CN1350585A

CN1350585A - i(Neisseria meningitidis) polypeptide BASBO52

Info

Publication number: CN1350585A
Application number: CN00804978A
Authority: CN
Inventors: J·－L·吕勒
Original assignee: SmithKline Beecham Biologicals SA
Current assignee: GlaxoSmithKline Biologicals SA
Priority date: 1999-01-15
Filing date: 2000-01-10
Publication date: 2002-05-22
Also published as: WO2000042192A1; CA2359486A1; AU2289000A; JP2004537954A; EP1144645A1

Abstract

The invention provides BASB052 polypeptides and polynucleotides encoding BASB052 polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are diagnostic, prophylactic and therapeutic uses.

Description

I (Neisseria meningitidis) polypeptide BASBO 52

Invention field

The present invention relates to polynucleotide (referring to " BASB052 polynucleotide " here), their encoded polypeptides (referring to " BASB052 " or " BASB052 polypeptide " here), reorganization material and their preparation method.In yet another aspect, the present invention relates to use this peptide species and polynucleotide to comprise the method for the vaccine of resisting infectation of bacteria.Again on the one hand, the present invention relates to detect the test of special pathogen diagnosis of infection.

Background of invention

Neisseria meningitidis (meningococcus) is a kind of gram negative bacterium that can be often be separated to from people's the upper respiratory tract.It can cause invasive bacterial disease such as mass formed by blood stasis and meningitis once in a while.The meningitis disease demonstrate geography, season and annual difference (Schwartz, B.Moore, P.S., Broome, C.V.; " Clinical microorganism summary " 2 (supplementary issues), S18-S24,1989).In temperate zone country, numerous diseases causes by the serotypes B bacterial strain, and annual morbidity changes between 100,000 of total number of persons/to 10/100000ths, reach higher numerical value (Kaczmarski sometimes, E.B. (1997), Commun.Dis.Rep.Rev.7:R55-9,1995; Scholten, R.J.R.M., Bijlmer, H.A., Poolman, J.T. etc. " clinical infectious disease " 16:237-246,1993; Cruz, C., Pavez, G., Aguilar, E. waits " infection epidemiology " 105:119-126,1990).

Prevailing disease by the serotype A meningococcus causes mainly in the Central Africa, can reach annual 1000/100000ths (Schwartz, B., Moore, P.S., Broome, C.V. " Clinical microorganism summary " 2 (supplementary issues), S18-S24,1989) sometimes.With regard to the meningitis disease on the whole, nearly all case is all caused by serotype A, B, C, W-135 and Y meningococcus, and can use polysaccharide vaccine (Armand, the J. of a kind of 4 valency A, C, W-135, Y, Arminjon, F., Mynard, M.C., Lafaix, C., " J.Biol.Stand " 10:335-339,1982).Polysaccharide vaccine is at present just by improving (Lieberman, J.M., Chiu, S.S., Wong, V.K. etc., JAMA 275:1499-1503,1996) with the mode of they and carrier proteins chemical coupling.

Do not obtain a kind of vaccine of serotypes B as yet,, be likely because it and host's composition have structural similarity (Wyle, F.A. because the capsular polysaccharide of B is found to be non-immunogenic, Artenstein, M.S., Brandt, M.L. etc., " transmissible disease magazine " 126:514-522,1972; Finne, J.M., Leinonen, M., M  kel , P.M. " lancet " ii:355-357,1983).

Be the vaccine of exploitation based on the meningococcus adventitia, people have made great efforts for many years (deMoraes, J.C., Perkins, B., Camargo, M.C. etc. " lancet " 340:1074-1078,1992; Bjune, G., Hoiby, E.A.Gronnesby, J.K. etc., 338:1093-1096,1991).This vaccine has proved that the validity in big children (＞4 years old) and grownup is 57%-85%.

There are many bacterial outer membrane components in these vaccines, for example PorA, PorB, Rmp, Opc, Opa, FrpB, the contribution that these compositions are done observed provide protection still needs further to determine.Use animal or human's antibody to determine other bacterial outer membrane components that may be relevant, for example TbpB and NspA (Martin, D., Cadieux with inducing of protective immunity, N., Hamel, J., Brodeux, B.R., " The Journal of Experimental Medicine " 185:1173-1183,1997; Lissolo, L., Maitre-Wilmotte, C., Dumas, P etc., Inf.Immun.63:884-890,1995).The mechanism of protective immunity will be referred to antibody-mediated fungicidal activity and engulfs opsonization.

A kind of bacterium blood animal model has been used to make up all antibody-mediated mechanism (Saukkonen, K., Leinonen, M., Abdillahi, H.Poolman, J.T. " vaccine " 7:325-328,1989).It is generally acknowledged that the bactericidal mechanism of complement component mediation in late period is concerning the immunity of opposing meningitis disease most important (Ross, S.C., Rosenthal P.J., Berberic, H.M., Densen, P.J. " transmissible disease magazine " 155:1266-1275,1987).

In the past few decades, the sickness rate of Neisseria meningitidis is greatly improved.This has been laid the blame on for the appearance of multiple strains and has been had the increase of more weak immune people colony.Separation no longer is unusual to the bacterial strain that some or all standard antibiotic have resistance.This phenomenon just makes us produce demand at the The Insatiable of novel antimicrobial reagent, vaccine, drug screening method and the diagnostic test of this microorganism.

The invention summary

The present invention relates to BASB052 particularly BASB052 polypeptide and BASB052 polynucleotide, reorganization material and their preparation method.In yet another aspect, the present invention relates to use the method for this peptide species and polynucleotide, comprise the prevention of microbial diseases and treatment and other.One further aspect, the present invention relates to detect the diagnostic test with infected by microbes diseases associated and the symptom relevant with such infection, for example detect BASB052 polynucleotide or polypeptide expression or active test.

By reading following description and other parts of the present disclosure, those skilled in the art will be readily appreciated that various changes and the modification within spirit and scope disclosed by the invention.

Detailed Description Of The Invention

Detailed introduction as hereinafter the present invention relates to BASB052 polypeptide and polynucleotide.Specifically, the present invention relates to polypeptide and the polynucleotide of the BASB052 of Neisseria meningitidis, BASB052 and gonococcus tcp albumen have homology on aminoacid sequence.The invention particularly relates to have and list in SEQ ID NO:1 and the Nucleotide of SEQ ID NO:2 and the BASB052 of aminoacid sequence respectively.Should be appreciated that the sequence that in the sequence table of back, is enumerated as " DNA " represent one embodiment of the invention for example because those skilled in the art just know that such sequence is often used as polynucleotide, comprises polyribonucleotide.Polypeptide

In one aspect of the invention, provide the polypeptide of the Neisseria meningitidis that is called as " BASB052 " and " BASB052 polypeptide " here, and in biology, diagnosis, prevention, clinical or treatment useful variant, and the composition that contains them.The present invention further provides: the isolated polypeptide that (a) contains a kind of like this aminoacid sequence, the aminoacid sequence of this aminoacid sequence and SEQ IDNO:2 has 85% identity at least, more preferably at least 90% identity, more preferably at least 95% identity, the most preferably identity of 97-99% or identical at least; (b) by the polypeptide of the separation polynucleotide encoding that contains following a kind of polynucleotide sequence, this polynucleotide sequence has at least 85% identity with the whole length of SEQ ID NO:1 respectively, more preferably at least 90% identity, more preferably at least 95% identity, the more preferably identity of 97-99% or identical at least; Perhaps (c) is by the polypeptide of the separation polynucleotide encoding that contains following a kind of polynucleotide sequence, the such peptide species of this polynucleotide sequence coding, the aminoacid sequence of this peptide species and SEQ ID NO:2 has 85% identity at least, more preferably at least 90% identity, more preferably at least 95% identity, the most preferably identity of 97-99% or identical at least;

The BASB052 polypeptide that provides among the SEQ ID NO:2 is the BASB052 polypeptide from Neisseria meningitidis strains A TCC13090.

The present invention also provides a kind of immunogenic fragments of BASB052 polypeptide, i.e. the BASB052 polypeptide successive part, and it has identical or substantially the same immunogenicity activity with the polypeptide of the aminoacid sequence that contains SEQ ID NO:2.That is to say that this fragment (in case of necessity can with a kind of carrier coupling) can produce the immunne response that can discern the BASB052 polypeptide.This immunogenic fragments can comprise such as lacking the terminal leader sequence of N-and/or striding diaphragm area and/or the BASB052 polypeptide of the terminal anchor region of C-.One preferred aspect, immunogenic fragments according to BASB052 of the present invention comprises all ectodomains of a peptide species basically, wherein this polypeptide and SEQ ID NO:2 have 85% identity at least on the whole length of SEQ ID NO:2, more preferably at least 90% identity, more preferably at least 95% identity, the most preferably identity of 97-99% at least.

Fragment is such peptide species, and it has a part rather than whole identical aminoacid sequence with any aminoacid sequence of any polypeptide of the present invention.With regard to the BASB052 polypeptide, fragment can be " the independent existence ", or is in the bigger polypeptide, forms a part or zone, preferably a kind of one successive zone in an independent big polypeptide.

Preferred fragment comprises, such as polypeptide or its variant of the brachymemma of a part of the aminoacid sequence with SEQ ID NO:2, for example comprises N-terminal and or the continuous residue series of C-terminal aminoacid sequence.With or the degraded form of the polypeptide of the present invention that in a kind of host cell, prepares also be preferred.The fragment that more preferably has structure or functional character for example contains α spiral and α spiralization zone, βZhe Die and βZhe Die and forms zone, corner and corner and form zone, a ball of string and a ball of string and form the fragment that zone, hydrophilic region, hydrophobic region, the amphipathic zone of α, the amphipathic zone of B, flexible region, surface form zone, substrate calmodulin binding domain CaM and high antigenicity zone.

Preferred fragment comprises the isolated polypeptide that contains following aminoacid sequence, and this aminoacid sequence has at least 15,20,30,40,50 or 100 continuous amino acids of the aminoacid sequence of SEQ ID NO:2; Or containing the isolated polypeptide of following aminoacid sequence, this aminoacid sequence has at least 15,20,30,40,50 or 100 continuous amino acids from the aminoacid sequence brachymemma of SEQ ID NO:2 or disappearance.

Polypeptide fragment of the present invention can be used for by the corresponding full-length polypeptide of the synthetic preparation of peptide; Therefore, these fragments can be used as the intermediate for preparing full-length polypeptide of the present invention.

Particularly preferably be variant, wherein replace, lack or added several, 5-10,1-5,1-3,1-2 or 1 amino acid with any array mode.

Polypeptide of the present invention or immunogenic fragments can be " maturation " albumen forms, perhaps can be a kind of parts than large protein such as precursor or fusion rotein.Usually preferably comprise and contain secretion or leader sequence, presequence, the sequence that can assist purifying such as polyhistidine residue, or can in the reorganization preparation process, play the additional sequences of stabilization.And, also consider to add exogenous polypeptid or lipid tail or polynucleotide sequence to increase the immunogenicity ability of final molecule.

In one aspect, the present invention relates to the soluble fusion protein of genetic engineering preparation, this fusion rotein contains the various parts of the constant region of the heavy chain of a peptide species of the present invention or its fragment and each subclass immunoglobulin (Ig) (IgG, IgM, IgA, IgE) or light chain.Preferably, immunoglobulin (Ig) is the particularly constant region part of IgG1 of human IgG, occurs in hinge area and merge.In a special embodiment, the Fc part can be removed by introducing a cutting sequence simply, and this cutting sequence can be cut with blood clotting factor Xa.

And, the present invention relates to prepare the method for these fusion roteins, and relate to their application in drug screening, diagnosis and treatment by genetic engineering.A further aspect of the present invention also relates to the polynucleotide of this fusion rotein of encoding.The example of fusion protein technology can be found in international patent application Nos.WO94/29458 and WO94/22914.

Protein can engage by chemical process, or with the formal representation of recombination fusion protein, this fusion rotein form can prepare with higher level at a kind of expression system than non-fusion rotein.Merge part and can help to provide T to assist epi-position (immunity fusion part), preferably can be by the auxiliary epi-position of the T that the people discerns; The auxiliary epi-position (expression enhanser) of T that perhaps can help albumen to express with high yield than original recombinant protein.Merging part preferably is that a kind of immunity merges part, is again to express the enhanser part.

Merging part comprises from the protein D of hemophilus influenzae with from the non-structural protein NS 1 (hemagglutinin) of influenza virus.Another kind of fusion part is the albumen that is called as LytA.The preferred C-terminal part of using this molecule.LytA is from streptococcus pneumoniae, its synthetic a kind of N-acetyl-L-ala amide enzyme---LytA Ntn hydrolase (by lytA genes encoding { " gene " 43 (1986) 265-272 pages or leaves }), the latter is a kind of autolysin, its some key in the peptidoglycan skeleton of degrading specifically.The affinity with choline or some cholinomimetic such as DEAE is responsible in the proteic C-terminal of LytA zone.This specific character has been used to develop the intestinal bacteria C-LytA expression plasmid that can be used for expressing fusion protein.The purifying that contains the segmental hybrid protein of C-LytA at its aminoterminal has had introduction { " biotechnology " 10 volume (1992) 795-798 pages or leaves }.Can use in the LytA molecule repeating part of finding at C-terminal, from residue 178, residue 188-305 for example.

The present invention also comprises above-mentioned variant polypeptides, promptly replaces the polypeptide different with object of reference by conservative amino acid, and wherein a kind of residue is replaced by the residue that another kind has similar features.Typical this being substituted between the following amino acid: Ala, Val, Leu and Ile; Ser and Thr; Acidic residues Asp and Glu; Asn and Gln; Alkaline residue Lys and Arg; Or aromatic moieties Phe and Tyr.

Polypeptide of the present invention can be prepared in any suitable manner.This peptide species comprises the polypeptide of the polypeptide of isolating natural generation, the polypeptide of reorganization preparation, synthetic preparation or with the polypeptide of the combined preparation of these methods.The method for preparing this peptide species is known in this area.

Polypeptide of the present invention is more preferably from Neisseria meningitidis, but it also preferably can be available from the other biological body of same category genus.Polypeptide of the present invention also can be available from such as same category subject or purpose organism.Polynucleotide

An object of the present invention is to provide the polynucleotide of coding BASB052 polypeptide, particularly coding is called as the polynucleotide of the polypeptide of BASB052 here.

In particularly preferred embodiment of the present invention, polynucleotide comprise the zone of a coding BASB052 polypeptide, and this zone comprises a sequence of listing in SEQ ID NO:1, and it comprises gene or its variant of a total length.

The BASB052 polynucleotide that provide among the SEQ ID NO:1 are the BASB052 polynucleotide from Neisseria meningitidis strains A TCC13090.

A further aspect of the present invention provides coding and/or has expressed the BASB052 polypeptide and polynucleotide particularly the BASB052 polypeptide of Neisseria meningitidis and the isolated nucleic acid molecule of polynucleotide, comprises such as unprocessed RNAs, ribozyme rna s, mRNAs, cDNAs, genomic dna s, B-and Z-DNAs.That further embodiment of the present invention is included in is biological, diagnostic, preventative, clinical or polynucleotide and polypeptide that therapeutic is useful and the composition that contains them.

Another aspect of the present invention relates to the separation polynucleotide that comprise a full-length gene at least, a kind of BASB052 with putative amino acid sequence of SEQ ID NO:2 of this genes encoding, and polynucleotide closely-related with it and variant thereof.

In another particularly preferred embodiment of the present invention, a kind of BASB052 polypeptide from Neisseria meningitidis is provided, it comprises or is made up of aminoacid sequence or its variant of SEQ ID NO:2.

Here the information that provides is provided, the polynucleotide sequence listed of SEQ ID NO:1 for example, the polynucleotide of coding BASB052 polypeptide of the present invention can use the clone of standard and screening method to obtain, for example use those be used for method from bacterium clone and order-checking chromosomal dna fragment with Neisseria meningitidis as initial substance, then obtain a full-length clone.For example, for obtaining a kind of polynucleotide sequence of the present invention, the polynucleotide sequence that provides among the SEQ ID NO:1 for example, usually use a kind of radiolabeled oligonucleotide from partial sequence, preferred 17-mer or longer screens the chromosomal DNA clone library of the Neisseria meningitidis in intestinal bacteria or other suitable host.The clone who carries with the probe same DNA then distinguishes with tight hybridization conditions.By using sequencing primer that the single clone who identifies with hybridizing method is like this checked order, just might extend polynucleotide sequence, thereby determine the gene order of total length at both direction according to original polypeptide or polynucleotide sequence design.Sort sequencer is conventional to use the double-stranded DNA such as the sex change that is prepared by plasmid clone to carry out.Suitable technique is seen Maniatis, T., Fritsch, " molecular cloning lab guide, second edition " Cold Spring Harbor Laboratory Press such as E.F. and Sambrook, Cold Spring Harbor, NewYork (1989).(specifically seeing screening by hybridization 1.90 and denatured double stranded dna template order-checking 13.70).Also can use direct genomic dna checks order and obtains the gene order of total length.Be explanation the present invention, each polynucleotide that SEQ ID NO:1 lists are all found from the DNA library from Neisseria meningitidis.

And, each dna sequence dna of listing among the SEQ ID NO:1 all contains a following proteic open reading frame of coding, this albumen contains the amino-acid residue number of listing among the SEQ ID NO:2, and the molecular weight of its derivation can use the values for molecular weight of amino-acid residue well known to those skilled in the art to calculate.

The polynucleotide of SEQ ID NO:1 are at the Nucleotide number 1 of the initiator codon of SEQ ID NO:1 with between the terminator codon of Nucleotide number 1066 beginnings, the polypeptide of coding SEQ IDNO:2.

One further aspect, the invention provides a kind of isolating polynucleotide, this Nucleotide comprises or is made up of following: (a) a kind of polynucleotide sequence, it and SEQ ID NO:1 have 85% identity at least respectively on the whole length of SEQ ID NO:1, more preferably at least 90% identity, more preferably at least 95% identity, the most preferably identity of 97-99% or identical at least; Or (b) a kind of polynucleotide sequence of coding one peptide species, the aminoacid sequence of this polypeptide and SEQ ID NO:2 has at least 85% identity respectively on the whole length of SEQ ID NO:2, more preferably at least 90% identity, more preferably at least 95% identity, the more preferably identity of 97-99% or 100% identical at least.

The encode polynucleotide of a peptide species of the present invention, comprise homologue or homology evolution thing from the kind except that Neisseria meningitidis, can obtain by a kind of method that may further comprise the steps: under tight hybridization conditions, (for example use 45-65 ℃ temperature range, the concentration of SDS is 0.1-1%) with probe a kind of mark or detectable suitable library is screened, its middle probe comprises or is made up of a sequence or the one fragment of SEQ ID NO:1, and separates full-length gene and/or the genomic clone that contains said polynucleotide sequence.

The invention provides a kind of on its whole length with the identical polynucleotide sequence of encoding sequence (open reading frame) of SEQ ID NO:1.The present invention also provide independent mature polypeptide or its segmental encoding sequence and with another encoding sequence at mature polypeptide or its a segmental encoding sequence of reading in the frame, the example of another encoding sequence for a kind of leading or secretion sequence of coding, preceding-, former-or preceding former-proteic sequence.Polynucleotide of the present invention also can contain at least a non-coding sequence, include but not limited to, such as at least a noncoding 5 ' and 3 ' sequence, for example transcribe but sequence, intron and the polyadenylation signal of the sequence do not translated, termination signal (for example rho-relies on and the termination signal of non-rho-dependence), ribosome bind site, Kozak sequence, stable mRNA.Polynucleotide sequence also can comprise the extra encoding sequence of the additional amino acid of encoding.For example, a kind of flag sequence that can promote the fusion polypeptide purifying of can encoding.In certain embodiments of the invention, flag sequence is a kind of 6 Histidine peptides, is provided by the pQE carrier that (Qiagen Inc.), sees " PNAS " 86:821-824 such as Gentz, the introduction in (1989); Or a kind of HA peptide tail (Wilson etc., " cell " 37:767 (1984); Two kinds of flags sequence all can be used for the polypeptide of purifying and their fusions.Polynucleotide of the present invention also include but not limited to, comprise the sequence of a kind of structure gene and its natural controlling gene expression that links to each other.

The nucleotide sequence of the BASB052 polypeptide of coding SEQ ID NO:2 can be identical with the contained polypeptid coding sequence of the Nucleotide 1 to 1065 of SEQ IDNO:1.In addition, it can be a kind of like this sequence, i.e. the polypeptide of SEQ ID NO:2 because genetic code Feng Yu (degeneracy) also encodes.

Be used for herein, " polynucleotide of the peptide species of encoding " speech relates to the polynucleotide of the sequence that comprises a kind of polypeptide of the present invention of encoding, polypeptide of the present invention is a kind of bacterial peptide specifically, the Neisseria meningitidis BASB052 polypeptide of more specifically saying so, it has the listed aminoacid sequence of SEQ ID NO:2.This speech also relates to a kind of like this polynucleotide, it comprises a single successive zone of this peptide of encoding or discontinuous zone (the carrier sequence of the phage that for example is integrated, the insertion sequence of integration, integration, the transposon sequence of integration or because of rna editing or genomic dna reset the polynucleotide that interrupted) and other zone, and coding and/or non-coding sequence also can be contained in this other zone.

The invention further relates to here the variant of the polynucleotide of introducing, can the encode variant polypeptides of putative amino acid sequence of this variant with SEQ ID NO:2.The fragment of polynucleotide of the present invention can be used for such as synthetic total length polynucleotide of the present invention.

Particularly preferred embodiment is the polynucleotide of coding BASB052 variant more, that this variant has is several in SEQ ID NO:2, minority, 5 to 10,1 to 5,1 to 3,2,1 or do not have that amino acid is replaced with any array mode, modifies, disappearance and/or the BASB052 amino acid sequence of polypeptide of adding.Especially preferred in them is characteristic and active reticent replacement, interpolation and the disappearance that does not change the BASB052 polypeptide.

The polynucleotide that preferred embodiment of the present invention is with coding has a BASB052 polypeptide of the aminoacid sequence that SEQ ID NO:2 lists have 85% identical polynucleotide at least on its whole length; And with this polynucleotide complementary polynucleotide.Thus, particularly preferably be the polynucleotide that on whole length, have 90% identity at least, and in these special preferred polynucleotides, have at least the polynucleotide of 95% identity more preferred, and have at least in the polynucleotide of 95% identity at these, have 97% identical polynucleotide at least more preferably, equally in them, have at least 98% with have at least 99% identical polynucleotide especially to be more preferably, wherein have 99% identical polynucleotide at least more preferably.

Embodiment preferred is the encode identical biological function of mature polypeptide of the dna encoding that kept SEQ ID NO:1 basically or the polynucleotide of active polypeptide.

According to particular preferred embodiment of the present invention, the invention provides can with BASB052 polynucleotide sequence for example the polynucleotide sequence hybridization of SEQ ID NO:1, the especially polynucleotide of under stringent condition, hybridizing.

The invention further relates to can with the polynucleotide sequence of the polynucleotide sequence that provides here hybridization.Thus, The present invention be more particularly directed to can be under stringent condition and the polynucleotide of the multi-nucleotide hybrid of introducing here.Be used for herein, the meaning of " stringent condition " and " tight hybridization conditions " is that hybridization only occurs in and has at least 95% preferably to have 97% at least when identical between the sequence.An object lesson of tight hybridization conditions is to be incubated overnight in a kind of solution at 42 ℃, this solution comprises: salmon sperm DNA is sheared in the sex change of 50% methane amide, 5x SSC (150mM NaCl, 15mM Trisodium Citrate), 50mM sodium phosphate (pH7.6), 5x Denhardt ' s solution, 10% dextran sulfate and 20 micrograms/ml, then washs the hybridization upholder in 0.1x SSC in the time of about 65 ℃.Hybridization and wash conditions are well-known, and at " molecular cloning lab guide, second edition " Cold Spring Harbor Laboratory Press such as Sambrook, ColdSpring Harbor particularly gives an example in 11 chapters among the New York (1989).The also available polynucleotide sequence provided by the invention of solution hybridization carries out.

The present invention also provides the polynucleotide that contain or be made up of a kind of like this polynucleotide sequence, this polynucleotide sequence obtains by screening a kind of suitable library with a kind of probe and separate described polynucleotide sequence under tight hybridization conditions, its Chinese library contains the complete genome of the polynucleotide sequence that SEQ ID NO:1 lists, and probe has the sequence of the said polynucleotide sequence that SEQ ID NO:1 lists.The fragment that can be used for obtaining a kind of like this polynucleotide comprises probe and the primer of introducing in detail such as other parts of this paper.

As of the discussion of the present invention's other parts here to the polynucleotide test, for example, the hybridization probe that polynucleotide of the present invention can be used as RNA, cDNA and genomic dna separates full-length cDNA s and the genomic clone of the BASB052 that encodes, and separation and BASB052 gene have the cDNA and the genomic clone of other genes of the particularly high sequence identity of high identity.This probe generally has at least 15 nucleotide residues or base pair, and this probe preferably has at least 30 nucleotide residues or base pair, also can have at least 50 nucleotide residues or base pair.Particularly preferred probe has at least 20 nucleotide residues or base pair, and is less than at least 30 nucleotide residues or base pair.

The coding region of BASB052 gene can be screened by the synthetic a kind of oligonucleotide probe of the dna sequence dna that uses SEQ ID NO:1 to provide and be separated.Use with gene order complementary labeled oligonucleotide of the present invention then and screen a kind of cDNA, genomic dna or mRNA library, determine the library member of this probe hybridization.

To one skilled in the art, there is several method to use and to know, can be used for obtaining full length DNA s or extend short DNAs, for example (be seen in as Frohman etc. based on the method for terminal rapid amplifying (RACE) method of cDNA, " PNAS " 85:8998-9002,1988).The nearest modification of this technology, for example MarathonTM technology (Clontech Laboratories Inc.) has obviously been simplified the searching to longer cDNAs.In the MarathonTM technology, cDNAs extractive mRNA preparation from a kind of tissue of selection, and connect a kind of " joint " at each end.Use gene specific then with the increase 5 ' end of DNA of " losing " of special oligonucleotide combination the carrying out nucleic acid amplification (PCR) of joint.Use " nido " primer to repeat the PCR reaction then, nested primer promptly design with the inside annealed primer of amplified production (be generally with a kind of joint Auele Specific Primer of 3 ' annealed of joint sequence and with a kind of gene-specific primer of 5 ' annealed of selected gene order).With dna sequencing the product of this reaction is analyzed then, produced a complete sequence, perhaps use the new sequence information of design 5 ' primer to carry out an independent total length PCR, make up the DNA of a total length by product directly is connected with existing DNA.

Polynucleotide of the present invention and polypeptide can be used as such as finding disease particularly human disease's treatment and Studies on Diagnosis reagent and material, as testing relevant discussion with polynucleotide here.

Polynucleotide of the present invention are the oligonucleotide derived from SEQ ID NO:1-2 sequence, the method that they can be used for introducing here, but more preferably be used for PCR, be used for determining here whether all or part of can in bacterium or infected tissue, transcribing of identified polynucleotides.Should be appreciated that this sequence also can be used for the stage of diagnose infections and the infection type of the pathogenic agent that obtains.

The present invention also provides the polynucleotide of the such peptide species of coding, and this peptide species is the amino acid (for example, when ripe form contains more than one polypeptide chain) that a kind of maturation protein adds other amino or C-terminal amino acid or mature polypeptide inside.This sequence can work a kind of albumen when precursor is processed into mature form, can allow albumen transportation, can prolong or shorten proteic half life, the operation in the time of maybe can being convenient to albumen and being used to test or preparing.Usually in vivo the time, other amino acid can be removed from maturation protein processing by cellular enzymes.

For each and all polynucleotide of the present invention, all provide a kind of polynucleotide of complementary with it.These complementary polynucleotide are preferably complementary fully with each polynucleotide of their complementary.

The precursor protein that a kind of mature form that contains polypeptide and one or more former sequences merge may be the inactive form of this polypeptide.When removing former sequence, the precursor of this non-activity generally is activated.Before activation, some or all of former sequences can be removed.This precursor is commonly referred to as proteinogen.

Nucleotide is except A, G, C, T/U with standard represent, " N " also can be used to describe specific polynucleotide of the present invention." N " refer to 4 kinds of DNA or RNA Nucleotide any one all can appear at this appointment site of DNA or RNA sequence, but preferred N is not a kind of nucleic acid, when it and adjacent nucleotide position are combined, when reading, read the terminator codon before a kind of maturation of generation in the frame at this by correct reading frame.

Generally speaking, a kind of maturation protein of polynucleotide codified of the present invention, a kind of maturation protein add a kind of leader sequence (it can be called as a kind of before albumen), contain one or more non-before former albumen before the precursor of maturation protein of former sequence of proteic leader sequences or the former proteic precursor, it has a kind of leader sequence and one or more former sequences, and they are removed in the course of processing of activity that produces polypeptide and mature form usually.

According to an aspect of the present invention, provide polynucleotide of the present invention in the particularly application aspect the inherited immunity of therapeutic or preventative purpose.

A kind of suitable transmission method is preferably used in the application of polynucleotide of the present invention aspect inherited immunity, for example directly plasmid DNA is injected into muscle (Wolff etc. " human molecular genetics " (1992) 1:363, Manthorpe etc., " human gene therapy " (1983) 4:419); DNA and specific proteins carrier are transmitted (Wu etc., " journal of biological chemistry " (1989) 264:16985) after compound, with DNA and coprecipitation of calcium phosphate (Benvenisty﹠amp; Reshef, " PNAS " (1986) 83:9551); With DNA with various forms of liposome (Kaneda etc., " science " (1989) 243:375); Particle bombardment (Tang etc., " nature " (1992) 356:152, Eisenbraun etc. " DNA and cytobiology " (1993) 12:791) and use clone's reverse transcription carrier to carry out infecting in the body (Seeger etc., " PNAS " 81:5949).Carrier, host cell, expression system

The invention still further relates to the carrier that contains polynucleotide of the present invention, carried out genetically engineered host cell and prepared polypeptide of the present invention by recombinant technology with carrier of the present invention.Cell free translation system also can be used for using the RNAs from dna structure thing of the present invention to prepare such albumen.

Recombinant polypeptide of the present invention can use method well known to those skilled in the art to be prepared by the genetically engineered host cell that contains expression system.Therefore, one further aspect, the present invention relates to contain polynucleotide of the present invention expression system, prepare polypeptide of the present invention with the genetically engineered host cell of this expression system and with recombinant technology.

For carrying out the reorganization preparation of polypeptide of the present invention, host cell can come integrative gene expression system or its part or polynucleotide of the present invention by genetically engineered.Polynucleotide are imported host cell can be finished by the method for multiple standards laboratory manual introduction, " molecular cloning lab guide such as Davis etc. " molecular biology basic skills " (1986) and Sambrook for example, second edition " Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NewYork (1989), for example calcium phosphate transfection, the transfection of DEAE-dextran mediation, carrier transfection (transvection), microinjection, the transfection of cationic lipid mediation, electroporation, transduction, the cut inoculation, bombardment imports and infects.

The representative example of suitable host comprises the cell of bacterial cell such as suis, staphylococcus, faecalis, intestinal bacteria, streptomycete, cyanobacteria, subtilis, morazella catarrhalis, hemophilus influenzae and Neisseria meningitidis; The cell of basidiomycetes such as yeast such as fungal cell such as Crewe Vickers yeast, sugar yeast, white candiyeast and aspergillus; The cell of insect cell such as fruit bat S2 and fall army worm Sf9; Zooblast such as CHO, COS, HeLa, C127,3T3, BHK, 293, CV-1 and Bowes melanoma cells; And vegetable cell such as gymnosperm or angiospermous cell.

Multiple expression system can be used for preparing polypeptide of the present invention.This carrier comprises chromosomal pattern, free type, virus type derivative vector, for example by bacterial plasmid, bacteriophage, transposon, yeast episome, insertion element, yeast chromosomal element, virus as baculovirus, papovavirus such as SV40, vaccinia virus, adenovirus, fowlpox virus, Pseudorabies virus, picornavirus, retrovirus and first C-type virus C deutero-carrier or by their composition deutero-carrier, for example by plasmid and phage genetic elements deutero-carrier, as clay and phagemid.The expression system works can contain the control area of regulating and causing expression.With regard to this aspect in general, any system or carrier that is suitable for keeping, breed or express polynucleotide and/or expresses a peptide species in a kind of host all can be used for expressing.Suitable dna sequence dna can be inserted in the expression system by any technology with routine that know, for example the technology listed in (above) of Sambrook etc. " molecular cloning lab guide ".

In the eukaryotic cell recombinant expression system,, suitable secretion signal can be incorporated in the polypeptide expressed for making the translation protein excretion in the endoplasm stratum reticulare, between pericentral siphon or in born of the same parents' external environment.These signals can be endogenic concerning polypeptide, perhaps also can be heterologys.

Polypeptide of the present invention can reclaim and purifying from the reconstitution cell culture by the technology of knowing, and these technology comprise ammonium sulfate or ethanol sedimentation, sour extracting, negatively charged ion or cation-exchange chromatography, phosphorylated cotton chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography.More preferably metal ion affinity chromatography (IMAC) is used for purifying.The folding again technology of knowing of albumen that is used for can be used for when polypeptide synthesizes in born of the same parents, the regeneration of activity conformation when separation and/or purge process sex change.

Expression system is a kind of live microorganism of reorganization also, for example a kind of virus or bacterium.Target gene can be inserted in the genome of recombinant virus alive or bacterium.The carrier that infects this work in inoculation and the body will cause antigenic expression in vivo and immune response inducing.The virus and the bacterium that are used for this purpose are such as poxvirus (for example vaccinia virus, fowlpox virus, canary avipoxvirus), Alphavirus (sindbis alphavirus, Semliki Forest virus, peste loca virus), adenovirus, adeno associated virus, picornavirus (poliovirus, rhinovirus), simplexvirus (varicella zoster virus etc.), listeria spp, Salmonellas, Shigellae, Neisseria, BCG.These are viral and bacterium can be toxic or the attenuation that ins all sorts of ways obtains a kind of living vaccine.This living vaccine also is a part of the present invention.Diagnosis, prediction, serotype and mutant test

The invention still further relates to BASB052 polynucleotide of the present invention and polypeptide are used as diagnostic reagent.Eukaryotic cell particularly mammalian cell especially detect in people's cell BASB052 polynucleotide and/or polypeptide can be diagnose the illness, disease stage or infection biological body provide a kind of diagnostic method to replying of medicine.Eukaryotic cell, mammalian cell particularly, especially people's cell, particularly those infection or suspection have infected the cell that contains BASB052 gene or proteic organism, can detect by multiple technology of knowing and the method that provides here at nucleic acid or amino acid levels.

Be used to predict, diagnose or the polypeptide of other analyses and polynucleotide can infect and/or the body material of infected individuals available from supposition.Can be directly used in detection from polynucleotide in any of these source especially DNA or RNA, perhaps can before analysis, use PCR or other any amplification techniques to carry out enzymatic amplification.RNA particularly mRNA, cDNA and genomic dna also can use in the same manner.Use amplification method, analyze, can identify the kind of settling down organism and the bacterial strain that exist in infectivity or the individuality by genotype to the selected polynucleotide of organism.Disappearance can detect by the size variation that amplified production is compared with a kind of genotype that is selected from the reference sequences of related organisms with inserting, and preferably compares with the not homophyletic of the same race or mutually not of the same race of same genus.Point mutation can be identified by the BASB052 polynucleotide sequence of DNA amplification and mark is hybridized.For DNA or RNA, by DNase or RNase digestion respectively can be very or obviously the sequence of coupling make a distinction with coupling binary not good or obvious mispairing, perhaps distinguish by the difference of detection melting temp or reannealing kinetics.The change of the electrophoretic migration that polynucleotide sequence difference also can be compared with a kind of reference sequences in gel by polynucleotide passage detects.This can use or not use sex change reagent.Polynucleotide difference also can check order by direct DNA or RNA and detect.Be seen in as Myers etc., " science " 230:1242 (1985).The sequence of specific position changes also and can show by nuclease protection test such as RNase, V1 and S1 protection test or chemical cracking method.Be seen in as " PNAS " 85:4397-4401 (1985) such as Cotton.

In another embodiment, can make up a series of BASB052 of containing nucleotide sequences or its segmental oligonucleotide probe, come effectively screening such as genetic mutation, serotype, classification or evaluation.The parallel techniques method is well-known, has versatility, can be used to solve the various problems in the molecular genetics, comprises that genetic expression, genetic linkage and heritable variation (are seen in as " science " 274:610 (1996) such as Chee.

Therefore, in yet another aspect, the present invention relates to a kind of diagnostic kit, it comprises

(a) a kind of polynucleotide of the present invention, nucleotide sequence or its fragment of preferred SEQ ID NO:1;

(b) with (a) nucleotide sequence complementary nucleotide sequence;

(c) peptide species of the present invention, polypeptide or its fragment of preferred SEQ ID NO:2; Or

(d) at the antibody of polypeptide of the present invention, preferred pin is to the antibody of the polypeptide of SEQ ID NO:2.

In any a kind of like this test kit, (a) and (b), (c) or (d) can preferably contain a kind of basal component.This test kit can be used for disease or to the diagnosis of the susceptibility of disease or other.

The invention still further relates to polynucleotide of the present invention as diagnostic reagent.To the preferred SEQ ID of polynucleotide of the present invention NO:1 and detection a kind of disease or pathogenic relevant mutant form, a kind of diagnostic means can be provided, and this diagnostic means can increase or limit low expression owing to these polynucleotide, overexpression or express the determining of susceptibility of prediction, disease stage or the disease of the diagnosis that changes a kind of disease that causes, lysis.The organism that has sudden change in such polynucleotide particularly infectious organisms can detect in the polynucleotide level by the technology that various technology are for example introduced here Anywhere.

The cell that comes to have the organism of sudden change or polymorphism (equipotential sudden change) in comfortable polynucleotide of the present invention and/or the polypeptide also can be used to use multiple technologies to detect at polynucleotide or polypeptide level, thereby carries out such as serological typing.For example, RT-PCR can be used to detect the sudden change among the RNA.Preferably RT-PCR is combined with automatic checkout system such as GeneScan and use.RNA, cDNA or genomic dna also can be used for identical purpose---PCR.For example, the polynucleotide complementary PCR primer with coding BASB052 polypeptide can be used to identify and analyze sudden change.

The present invention further provides the primer of removing 1,2,3 or 4 Nucleotide from 5 ' and/or 3 ' end.These primers can be used from isolating BASB052 DNA and/or RNA a kind of sample that amplification obtains from individuality such as the body material with other materials one.These primers can be used for amplification isolating a kind of polynucleotide from infected individuals, and like this, these polynucleotide can be illustrated polynucleotide sequence by various technology subsequently.By this method, can detect the sudden change in the polynucleotide sequence, and be used for diagnosis and/or prediction infection or its stage or process, or carry out the serological typing and/or the classification of infectant.

The present invention further provides and diagnose the illness, infectation of bacteria preferably, be more preferably the method for the infection that is caused by Neisseria meningitidis, this is included in the raising that detects the expression level of the polynucleotide with SEQ ID NO:1 sequence from the sample of individuality acquisition such as a kind of body material.Increase that the BASB052 polynucleotide are expressed or minimizing can use any known polynucleotide quantitative methods that is used in this area to detect, for example amplification, PCR, RT-PCR, RNase protection, Northern trace, spectrophotometric and other hybridizing methods.

In addition, be used to detect the BASB052 polypeptide and compare the existence that the diagnostic test of overexpression can be used to test example such as a kind of infection with the normal control tissue sample according to of the present invention.Can be used to measure the BASB052 polypeptide is knowing to one skilled in the art from the experimental technique of the level in a kind of host such as a kind of body material.This test method comprises that radioimmunoassay, competition are in conjunction with test, Western trace, antibody sandwich test, antibody test and ELISA test.

Polynucleotide of the present invention can be used as polynucleotide array preferably the high-density array or the component of carrying net.These high-density arrays are particularly useful for diagnosis and prediction purpose.For example, a cover point that respectively contains a kind of different genes and further contain polynucleotide of the present invention can be used for probe in detecting, for example use hybridization or nucleic acid amplification, use from or derived from a kind of probe of body sample, detect the existence in body one by one of a kind of special oligonucleotide sequence or correlated series.This existence can illustrate the especially existence of Neisseria meningitidis of a kind of pathogenic agent, and can be used to diagnosis and/or prediction disease or lysis.Preferably a kind of variant of the nucleotide sequence that contains multiple SEQ IDNO:1 carry net.Contain coding SEQ ID NO:2 peptide sequence polynucleotide sequence multiple variant to carry a net also be preferred.Antibody

Polypeptide of the present invention and polynucleotide or its variant or the cell of expressing them can be used as the original preparation of immunity respectively at the antibody of this peptide species or polynucleotide.

In particular preferred embodiment of the present invention, provide antibody at BASB052 polypeptide or polynucleotide.

Antibody at polypeptide of the present invention or polynucleotide preparation can obtain by using traditional method, be about to one of polypeptide of the present invention and/or polynucleotide or they or both fragments of carrying epi-position, they one of or both analogue express they one of or both cell deliver medicine to a kind of animal, preferably a kind of inhuman animal.Any technology of the antibody for preparing with the continuous cell line cultivation that can provide in this area can be used.Example comprises various technology, Kohler for example, G. and Milstein, C. " nature " 256:495-497 (1975); " immunology today " 4:72 such as Kozbor (1983); Cole etc. " monoclonal antibody and cancer therapy ", AlanR.Liss, the 77-96 page or leaf of Inc. (1985).

The technology (U.S. Patent No. 4,946,778) of preparation single-chain antibody can be revised the single-chain antibody for preparing at polypeptide of the present invention or polynucleotide.Transgenic mouse or other biological body or animal such as other Mammalss also can be used to express to polypeptide of the present invention or the special humanized antibody of polynucleotide immunity.

In addition, phage display technology can be used to select polypeptide of the present invention is had in conjunction with active antibody gene (McCafferty etc. (1990) " nature " 348:552-554 from the V-gene library of the pcr amplification of the human lymphocyte that contains anti-BASB052 or natural library; Marks etc. (1992) " biotechnology " 10:779-783).The affinity of these antibody also can be by improving (Clackson etc., (1991) " nature " 352:628) such as the strand displacement technology.

The antibody of introducing above can be used to separate or identify the clone that can express polypeptide of the present invention or polynucleotide, passes through such as these polypeptide of affinitive layer purification or polynucleotide.

These antibody and other antibody at BASB052 polypeptide or BASB052 polynucleotide can be used to treatment and infect, particularly infectation of bacteria.

Polypeptide variants comprises the variant of antigenicity, epi-position or immunity equivalence, and they have formed a special aspect of the present invention.

Antibody or its fragment preferably by modifying, make it to have less immunogenicity in individuality.For example, if individuality is the people, antibody can be more preferably the antibody of a kind of " humanization ", wherein the complementary determining region of hybridoma source antibody is transplanted in the human monoclonal antibodies, (1986) " nature " 321 such as Jones for example, 522-525 or Tempest etc. (1991) " biotechnology " 9, the introduction among the 266-273.Antagonist and agonist---test and molecule

Polypeptide of the present invention and polynucleotide also can be used to estimate small molecules substrate and part such as combining in cell, cell-free extract, chemical library and the natural product mixture.These substrates and part can be natural substrate and part, perhaps can be structure or functional analogue thing, are seen in as " Immunization Update methods " 1 (2) such as Coligan: 5 chapters (1991).

Screening method can be measured candidate compound and polypeptide or polynucleotide simply or carry polypeptide or the combining of the fusion rotein of the cell of polynucleotide or film or polypeptide by a kind of mark that directly or indirectly is connected with candidate compound.In addition, screening method can comprise that the competition thing with a kind of mark is at war with.And these screening methods can detect candidate compound and whether cause a kind of signal that is produced by the activation of polypeptide or polynucleotide, and this can use the detection system that suits with the cell that contains polypeptide or polynucleotide to carry out.The activatory inhibitor is generally tested existing under a kind of situation of known agonist, and observes the influence of candidate compound to the agonist activation.The polypeptide and the polynucleotide of constitutive activity polypeptide and/or constitutive expression can be as required, under the situation that does not have a kind of agonist or antagonist, be used to reverse the screening method of agonist or inhibitor, whether this can cause the activatory of polypeptide or polynucleotide to suppress to carry out by detecting candidate compound.And screening method can may further comprise the steps simply, and a kind of candidate compound is mixed with the solution that contains a peptide species of the present invention or polynucleotide, forms a kind of mixture; The activity of BASB052 polypeptide and/or polynucleotide in the detection mixture; Activity and a kind of standard of BASB052 polypeptide and/or polynucleotide in the mixture are compared.Fusion rotein also can be used for high-throughout shaker test as the Fc part introduced and the fusion rotein of BASB052 polypeptide preparation here, is used for identifying polypeptide relevant on the antagonist of polypeptide of the present invention and phylogeny and/or the function (seeing " journal of biological chemistry " 270 (16): 9459-9471 (1995) such as " molecular recognition magazine " 8:52-58 (1995) such as D.Bennett and K.Johanson).

Can combine with a peptide species of the present invention and/or interactional polynucleotide, polypeptide and antibody also can be used for designing the screening method that detects the influence that mRNA in the compound pair cell that adds and/or polypeptide produce.For example, can design a kind of ELISA test, use mono-clonal and polyclonal antibody, according to the standard method of this area, the secretion of measurement polypeptide or cell are in conjunction with level.This can be used to find to suppress or to strengthen the reagent (being also referred to as antagonist or agonist respectively) that polypeptide produces in suitable manipulating cells or tissue.

The present invention also provides a kind of method of SCREENED COMPOUND, is used for identifying the compound of the effect that can strengthen (agonist) or blocking-up (antagonist) BASB052 polypeptide or polynucleotide, especially can antibacterial and/or germ-resistant compound.This screening method can use high-throughput techniques.For example, be screening agonist or antagonist, with a kind of synthesis reaction mixture, a kind of cellular component such as film, cell envelope or cell walls or their any prepared product, comprise the part of BASB052 polypeptide and a kind of labeled substrate or this peptide species, a kind of may be the candidate molecules of BASB052 agonist or antagonist exist or non-existent condition under, carry out incubation.The ability of candidate compound excitement or antagonism BASB052 polypeptide be reflected in tagged ligand in conjunction with reducing or the generation of the product of this substrate reduces.Invalid combination does not promptly induce the molecule of the effect of BASB052 polypeptide to be likely outstanding antagonist.According to circumstances, in conjunction with good and to increase speed that product produces by substrate, increase signal transduction or increase the active molecule of chemical access promptly be agonist.According to circumstances, product can be strengthened by using a kind of reporting system by the detection of substrate generation, signal conduction or active speed of chemical access or level.Can be used for this purpose reporting system and include but not limited to that colorimetric, labeled substrate are transformed into product, a kind of reporter gene and combination test well known in the art that the variation of BASB052 polynucleotide or polypeptide is replied.

Another example of BASB052 agonist test is a kind of competition experiments, BASB052 and a kind of possible agonist and BASB052 binding molecule, the stand-in of recombinate BASB052 binding molecule, natural substrate or part or substrate or part are combined in this test, and inhibition test is at war with under appropriate condition.BASB052 can carry out mark, for example with radioactivity or colorimetric compound mark, the number that is attached on a kind of binding molecule or changes into the BASB052 molecule of product can accurately be measured, and estimates the effect of possible antagonist.

Possible antagonist comprises and can combine with polynucleotide of the present invention and/or polypeptide and thereby suppress or suppress its active or little organic molecule, peptide, polypeptide and the antibody of expressing and other.Possible antagonist also can be so little organic molecule, peptide, polypeptide such as closely-related albumen or antibody, they are combined in the same loci of binding molecule, for example a kind of binding molecule, but do not induce BASB052 inductive activity, thereby by stoping BASB052 polypeptide and/or polynucleotide in conjunction with the activation or the expression that suppress BASB052 polypeptide and/or polynucleotide.

Possible antagonist comprises such small molecules, their combinations and occupy the binding site of polypeptide, thereby stop it to combine with the cell binding molecule, thus suppress normal biological activity.Micromolecular example includes but not limited to little organic molecule, peptide or peptide sample molecule.Other possible antagonists comprise that antisense molecule (sees Okano, " neurochemistry magazine " 56:560 (1991); " oligodeoxynucleotide is as the antisense inhibitor of genetic expression " CRC Press, Boca Raton, FL (1988) is to the introduction of these molecules).Preferred possible antagonist comprises the compound relevant with BASB052 and variant thereof.

One further aspect, the present invention relates to the soluble fusion protein of genetic engineering, this albumen comprises a peptide species of the present invention or its fragment and the heavy chain of various subclass immunoglobulin (Ig) (IgG, IgM, IgA, IgE) or the various parts of constant region of light chain.Preferred immunoglobulins is the particularly constant region part of IgG1 heavy chain of human IgG, wherein merges to betide hinge area.In a special embodiment, the Fc part can be removed by the following method simply: introduce a cutting sequence, this sequence available blood Rh factor Xa.And, the present invention relates to prepare the method for these fusion roteins, and relate to and use it for drug screening, diagnosis and treatment by genetic engineering.A further aspect of the present invention also relates to the polynucleotide of this fusion rotein of encoding.The example of fusion protein technology is found in international patent application Nos.WO94/29458 and WO94/22914.

Here each polynucleotide sequence that provides can be used for finding and the exploitation antimicrobial compounds.Proteins encoded can be used as the target that screens antibacterials by expression.In addition, the encode SD of coded proteic amino terminal region or corresponding mRNA or other translations promotes the polynucleotide sequence in zone to can be used for making up the expression that antisense sequences comes the controlled target encoding sequence.

The present invention also provides polypeptide of the present invention, polynucleotide, agonist or antagonist is used to disturb a kind of pathogenic agent or multiple pathogenic agent and a kind of preferably initial physiology interaction between the mammalian hosts of eukaryote to infecting the secondary disease sensitivity.Specifically, molecule of the present invention can be used for: suppress bacterium and be specifically Gram-positive and/or gram negative bacterium adhere to eukaryote preferably mammiferous buried device extracellular matrix protein or adhere to the extracellular matrix protein of wound; Bacillary adhesion between blocking-up preferably mammiferous extracellular matrix protein of eukaryote and the bacterium BASB052 albumen, the latter mediates tissue injury; And/or blocking-up is no matter be that buried device is implanted or the normal pathogenic course of the infection that other surgical technics cause.

According to another aspect of the present invention, provide BASB052 agonist and antagonist, preferably antibacterial or bactericidal properties agonist and antagonist.

Antagonist of the present invention and agonist can be used for for example stoping, suppress and/or treat disease.

One further aspect, the present invention relates to the mimic epitopes of polypeptide of the present invention.Mimic epitopes is a kind of peptide sequence, enough similar to native peptides (on the sequence or on the structure), and it can be identified the antibody recognition of native peptides, or can produce the antibody of identification native peptides with a kind of suitable carrier coupling the time.

The peptide mimic epitopes can be used for special purpose by adding, lacking or replace selected amino acid.Therefore, peptide can be modified being easy to and is connected with a kind of protein carrier.For example, for some Chemical bond method, need contain a terminal cysteine.In addition, combine with a kind of protein carrier, need comprise one, make the unconjugated free-end maintenance of peptide and the interaction on carrier proteins surface away from the hydrophobic end of peptide in conjunction with end for peptide.Thereby make peptide have a kind of conformation, the conformation that this conformation and peptide have in whole natural molecule structure is closely similar.For example, peptide can be modified and have a N-terminal halfcystine and the amidated tail that C-terminal is hydrophobic.In addition, can carry out the interpolation or the replacement of one or more amino acid whose D steric isomers, prepare a kind of useful derivative, be used for for example strengthening the stability of peptide.

In addition, the peptide mimic epitopes can be by using and can identify with polypeptide bonded antibody of the present invention such as phage display technology (EP 0 552 267 B1).This technology produces the peptide sequence of the structure of a large amount of simulation native peptides, and these peptide sequences therefore can be in conjunction with the antibody of anti-native peptides, but may be not enough to have the obvious sequence homology with natural polypeptides.Vaccine

Another aspect of the present invention relates in the individuality Mammals method of induce immune response among the people preferably particularly; this method comprises with BASB052 polynucleotide and/or polypeptide or its fragment or variant inoculation individuality; they are enough to produce antibody and/or T cellullar immunologic response; the protection individuality is not infected; particularly infectation of bacteria, especially Neisseria meningitidis infect.The present invention also provides and produces this immunne response to delay the method that bacterium is duplicated.Another aspect of the present invention relates to the method for induce immune response in individuality; comprise the nucleic acid carrier that gives a kind of BASB052 of guidance polynucleotide of this individuality and/or expression of polypeptides or its fragment or variant expression; sequence or ribozyme; express BASB052 polynucleotide and/or expression of polypeptides or its fragment or variant in vivo; thereby induce a kind of immunne response; for example produce antibody and/or T cellullar immunologic response; comprise such as producing cytokine T cell or cytotoxic T cell; protect described individuality; preferably the people avoid ill, and no matter this disease is to exist in individuality or do not exist.An example of gene drug delivery is to promote it to enter required cell its encrusting substance as particle or other materials.This nucleic acid carrier can comprise nucleic acid, DNA/RNA heterozygote, DNA albumen composition or the RNA-albumen composition of DNA, RNA, ribozyme, modification.

A further aspect of the present invention relates to a kind of immune composition, and said composition can be induced a kind of immunne response importing preferably man-hour of individuality, promptly induces in this individuality at the BASB052 polynucleotide and/or by the immunne response of its encoded polypeptides.Wherein composition comprises the BASB052 polynucleotide of reorganization and/or by its encoded polypeptides; And/or comprise can encode and express described BASB052 polynucleotide, by the antigenic DNA and/or the RNA of its encoded polypeptides or other polypeptide of the present invention.Immunne response can be used for treatment or prevention purpose, and can adopt the form of antibody mediated immunity and/or cellular immunization, for example cellular immunization that is caused by CTL or CD4+T cell.

Therefore; BASB052 polypeptide or its fragment can merge with accessory protein or chemical half point; accessory protein or chemical half point can or can not self produce antibody; but can make first kind protein stabilized; and produce albumen a kind of fusion or that modify; the latter has antigenicity and/or immunogenicity, preferably protectiveness.Such fusion recombinant protein preferably further contains a kind of antigenicity accessory protein, for example from lipoprotein D, glutathione-S-transferase (GST) or the beta-galactosidase enzymes of hemophilus influenzae or other are any can stabilize proteins and promote the big accessory protein of its preparation and purifying.And accessory protein can be used as a kind of adjuvant when providing a kind of standard stimulus to the immunity system of accepting this proteic organism.Accessory protein can be connected first kind of proteic aminoterminal or carboxyl terminal.

In vaccine composition of the present invention, BASB052 polypeptide and/or polynucleotide or its fragment, mimic epitopes (mimotope) or variant may reside in a kind of carrier, as live-weight group carriers such as above-mentioned bacteria carriers alive.

The no life carrier of BASB052 polypeptide also is suitable, for example bacterial outer membrane vesica or " vesicle (bleb) ".The OM vesicle is derived from the adventitia of gram negative bacterium duplicature, multiple gram negative bacterium comprised report among C.trachomatis and the C.psittaci (Zhou, L etc., 1998, the communication of FEMS microorganism, 163:223-228).The limiting examples that produces the bacterial pathogens of vesicle according to reports also comprises: Bordetella pertussis, B. burgdorferi, Bacterium melitense, Brucella melitensis, intestinal bacteria, hemophilus influenzae, legionella pneumophilia, gonococcus, Neisseria meningitidis, Pseudomonas aeruginosa and yersinia entero-colitica.

The advantage of vesicle is to provide outer membrane protein with native conformation, and is therefore particularly useful in vaccine.Vesicle also can be used for vaccine with the expression that changes one or more molecules on the adventitia by engineered bacterium.Therefore, for example can introduce or raise of the expression of required immunogenic protein such as (as by changing promotor) BASB052 polypeptide at adventitia.Another kind of mode or further, can be reduced the comprising of outer membrane molecule of uncorrelated (as non-protective antigen or immunodominance but variable albumen) or harmful (as toxicity molecules such as LPS, or the potential inductor of autoimmune response).These modes are following to be described in detail.

The non-coding flanking region of BASB052 gene contains controlling element important in the genetic expression.This regulation and control not only are present in transcriptional level but also be present in translation skill.The sequence in these zones (no matter being the upstream or the downstream of gene open reading frame) can obtain by dna sequencing.This sequence information can be determined potential regulation and control primitive, but as different promoter elements, terminator sequence induced sequence element, repressor, the responsible element that changes mutually, ribosome binding sequence, have the potential secondary structure of the regulation and control of participating in, and the regulation and control primitive or the sequence of other type.This sequence is another aspect of the present invention.

This sequence information allows to regulate the natural expression of BASB052 gene.The rise of genetic expression also can be by changing promotor, ribosome binding sequence, potential repressor or operator gene element or any other relevant elements.Equally, the downward modulation of expression can be by similarly regulation and control realization.Perhaps, change sequence mutually by changing, expression of gene can place and change mutually under the control, perhaps can with this regulation and control uncoupling.Another kind of approach is that this expression of gene can place one or more permissions to regulate under the control of the induced element of expressing.The example of this adjusting includes but not limited to by the inducing of temperature transition, and adds selected carbohydrate or derivatives thereof etc. and induces substrate, trace elements, VITAMIN, cofactor, metal ion etc.

Above-mentioned change can import by several different modes.Changing the sequence that participates in genetic expression can select desired phenotype to realize by random mutagenesis in the body then.Another kind of approach is the separate targets zone, changes it by random mutagenesis or fixed point displacement, insertion or deletion mutagenesis.The zone that changes can import bacterial genomes again by homologous recombination, and the effect of genetic expression can be estimated.Another kind of approach is that the sequence knowledge of target area can be used to replace or lack all or part of of natural regulating and controlling sequence.In this case, separate and change target control region, to comprise controlling element from other gene, from the combination of heterogeneic controlling element, synthetic control region, or any other control region, or the selected part of disappearance wild-type regulating and controlling sequence.The sequence of these changes can be imported in the bacterial genomes again by homologous recombination.The limiting examples that can be used for the preferred promoter that up-regulated gene expresses comprises the promotor proA from Neisseria meningitidis or gonococcus, proB, 1bpB, tbpB, p110,1st, hpuAB; TbpB from M.Catarrhalis; From the p1 of hemophilus influenzae, p2, p4, p5, p6,1pD, tbpB, D15, Hia, Hmw1, Hmw2.

In an example, genetic expression can be by changing its promotor into stronger promotor (by separating the upstream sequence of this gene, this sequence of external modification is by homologous recombination again in the quiding gene group) change.The expression of raising can realize in bacterium and the outer membrane vesicles from this bacterium.

In an example, above-mentioned approach can be used for producing the improved recombinant bacteria bacterial strain of performance in the vaccine application.These can be attenuated strains, the bacterial strain that selected antigenic expression increases, and the bacterial strain of the gene knockout of intervention immunne response (or express and reduce), the bacterial strain that the proteic expression of immunodominance changes, the bacterial strain that comes off and change of outer membrane vesicles, but be not limited thereto.

Therefore, the present invention also provides the upstream of the change of BASB052 gene, and the allos controlling element is also contained in the zone of these changes, and its change is positioned at the proteic expression level of BASB052 of outer membrane protein.The upstream of this respect of the present invention comprises the upstream sequence of BASB052 gene.Upstream starts from the upstream of BASB052 gene, extends to the ATG upstream from start codon usually less than about 1000bp place.When gene was arranged in polycistron sequence (operon), upstream can just start from before the goal gene, or before first gene of operon.Preferably, the change upstream of this respect of the present invention contains and is positioned at ATG upstream 500 allogeneic promoter to the 700bp position.

Therefore, the invention provides BASB052 polypeptide in the bacterium vesicle that changes.The present invention also provides the modification host cell that can produce based on the vesicle carrier of non-life film.The present invention also provides the nucleic acid carrier that contains the BASB052 gene, and described gene has the change upstream that contains the allos controlling element.

The present invention also provides the method for preparation host cell of the present invention and bacterium vesicle.

The present invention also provides and contains polypeptide of the present invention and/or polynucleotide and immunomodulatory dna sequence dna such as Sato, the composition, particularly vaccine composition of the dna sequence dna of introducing among Y. " science " 273:352 (1996), and method.

The present invention also is provided in the polynucleotide works used in this inherited immunity experiment of Neisseria meningitidis animal models infected and uses polynucleotide or its special segmental method of being introduced, wherein polynucleotide or its special fragment non-variable region of code displaying bacterial cell surface protein.This experiment can be used in particular for identifying and can excite albumen epi-position preventative or that therapeutic immunization is replied.Can believe, this method will can be used in subsequently by the essential organ of successfully having resisted or removed infected animals, preparation has the monoclonal antibody of special value, is used to develop particularly people's the treatment bacterial infection preventative reagent or the therapeutic agent that infect of Neisseria particularly of Mammals.

The present invention also comprises vaccine preparation, and this preparation comprises a kind of immunogenicity recombinant polypeptide of the present invention and/or polynucleotide and a kind of suitable carriers, for example a kind of pharmaceutically acceptable carrier.Because polypeptide and polynucleotide may can be interrupted under one's belt, they are all preferably through the enteron aisle external administration, comprise such as subcutaneous, muscle, vein or intracutaneous, the preparation that is applicable to the enteron aisle external administration comprises the aseptic injectable solution of water and non-water, they can contain antioxidant, damping fluid, fungistat and can make the preferably isoosmotic solvent of blood of preparation and individual body fluid, and the water and the non-water sterile suspension that can comprise suspension agent or thickening material.Preparation can place in the container of single dose or multiple doses, Mi Feng peace bottle and bottle for example, and can be stored in cryodesiccated environment, only need add aseptic liquid vehicle before use.

Vaccine preparation of the present invention also can comprise adjuvant system, is used for strengthening the immunogenicity of preparation.The preferred adjuvant system preferentially causes the TH1 type and replys.

Immunne response can be divided into two typical kinds substantially, i.e. body fluid or cell-mediated immune responses (generally distinguishing with the antibody and the cytological effect mechanism of its provide protection respectively).These are replied kind and are called as the TH1 type and reply (cell-mediated replys) and TH2 type immunne response (humoral response).

The feature of typical TH1 type immunne response is cytotoxic T cell and the natural killer type cell response that produces the haplotype restriction of antigen-specific.In mouse, the feature that the TH1 type is replied normally produces the antibody of IgG2a hypotype, and in the people, their counterpart is an IgG1 type antibody.The feature of TH2 type immunne response is the immunoglobulin (Ig) isotype that produces wide spectrum, comprises IgG1, IgA and IgM in mouse.

Can imagine to obtain that the motivating force after this immunne response of two types is a cytokine.High-caliber TH1 cytokines is preferentially induced at the antigenic cell-mediated immune responses of give, and high-caliber TH2 cytokines is preferentially induced at antigenic humoral immunoresponse(HI).

The differentiation of TH1 and TH2 type immunne response is not absolute.In fact, body can be supported the immunne response of a kind of TH1 of being described to advantage or TH2 advantage one by one.But, usually easily according to Mosmann and Coffman the introduction in mouse CD4+ve T cell clone consider the (Mosmann of family of cytokine, T.R. and Coffman, R.L. (1989), TH1 and TH2 cell: the different secretion patterns of lymphokine cause different functional performances." immunology yearbook " 7 volumes, the 145-173 page or leaf).Usually, the TH1 type is replied relevant with the IL-2 cytokine with T lymphocyte generation INF-γ.Other usually directly can't help the generation of T cell with TH1 type immune response inducing related cytokine such as IL-12.On the contrary, the TH2 type is replied relevant with the secretion of IL-4, IL-5, IL-6 and IL-13.

Known particular vaccine adjuvant is particularly suitable for stimulating TH1 or TH2 cytokines to reply.The TH1 that vaccine inoculation or premunition are replied: TH2 equilibrated optimal parameter is usually included in external use antigen stimulates the back directly to measure TH1 or the TH2 cytokine that the T lymphocyte produces again, and/or measures the IgG1 of the antibody response of antigen-specific: the IgG2a ratio.

Therefore, TH1 type adjuvant is the adjuvant that the preferential immunoglobulin (Ig) that stimulates isolating T cell colony to produce high-caliber TH1 cytokines and promotion CD8+ cytotoxic T cell and the antigen-specific relevant with TH1 type isotype is replied generation when external use antigen stimulates again.

Can preferentially stimulate the adjuvant of TH1 cell response in international patent application No.WO94/00153 and WO95/17209, to introduce.

The single phosphinylidyne lipoid A of 3 De-O-acidylates (3D-MPL) are a kind of such adjuvants.This can be known by GB2220211 (Ribi).It is at the mixture that chemically is single phosphinylidyne lipoid A of 3 De-O acidylates and 4,5 or 6 acyl chains, and by Ribi Immunochem, Montana makes.A kind of preferred form of the single phosphinylidyne lipoid of 3De-O-acidylate A is open in European patent 0 689 454 (SmithKline Beecham Biologicals SA).

The 3D-MPL particle preferably is small enough to carry out filtration sterilization (European patent 0 689 454) by one 0.22 μ m millipore filtration.

3D-MPL is with every dose 10 μ g-100 μ g, and preferably the scope of 20-25 μ g exists, and antigen exists with the scope of every dose of 2-50 μ g usually.

Another preferred adjuvants comprises QS21, and it is a kind of nontoxic component of Hplc purifying of the stem from Quillaja SaponariaMolina.Its optional being used for is mixed with the single phosphinylidyne lipoid A of 3 De-O-acidylates (3D-MPL), and can use with a kind of carrier.

The method for preparing QS21 is in U.S. Patent No. 5,057, and is open in 540.

Contain the former existing introduction of non-reacted adjuvant formulation (WO96/33739) of QS21.Contain QS21 and cholesteric this preparation and when preparing, shown it is that successful TH1 stimulates adjuvant with antigen.

The further adjuvant of preferential stimulation TH1 type cell response comprises the oligonucleotide of immunomodulatory, and for example non-methylated CpG sequence is as disclosing among the WO96/02555.

The combination of the adjuvant that different TH1 pungency adjuvants are introduced as mentioned also is considered to provide a kind of adjuvant of preferential stimulation TH1 type cell response.For example, QS21 can with the 3D-MPL formulated in combination.The ratio of OS21:3D-MPL is generally 1: 10 to 10: 1, is preferably 1: 5 to 5: 1, and is typically about 1: 1.Preferred best of breed scope is 2.5: 1 to 1: 1 3D-MPL:QS21.

According to the present invention, vaccine composition also preferably contains a kind of carrier.Carrier can be a kind of oil-in-water emulsion, or a kind of aluminium salt such as aluminum phosphate or aluminium hydroxide.

Oil-in-water emulsion preferably comprises a kind of metabolizable oil, for example squalene, alpha-tocopherol and Tween 80.One particularly preferred aspect, according to the present invention, antigen in the vaccine composition and QS21 and 3D-MPL make up in a kind of like this emulsion.In addition, this oil-in-water emulsion can contain span 85 and/or Yelkin TTS and/or tricaprylin.

To people's administration the time, the scope of QS21 and 3D-MPL is every dose 1 μ g-200 μ g in the vaccine, for example 10-100 μ g, preferably 10 μ g-50 μ g.Oil-in-water contains 2 to 10% squalene, 2 to 10% alpha-tocopherol and 0.3 to 3% Tween 80 usually.When providing with more stable emulsion form, the ratio of squalene: alpha-tocopherol: Tween 80 is preferred identical or be less than 1.The Span85 that also can comprise 1% level.In some cases, vaccine of the present invention preferably further contains a kind of stablizer.

Atoxic oil-in-water emulsion preferably contains a kind of atoxic oil such as squalane or squalene, a kind of emulsifying agent such as Tween 80 in a kind of water carrier.Water carrier can be a phosphate-buffered saline for example.

Introduced a kind of strong especially adjuvant formulation among the WO95/17210, it contains QS21,3D-MPL and tocopherol in a kind of oil-in-water emulsion.

The present invention also provides a kind of polyvalent vaccine composition, and it contains vaccine preparation of the present invention and other antigen, particularly to treatment cancer, autoimmune disease and the useful antigen of associated conditions.A kind of like this multivalent vaccine composition can comprise a kind of foregoing TH1 induction type adjuvant.

Though the present invention introduces specific BASB052 polypeptide and polynucleotide, but be to be understood that polypeptide that they have covered natural generation and polynucleotide and the similar polypeptide and the polynucleotide that contain interpolation, disappearance or replace, these add, disappearance or replace the immunogen feature that does not influence recombinant polypeptide or polynucleotide basically.

Antigen also can intact bacterial (dead or live) or the form of subcellular components is transmitted, and these possibilities comprise Neisseria meningitidis self.Composition, test kit and administration

Of the present invention one further aspect, provide to contain to be useful on to the BASB052 polynucleotide of a cell or a multicellular organisms administration and/or the composition of BASB052 polypeptide.

The invention still further relates to the composition that contains polynucleotide discussed here and/or polypeptide or their agonist or antagonist.Polypeptide of the present invention and polynucleotide can or be used for the carrier of cell, tissue or organism with a kind of non-sterile or sterile carrier, for example a kind ofly are applicable to that the pharmaceutical carrier to individual administration is used in combination.This composition comprises for example a kind of medium additive or a kind of polypeptide of the present invention and/or polynucleotide and a kind of pharmaceutically acceptable carrier or vehicle for the treatment of effective dose.This carrier can include but not limited to salt solution, buffer saline, glucose, water, glycerine, ethanol and their composition.Preparation should be suitable for mode of administration.The invention further relates to diagnosis and pharmaceutical pack and test kit, they contain one or more containers, and one or more compositions above-mentioned of the present invention are housed in the container.

Polypeptide of the present invention, polynucleotide and other compounds can be separately or with other compounds for example therapeutic compound use.

Medicinal compositions can carry out administration by any effective, traditional mode, comprise such as by in part, mouth, anus, vagina, intravenously, intraperitoneal, muscle, subcutaneous, the nose or intradermal routes and other approach carry out administration.

When being used for the treatment of or prevent, the form administration that active agent can a kind of injectable composition is in individuality, for example with the preferably isoosmotic form administration of aseptic water dispersion.

One further aspect, the invention provides medicinal compositions, it comprises a kind of polypeptide of effective dose and/or soluble form, agonist or antagonist or micromolecular compound and a kind of pharmaceutically acceptable carrier or vehicle of polynucleotide polypeptide for example of the present invention and/or polynucleotide for the treatment of.This carrier includes but not limited to salt solution, buffer saline, glucose, water, glycerine, ethanol and their composition.The invention further relates to pharmaceutical pack and test kit, they contain one or more containers, and one or more compositions above-mentioned of the present invention are housed in the container.Polypeptide of the present invention, polynucleotide and other compounds can be separately or with other compounds for example therapeutic compound use.

Composition should adopt a kind of route of administration, for example by a kind of general or per os approach.The preferred form of general administration comprises injection, usually by intravenous injection.Other injecting pathway is for example subcutaneous, muscle or peritonaeum can adopt.The additive method of general administration comprises and uses permeate agent such as cholate or fusidinic acid or other stain removers through mucous membrane and percutaneous drug delivery.In addition, if a peptide species of the present invention or other compounds can be mixed with a kind of enteric or a kind of capsule preparations, the per os approach also can use.The administration of these compounds also can be used with forms such as ointment, obedient cream, gel, solution, powder through epidermis and/or part.

For to Mammals particularly the people carry out administration, dosage level every day of active agent should be generally about 1mg/kg from 0.01mg/kg to 10mg/kg.Under any circumstance, the doctor should determine to be suitable for most individual actual dose, and according to age, body weight and replying of special entity and different.Certainly, when individual instances, can use higher or lower dosage range, these also all within the scope of the invention.

Required dosage range depends on the character of selected peptide, route of administration, preparation, the character of medication person's illness and doctor's judgement.But proper dosage is the scope of every kg 0.1-100 μ g.

Vaccine composition generally adopts injection form.Traditional adjuvant can be used to enhancing immunity and replys.The appropriate units dosage of vaccine inoculation is the antigen of 0.5-5 μ g/kg, the preferred administration of such dosage 1-3 time, interval 1-3 week.For specified dosage range, compound of the present invention is delivering medicine to suitablely when individual, can not observe toxic side effect.

But consider and to use different compounds and different way of administration can produce different effects that required dosage has variation widely.For example, oral route needs higher dosage than intravenous injection.The variation of these dosage levels can use the optimization of standard to put into practice approach adjustment according to method well known in the art.Sequence in sequence library, the tangible medium and algorithm

Polynucleotide and peptide sequence have been formed valuable information source, are used for determining their 2 and 3 dimensional organization and further identifying similar homologous sequence.These methods can be undertaken by following step easily, are about to them and deposit computer-readable medium in, then data are deposited in research tool such as sequence library of GCG routine package search that a known macromolecular structure program or use are known.

The present invention also provides the particularly method of gene order or coding protein sequence of analytical characteristic sequence or chain.Preferred sequencing technique comprises: such as sequence homology analysis method such as identity with similarity analysis, DNA, RNA and protein structure analysis, series arrangement, evolutionary analysis, sequence motif analysis, open reading frame are determined, nucleic acid base calling, codon operational analysis, nucleic acid base arrangement and the analysis of sequence chromatographic peak.

The invention provides a kind of method, be used to carry out homology and identify based on computer.This method may further comprise the steps: be provided at first polynucleotide sequence that contains a kind of polynucleotide sequence of the present invention in a kind of computer-readable medium, described first polynucleotide and at least a second polynucleotide or peptide sequence are compared, determine homology.

The invention provides a kind of method, be used to carry out homology and identify based on computer.Said method comprising the steps of: be provided at first peptide sequence that contains a kind of peptide sequence of the present invention in a kind of computer-readable medium, described first peptide sequence and at least a second polynucleotide or peptide sequence are compared, determine homology.

All publications and the reference quoted in the application's book include but not limited to patent and patent application, here all are incorporated herein by reference in full, all list in full at this especially and individually separately as them.The application requires any patent application of right of priority also to be incorporated herein by reference in full at this to publication with reference to the same way as of being introduced with top.Definition

" identity " is the dependency between two or more peptide sequences or two or more polynucleotide sequences as known in the art, as the case may be, can relatively come to determine by sequence.In this area, " identity " also refers to the degree that sequence between polypeptide or the polynucleotide sequence is relevant, and as the case may be, the coupling of chain that can be by these sequences is determined." identity " can use known method to calculate easily, includes but not limited to that (A.M. edits for " calculating molecular biology ", Lesk, Oxford University Press, New York, 1988; " biocomputer: information and genome plan " Smith, D.W. edits, Academic Press, New York, 1993; " Computer Analysis of sequence data " I part, Griffin, A.M. and Griffin, H.G. edits, HumanaPress, New Jersey, 1994; " sequential analysis in the molecular biology " von Heine, G., Academic Press, 1987; " sequence analysis primer " Gribskov, M. and Devereux, J. edits, M Stockton Press, New York, 1991; And Carillo, H. and Lipman, D., " SIAM J.Applied Math " 48:1073 (1988).The method of measuring identity is designed to provide the maximum match that detects between the sequence.And, measure the method for identity and in the obtainable computer program of the public, encode.The computer program means that is used for measuring two kinds of identity between the sequence includes but not limited to the GAP program (Devereux in the GCG routine package, " nucleic acids research " 12 (1) such as J.: 387 (1984), BLASTP, BLASTN (Altschul, " molecular biology magazine " 215:403-410 such as S.F. (1990) and FASTA (Pearson and Lipman " PNAS " 85:2444-2448 (1988).Blast program family can obtain from NCBI and other source (" BLAST handbook Altschul, S. etc., NCBI NLM NIH Bethesda, MD 20894; Altschul, " molecular biology magazine " 215:403-410 such as S. (1990).Famous Smith Waterman algorithm also can be used to measure identity.

The parameter of peptide sequence comparison comprises following:

Algorithm: Needleman and Wunsch, " molecular biology magazine " 48:443-453 (1970)

The BLOSSUM62 of comparator matrix: Henikoff and Henikoff

" PNAS " 89:10915-10919 (1992)

Breach compensation: 8

Notch length compensation: 2

Use the program of these parameters can be from Genetics Computer Group, Madison WI obtains with the form of " breach " program.Above-mentioned parameter is a polypeptide default parameter (uncompensated to terminal breach) relatively.

The parameter of polynucleotide sequence comparison comprises following:

Comparator matrix: coupling=+ 10, do not match=0

Breach compensation: 50

Notch length compensation: 3

Source: Genetics Computer Group, " breach " program of Madison WI.Above-mentioned parameter is a nucleic acid default parameter relatively.

The preferred meaning of " identity " of polynucleotide and polypeptide provides in (1) and (2) according to circumstances below.

(1) the polynucleotide embodiment further comprises the separation polynucleotide that contain following nucleotide sequence, the reference sequences of this nucleotide sequence and SEQ ID NO:1 has 50 at least, 60,70,80,85,90,95,97 or 100% identity, wherein said polynucleotide sequence can be identical with the reference sequences of SEQ ID NO:1, perhaps compare with reference sequences, the Nucleotide that can comprise some amount changes, wherein this change can be selected from least one nucleotide deletion, displacement (comprising conversion and transversion) or insertion, wherein said change can occur in reference to any position between 5 of polynucleotide sequence ' or 3 ' terminal or two ends, be dispersed in respectively between the Nucleotide of canonical sequence, perhaps be dispersed in the canonical sequence with one or more contigs, wherein the quantity of Nucleotide change is following determines: the total nucleotide number among the SEQ ID NO:1 multiply by corresponding identity percentage ratio (divided by 100), then its result is reduced from the total nucleotide number of SEQ ID NO:1, perhaps:

n _n≤x _n-(x _n·y)

N wherein _nBe the number that Nucleotide changes, x _nIt is the Nucleotide sum among the SEQ ID NO:1, v is 0.50 to 50%, to 60% being 0.60, is 0.70 to 70%, to 80% is 0.80, to 85% being 0.85,, be 0.95 to 95% to 90% being 0.90, to 97% is 0.97, to 100% being 1.00, but the symbol of multiplying, to x _nWith the result of y be not integer, then it is rounded up and gets nearest integer, again from x _nMiddle subduction.The change of the polynucleotide of the polypeptide of coding SEQ ID NO:2 may produce nonsense, missense or phase shift mutation in this encoding sequence, the polypeptide of the polynucleotide encoding after therefore changing can change.

For example, polynucleotide sequence of the present invention can be identical with the canonical sequence of SEQ ID NO:1, and promptly identity is 100%, perhaps compare the Nucleotide that comprises some amount with canonical sequence and change, so identity is less than 100%.This change can be selected from least one nucleotide deletion, displacement (comprising conversion and transversion) or insert, wherein said change can occur in reference to any position between 5 of polynucleotide sequence ' or 3 ' terminal or two ends, be dispersed in respectively between the Nucleotide of canonical sequence, perhaps be dispersed in the canonical sequence with one or more contigs.The quantity that Nucleotide changes is following to be determined: the total nucleotide number among the SEQ ID NO:1 multiply by corresponding identity percentage ratio (divided by 100), then its result is reduced from the total nucleotide number of SEQ ID NO:1, perhaps:

n _n≤x _n-(x _n·y)

N wherein _nFor Nucleotide changes quantity, x _nIf be the total nucleotide number of SEQ ID NO:1, the v value was 0.70 at 70% o'clock, was 0.80 at 80% o'clock, was 0.85 at 85% o'clock, and the rest may be inferred, is the symbol of multiplying, wherein x _nWith the result of y be not integer, then it is rounded up and gets nearest integer, again from x _nMiddle subduction.

(2) the polypeptide embodiment further comprises the isolated polypeptide that contains following peptide sequence, the reference sequences of this peptide sequence and SEQ ID NO:2 has 50 at least, 60,70,80,85,90,95,97 or 100% identity, wherein said peptide sequence can be identical with the reference sequences of SEQ IDNO:2, perhaps compare with reference sequences, the amino acid change that can comprise some amount, wherein said change is selected from least one aminoacid deletion, replace (comprising that conservative property or non-conservation replace) or insertion, and described change can occur between the amino of reference polypeptide sequence or C-terminal position or these terminal positions Anywhere, be dispersed in respectively in the amino acid of reference sequences or and be dispersed in the reference sequences with one or more contigs, and the number of described amino acid change is following to be determined: the total amino acid number among the SEQ ID NO:2 multiply by corresponding identity percentage ratio (divided by 100), then its result is reduced from the total amino acid number of SEQ ID NO:2, perhaps:

n _a≤x _a-(x _a·y)

N wherein _aBe amino acid change quantity, x _aBe the amino acid sum among the SEQ ID NO:2, v is 0.50 to 50%, to 60% being 0.60, is 0.70 to 70%, to 80% is 0.80, to 85% being 0.85,, be 0.95 to 95% to 90% being 0.90, to 97% is 0.97, to 100% being 1.00, but the symbol of multiplying, if x _aWith the result of y be not integer, then it is rounded up and gets nearest integer, again from x _aMiddle subduction.

For example, peptide sequence of the present invention can be identical with the canonical sequence of SEQ ID NO:2, and promptly identity is 100%, perhaps compare the amino acid change that comprises some amount with canonical sequence, so identity is less than 100%.This change can be selected from least one aminoacid deletion, displacement (comprising conservative and non-conservative substitution) or insert, wherein said change can occur in reference to any position between the amino of peptide sequence or C-terminal or two ends, be dispersed in respectively between the amino acid of canonical sequence, perhaps be dispersed in the canonical sequence with one or more contigs.The quantity of identity percentage ratio one timing amino acid change is following to be determined: the total amino acid number among the SEQ ID NO:2 multiply by corresponding identity percentage ratio (divided by 100), then its result is reduced from the total amino acid number of SEQ ID NO:2, perhaps:

n _a≤x _a-(x _a·y)

N wherein _aBe amino acid change quantity, x _aBe the total amino acid number of SEQ ID NO:2, the v value was 0.70 at 70% o'clock, was 0.80 at 80% o'clock, was 0.85 at 85% o'clock, and the rest may be inferred, but the symbol of multiplying, wherein if x _aWith the result of y be not integer, then it is rounded up and gets nearest integer, again from x _aMiddle subduction.

" individuality " is meant a kind of many cells eukaryote when being used for indicating a kind of organism herein, include but not limited to metazoan, Mammals, oviparous animal, Bovidae, ape, primate and people.

" isolating " expression " by artificial " changes its native state, if promptly " isolating " composition or material exist at occurring in nature, then it changes or take out or taken out and change from its primal environment.For example, when using this term in this article, polynucleotide or the polypeptide that exists in moving object is not " isolating ", but with native state under the material that coexists the as much Nucleotide or the polypeptide that separate be exactly " isolating ".And a kind of polynucleotide or polypeptide are when importing a kind of organism by conversion, genetic manipulation or any other recombination method, even it still is present in the described organism, it also is " separation ", and this organism can be that live or dead.

" polynucleotide " refer generally to any polyribonucleotide or polydeoxyribonucleotide, and it can be the RNA or the DNA of modification or non-modification, comprises strand and double-stranded region.

" variant " refers to be different from reference to polynucleotide or polypeptide but kept the polynucleotide or the polypeptide of fundamental characteristics.The nucleotide sequence of general polynucleotide variant is with different with reference to polynucleotide.The variation of variant nucleotide sequence may change or not change the amino acid sequence of polypeptide by the reference polynucleotide encoding.As described below, Nucleotide changes may cause amino acid whose displacement, interpolation, disappearance, fusion and brachymemma in the canonical sequence encoded polypeptides.The aminoacid sequence of general polypeptide variants is with different with reference to polypeptide.Usually, difference is limited, and therefore the sequence with reference to polypeptide and variant is extremely proximate on the whole, is identical in many zones.Variant and one or more displacements, the interpolation of any array configuration, the difference of disappearance can be arranged on aminoacid sequence with reference to polypeptide.Displacement or the amino-acid residue that inserts can yes or no genetic code amino acids coding.Polynucleotide or variant polypeptides can be naturally occurring, as allelic variant, or the non-natural existence.Polynucleotide that non-natural exists and polypeptide variants can be by induced-mutation technique or directly synthetic preparations.

" disease " is meant and anyly caused or diseases associated with it by infectation of bacteria, comprise such as upper respiratory tract infection, aggressive bacterial disease such as microbemia and meningitis.

Embodiment

The following examples use standard techniques to carry out, these technology be to one skilled in the art know with routine, unless they are introduced in detail at elsewhere.Embodiment is illustrative, can not limit the present invention.Embodiment 1: the BASB052 sequence among the meningitis Nai Seshi serotypes B strains A TCC13090

The BASB052 gene order of Neisseria meningitidis strains A TCC13090 is shown in SEQID NO:1.The translation result of the BASB052 polynucleotide sequence shown in the SEQ ID NO:2 and gonococcus tcp albumen have amino acid sequence homology.The BASB052 polypeptide contains the distinctive signal sequence of lipoprotein.Embodiment 2: the clone of the structure A:BASB052 of the plasmid of express recombinant BASB052

With NdeI and XhoI restriction site insert respectively forward Lip10-Fm/p (5 '-AGG CAGAGG CAT ATG TCC GAA AAC AAA CAA AAC GAA GTC-3 ', SEQ IDNO:3) and oppositely Lip10-RCf/p (5 '-AGG CAG AGG CTC GAG TTC ATT CGTTAC CTG ACC GGC GAT GCC GTG-3 ', SEQ ID NO:4) amplimer, they allow BASB052 PCR product directed cloning to low copy colibacillus expression plasmid pTLZ2, make BASB052 albumen can be expressed as the fusion rotein that C-terminal contains (His) 6 affinity chromatography signs.BASB052 PCR product uses silica gel base spin post (QiaGen) to illustrate by purifying in the amplified production according to manufacturer.In order to produce clone required NdeI and XhoI end, the PCR product of purifying illustrates that according to manufacturer (Life Technologies) digestion fully successively with NdeI and XhoI restriction enzyme.

After the restriction digestion for the first time, the PCR product is the same to desalt to remove by the spin column purification, before the enzymic digestion second time, uses the sterilized water wash-out.The dna fragmentation of digestion with the post purifying once more that spins of use silica gel base before the pTLZ2 plasmid is connected.B: expression vector preparation

In order to prepare the expression plasmid pTLZ2 that connects usefulness, it is complete with NdeI and the same digestion of XhoI, handle to prevent that self from connecting with Roll Phosphoric acid esterase (CIP, about 0.02 unit/picomole 5 ' end, Life Technologies) according to manufacturer's explanation then.The digestion fragment of the about 5 times of molar excess of carrier of preparation relatively is used for carrying out ligation.20 μ l ligations of standard (about 16 ℃, about 16 hours) utilize technology known in the art to use T4 dna ligase (about 2.0 unit/reactions, Life Technologies) to carry out.The aliquots containig of ligation (about 5 μ l) is used for transforming electroreception attitude JM109 cell according to technology known in the art.Growth is after about 2-3 hour under 37 ℃ in about 1.0 milliliters of LB nutrient solutions, and transformant is coated on the LB agar plate that contains penbritin (100 μ g/ml).In selecting substratum, comprise that microbiotic carries pTLZ2 plasmid (ApR) to guarantee all transformants.Dull and stereotyped about 16 hours of 37 ℃ of incubations.Independent ApR bacterium colony is chosen with aseptic toothpick, the dull and stereotyped and about 1.0ml LB ApRR nutrient solution of the fresh LB ApR of " sheet " inoculation.Dull and stereotyped and the nutrient solution 37 ℃ of incubations on standard incubation case (flat board) or shaking bath of sheet.

Carry out full cell pcr analysis and contain BASB052 DNA inset with the conclusive evidence transformant.About 1.0 milliliters of LB Ap overnight culture are transferred in the 1.5 ml polypropylene pipes, and centrifugal collecting cell in the Beckman whizzer (about 3 minutes, room temperature, about 12000 * g).Cell precipitation is resuspended in about 200 μ l sterilized waters, and about 10 μ l aliquots containigs are used to carry out the PCR reaction of the about 50 μ l of final volume, wherein contains BASB052 forward and reverse amplimer.The final concentration of PCR reacted constituent basically with embodiment 2 in identical, but use about 5 Taq of unit polysaccharases.95 ℃ of denaturing steps of beginning are increased to 3 minutes with the thermo-cracking of guaranteeing bacterial cell and the release of plasmid DNA.Use ABI 9700 type thermal cyclers, carry out the 3 step thermal cyclings of 32 round-robin, cycling condition is: 95 ℃ 45 seconds; 55-58 ℃ 45 seconds; 72 ℃ 1 minute, with the BASB052 PCR fragment of amplification cracked transformant sample.After the thermal cycling, get about 20 μ l reaction aliquot and analyze by agarose gel electrophoresis (0.8% agarose in Tris-acetate-EDTA (TAE) damping fluid).Use UV irradiation and ethidium bromide staining to show dna fragmentation after the gel electrophoresis.The parallel electrophoresis with sample of dna molecular amount standard (1Kb gradient, Life Technologies) is used to estimate the size of PCR product.The transformant that produces expection PCR product is accredited as the bacterial strain that contains the BASB052 expression construct.Analyze the abduction delivering of reorganization BASB052 in the bacterial strain that contains expression plasmid then.The expression analysis of C:PCR positive transformant

For each PCR positive transformant of above evaluation, the cell inoculation of about 5 milliliters of LB nutrient solutions that contain penbritin (100 μ g/ml) from the sheet flat board, 37 ℃ of vibrations (about 250rpm) grow overnight.Aliquots containig (about 1.0ml) inoculation of inoculum of spending the night contains in 125 ml flasks of the 25 milliliters of LB Ap nutrient solutions of having an appointment, 37 ℃ of vibrations (about 250rpm) grow overnight, reach O.D.600 until the culture turbidity and be about 0.5, promptly middle logarithmic phase (needing about 1.5 to 2.0 hours usually).At this moment, general approximately culture (about 12.5ml) is transferred in second 125 ml flasks, and (the 1.0M stoste for preparing in the sterilized water Sigma) is induced the proteic expression of reorganization BASB052 to final concentration 1.0mM by adding IPTG.IPTG induces and inducing culture thing about 4 hours of the incubation that vibrates again under 37 ℃ not.Induce and not inducing culture matter sample (about 1.0 milliliters) after inductive phase, take out, under the room temperature in whizzer centrifugal about 3 minutes collecting cells.Independent cell precipitation is suspended in about 50 μ l sterilized waters, mixes with 2 * Laemmli SDS-PAGE sample buffer that equal-volume contains 2 mercapto ethanol then, places about 3 minutes of boiling water bath with metaprotein.Equal-volume (about 15 μ l) IPTG induce and not the thick lysate of inducing cell be splined on two 12% Tris/ glycine polyacrylamide gels (the thick Mini-gels of 1mm, Novex).(SeeBlue, Novex) next plays electrophoresis in normal condition, uses standard SDS/Tris/ glycine electrophoretic buffer (BioRad) to induce and do not induce lysate sample and prestained molecular weight marker.Behind the electrophoresis, a clotting glue decolours to show new BASB052 IPTG inducible protein then with coomassie brilliant blue R250 (BioRad) dyeing.The second clotting glue use BioRad Mini-Protean II blotter and Towbin methyl alcohol (20%) transfering buffering liquid 4 ℃ of following electroblottings transferred in about 2 hours pvdf membrane (0.45 micron pore size, Novex).The sealing of film and antibody incubation carry out according to technology known in the art.Using monoclonal anti (His) 5 antibody, is the anti-mouse antibodies of rabbit that stops and close HRP (QiaGen) then, with conclusive evidence BASB052 Recombinant Protein Expression and identity.Use the insoluble substrate of ABT or use the Hyperfilm of Amersham ECL chemiluminescence system to show anti-His antibody response feature.Embodiment 3: reorganization BASB052 fatization (lipidation) state

In order to determine whether fatization of BASB052 recombinant protein, use ³H-glycerine and ³The H-palmitinic acid mixes mark as the fat specificity and carries out the radio-labeled experiment.The BL21DE3 coli strain culture that contains expression plasmid is grown on the M9 minimum medium, with 50 μ Ci ³H-glycerine or ³The H-palmitinic acid carries out pulse labelling at 3 hours IPTG between inductive phase.After uncorporated isotropic substance was removed in washing, the full cell lysate of radio-labeled culture was gone up electrophoresis at gradient polyacrylamide gel (4-20%), and is fixing, dry then.At 70 ℃ xerogel was produced autoradiogram(ARGM) in about 7 days to Hyperfilm (Amersham) exposure.Two kinds of marks have all produced and the IPTG inductive Xylene Brilliant Cyanine G identical visible band of albumen size that can dye.Be incorporated into the IPTG inducible protein ³BASB052 albumen has the conclusion of fatization to a certain degree in the H-mark support intestinal bacteria.Embodiment 4: produce reorganization BASB052

Bacterial isolates

The recombinant expressed cell colony that is used to produce purification of recombinant proteins of e. coli jm109 that contains the pTLZ2 plasmid of coding Neisseria meningitidis BASB052.Expression strain is cultivated on the LB agar plate, contains 100 μ g/ml penbritins (Ap) in the flat board and exists to guarantee plasmid.For-80 ℃ of down refrigerations, bacterial strain is bred in containing the antibiotic LB nutrient solution of same concentrations, mixes with the LB nutrient solution that equal-volume contains 30% (w/v) glycerine then.

Substratum

The fermention medium that is used to produce recombinant protein is the 2X YT nutrient solution (Difco) that contains 100 μ g/ml Ap.Substratum in fermentor tank add defoamer to 0.25ml/L (Antifoam204, Sigma).In order to induce the BASB052 Recombinant Protein Expression, in fermentor tank, add IPTG (isopropyl ss-D-sulfo-semi-lactosi pyrans glycosides) (1mM, final concentration).

Fermentation

Contain 0.3 milliliter of freezing culture that thaws rapidly of inoculation in 500 milliliters of seed culture flasks of 50 milliliters of working volumes or come several bacterium colonies on the self-selectively agar plate, go up with 150rpm about 12 hours of 37 ± 1 ℃ of incubations at shaking table (Innova 2100, New Brunswick Scientific).This inoculum is used for inoculating that to contain 2X YT nutrient solution and Ap, working volume be 5 liters fermentor tank.The operational conditions of fermentor tank (Bioflo 3000, New Brunswick Scientific) is 37 ± 1 ℃, 0.2-0.4 VVM ventilation, Rushton impeller 250rpm.PH in flask inoculum and the fermentor tank need not control.Between yeast phase, the pH in the fermentor tank is 6.5 to 7.3.(O.D.600 is about 0.7 unit) adds IPTG (1.0M stoste prepares) in sterilized water in fermentor tank when culture reaches the middle logarithmic phase of growth.Inducing cell 2-4 hour, then, use 28RS Heraeus (Sepatech) or RC5C ultracentrifuge (Srovall Instruments) centrifugal collecting cell.Cell precipitation be kept at-20 ℃ stand-by.

Purifying

Imidazoles and biotechnology level or higher category reagent are all available from Ameresco Chemical, Solon, Ohio.Triton X-100 (uncle's octylphenoxy polyethoxy ethanol), Triton X-114, monovalence sodium phosphate, urea are SILVER REAGENT or more top grade is other, all available from Sigma ChemicalCompany, St.Louis, Missouri.(1 * PBS) available from Quality Biologicai, Inc., Gaithersburg, Maryland for Dulbecco ' s phosphate-buffered saline.(10 * PBS) available from BioWhittaker, Walkersville, Maryland for Dulbecco ' s phosphate-buffered saline.The five His antibody of no BSA are available from QiaGen, Valencia, California.Peroxidase stops the affine pure level goat anti-mouse igg that closes available from Jackson Immuno Research, West Grove, Penn.All other reagent are SILVER REAGENT or higher category.

Ni chelating Sepharose speed flows resin available from Pharmacia, Sweden.Prefabricated Tris-glycine 4-20% and 10-20% polyacrylamide gel, all electrophoretic buffers and solution, SeeBlue prestain standard, multiple labeling polychrome standard and PVDF transfer film be available from Novex, San Diego, California.SDS-PAGE silver transfection reagent box is available from Daiichi PureChemicals Company Limtid, Toyko, Japan.The coomassie dyeing solution is available from Bio-RadLaboratories, Hercules, California.Acrodisc  PF 0.2m syringe filter is available from Pall Gelman Science, Ann Arbor, Michigan.GD/X 25mm disposable syringe formula filter is available from Whatman Inc., Clifton, New Jersey.Dialysis tubing 8,000 MWCO are available from BioDesign Inc.Od New York, Carmak New York.BCA analysis of protein reagent and Snake Skin dialysis tubing 3,500 MWCO are available from Pierce Chemical Co.Rockford, Illinois.

Extraction step

Cell precipitation thawed 30 to 60 minutes in room temperature.Take by weighing in 5 to 6 gram material to the 50 milliliter disposable centrifuge tubes.Reorganization BASB052 antigen handles by N,O-Diacetylmuramidase and supersound process is extracted.The material of gained is centrifugal, and precipitation is suspended in 50 mM Tris pH of buffer 7.5 by supersound process, contains 8M urea, 20% glycerine and 0.005% triton X100.This material recentrifuge, precipitation is by nickel chelating Sepharose speed fluidization tower.Use 200 mM imidazoles eluted proteins then,, obtain to surpass 90% pure protein with the albumen of affinity purification histidine mark.This fraction that contains eluted protein is to containing PBS (pH7.4) dialysis of 0.5M arginine and 0.1% Triton X100.

Final preparation

BASB052 is to 0.1% Triton X-100 and 1 * PBS, and the pH7.4 dialysed overnight is changed 3 times dialyzate.Purification Identification albumen also as described belowly is used to produce antibody.

Biological chemistry is identified: SDS-PAGE and western blot analysis

The reorganization purifying protein separates on the 4-20% polyacrylamide gel, as previously mentioned under 100V 1 hour electrotransfer to pvdf membrane (Thebaine etc., 1979, institute of NAS periodical, 76:4350-4354).Pvdf membrane contains the Dulbecco ' s phosphate-buffered salt water pretreatment of 5% alipoidic milk power then with 25ml.All further incubations all use this pre-treatment damping fluid to carry out.

Pvdf membrane was with anti-His tail antibody diluent room temperature incubation 1 hour.Use lavation buffer solution (20 mM Tris damping fluids, pH7.5 contains 150mM sodium-chlor and 0.05%Tween-20) to wash film twice then.Stop compound room temperature incubation 30 minutes of the peroxidase labelling specific specificity of 25 milliliters of dilutions in 1: 5000 of pvdf membrane using then.Pvdf membrane is then with lavation buffer solution washing 4 times, with Zymed (San Francisco, the 3-that CA) provides amino-9-ethyl carbazole and urea peroxide colour developing.

SDS-PAGE result (Figure 1A) shows a purity greater than about 49kDa albumen of 90%, itself and anti-four His antibody responses in the Western blot of SDS-PAGE (Figure 1B).Embodiment 5: with reorganization BASB052 immune mouse

The partially purified injection at the 0th day, 14 days and 28 days three times at the reorganization BASB052 of expression in escherichia coli albumen given BalB/C mouse (10 animal/groups).Animal is injected by subcutaneous route, every dosage is 5 μ g left and right sides antigens, described antigen has two kinds of different dosage forms: perhaps be adsorbed on the 100 μ g aluminum phosphates, perhaps be formulated in the SBAS2 emulsion (every dose of SB62 emulsion that contains 5 μ g MPL and 1 μ g QS21).Also add negative control group in experiment, it is by only forming with SBAS2 emulsion mice immunized.Mouse is in the 28th day (14 days Post II) and (7 days Post III) bloodletting in 35 days, with the anti-BASB052 antibody of detection specificity.The anti-BASB052 antibody of specificity is measured by the Elisa of partially purified BASB052 albumen and e. coli protein.Antibody response is also undertaken by the Western blot to different Neisseria meningitidis B bacterial strains.Come the pooled serum (from 10 mouse/groups) of self-preparing agent group (only 7 days Post III) in these experiments, to measure.Result shown in Figure 2 clearly illustrates that antibody response is splendid, and Chinese People's Anti-Japanese Military and Political College's enterobacteria antibody response (Fig. 3) is replied (Fig. 2) low about 10 times although also be positive than specific b ASB052.The aluminum phosphate preparation is induced the highest antibody horizontal.Western blot shown in the Figure 4 and 5 confirms that BASB052 albumen is high-visible at the 50kDa MW place of expection.

By Western blot the BASB052 epi-position on the different Neisseria meningitidis bacterial strains is discerned

In this test, discern with the BASB052 epi-position on Neisseria meningitidis B bacterial strain different and the partially purified reorganization BASB052 albumen through the serum (pooled serum) of immune mouse by the Western blot detection 7 strains, 7 strain bacterial strains are: H44/76 (B:15:P1.7, pedigree ET-5), M97 250987 (B:4:P1.15), BZ10 (B:2b:P1.2, pedigree A4), BZ198 (B:NT ^*:-, pedigree 3), EG328 (B:NT ^*, pedigree ST-18), NGP165 (B:2a:P1.2,37 bunches of ET) and ATCC 13090 (B:15:P1.15) Neisseria meningitidis B bacterial strain.(*: NT: not somatotype).

In brief, will handle 10 μ l (＞10 of every kind of sample of (95 ℃ 10 minutes) with sample buffer ⁸Cell/swimming lane) places SDS-PAGE gradient gel (Tris-glycine 4-20%, Novex, code n ° EC60252).At 125 volts of electrophoresis 90 minutes (35mA/ gel).Use Bio-rad transferring system (code n ° 170-3930) through 1: 30 hour albumen to be transferred to nitrocellulose filter (0.45 μ m, code n ° 162-0114) then at 100 volts.Filter membrane spends the night in the room temperature sealing with PBS-0.05% Tween 20, and with the mice serum incubation that contains anti-BASB052 antibody, described serum source is from aluminum phosphate and SBAS2 preparation then.These serum dilute 100 times in PBS-0.05% Tween 20, and the incubation 2 hours of at room temperature vibrating on nitrocellulose filter.Nitrocellulose filter was washed 5 minutes in PBS-0.05% Tween 20, repeat this step 3 time, then with nitrocellulose filter with in identical lavation buffer solution by at room temperature vibrate gently incubation 1 hour of the suitable compound that stops (biotinylated goat anti-mouse Ig antibody, n ° of RPN1001 of Amersham code) of 1/500 dilution.By the introduction of front with film washing 3 times, and with in lavation buffer solution by the streptavidin-superoxide enzyme complex of 1/1000 dilution (Amersham code n ° 1051) vibration incubation 30 minutes.Through last 3 multiple washing steps, containing 30mg 4-chloro-1-naphthols (Sigma), 10ml methyl alcohol, 40ml PBS and 30 μ l H ₂O ₂50ml solution in incubation developed the color in 20 minutes.Film is washed color development stopping for several times in distilled water.

Result in the Figure 4 and 5 shows that all detect bacterial strain and all with about about 10kDa mainly are with reaction, and other has the band of minority higher molecular weight, and this may be also relevant with antigen.This explanation BASB052 albumen may all expressed in the Neisseria meningitidis serotypes B bacterial strain.In Fig. 4 and Fig. 5, the reorganization BASB052 albumen of mice serum identification has identical molecular weight.But, the minimum band of mice serum identification (＜9kDa) as if relevant with e. coli contamination, this can be confirmed by last swimming lane (Fig. 4 right-hand component) of intestinal bacteria extract.

BASB052SEQ ID NO：1ATCC13090BASB052ATGTCCGAAAACAAACAAAACGAAGTCCTGAGCGGTTACGAACAACTCAAACGGCGCAACCGCCGCCGTCTCGTAACGGCAAGCTGCCTGGTTGCCGCCTCCTGCCTGCTGCTGGCGGCCGCCCTCAGTTCCGATCCTGCCGAAAATCCCCCTGCCGCACCGACTGACGAAACAGGCAGCATGGAAAACCAAGCGGCAAGCACGGTACAAACCCCGACCTTAAAATCCGCCTCAGAAGGTGTTGAACCCGCCGCCGACAAACCTCAAGACCTGGCCAGTGAAGAACAAGCACCTGCCGCCGACAATGGAATCAGCGAACCTGAAGACGTAGGCGCACCATTGGTCATCATCAACGACCGACTCGACGACAGCAATATCAAAGGCTTGGAGTCATCCGCAAAACCGAAACAGGGAGAAGCTGCCGAGAAACCGCAGCAGGCAGAAACTGCCAAAACCGCACCGAAGCAGGCAAAACAACGCGCTGCCGAAAAAGTGTCGGCAACTGCCGACAGTACGGATACGGTAGCGGTTGAAAAACCGAAACGCACTGCCGAAACAAAACCGCAAAAAGCGGAACGCACTGCCGAAGCCAAGCCCAAAGCCAAAGAAACCAAAACCGCCGAAAAAGTTGCCGACAAACCGAAAACTGCCGCCGAAAAAACCAAACCGGATACGGCAAAATCCGACAGCGCGGTAAAAGAAGCGAAAAAAGCCGACAAGGCTGAAGGCAAAAAAACAGCCGAAAAAGACCATTCGGACGGGAAAAAACACGAAACGGCACAAAAAAGCGACAAAGCGGACAAGACCAAAACCGCCGAAAAAGAAAAATCCGAAAATTCCGGCAAAAAAGCCGCCATTCAGGCAGGTTATGCCGAAAAAGAACGCGCCTTGAGCCTCCAGCGCAAAATGAAAGCGGCGGGTATCGATTCGACCATTACTGAAATCATGACCGACAACGGCAAAGTTTACCGCGTCAAATCAAGCAACTATAAAAACGCAAGGGATGCCGAGCGCGATTTGAACAAACTGCGCGTGCACGGCATCGCCGGTCAGGTAACGAATGAATAASEQ ID NO：2SEQ ID NO：1BASB052MSENKQNEVLSGYEQLKRRNRRRLVTASCLVAASCLLLAAALSSDPAENPPAAPTDETGSMENQAASTVQTPTLKSASEGVEPAADKPQDLASEEQAPAADNGISEPEDVGAPLVIINDRLDDSNIKGLESSAKPKQGEAAEKPQQAETAKTAPKQAKQRAAEKVSATADSTDTVAVEKPKRTAETKPQKAERTAEAKPKAKETKTAEKVADKPKTAAEKTKPDTAKSDSAVKEAKKADKAEGKKTAEKDHSDGKKHETAQKSDKADKTKTAEKEKSENSGKKAAIQAGYAEKERALSLQRKMKAAGIDSTITEIMTDNGKVYRVKSSNYKNARDAERDLNKLRVHGIAGQVTNESEQ ID NO：3AGG CAG AGG CAT ATG TCC GAA AAC AAA CAA AAC GAA GTCSEQ ID NO：4AGG CAG AGG CTC GAG TTC ATT CGT TAC CTG ACC GGC GAT GCC GTG

Preserved material

The preservation thing that contains Neisseria meningitidis serotypes B bacterial strain carries out preservation on June 22nd, 1997 in American type culture collection (being expressed as " ATCC " here), and preserving number is 13090.This preservation thing is Neisseria meningitidis (Albrecht and Ghon), is that a kind of 1.5-2.9kb that makes up from the Neisseria meningitidis isolate inserts the lyophilized products in library.This preservation thing is introduced in " Iht.Bull.Bacteriol.Nomencl.Taxon. " 8:1-15 (1958).

This Neisseria meningitidis bacterial strain preservation thing is referred to herein as " preservation strain " or " DNA of preservation strain ".

Preservation strain contains the BASB052 gene of total length.When the sequence description of any amino acid sequence of polypeptide of contained polynucleotide and coding and this paper is inconsistent in the preservation strain, be as the criterion with the former.

The preservation of preservation strain is that the clause of basis " international recognition is used for the budapest treaty of the microbial preservation of patented procedure " carries out.When patent was awarded, this bacterial strain can unconditionally be provided to the public without restriction.Provide this preservation strain only for making things convenient for those skilled in the art, enforcement of the present invention might not need this preserved material, as the requirement of 35U.S.C. ξ 112..

Applicant or attorney docket

International application no

Explanation about microbial preservation

(detailed rules and regulations 13 two)

A. to the 42nd page in specification sheets. the explanation of the capable described microorganism of 1-16:
		B. the preservation item other be deposited in the supplementary page two
Depositary institution's title American type culture collection
		Depositary institution address (comprising postcode and name of the country) 10801 University Blvd, Manassas, Virginia 20110-2209.U.S.A
Preservation date on June 22nd, 1997	Deposit number 13090
Preservation date on June 22nd, 1997	Deposit number 13090	C. have supplementary page two in supplementary notes (in case of necessity) this column
This invention is specified when seeking the European patent protection; the microorganism of preservation will the present invention be awarded Europe specially to or the application be rejected or abandon Shi Caike and obtain, and only to the specified expert's granting of people that requires to obtain sample.
		D. this explanation is done (if explanation is done for all designated states down) for following designated state

		E. remark additionally (in case of necessity)
Following explanation will provide (writing out the classification of explanation, for example: " numbering of preservation ") to international office subsequently		E. remark additionally (in case of necessity)

PCT/RO/134 shows (in July, 1002)

Sequence table

<110>SmithKline?Beecham?Biologicals

＜120〉new compound

<130>BM45350

<160>4

<170>FastSEQ?for?Windows?Version?3.0

<210>1

<211>1068

<212>DNA

＜213〉Neisseria meningitidis (Neisseria meningitidis)

<400>1atgtccgaaa?acaaacaaaa?cgaagtcctg?agcggttacg?aacaactcaa?acggcgcaac??????60cgccgccgtc?tcgtaacggc?aagctgcctg?gttgccgcct?cctgcctgct?gctggcggcc?????120gccctcagtt?ccgatcctgc?cgaaaatccc?cctgccgcac?cgactgacga?aacaggcagc?????180atggaaaacc?aagcggcaag?cacggtacaa?accccgacct?taaaatccgc?ctcagaaggt?????240gttgaacccg?ccgccgacaa?acctcaagac?ctggccagtg?aagaacaagc?acctgccgcc?????300gacaatggaa?tcagcgaacc?tgaagacgta?ggcgcaccat?tggtcatcat?caacgaccga?????360ctcgacgaca?gcaatatcaa?aggcttggag?tcatccgcaa?aaccgaaaca?gggagaagct?????420gccgagaaac?cgcagcaggc?agaaactgcc?aaaaccgcac?cgaagcaggc?aaaacaacgc?????480gctgccgaaa?aagtgtcggc?aactgccgac?agtacggata?cggtagcggt?tgaaaaaccg?????540aaacgcactg?ccgaaacaaa?accgcaaaaa?gcggaacgca?ctgccgaagc?caagcccaaa?????600gccaaagaaa?ccaaaaccgc?cgaaaaagtt?gccgacaaac?cgaaaactgc?cgccgaaaaa?????660accaaaccgg?atacggcaaa?atccgacagc?gcggtaaaag?aagcgaaaaa?agccgacaag?????720gctgaaggca?aaaaaacagc?cgaaaaagac?cattcggacg?ggaaaaaaca?cgaaacggca?????780caaaaaagcg?acaaagcgga?caagaccaaa?accgccgaaa?aagaaaaatc?cgaaaattcc?????840ggcaaaaaag?ccgccattca?ggcaggttat?gccgaaaaag?aacgcgcctt?gagcctccag?????900cgcaaaatga?aagcggcggg?tatcgattcg?accattactg?aaatcatgac?cgacaacggc?????960aaagtttacc?gcgtcaaatc?aagcaactat?aaaaacgcaa?gggatgccga?gcgcgatttg????1020aacaaactgc?gcgtgcacgg?catcgccggt?caggtaacga?atgaataa?????????????????1068

<210>2

<211>355

<212>PRT

＜213〉Neisseria meningitidis (Neisseria meningitidis)

<400>2Met?Ser?Glu?Asn?Lys?Gln?Asn?Glu?Val?Leu?Ser?Gly?Tyr?Glu?Gln?Leu?1????????????5???????????????10???????????????15Lys?Arg?Arg?Asn?Arg?Arg?Arg?Leu?Val?Thr?Ala?Ser?Cys?Leu?Val?Ala

20???????????????25???????????????30Ala?Ser?Cys?Leu?Leu?Leu?Ala?Ala?Ala?Leu?Ser?Ser?Asp?Pro?Ala?Glu

35???????????????40???????????????45Asn?Pro?Pro?Ala?Ala?Pro?Thr?Asp?Glu?Thr?Gly?Ser?Met?Glu?Asn?Gln???50???????????????55???????????????60Ala?Ala?Ser?Thr?Val?Gln?Thr?Pro?Thr?Leu?Lys?Ser?Ala?Ser?Glu?Gly65???????????????70???????????????75???????????????80Val?Glu?Pro?Ala?Ala?Asp?Lys?Pro?Gln?Asp?Leu?Ala?Ser?Glu?Glu?Gln

85???????????????90???????????????95Ala?Pro?Ala?Ala?Asp?Asn?Gly?Ile?Ser?Glu?Pro?Glu?Asp?Val?Gly?Ala

100??????????????105??????????????110Pro?Leu?Val?Ile?Ile?Asn?Asp?Arg?Leu?Asp?Asp?Ser?Asn?Ile?Lys?Gly

115???????????????120??????????????125Leu?Glu?Ser?Ser?Ala?Lys?Pro?Lys?Gln?Gly?Glu?Ala?Ala?Glu?Lys?Pro???130??????????????135???????????????140Gln?Gln?Ala?Glu?Thr?Ala?Lys?Thr?Ala?Pro?Lys?Gln?Ala?Lys?Gln?Arg145??????????????150??????????????155??????????????160Ala?Ala?Glu?Lys?Val?Ser?Ala?Thr?Ala?Asp?Ser?Thr?Asp?Thr?Val?Ala

165??????????????170???????????????175Val?Glu?Lys?Pro?Lys?Arg?Thr?Ala?Glu?Thr?Lys?Pro?Gln?Lys?Ala?Glu

180??????????????185??????????????190Arg?Thr?Ala?Glu?Ala?Lys?Pro?Lys?Ala?Lys?Glu?Thr?Lys?Thr?Ala?Glu

195??????????????200??????????????205Lys?Val?Ala?Asp?Lys?Pro?Lys?Thr?Ala?Ala?Glu?Lys?Thr?Lys?Pro?Asp???210??????????????215??????????????220Thr?Ala?Lys?Ser?Asp?Ser?Ala?Val?Lys?Glu?Ala?Lys?Lys?Ala?Asp?Lys225??????????????230??????????????235??????????????240Ala?Glu?Gly?Lys?Lys?Thr?Ala?Glu?Lys?Asp?His?Ser?Asp?Gly?Lys?Lys

245??????????????250???????????????255His?Glu?Thr?Ala?Gln?Lys?Ser?Asp?Lys?Ala?Asp?Lys?Thr?Lys?Thr?Ala

260??????????????265??????????????270Glu?Lys?Glu?Lys?Ser?Glu?Asn?Ser?Gly?Lys?Lys?Ala?Ala?Ile?Gln?Ala

275??????????????280??????????????285Gly?Tyr?Ala?Glu?Lys?Glu?Arg?Ala?Leu?Ser?Leu?Gln?Arg?Lys?Met?Lys???290??????????????295???????????????300Ala?Ala?Gly?Ile?Asp?Ser?Thr?Ile?Thr?Glu?Ile?Met?Thr?Asp?Asn?Gly305??????????????310??????????????315???????????????320Lys?Val?Tyr?Arg?Val?Lys?Ser?Ser?Asn?Tyr?Lys?Asn?Ala?Arg?Asp?Ala

325??????????????330???????????????335Glu?Arg?Asp?Leu?Asn?Lys?Leu?Arg?Val?His?Gly?Ile?Ala?Gly?Gln?Val

340??????????????345??????????????350Thr?Asn?Glu

355

<210>3

<211>39

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>3aggcagaggc?atatgtccga?aaacaaacaa?aacgaagtc?????????????????????????39

<210>4

<211>45

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>4aggcagaggc?tcgagttcat?tcgttacctg?accggcgatg?ccgtg??????????????????45

Claims

1. the isolated polypeptide that contains following aminoacid sequence, the aminoacid sequence of this aminoacid sequence and SEQ IDNO:2 has at least 85% identity.

2. the isolated polypeptide of claim 1, wherein the aminoacid sequence of aminoacid sequence and SEQ ID NO:2 has at least 95% identity.

3. the isolated polypeptide of claim 1 contains the aminoacid sequence of SEQ ID NO:2.

4.SEQ the isolated polypeptide of ID NO:2.

5. each the immunogenic fragments of polypeptide of claim 1-4, the immunogen activity of the wherein said immunogenic fragments polypeptide with SEQ ID NO:2 basically is identical.

6. the separation polynucleotide of nucleotide sequence that contain the polypeptide of coding SEQ ID NO:2.

7. the separation polynucleotide that contain the polynucleotide of SEQ ID NO:1.

8. contain the separation polynucleotide of nucleotide sequence of the polypeptide of coding SEQ ID NO:2, it can obtain with the suitable library of label probe screening with SEQ ID NO:1 or its fragments sequence by under tight hybridization conditions.

9. contain claim 6-8 each the expression vector of separation polynucleotide or the live microorganism of reorganization.

10. express each the method for polynucleotide of claim 6-8, comprise, and cultivate described host cell being enough to express under the condition of any said polynucleotide with the expression vector transformed host cell that contains at least a described polynucleotide.

11. contain significant quantity claim 1 to 5 each polypeptide and the vaccine composition of pharmaceutically acceptable carrier.

12. contain significant quantity claim 6 to 8 each polynucleotide and the vaccine composition of pharmaceutically acceptable carrier.

13. claim 11 or 12 each vaccine compositions, wherein said composition contains a kind of other Neisseria meningitidis antigen at least.

14. to each polypeptide or the antibody of immunity fragment of claim 1 to 5 with immunologic opsonin.

15. the method that the diagnosis of meningitis Neisseria infects, comprise evaluation have from suspection the claim 1-5 that exists in the biological sample of this infected animals each polypeptide or the antibody that described polypeptide is had immunologic opsonin.

16. containing each the composition of polypeptide of claim 1-5 of immune significant quantity is used for producing purposes in the medicine of immunne response animal in preparation.

17. containing each the composition of polynucleotide of claim 6-8 of immune significant quantity is used for producing purposes in the medicine of immunne response animal in preparation.

18. be used for the treatment of the people's who suffers from the Neisseria meningitidis disease therapeutic composition, it comprises the antibody of polypeptide of at least a anti-claim 1-5 and suitable pharmaceutical carrier.