTAGCC CAaATTTTTT TAAAATGTTA ATTTTTATTG

401	tTTTATTGGT ATTGGCtCTG AAAATCGGGC AttCGGGTTT AAtCAAACTT

451	TAA

This corresponds to the amino acid sequence <SEQ ID 74; ORF51>:


1	HAIITLYYSV NGILNVCAKA KNIQVVANNK NMVLFGFLXX IICGSTNANS

51	PILLIFLLSE TENKNRIVKS SNLCYLLAKI VQIYMLRDQY WLLNKSEYXL

101	IFLLSVLSVI GLYVGIRLRT KISPNFFKML IFIVLLVLAL KIGHSGLIKL

151	*

Further work revealed the complete nucleotide sequence <SEQ ID 75>:


1	ATGCAAGAAA TAATGCAATC TATCGTTTTT GTTGCTGCCG CAATACTGCA

51	CGGAATTACA GGCATGGGAT TTCCGATGCT CGGTACAACC GCATTGGCTT

101	TTATCATGCC ATTGTCTAAG GTTGTTGCCT TGGTGGCATT ACCAAGCCTG

151	TTAATGAGCT TGTTGGTTCT ATGCAGCAAT AACAAAAAGG GTTTTTGGCA

201	AGAGATTGTT TATTATTTAA AAACCTATAA ATTGCTTGCT ATCGGCAGCG

251	TCGTTGGCAG CATTTTGGGG GTGAAGTTGC TTTTGATACT TCCAGTGTCT

301	TGGCTGCTTT TACTGATGGC AATCATTACA TTGTATTATT CTGTCAATGG

351	TATTTTAAAT GTATGTGCAA AAGCAAAAAA TATTCAAGTA GTTGCCAATA

401	ATAAGAATAT GGTTCTTTTT GGGTTTTTGG CAGGCATCAT CGGCGGTTCA

451	ACCAATGCCA TGTCTCCCAT ATTGTTAATA TTTTTGCTTA GCGAAACAGA

501	AAATAAAAAT CGTATCGTAA AATCAAGCAA TCTATGCTAT CTTTTGGCGA

551	AAATTGTTCA AATATATATG CTAAGAGACC AGTATTGGTT ATTAAATAAG

601	AGTGAATACG GTTTAATATT TTTACTGTCC GTATTGTCTG TTATTGGATT

651	GTATGTTGGA ATTCGGTTAA GGACTAAGAT TAGCCCAAAT TTTTTTAAAA

701	TGTTAATTTT TATTGTTTTA TTGGTATTGG CTCTGAAAAT CGGGCATTCG

751	GGTTTAATCA AACTTTAA

This corresponds to the amino acid sequence <SEQ ID 76; ORF51-1>:


1	MQEIMQSIVF VAAAILHGIT GMGFPMLGTT ALAFIMPLSK VVALVALPSL

51	LMSLLVLCSN NKKGFWQEIV YYLKTYKLLA IGSVVGSILG VKLLLILPVS

101	WLLLLMAIIT LYYSVNGILN VCAKAKNIQV VANNKNNVLF GFLAGIIGGS

151	TNAMSPILLI FLLSETENKN RIVKSSNLCY LLAKIVQIYN LRDQYWLLNK

201	SEYGLIFLLS VLSVIGLYVG IRLRTKISPN FFKMLIFIVL LVLALKIGHS

251	GLIKL*

Computer analysis of this amino acid sequence reveals three putative transmembrane domains. A corresponding ORF from strain A of N. meningitidis was also identified:
Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF51 shows 96.7% identity over a 150 aa overlap with an ORF (ORF51a) from strain A of N. meningitidis:


	10 20 30
orf51.pep	MAIITLYYSVNGILNVCAKAKNIQVVANNK
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf51a	YKLLAIGSVVGSILGVKLLLILPVSWLLLLMAIITLYYSVNGILNVCAKAKNIQVVANNK
	80 90 100 110 120 130

	40 50 60 70 80 90
orf51.pep	NMVLFGFLXXIIGGSTNAMSPILLIFLLSETENKNRIVKSSNLCYLLAKIVQIYMLRDQY
	\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf51a	NMVLFGFLAGIIGGSTNAMSPILLIFLLSETENKNRIAKSSNLCYLLAKIVQIYMLRDQY
	140 150 160 170 180 190

	100 110 120 130 140 150
orf51.pep	WLLNKSEYXLIFLLSVLSVIGLYVGIRLRTKISPNFFKMLIFIVLLVLALKIGHSGLIKL
	\|\|\|\|\|\|\|\| \|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|
orf51a	WLLNKSEYGLIFLLSVLSVIGLYVGIRLRTKISPNFFKMLIFIVLLVLALKIGYSGLIKL
	200 210 220 230 240 250

ORF51-1 and ORF51a show 99.2% identity in 255 aa overlap:


orf51a.pep	MQEIMQSIVFVAAAILHGITGMGFPMLGTTALAFIMPLSKVVALVALPSLLMSLLVLCSN
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf51-1	MQEIMQSIVFVAAAILHGITGMGFPMLGTTALAFIMPLSKVVALVALPSLLMSLLVLCSN

orf51a.pep	NKKGFWQEIVYYLKTYKLLAIGSVVGSILGVKLLLILPVSWLLLLMAIITLYYSVNGILN
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf51-1	NKKGFWQEIVYYLKTYKLLAIGSVVGSILGVKLLLILPVSWLLLLMAIITLYYSVNGILN

orf51a.pep	VCAKAKNIQVVANNKNMVLFGFLAGIIGGSTNAMSPILLIFLLSETENKNRIAKSSNLCY
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|
orf51-1	VCAKAKNIQVVANNKNMVLFGFLAGIIGGSTNAMSPILLIFLLSETENKNRIVKSSNLCY

orf51a.pep	LLAKIVQIYMLRDQYWLLNKSEYGLIFLLSVLSVIGLYVGIRLRTKISPNFFKMLIFIVL
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf51-1	LLAKIVQIYMLRDQYWLLNKSEYGLIFLLSVLSVIGLYVGIRLRTKISPNFFKMLIFIVL

orf51a.pep	LVLALKIGYSGLIKLX
\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|
orf51-1	LVLALKIGHSGLIKLX

The complete length ORF51a nucleotide sequence SEQ D 77> is:


1	ATGCAAGAAA TAATGCAATC TATCGTTTTT GTTGCTGCCG CAATACTGCA

51	CGGAATTACA GGCATGGGAT TTCCGATGCT CGGTACAACC GCATTGGCTT

101	TTATCATGCC ATTGTCTAAG GTTGTTGCCT TGGTGGCATT ACCAAGCCTG

151	TTAATGAGCT TGTTGGTTCT ATGCAGCAAT AACAAAAAGG GTTTTTGGCA

201	AGAGATTGTT TATTATTTAA AAACCTATAA ATTGCTTGCT ATCGGCAGCG

251	TCGTTGGCAG CATTTTGGGG GTGAAGTTGC TTTTGATACT TCCAGTGTCT

301	TGGCTGCTTT TACTGATGGC AATCATTACA TTGTATTATT CTGTCAATGG

351	TATTTTAAAT GTATGTGCAA AAGCAAAAAA TATTCAAGTA GTTGCCAATA

401	ATAAGAATAT GGTTCTTTTT GGGTTTTTGG CAGGCATCAT CGGCGGTTCA

451	ACCAATGCCA TGTCTCCCAT ATTGTTAATA TTTTTGCTTA GCGAAACAGA

501	GAATAAAAAT CGTATCGCAA AATCAAGCAA TCTATGCTAT CTTTTGGCAA

551	AAATTGTTCA AATATATATG CTAAGAGACC AGTATTGGTT ATThAATAAG

601	AGTGAATACG GTTTAATATT TTTACTGTCC GTATTGTCTG TTATTGGATT

651	GTATGTTGGA ATTCGGTTAA GGACTAAGAT TAGCCCAAAT TTTTTTAAAA

701	TGTTAATTTT TATTGTTTTA TTGGTATTGG CTCTGAAAAT CGGGTATTCA

751	GGTTTAATCA AACTTTAA

This encodes a protein having amino acid sequence <SEQ ID 78>:


1	MQEIMQSIVF VAAAILHGIT GMGFPNLGTT ALAFIMPLSK VVALVALPSL

51	LMSLLVLCSN NKKGFWQEIV YYLKTYKLLA IGSVVGSILG VKLLLILPVS

101	WLLLLMAIIT LYYSVNGILN VCAKAKNIQV VANNKNMVLF GFLAGIIGGS

151	TNAMSFILLI FLLSETENKN RIAXSSNLCY LLAKIVQIYM LRDQYWLLNK

201	SEYGLIFLLS VLSVIGLYVG IRLRTKISPN FFKMLIFIVL LVLALKIGYS

251	GLIKL*

Example 18

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 79>


1	ATGAGACATA TGAAAATACA AAATTATTTA CTAGTATTTA TAGTTTTACA

51	TATAGCCTTG ATAGTAATTA ATATAGTGTT TGGTTATTTT GTTTTTCTAT

101	TTGATTTTTT TGCGTTTTTG TTTTTTGCAA ACGTCTTTCT TGCTGTAAAT

151	TTATTATTTT TAGAAAAAAA CATAAAAAAC AAATTATTGT TTTTATTGCC

201	GATTTCTATT ATTATATGGA TGGTAATTCA TATTAGTATG ATAAATATAA

251	AATTTTATAA ATTTGAGCAT CAAATAAAGG AACAAAATAT ATCCTCGATT

301	ACTGGGGTGA TAAAACCACA TGATAGTTAT AATTATGTTT ATGACTCAAA

351	TGGATATGCT AAATTAAAAG ATAATCATAG ATATGGTAGG GTAATTAGAG

401	AAACACCTTA TATTGATGTA GTTGCATCTG ATGTTAAAAA TAAATCCATA

451	AGATTAAGCT TGGTTTGTGG TATTCATTCA TATGCTCCAT GTGCCAATTT

501	TATAAAATTT GTCAGG..

This corresponds to the amino acid sequence <SEQ ID 80; ORF82>:


1	MRHMKIQNYL LVFIVLHIAL IVINIVFGYF VFLFDFFAFL FFANVFLAVN

51	LLFLEKNIKN KLLFLLPISI IIWMVIHISM INIKFYKFEH QIKEQNISSI

101	TGVIKPNDSY NYVYDSNGYA KLKDWHRYGR VIRETPYIDV VASDVKNKSI

151	RLSLVCGIHS YAPCANFIKF VR..

Further work revealed the complete nucleotide sequence SEQ ID 81>:


1	ATGAGACATA TGAAAAATAA AAATTATTTA CTAGTATTTA TAGTTTTACA

51	TATAGCCTTG ATAGTAATTA ATATAGTGTT TGGTTATTTT GTTTTTCTAT

101	TTGATTTTTT TGCGTTTTTG TTTTTTGCAA ACGTCTTTCT TGCTGTAAAT

151	TTATTATTTT TAAAAAAAAA CATAAAAAAC AAATTATTGT TTTTATTGCC

201	GATTTCTATT ATTATATGGA TGGTAATTCA TATTAGTATG ATAAATATAA

251	AATTTTATAA ATTTGAGCAT CAAATAAAGG AACAAAATAT ATCCTCGATT

301	ACTGGGGTGA TAAAACCACA TGATAGTTAT AATTATGTTT ATGACTCAAA

351	TGGATATGCT AAATTAAAAG ATAATCATAG ATATGGTAAG GTAATTAGAG

401	AAACACCTTA TATTGATGTA GTTGCATCTG ATGTTAAAAA TAAATCCATA

451	AGATTAAGCT TGGTTTGTGG TATFCATTCA TATGCFCCAT GTGCCAATTT

501	TATAAAATTT GCAAAAAAAC CTGTTAAAAT TTATTTTTAT AATCAACCTC

551	AAGGAGATTT TATAGATAAT GTAATATTTG AAATTAATGA TGGAAACAAA

601	AGTTTGTACT TGTTAGATAA GTATAAAACA TTTTTTCTTA TTGAAAACAG

651	TGTTTGTATC GTATTAATTA TTTTATATTT AAAATTTAAT TTGCTTTTAT

701	ATAGGACTTA CTTCAATGAG TTGGAATAG

This corresponds to the amino acid sequence <SEQ ID 82; ORF82-1>:


1	MRHMKNKNYL LVFIVLHIAL IVINIVFGYF VFLFDFFAFL FFANVFLAVN

51	LLFLEKNIKH KLLFLLPISI IIWMVIHISH INIKFYKFEH QIKEQNISSI

101	TGVIKPHDSY NYVYDSNGYA KLKDNHRYGR VIRETPYIDV VASDVKNKSI

151	RLSLVCGIHS YAPCANFIKF AKKPVKIYFY NQPQGDFIDN VIFEINDGNK

201	SLYLLDKYKT FFLIENSVCI VLIILYLKFN LLLYRTYFNE LE*

Computer analysis of this amino acid sequence reveals a predicted leader peptide.
A corresponding ORF from strain A of N. meningitidis was also identified:
Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF82 shows 97.1% identity over a 172 aa overlap with an ORF (ORF82a) from strain A of N. meningitidis:


	10 20 30 40 50 60
orf82 pep	MRHMKIQNYLLVFIVLHIALIVINIVFGYFVFLFDFFAFLFFANVFLAVNLLFLEKNIKN
	\|\|\|\|\| :\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf82a	MRHMKIKNYLLVFIVLHITLIVINIVFGYFVFLFDFFAFLFFANVFLAVNLLFLEKNIKN
	10 20 30 40 50 60

	70 80 90 100 110 120
orf82 pep	KLLFLLPISIIIWMVIHISMINIKFYKFEHQIKEQNISSITGVIKPHDSYNYVYDSNGYA
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf82a	KLLFLLPISIIIWMVIHISMINIKFYKFEHQIKEQNISSITGVIKFHDSYNYVYDSNGYA
	70 80 90 100 110 120

130 140 150 160 170
orf82 pep	KLKDNHRYGRVIRETPYIDVVASDVKNKSIRLSLVCGIHSYAPCANFIKFVR
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|::
orf82a	KLKDNHRYGRVIRETPYIDVVASDVKNKSIRLSLVCGIHSYAPCANFIKFAKKPVKIYFY
	130 140 150 160 170

orf82a.pep	MRHMKNKNYLLVFIVLHITLIVINIVFGYFVFLFDFFAFLFFANVFLAVNLLFLEKNIKN
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf82-1	MRHMKNKNYLLVFIVLHIALIVINIVFGYFVFLFDFFAFLFFANVFLAVNLLFLEKNIKN

orf82a.pep	KLLFLLPISIIIWMVIHISMINIKFYKFEHQIKEQNISSITGVIKPHDSYNYVYDSNGYA
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf82-1	KLLFLLPISIIIWMVIHISMINIKFYKFEHQIKEQNISSITGVIKPHDSYNYVYDSNGYA

orf82a.pep	KLKDNHRYGRVIRETPYIDVVASDVKNKSIRLSLVCGIHSYAPCANFIKFAKKPVKIYFY
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf82-1	KLKDNHRYGRVIRETPYIDVVASDVKNKSIRLSLVCGIHSYAPCANFIKFAKKPVKIYFY

orf82a.pep	NQPQGDFIDNVIFEINKGKKSLYLLDKYKTFFLIENSVCIVLIILYLKFNLLLYRTYFNE
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf82-1	NQPQGDFIDNVIFEINKGKKSLYLLDKYKTFFLIENSVCIVLIILYLKFNLLLYRTYFNE

orf82a.pep	LEX
	\|\|\|
orf82-1	LEX

ORF82a and ORF82-1 show 99.2% identity in 242 aa overlap:


orf82a.pep	MRHMKNKNYLLVFIVLHITLIVINIVFGYGVFLFDFFAFLFFANVFLAVNLLFLEKNIKN
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf82-1	MRHMKNKNYLLVFIVLHIALIVINIVFGYGVFLFDFFAFLFFANVFLAVNLLFLEKNIKN

orf82a.pep	KLLFLLPISIIIWMVIHISMINIKFYKFEHQIKEQNISSITGVIKPHDSYNYVYDSNGYA
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf82-1	KLLFLLPISIIIWMVIHISMINIKFYKFEHQIKEQNISSITGVIKPHDSYNYVYDSNGYA

orf82a.pep	KLKDNHRYGRVIRETPYIDVVASDVKNKSIRLSLVCGIHSYAPCANFIKFAKKPVKIYFY
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf82-1	KLKDNHRYGRVIRETPYIDVVASDVKNKSIRLSLVCGIHSYAPCANFIKFAKKPVKIYFY

orf82a.pep	NQPQGDFIDNVIFEINDGKKSLYLLDKYKTFFLIENSVCIVLIILYLKFNLLLYRTYFNE
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf82-1	NQPQGDFIDNVIFEINDGNKSLYLLDKYKTFFLIENSVCIVLIILYLKFNLLLYRTYFNE

orf82a.pep	LEX
	\|\|\|
orf82-1	LEX

The complete length ORF82a nucleotide sequence <SEQ D 83> is:


1	ATGAGACATA TGAAAAATAA AAATTATTTA CTAGTATTTA TAGTTTTACA

51	TATAACCTTG ATAGTAATTA ATATAGTGTT TGGTTATTTT GTTTTTCTAT

101	TTGATTTTTT TGCGTTTTTG TTTTTTGCAA ACGTCTTTCT TGCTGTAAAT

151	TTATTATTTT TAGAAAAAAA CATAAAAAAC AAATTATTGT TTTTATTGCC

201	GATTTCTATT ATTATATGGA TGGTAATTCA TATTAGTATG ATAAATATAA

251	AATTTTATAA ATTTGAGCAT CAAATAAAGG AACAAAATAT ATCCTCGATT

301	ACTGGGGTGA TAAAACCACA TGATAGTTAT AATTATGTTT ATGACTCAAA

351	TGGATATGCT AAATTAAAAG ATAATCATAG ATATGGTAGG GTAATTACAG

401	AAACACCTTA TATTGATGTA GTIGCATCTG ATGTTAAAAA TAAATCCATA

451	AGATIAAGCT TGGTTTGTGG TATTCATTCA TATGCTCCAT GTGCCAATTT

501	TATAAAATTT GCAAAAAAAC CTGTTAAAAT TTATTTTTAT AATCAACCTC

551	AAGGAGATTT TATAGATAAT GTAATATTTG AAATTAATGA TGGAAAAAAA

601	AGTTTGTACT TGTTAGATAA GTATAAAACA TTTTTTCTTA TTGAAAACAG

651	TGTTTGTATC GTATTAATTA TTTTATATTT AAAATTTAAT TTGCTTTTAT

701	ATAGGACTTA CTTCAATGAG TTGGAATAG

This encodes a protein having amino acid sequence <SEQ ID 84>:


1	MRHMKNKNYL LVFIVLHITL IVINIVFGYF VFLFDFFAFL FFANVFLAVN

51	LLFLEKNIKN KLLFLLPISI IIWMVIHISM INIKFYKFEH QIKEQNISSI

101	TGVIKPHDSY NYVYDSNGYA KLKDNHRYGR VIRETPYIDV VASDVIQKSI

151	RLSLVCGIHS YAPCANFIKF AXKPVKIYFY NQPQGDFXDN VIFEINDGKK

201	SLYLLDKYKT FFLIENSVCI VLIILYLKFN LLLYRTYFNE LE*

Example 19

The following partial DNA sequence was identified in N. meningitidis <SEQ ID 85>


1	..ACCCCCAACA GCGTGACCGT CTTGCCGTCT TTCGGCGGAT TCGGGCGTAC

51	CGGCGCGACC ATCAATGCAG CAGGCGGGGT CGGCATGACT GCCTTTTCGA

101	CAACCTTAAT TTCCGTAGCC GAGGGCGCGG TTGTAGAGCT GCAGGCCGTG

151	AGAGCCAAAG CCGTCAATGC AACCGCCGCT TGCATTTTTA CGGTCTTGAG

201	TAAGGACATT TTCGATTTCC TTTTTATTTT CCGTTTTCAG ACGGCTGACT

251	TCCGCCTGTA TTTTCGCCAA AGCCATGCCG ACAGCGTGCG CCTTGACTTC

301	ATATTTAAAA GCTTCCGCGC GTGCCAGTTC CAGTTCGCGC GCATAGTTTT

351	GAGCCGACAA CAGCAGGGCT TGCGCCTTGT CGCGCTCCAT CTTGTCGATG

401	ACCGCCTGCA GCTTCGCAAA TGCCGACTFG TAGCCTTGAT GGTGCGACAC

451	AGCCAAGCCC GTGCCGACAA GCGCGATAAT GGCAATCGGT TGCCAGTAAT

501	TCGCCAGCAG TTTCACGAGA TTCATTCTCG ACCTCCTGAC GCTTCACGCT

551	GA

This corresponds to the amino acid sequence <SEQ ID 86; ORF124>:


1	..TPNSVTVLPS FGGFGRTGAT INAAGGVGMT AFSTTLISVA EGAVVELQAV

51	RAKAVNATAA CIFTVLSKDI FDFLFIFRFQ TADFRLYFRQ SHADSVRLDF

101	IFKSFRACQF QFARIVLSRQ QQGLRLVALH LVDORLQLRX CRLVALMVRH

151	SQARADKRDN GNRLPVIRQQ FHEIHSRPPD ASR*

Computer analysis of this amino acid sequence predicts a transmembrane domain.

Further work revealed the complete nucleotide sequence SEQ ID 87>:


1	ATGACTGCCT TTTCGACAAC CTTAATTTCC GTAGCCGAGG GCGCGGTTGT

51	AGAGCTGCAG GCCGTGAGAG CCAAAGCCGT CAATGCAACC GCCGCTTGCA

101	TTTTTACGGT CTTGAGTAAG GACATTTTCG ATTTCCTTTT TATTTTCCGT

151	TTTCAGACGG CTGACTTCCG CCTGTTTTTT CGCCAAAGCC ATGCCGACAG

201	CGTGCGCCTT GACTTCATAT TTTTTAGCTT CCGcGCGTGC CAGTTCCAGT

251	TCGCGCGCAT AGTTTTGAGC CGACAACAGC AGGGCTTGCG CCTTGTCGCG

301	CTCCATCTTG TCGATGACCG CCTGCTGCTT CGCAAATGCC GACTTGTAGC

351	CTTGATGGTG CGACACAGCC AAGCCCGTGC CGACAAGCGC GATAATGGCA

401	ATCGGTTGCC AGTTATTCGC CAGCAGTTTC ACGAGATTCA TTCTCGACCT

451	CCTGACGCTT CACGCTGA

This corresponds to the amino acid sequence SEQ ID 88; ORF124-1>:


1	MTAFSTTLIS VAEGAVVELQ AVRAKAVNAT AACIFTVLSK DIFDFLFIFR

51	FQTADFRLFF RQSHADSVRL DFIFFSFRAC QFQFARIVLS RQQQGLRLVA

101	LHLVDDRLLL RKCRLVAIMV RHSQARADKR DNGNRLPVIR QQFHEIHSRP

151	PDASR*

A corresponding ORF from strain A of N. meningitidis was also identified:
Homology with a Predicted ORF from N. meningitidis (Strain A)

ORF124 shows 87.5% identity over a 152 aa overlap with an ORF (ORF124a) from strain A of N. meningitidis:


	10 20 30 40 50 60
orf124.pep	TPNSVTVLPSFGGFGRTGATINAAGGVGMTAFSTTLISVAEGAVVELQAVRAKAVNATAA
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\| \|\|\|\|\|:\|\|\|
orf124a	MTAFSTTLISVAEGALVELQAVMAKAVNTTAA
	10 20 30

	70 80 90 100 110 120
orf124.pep	CIFTVLSKDIFDFLFIFRFQTADFRLYFRQSHADSVRLDFIFKSFRACQFQFARIVLSRQ
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\|\|:\|\|\|\|\|\|\| \|\|\|: \|\|\|\| :\|\|\|\|\|
orf124a	CIFTVLSKDIFDFLFIFRFQTADFRLFFRQSHADGVRLDFIFFSFRTRLFQFAGVVLSRQ
	40 50 60 70 80 90

	130 140 150 160 170 180
orf124.pep	QQGLRLVALHLVDDRLQLRKCRLVALMVRHSQARADKRDNGNRLPVIRQQFHEIHSRPPD
	\|\|\|\|\|\|\|\|\|\|:::\|\|\| \|\|\| \|\|\|\|\|\|\|\|\| \|:\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf124a	QQGLRLVALHFLNDRLLLRKSRLVALMVRHRQTRADKRDDGNRLPVIRQQFHEIHSRPPD
	100 110 120 130 140 150

orf124.pep	ASRX
	:
orf124a	VX

ORF124a and ORF124-1 show 89.5% identity in 152 aa overlap:


orf124-1.pep	MTAFSTTLISVAEGAVVELQAVRAKAVNATAACIFTVLSKDIFDFLFIFRFQTADFRLFF
	\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:\|\|\|\|\|\| \|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|
orf124a	MTAFSTTLISVAEGALVELQAVMAKAVNTTAACIFTVLSKDIFDFLFIFRFQTADFRLFF

orf124-1.pep	RQSHADSVRLDFIFFSFRACQFQFARIVLSRQQQGLRLVALHLVDDRLLLRKCRLVALMV
	\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|: \|\|\|\| :\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:::\|\|\|\|\|\|\| \|\|\|\|\|\|\|
orf124a	RQSHADGVRLDFIFFSFRTRLFQFAGVVLSRQQQGLRLVALHFLNDRLLLRKSRLVALMV

orf124-1.pep	RHSQARADKRDNGNRLPVIRQQFHEIHSRPPDASRX
	\|\| \|:\|\|\|\|\|\|:\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|\|:
orf124a	RHRQTRADKRDDGNRLPVIRQQFHEIHSRPPDVX

The complete length ORF124a nucleotide sequence <SEQ ID 89> is:


1	ATGACCGCCT TTTCGACAAC CTTAATTTCC GTAGCCGAGG GCGCGCTTGT

51	AGAGCTGCAA GCCGTGATGG CCAAAGCCGT CAATACAACC GCCGCCTGCA

101	TTTTTACGGT CTTGAGTAAG GACATTTTCG ATTTCCTTTT TATTTTCCGT

151	TTTCAGACGG CTGACTTCCG CCTGTTTTTT CGCCAAAGCC ATGCCGACGG

201	CGTGCGCCTT GACTTCATAT TTTTTAGCTT CCGCACGCGC CTGTTCCAGT

251	TCGCGGGCGT AGTTTTGAGC CGACAACAGC AGGGCTTGCG CCTTGTCGCG

301	CTTCATTTTC TCAATGACCG CCTGCTGCTT CGCAAAAGCC GACTTGTAGC

351	CTTGATGGTG CGACACCGCC AAACCCGTGC CGACAAGCGC GATGATGGCA

401	ATCGGTTGCC AGTTATTCGC CAGCAGTTTC ACGAGATTCA TTCTCGACCT

451	CCTGACGTTT GA

This encodes a protein having amino acid sequence <SEQ ID 90>:


1	MTAFSTTLIS VAEGALVELQ AVNAXAVNTT AACIFTVLSK DIFDFLFIFR

51	FQTADFRLFF RQSHADGVRL DFIFFSFRTR LFQFAGVVLS RQQQGLRLVA

101	LHFLNDELLL RKSRLVALHV RHRQTRADKR DDGNRLPVIR QQFHEIHSRP

151	PDV*

ORF124-1 was amplified as described above. FIG. 7 shows plots of hydrophilicity, antigenic index, and AMPHI regions for ORF124-1.
Based on this analysis, it is predicted that this protein from N. meningitidis, and its epitopes, could be useful antigens for vaccines or diagnostics.

Example 20

Table III lists several Neisseria strains which were used to assess the conservation of the sequence of ORF 40 among different strains.

TABLE III


List of Neisseria Strains Used for Gene Variability Study of ORF 40

Identification
number	Strains	Source/reference

Group B

zn02_1	BZ198	R. Moxon/Seiler et al., 1996
zn03_1	NG3/88	R. Moxon/Seiler et al., 1996
zn04_1	297-0	R. Moxon/Seiler et al., 1996
zn06_1	BZ147	R. Moxon/Seiler et al., 1996
zn07_1	BZ169	R. Moxon/Seiler et al., 1996
zn08_1	528	R. Moxon/Seiler et al., 1996
zn10_1	BZ133	R. Moxon/Seiler et al., 1996
zn11_1ass	NGE31	R. Moxon/Seiler et al., 1996
zn14_1	NGH38	R. Moxon/Seiler et al., 1996
zn16_1	NGH15	R. Moxon/Seiler et al., 1996
zn18_1	BZ232	R. Moxon/Seiler et al., 1996
zn19_1	BZ83	R. Moxon/Seiler et al., 1996
zn20_1	44/76	R. Moxon/Seiler et al., 1996
zn21_1	MC58	R. Moxon

Group A

zn22_1	205900	R. Moxon
zn23_1	F6124	R. Moxon
z2491_1	Z2491	R. Moxon/Maiden et al., 1998

Group C

zn24_1

	90/18311	R. Moxon
zn25_1ass	93/4286	R. Moxon

Others

zn28_1ass	860800 (group Y)	R. Moxon/Maiden et al., 1998
zn29_1ass	E32 (group Z)	R. Moxon/Maiden et al., 1998

References:
Seiler A. et al., Mol. Microbiol., 1996, 19(4): 841-856.
Maiden et al., Proc. Natl. Acad. Sci. USA, 1998, 95: 3140-3145.

The amino acid sequences for each listed strain are as follows:


>Z2491 <SEQ ID 91>
MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVKTAVLATLLFATVQANATDEDEEEEL
ESVQRSVVGSIQASMEGSGELETISLSMTNDSKEFVDPYIVVTLKAGDNLKIKQNTNENT
NASSFTYSLKKDLTGLINVETEKLSFGANGKKVNIISDTKGLNFAKETAGTNGDTTVHLN
GIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSENVDF
VRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSS
TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV
SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKISGNVSPSKADEMDETV
NINAGNNIEISRNGKNIDIATSNAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDANKPV
RINVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVWDGNARAGIAQAIATAGLVQAYLPGKS
MMAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW*

>ZN02_1 <SEQ ID 92>
MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVATAVLATLLFATVQANATDDDDLYLE
PVQRTAVLSFRSDKEGTGEKEGTEDSHGGAVYFDEKRVLKAGAITLKAGDNLKIKQNTNE
NTNDSSFTYSLKKDLTDLTSVETEKLSFGAAGNKVNITSDTKGLNFAKETAGTAGDPTVH
LNGIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTASDNV
DFVRTYDTVEFLSADTKTTTNVSKDNGKKTEVICIGAIVTSVIKEKDGKLVTGKGKDENG
SSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTA
TVSKODQGNITVRYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGRMDE
TVNINAGNNIEITRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDTNK
PVRITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPG
KSMMAIGGDTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW*

>ZN03_1 <SEQ ID 93>
MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVATAVLATLLFATVQASTTDDDDLYLE
PVQRTAPVLSFHADSEGTGEKEVTEDSNWGVYFDKKGVLTAGTITLKAGDNLKIKQNTDE
NTNDSSFTYSLKKDLTDLTSVETEKLSFGANGNKVNITSDTKGLNFAKETAGTNGDPTVH
LNGIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTASDNV
DFVRTYDTVEFLSADTRTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTGKGKDENG
SSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTA
TVSKDDQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDE
TVNINAGNNIEITRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDTNK
PVRITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPG
KSMMAIGGDTYRGEAGYAIGYSSISDGGNMIIKGTASGNSRGHFGASASVGYQW*

>ZN04_1 <SEQ ID 94>
MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVATAVLATLLFATVQASTTDDDDLYLE
PVQRTAPVLSFHADSEGTGEKEVTEDSNWGVYFDKKGVLTAGTITLKAGDNLKIKQNTDE
NTNDSSFTYSLKKDLTDLTSVETEKLSFGANGNKVNITSDTKGLNFAKETAGTNGDPTVH
LNGIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTASDNV
DFVRTYDTVEFLSADTRTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTGKGKDENG
SSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTA
TVSKDDQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDE
TVNINAGNNIEITRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDTNK
PVRITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPG
KSMMAIGGDTYRGEAGYAIGYSSISDGGNMIIKGTASGNSRGHFGASASVGYQW*

>ZN06_1 <SEQ ID 95>
MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVKTAVLATLLFATVQANATDEDEEEEL
ESVQRSVVGSIQASMEGSGELETISLSMTNDSKEFVDPYIVVTLKAGDNLKIKQNTNENT
NASSFTYSLKKDLTGLINVETEKLSFGANGKKVNIISDTKGLNFAKETAGTNGDTTVHLN
GIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSENVDF
VRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSS
TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV
SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKISGNVSPSKADEMDETV
NINAGNNIEISRNGKNIDIATSNAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDANKPV
RINVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVWDGNARAGIAQAIATAGLVQAYLPGKS
MMAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW*

>ZN07_1 <SEQ ID 96>
MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVKTAVLATLLFATVQANATDEDEEEEL
ESVQRSVVGSIQASMEGSGELETISLSMTNDSKEFVDPYIVVTLKAGDNLKIKQNTNENT
NASSFTYSLKKDLTGLINVETEKLSFGANGKKVNIISDTKGLNFAKETAGTNGDTTVHLN
GIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSENVDF
VRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSS
TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV
SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKISGNVSPSKADEMDETV
NINAGNNIEISRNGKNIDIATSNAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDANKPV
RINVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVWDGNARAGIAQAIATAGLVQAYLPGKS
MMAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW*

>ZN08_1 <SEQ ID 97>
MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVETAVLATLLFATVQANATDTDEDDEL
EPVVRSALVLQFMIDKEGNGEIESTGDIGWSIYYDDHNTLHGATVTLKAGDNLKIKQNTD
ENTNASSFTYSLKKDLTDLTSVGTEELSFGANGNKVNITSDTKGLNFAKKTAGTNGDTTV
HLNGIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSEN
VDFVRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGEN
GSSTEDGEGELVTAKEVIDAVNKAGWRMKTTANGQTGQADKFETVTSGTNVTFASGKGTT
ATVSKDDQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKISGNAVSPSKGKMD
ETVNINAGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPLTLSVDDEALNVGSKDAN
KPVRITNVAPGVKEGDVTNVAQLKGVAQNLNNHIDNVDGNARAGIAQAIATAGLVQAYLP
GKSMMAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW*

>ZN10_1 <SEQ ID 98>
MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVKTAVLATLLFATVQANATDEDEEEEL
ESVQRSVVGSIQASMEGSGELETISLSMTNDSKEFVDPYIVVTLKAGDNLKIKQNTNENT
NASSFTYSLKKDLTGLINVETEKLSFGANGKKVNIISDTKGLNFAKETAGTNGDTTVHLN
GIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSENVDF
VRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSS
TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV
SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDETV
NINAGNNIEISRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDANKPV
RITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPGKS
MMAIGGGYTRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW*

>ZN11_1 ASS <SEQ ID 99>
MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVATAVLATLLFATVQASTTDDDDLYLE
PVQRTAPVLSFHADSEGTGEKEVTEDSNWGVYFDKKGVLTAGTITLKAGDNLKIKQNTDE
NTNDSSFTYSLKKDLTDLTSVETEKLSFGANGNKVNITSDTKGLNFAKETAGTNGDPTVH
LNGIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTASDNV
DFVRTYDTVEFLSADTRTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTGKGKDENG
SSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTA
TVSKDDQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDE
TVNINAGNNIEITRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDTNK
PVRITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPG
KSMMAIGGDTYRGEAGYAIGYSSISDGGNMIIKGTASGNSRGHFGASASVGYQW*

>ZN14_1 <SEQ ID 100>
MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVETAVLATLLFATVQANATDTDEDDEL
EPVVRSALVLQFMIDKEGNGEIESTGDIGWSIYYDDHNTLHGATVTLKAGDNLKIKQNTD
ENTNASSFTYSLKKDLTDLTSVGTEELSFGANGNKVNITSDTKGLNFAKKTAGTNGDTTV
HLNGIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSEN
VDFVRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGEN
GSSTEDGEGELVTAKEVIDAVNKAGWRMKTTANGQTGQADKFETVTSGTNVTFASGKGTT
ATVSKDDQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKISGNAVSPSKGKMD
ETVNINAGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPLTLSVDDEALNVGSKDAN
KPVRITNVAPGVKEGDVTNVAQLKGVAQNLNNHIDNVDGNARAGIAQAIATAGLVQAYLP
GKSMMAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW*

>ZN16_1 <SEQ ID 101>
MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVATAVLATLLFATVQANATDDDDLYLE
PVQRTAVVLSFRSDKEGTGEKEGTEDSNWAVYFDEKRVLKAGAITLKAGDNLKIKQNTNE
NTNENTNDSSFTYSLKKDLTDLTSVETEKLSFGANGNKVNITSDTKGLNFAKETAGTNGD
PTVHLNGIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTA
SDNVDFVRTYDTVEFLSADTKTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTGKGK
DENGSSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTKVTFASGN
GTTATVSKDDQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKG
KMDETVNINAGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPTLSVDDEGALNVGSK
DANKPVRITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLAQA
YLPGKSMMAIGGGTYRGEAGYAIGYSSISDTGNWVIKGTASGNSRGHFGASASVGYQW*

>ZN18_1 <SEQ ID 102>
MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVATAVLATLLFATVQASTTDDDDLYLE
PVQRTAPVLSFHADSEGTGEKEVTEDSNWGVYFDKKGVLTAGTITLKAGDNLKIKQNTDE
NTNDSSFTYSLKKDLTDLTSVETEKLSFGANGNKVNITSDTKGLNFAKETAGTNGDPTVH
LNGIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTASDNV
DFVRTYDTVEFLSADTRTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTGKGKDENG
SSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTA
TVSKDDQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDE
TVNINAGNNIEITRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDTNK
PVRITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPG
KSMMAIGGDTYRGEAGYAIGYSSISDGGNMIIKGTASGNSRGHFGASASVGYQW*

>ZN19_1 <SEQ ID 103>
MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVKTAVLATLLFATVQASANNEEQEEDL
YLDPVQRTVAVLIVNSDKEGTGEKEKVEENSDWAVYFNEKGVLTAREITLKAGDNLKIKQ
NGTNFTYSLKKDLTDLTSVGTEKLSFSANGNKVNITSDTKGLNFAKETAGTNGDTTVHLN
GIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTASDNVDF
VRTYDTVEFLSADTKTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTGKDKGENGSS
TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV
SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDETV
NINAGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPTLSVDGDALNVGSKKDNKPVR
ITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPGKSM
MAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW*

>ZN20_1 <SEQ ID 104>
MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVKTAVLATLLFATVQASANNEEQEEDL
YLDPVQRTVAVLIVNSDKEGTGEKEKVEENSDWAVYFNEKGVLTAREITLKAGDNLKIKQ
NGTNFTYSLKKDLTDLTSVGTEKLSFSANGNKVNITSDTKGLNFAKETAGTNGDTTVHLN
GIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTASDNVDF
VRTYDTVEFLSADTKTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTGKDKGENGSS
TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV
SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDETV
NINAGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPTLSVDGDALNVGSKKDNKPVR
ITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPGKSM
MAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW*

>ZN21_1 <SEQ ID 105>
MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVKTAVLATLLFATVQASANNEEQEEDL
YLDPVQRTVAVLIVNSDKEGTGEKEKVEENSDWAVYFNEKGVLTAREITLKAGDNLKIKQ
NGTNFTYSLKKDLTDLTSVGTEKLSFSANGNKVNITSDTKGLNFAKETAGTNGDTTVHLN
GIGSTLTDTLLNTGATTNVTNDNVTDDEKKRAASVKDVLNAGWNIKGVKPGTTASDNVDF
VRTYDTVEFLSADTKTTTVNVESKDNGKKTEVKIGAKTSVIKEKDGKLVTGKDKGENGSS
TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV
SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDETV
NINAGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPTLSVDGDALNVGSKKDNKPVR
ITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPGKSM
MAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW*

>ZN22_1 <SEQ ID 106>
MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVKTAVLATLLFATVQANATDEDEEEEL
ESVQRSVVGSIQASMEGSGELETISLSMTNDSKEFVDPYIVVTLKAGDNLKIKQNTNENT
NASSFTYSLKKDLTGLINVETEKLSFGANGKKVNIISDTKGLNFAKETAGTNGDTTVHLN
GIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSENVDF
VRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSS
TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV
SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDETV
NINAGNNIEISRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDANKPV
RITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPGKS
MMAIGGGYTRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW*

>ZN23_1 <SEQ ID 107>
MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVKTAVLATLLFATVQANATDEDEEEEL
ESVQRSVVGSIQASMEGSGELETISLSMTNDSKEFVDPYIVVTLKAGDNLKIKQNTNENT
NASSFTYSLKKDLTGLINVETEKLSFGANGKKVNIISDTKGLNFAKETAGTNGDTTVHLN
GIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSENVDF
VRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSS
TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV
SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDETV
NINAGNNIEISRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDANKPV
RITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPGKS
MMAIGGGYTRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW*

>ZN24_1 <SEQ ID 108>
MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVATAVLATLLSATVQANATDTDEDEEL
ESVVRSALVLQFMIDKEGNGEIESTGDIGWSIYYDDHNTLHGATVTLKAGDNLKIKQSGK
DFTYSLKKELKDLTSVETEKLSFGANGNKVNITSDTKGLNFAKETAGTNGDPTVHLNGIG
STLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSENVDFVRT
YDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSSTDE
GEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTKVTFASGNGTTATVSKD
DQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDETVNIN
AGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPTLSVDDEGALNVGSKDANKPVRIT
NVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLAQAYLPGKSMMA
IGGGTYRGEAGYAIGYSSISDTGNWVIKGTASGNSRGHFGTSASVGYQW*

>ZN25_ASS <SEQ ID 109>
MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVATAVLATLLSATVQANATDTDEDEEL
ESVVRSALVLQFMIDKEGNGEIESTGDIGWSIYYDDHNTLHGATVTLKAGDNLKIKQSGK
DFTYSLKKELKDLTSVETEKLSFGANGNKVNITSDTKGLNFAKETAGTNGDPTVHLNGIG
STLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSENVDFVRT
YDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSSTDE
GEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTKVTFASGNGTTATVSKD
DQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDETVNIN
AGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPTLSVDDEGALNVGSKDANKPVRIT
NVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLAQAYLPGKSMMA
IGGGTYRGEAGYAIGYSSISDTGNWVIKGTASGNSRGHFGTSASVGYQW*

>ZN28_ASS <SEQ ID 110>
MNKIYRIIWNSALNAWVAVSELTRNHTKRASATVKTAVLATLLFATVQANATDEDEEEEL
ESVQRSVVGSIQASMEGSGELETISLSMTNDSKEFVDPYIVVTLKAGDNLKIKQNTNENT
NASSFTYSLKKDLTGLINVETEKLSFGANGKKVNIISDTKGLNFAKETAGTNGDTTVHLN
GIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTTGQSENVDF
VRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGKGKGENGSS
TDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTNVTFASGKGTTATV
SKDDQGNITVMYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKMDETV
NINAGNNIEISRNGKNIDIATSMAPQFSSVSLGAGADAPTLSVDDEGALNVGSKDANKPV
RITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPGKS
MMAIGGGYTRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW*

>ZN29_ASS <SEQ ID 111>
MNKIYRIIWNSALNAWVVVSELTRNHTKRASATVETAVLATLLFATVQANATDTDEDDEL
EPVVRTAPVLSFHSDKEGTGEKEEVGASSNLTVYFDKNRVLKAGTITLKAGDNLKIKQNT
NENTNENTNASSFTYSLKKDLTGLINVETEKLSFGANGKKVNIISDTKGLNFAKETAGTN
GDPTVHLNGIGSTLTDTLAGSSASHVDAGNQSTHYTRAASIKDVLNAGWNIKGVKTGSTT
GQSENVDFVRTYDTVEFLSADTKTTTVNVESKDNGKRTEVKIGAKTSVIKEKDGKLVTGK
GKGENGSSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVTSGTKVTFAS
GNGTTATVSKDDQGNITVKYDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPS
KGKMDETVNINAGNNIEITRNGKNIDIATSMTPQFSSVSLGAGADAPTLSVVEAGALNVG
SKDANKPVRITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLV
QAYLPGKSMMAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASGNSRGHFGASASVGYQW
*

FIG. 8 shows the results of aligning the sequences of each of these strains. Dark shading indicates regions of homology, and gray shading indicates the conservation of amino acids with similar characteristics. As is readily discernible, there is significant conservation among the various strains of ORF 40, further confirming its utility as an antigen for both vaccines and diagnostics.
It will be appreciated that the invention has been described by means of example only, and that modifications may be made whilst remaining within the spirit and scope of the invention.

Appendix 1



Scarlato, Continuation of U.S. App. Ser.
No. 10/695,499, filed herewith	Ruelle, U.S. Pat. No. 6,780,419

18. (New) An isolated polypeptide comprising	1. An isolated polypeptide comprising a
a member selected from the group consisting of	member selected from the group consisting of
(a) the amino acid sequence of SEQ ID	(a) the amino acid sequence of SEQ ID
NO: 4; and	NO: 2;
(b) an immunogenic fragment of at	(b) an immunogenic fragment of at least
least 15 contiguous amino acids of SEQ ID	15 contiguous amino acids of SEQ ID NO: 2;
NO: 4, wherein the immunogenic fragment,	wherein the immunogenic fragment, when
when administered to a subject in a suitable	administered to a subject in a suitable
composition which can include an adjuvant, or	composition which can include an adjuvant, or
a suitable carrier coupled to the polypeptide,	a suitable carrier coupled to the polypeptide,
induces an antibody or T-cell meditated	induces an antibody or T-cell meditated
immune response that recognizes the isolated	immune response that recognizes the isolated
polypeptide SEQ ID NO: 4.	polypeptide SEQ ID NO: 2.
19. (New) The isolated polypeptide of claim	2. The isolated polypeptide of claim 1,
18, wherein the polypeptide is according to (a).	wherein the polypeptide is according to (a).
20. (New) The isolated polypeptide of claim	3. The isolated polypeptide of claim 1,
18, wherein the polypeptide is according to (b).	wherein the polypeptide is according to (b).
21. (New) The isolated polypeptide of claim	4. The isolated polypeptide of claim 1,
18, wherein the immunogenic fragment of (b)	wherein the immunogenic fragment of (b)
comprises at least 20 contiguous amino acids of	comprises at least 20 contiguous amino acids of
SEQ ID NO: 4; wherein the immunogenic	SEQ ID NO: 2; wherein the immunogenic
fragment, when administered to a subject in a	fragment, when administered to a subject in a
suitable composition which can include an	suitable composition which can include an
adjuvant, or a suitable carrier coupled to the	adjuvant, or a suitable carrier coupled to the
polypeptide, induces an antibody or T-cell	polypeptide, induces an antibody or T-cell
meditated immune response that recognizes the	meditated immune response that recognizes the
isolated polypeptide SEQ ID NO: 4.	isolated polypeptide SEQ ID NO: 2.
22. (New) The isolated polypeptide of claim	5. The isolated polypeptide of claim 1,
18, wherein the isolated polypeptide consists of	wherein the isolated polypeptide consists of
SEQ ID NO: 4.	SEQ ID NO: 2.
23. (New) A fusion protein comprising the	6. A fusion protein comprising the isolated
isolated polypeptide of claim 18.	polypeptide of claim 1.
24. (New) An immunogenic composition	7. An immunogenic composition comprising
comprising the polypeptide of claim 18, and a	the polypeptide of claim 1, and a
pharmaceutically acceptable carrier.	pharmaceutically acceptable carrier.
25. (New) The isolated polypeptide of claim	9. The isolated polypeptide of claim 1,
18, wherein the isolated polypeptide is a	wherein the isolated polypeptide is a
recombinant polypeptide.	recombinant polypeptide.
26. (New) The isolated polypeptide of claim	10. The isolated polypeptide of claim 2,
19, wherein the isolated polypeptide is a	wherein the isolated polypeptide is a
recombinant polypeptide.	recombinant polypeptide.
27. (New) The isolated polypeptide of claim	11. The isolated polypeptide of claim 3,
20, wherein the isolated polypeptide is a	wherein the isolated polypeptide is a
recombinant polypeptide.	recombinant polypeptide.
28. (New) An immunogenic composition	12. An immunogenic composition comprising
comprising the isolated polypeptide of claim 19.	the isolated polypeptide of claim 2.
29. (New) An immunogenic composition	13. An immunogenic composition comprising
comprising the isolated polypeptide of claim 20.	the isolated polypeptide of claim 3.
30. (New) A fusion protein comprising the	14. A fusion protein comprising the isolated
isolated polypeptide of claim 19.	polypeptide of claim 2.
31. (New) A fusion protein comprising the	15. A fusion protein comprising the isolated
isolated polypeptide of claim 20.	polypeptide of claim 3.

Appendix 2



	Written Description Support
	in the Current Application	Written Description
	(Continuation of Application	Support in Application
Added Claim #	No. 10/695,499)	No. PCT/IB99/00103

Claims 18-31	Throughout the	Throughout the
	application and at least at	application and at least at
	the following citations:	the following citations:
	Page 3, lines 2-24;	Page 2, line 29 to page 3,
	Page 31, line 7 to page 34,	line 20;
	line 17;	Page 30, line 6 to page
	Page 52, lines 10-18;	33, line 11;
	Page 65, line 3 to page 70,	Page 50, lines 12-20;
	line 3.	Page 61, line 11 to page
		66, line 6.
Claims 23, 30,	Throughout the	Throughout the
and 31	application and at least at	application and at least at
	the following citations:	the following citations:
	Page 3, lines 24-27;	Page 3, lines 21-24;
	Page 9, line 26 to page 10,	Page 9, lines 11-18;
	line 4;	Page 20, line 6 to page
	Page 21, lines 1-22.	21, line 4.
Claims 25-27	Throughout the	Throughout the
	application and at least at	application and at least at
	the following citations:	the following citations:
	Page 3, lines 24-27;	Page 3, lines 17-20;
	Page 8, line 15 to page 28,	Page 8, line 1 to page 27,
	line 23.	line 25.

Appendix 3

Disclosure of Constructive Reductions to Practice within the Scope of the Interfering Subject Matter in Application No. GB 9800760.2, filed Jan. 14, 1998



Number of
Amino Acids	Location in ORF40 of	Location in SEQ 2
in Fragment	Application No. GB 9800760.2	of ‘419 Patent

25	Residues 85-109	Residues 127-151
16	Residues 111-126	Residues 153-168
98	Residues 131-228	Residues 173-270
16	Residues 230-245	Residues 272-287

Claims

1-17. (canceled)

18. An isolated polypeptide comprising a member selected from the group consisting of

(a) the amino acid sequence of SEQ ID NO: 4; and

(b) an immunogenic fragment of at least 15 contiguous amino acids of SEQ ID NO: 4, wherein the immunogenic fragment, when administered to a subject in a suitable composition which can include an adjuvant, or a suitable carrier coupled to the polypeptide, induces an antibody or T-cell meditated immune response that recognizes the isolated polypeptide SEQ ID NO: 4.

19. The isolated polypeptide of claim 18, wherein the polypeptide is according to (a).

20. The isolated polypeptide of claim 18, wherein the polypeptide is according to (b).

21. The isolated polypeptide of claim 18, wherein the immunogenic fragment of (b) comprises at least 20 contiguous amino acids of SEQ ID NO:4; wherein the immunogenic fragment, when administered to a subject in a suitable composition which can include an adjuvant, or a suitable carrier coupled to the polypeptide, induces an antibody or T-cell meditated immune response that recognizes the polypeptide SEQ ID NO: 4.

22. The isolated polypeptide of claim 18, wherein the isolated polypeptide consists of SEQ ID NO: 4.

23. A fusion protein comprising the isolated polypeptide of claim 18.

24. An immunogenic composition comprising the polypeptide of claim 18, and a pharmaceutically acceptable carrier.

25. The isolated polypeptide of claim 18, wherein the isolated polypeptide is a recombinant polypeptide.

26. The isolated polypeptide of claim 19, wherein the isolated polypeptide is a recombinant polypeptide.

27. The isolated polypeptide of claim 20, wherein the isolated polypeptide is a recombinant polypeptide.

28. An immunogenic composition comprising the isolated polypeptide of claim 19.

29. An immunogenic composition comprising the isolated polypeptide of claim 20.

30. A fusion protein comprising the isolated polypeptide of claim 19.

31. A fusion protein comprising the isolated polypeptide of claim 20.