CN1420930A - Novel compounds - Google Patents

Abstract

The invention provides Neisseria meningitidis BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 and BASB071 polypeptides, and polynucleotides encoding BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 and BASB071 polypeptides, and methods for producing such polypeptides by recombinant techniques. Also provided are diagnostic, prophylactic and therapeutic uses thereof.

Description

Novel compound

Invention field

The present invention relates to polynucleotide and (refer to the BASB051 polynucleotide herein, the BASB057 polynucleotide, the BASB060 polynucleotide, the BASB061 polynucleotide, the BASB063 polynucleotide, the BASB065 polynucleotide, BASB066 polynucleotide and BASB071 polynucleotide), the polypeptide of these nucleotide codings (refers to corresponding BASB051 herein, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 and BASB071, or corresponding BASB051 polypeptide, the BASB057 polypeptide, the BASB060 polypeptide, the BASB061 polypeptide, the BASB063 polypeptide, the BASB065 polypeptide, BASB066 polypeptide and BASB071 polypeptide), reorganization material and the method for producing these materials.On the other hand, the present invention relates to utilize these polypeptide and polynucleotide, comprise vaccine, prevent infectation of bacteria.Further, the present invention relates to diagnositc analysis to detect the infection of special pathogen.

Background of invention

Neisseria meningitidis (Neisseria meningitidis) is a kind of gram positive bacterium that can often be separated to from people's upper respiratory tract.It causes invasive bacterial disease such as microbemia and meningitis sometimes.The caused disease of Neisseria meningitidis have geographic season and annual difference (Schwartz, B., Moore, P.S., Broome, a C.V.; Clin.Microbiol.Rev.2 (Supplement), S18-24,1989).In the country that has a moderate climate, the disease that most Neisseria meningitidis causes is caused by serologic group B Neisseria meningitidis, and their incidence is at 1-10/100, change between total population in 000/, sometimes can reach a high value (Kaczmarski, E.B. (1997), Commun.Dis.Rep.Rev.7:R55-9,1995; Scholten, R.J.P.M., Bijlmer, H.A., Poolman, J.T.et al.Clin.Infect.Dis.16:237-246,1993; Cruz, C., Pavez, G., Aguilar, E., etal.Epidemiol.Infect.105:119-126,1990).The prevailing epiphytotics sickness rate of serologic group A type Neisseria meningitidis can be up to 1000/100,000/, and these prevailing disease mainly are (the Schwartz that Central African States meets with, B., Moore, P.S., Broome, C.V.Clin.Microbiol.Rev.2 (Supplement), S18-S24,1989).In terms of overall, nearly all Neisseria meningitidis disease is caused by serologic group A, C, W-135 and Y type Neisseria meningitidis, and has available quaternary A, C, W-135, Y polysaccharide vaccine (Armand, J., Arminjon, R., Mynard, M.C., Lafaix, C., J.Biol.Stand.10:335-339,1982).

These polysaccharide vaccines are now just by improving (Lieberman, J.M., Chiu, S.S., Wong, V.K., et al.JAMA275:1499-1503,1996) with their Chemical bond to the method on the carrier proteins.

Serologic group Type B vaccine does not obtain as yet, because find that B capsule polysaccharide right and wrong are immunogenic, most possibly is owing to it and host's moiety structural similarity (Wyle to be arranged, F.A., Artenstein, M.S., Brandt, M.L.et al.J.Infect.Dis.126:514-522,1972:Finne, J.M., Leinonen, M., M  kel , P.M.Lancetii.:355-357,1983).

Through effort for many years, vaccine (de Moraes, J.C. have been initiated and have set up based on the Neisseria meningitidis adventitia, Perkins, B., Camargo, M.C.et al.Lancet 340:1074-1078,1992:Bjune, G., Hoiby, E.A.Gronnesby, J.K.et al.338:1093-1096,1991).These vaccines are 57%-85% in the effect that big-age-child (＞4 years old) and teenager show on one's body.

The outer membrane component that in these vaccines, contains various bacteria, PorA for example, PorB, Rmp, Opc, Opa, FrpB, these components still await further determining to the contribution of visible protective effect.Induce potential related animal or human's antibody by utilizing with protective immunity,, determined other bacterial outer membrane components (Martin, D., Cadieux as TbpB and NSpA; N., Hamel, J., Brodeux, B.R.; J.Exp.Med.185:1173-1183,1997:Lissolo, L. -Wilmotte, C., Dumas, p.et al., Inf.Immun.63:884-890,1995).The mechanism of protective immunity comprises antibody-mediated fungicidal activity and opsonophagocytosis.

Utilized a kind of microbemia animal model to come in conjunction with all antibody-mediated mechanism of action (Saukkonen, K., Leinonen, M., Abdillahi, H.Poolman, J.T.Vaccine 7:325-328,1989).Generally accepted is play a crucial role in the Neisseria meningitidis disease prevention (Ross, S.C., Rosenthal P.J. of the bactericidal mechanism of complement component mediation in late period, Berberic, H.M., Densen, P.J.Infect.Dis.155:1266-1275,1987).

In the past few decades, the frequency that Neisseria meningitidis infects has theatrical raising.This will and have the immune crowd's of weakening increase gradually owing to the appearance of composite antibiotic resistant strain.Separation has been rare thing no longer to the Neisseria meningitidis of certain or all standard antibiotic tool resistances.This phenomenon has caused the demand to medical treatment, and to new anti-microbial agents, vaccine, drug screening method with to the demand of these biological diagnostic detection.

Summary of the invention

The present invention relates to BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 and BASB071, especially BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 and BASB071 polypeptide and BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 and BASB071 polynucleotide, reorganization material and the method for producing these materials.On the other hand, the present invention relates to use the method for these polypeptide and polynucleotide, comprise the disease that prevention and treatment are caused by microorganism, and other disease.Further, the present invention relates to detect with the infected by microbes diseases associated and with these diagnostic assays that infects the relevant state of an illness, for example detect the analytical method of BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 and BASB071 polynucleotide or polypeptide active and expression.

By reading other parts of following description and disclosure, for those of skill in the art, the marrow of invention disclosed and the variation within its scope and change are conspicuous.

Invention is described

The present invention relates to as detailed below BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 and BASB071 polypeptide and polynucleotide.The invention particularly relates to those contain respectively SEQ ID NO:1,3,5,7,9,11,13,15 and SEQID NO:2,4,6,8,10,12,14,16 in Nucleotide and BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 and the BASB071 of aminoacid sequence.Hereinafter the dna sequence dna described in the sequence table be interpreted as he be the representative a kind of embodiment of the present invention example because those those of ordinary skill think that all these sequences can effectively be applied to general polynucleotide, comprise the ribose polynucleotide.Polypeptide

One aspect of the present invention provides the polypeptide of Neisseria meningitidis, refer to BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 and BASB071 herein, and BASB051 polypeptide, BASB057 polypeptide, BASB060 polypeptide, BASB061 polypeptide, BASB063 polypeptide, BASB065 polypeptide, BASB066 polypeptide and BASB071 polypeptide, with and biologically, in the diagnosis, in the prevention, clinically or on treating effective variant and comprise the composition of these materials.

The present invention further provides:

(a) a kind of isolating peptide section, this peptide section comprises an aminoacid sequence, this sequence has 85% identity with SEQ ID NO:2 at least, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, most preferably with its 97-99% identity or the sequence of identity at least fully.

(b) a kind of polypeptide of isolating polynucleotide encoding is arranged, this polynucleotide contain a polynucleotide sequence, this sequence is with regard to the SEQID NO:1 of complete length, has 85% identity at least with SEQID NO:1, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, in addition more preferably with its 97-99% identity or the sequence of identity at least fully.

(c) by a kind of polypeptide of isolating polynucleotide encoding, the polynucleotide of this polypeptide of encoding contain a polynucleotide sequence, this sequence encoding one peptide species, the aminoacid sequence of this polypeptide and SEQ IDNO:2 has 85% identity at least, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, in addition more preferably with its 97-99% identity or identical sequence at least.

The BASB051 polypeptide that provides among the SEQ ID NO:2 is from Neisseria meningitidis strains A TCC13090.

In addition, the present invention also provides the immunogenic fragments of BASB051 polypeptide, also is the sequential portion of BASB051 polypeptide, this fragment and the identical or essentially identical immunogenicity of the polypeptide of the aminoacid sequence that contains SEQ ID NO:2.That is to say that (when with a kind of carrier when coupled, if necessary) this fragment just can cause the immune response of identification BASB051 polypeptide.Such fragment can comprise, for example lacks the BASB051 polypeptide of the terminal leader sequence of N-, and/or membrane-spanning domain, and/or C-end anchors localization.。According to the present invention, the immunogenic fragments that a kind of preferred viewpoint is BASB051 has comprised all extracellular domains of a peptide species basically, the sequence of this peptide species is with regard to the SEQ ID NO:2 of whole length, have with SEQ ID NO:2 and to have 85% identity at least, the sequence that preferably has 90% identity at least with it, yet the sequence that more preferably has 95% identity with it at least is most preferably with its 97-99% identity or identical sequence at least.

The present invention further provides:

(a) a kind of isolating peptide section, this peptide section comprises an aminoacid sequence, this sequence has 85% identity with SEQ ID NO:4 at least, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, most preferably with its 97-99% identity or identical sequence at least.

(b) a kind of polypeptide of isolating polynucleotide encoding is arranged, this polynucleotide contain a nucleotide sequence, this sequence is with regard to the SEQ ID NO:3 of complete length, has 85% identity at least with SEQ IDNO:3, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, in addition more preferably with its 97-99% identity or identical sequence at least.

(c) by a kind of polypeptide of isolating polynucleotide encoding, the polynucleotide of this polypeptide of encoding contain a polynucleotide sequence, this sequence encoding one peptide species, the aminoacid sequence of this polypeptide and SEQ IDNO:4 has 85% identity at least, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, in addition more preferably with its 97-99% identity or identical sequence at least.

The BASB057 polypeptide that provides among the SEQ ID NO:4 is from Neisseria meningitidis strains A TCC13090.

In addition, the present invention also provides the immunogenic fragments of BASB057 polypeptide, also is the sequential portion of BASB057 polypeptide, this fragment and the identical or essentially identical immunogenicity of the polypeptide of the aminoacid sequence that contains SEQ ID NO:4.That is to say that (when with a kind of carrier when coupled, if necessary) this fragment just can cause the immune response of identification BASB057 polypeptide.Such fragment can comprise, for example lacks the BASB057 polypeptide of the terminal leader sequence of N-, and/or membrane-spanning domain, and/or C-end anchors localization.According to the present invention, the immunogenic fragments that a kind of preferred viewpoint is BASB057 has comprised all extracellular domains of a peptide species basically, the sequence of this peptide species is with regard to the SEQ ID NO:4 of whole length, has 85% identity at least with SEQ ID NO:4, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, most preferably with its 97-99% identity or identical sequence at least.

The present invention further provides:

(a) a kind of isolating peptide section, this peptide section comprises an aminoacid sequence, this sequence has 85% identity with SEQ ID NO:6 at least, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, most preferably with its 97-99% identity or identical sequence at least.

(b) a kind of polypeptide of isolating polynucleotide encoding is arranged, this polynucleotide contain a nucleotide sequence, this sequence is with regard to the SEQ ID NO:5 of complete length, has 85% identity at least with SEQ IDNO:5, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, in addition more preferably with its 97-99% identity or identical sequence at least.

(c) by a kind of polypeptide of isolating polynucleotide encoding, the polynucleotide of this polypeptide of encoding contain a polynucleotide sequence, this sequence encoding one peptide species, the aminoacid sequence of this polypeptide and SEQ IDNO:6 has 85% identity at least, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, in addition more preferably with its 97-99% identity or identical sequence at least.

The BASB060 polypeptide that provides among the SEQ ID NO:6 is from Neisseria meningitidis strains A TCC13090.

In addition, the present invention also provides the immunogenic fragments of BASB060 polypeptide, also is the sequential portion of BASB060 polypeptide, this fragment and the identical or essentially identical immunogenicity of the polypeptide of the aminoacid sequence that contains SEQ ID NO:6.That is to say that (when with a kind of carrier when coupled, if necessary) this fragment just can cause the immune response of identification BASB060 polypeptide.Such fragment can comprise, for example lacks the BASB060 polypeptide of the terminal leader sequence of N-, and/or membrane-spanning domain, and/or C-end anchors localization.According to the present invention, the immunogenic fragments that a kind of preferred viewpoint is BASB060 has comprised all extracellular domains of a peptide species basically, the SEQ ID NO:6 of the sequence of this peptide species and whole length, has 85% identity at least with SEQ ID NO:6, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, most preferably with its 97-99% identity or identical sequence at least.

The present invention further provides:

(a) a kind of isolating peptide section, this peptide section comprises an aminoacid sequence, this sequence has 85% identity with SEQ ID NO:8 at least, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, most preferably with its 97-99% identity or identical sequence at least.

(b) a kind of polypeptide of isolating polynucleotide encoding is arranged, this polynucleotide contain a nucleotide sequence, this sequence is with regard to the SEQ ID NO:7 of complete length, has 85% identity at least with SEQ IDNO:7, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, in addition more preferably with its 97-99% identity or identical sequence at least.

(c) by a kind of polypeptide of isolating polynucleotide encoding, the polynucleotide of this polypeptide of encoding contain a polynucleotide sequence, this sequence encoding one peptide species, the aminoacid sequence of this polypeptide and SEQ IDNO:8 has 85% identity at least, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, in addition more preferably with its 97-99% identity or identical sequence at least.

The BASB061 polypeptide that provides among the SEQ ID NO:8 is from Neisseria meningitidis strains A TCC13090.

In addition, the present invention also provides the immunogenic fragments of BASB061 polypeptide, also is the sequential portion of BASB061 polypeptide, this fragment and the identical or essentially identical immunogenicity of the polypeptide of the aminoacid sequence that contains SEQ ID NO:8.That is to say that (when with a kind of carrier when coupled, if necessary) this fragment just can cause the immune response of identification BASB061 polypeptide.Such fragment can comprise, for example lacks the BASB061 polypeptide of the terminal leader sequence of N-, and/or membrane-spanning domain, and/or C-end anchors localization.According to the present invention, the immunogenic fragments that a kind of preferred viewpoint is BASB061 has comprised all extracellular domains of a peptide species basically, the SEQ ID NO:8 of the sequence of this peptide species and whole length, has 85% identity at least with SEQ ID NO:8, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, most preferably with its 97-99% identity or identical sequence at least.

The present invention further provides:

(a) a kind of isolating peptide section, this peptide section comprises an aminoacid sequence, this sequence has 85% identity with SEQ ID NO:10 at least, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, most preferably with its 97-99% identity or identical sequence at least.

(b) a kind of polypeptide of isolating polynucleotide encoding is arranged, this polynucleotide contain a nucleotide sequence, this sequence is with regard to the SEQ ID NO:9 of complete length, has 85% identity at least with SEQ IDNO:9, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, in addition more preferably with its 97-99% identity or identical sequence at least.

(c) by a kind of polypeptide of isolating polynucleotide encoding, the polynucleotide of this polypeptide of encoding contain a polynucleotide sequence, this sequence encoding one peptide species, the aminoacid sequence of this polypeptide and SEQ IDNO:10 has 85% identity at least, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, in addition more preferably with its 97-99% identity or identical sequence at least.

The BASB063 polypeptide that provides among the SEQ ID NO:10 is from Neisseria meningitidis strains A TCC13090.

In addition, the present invention also provides the immunogenic fragments of BASB063 polypeptide, also is the sequential portion of BASB063 polypeptide, this fragment and the identical or essentially identical immunogenicity of the polypeptide of the aminoacid sequence that contains SEQID NO:10.That is to say that (when with a kind of carrier when coupled, if necessary) this fragment just can cause the immune response of identification BASB063 polypeptide.Such fragment can comprise, for example lacks the BASB063 polypeptide of the terminal leader sequence of N-, and/or membrane-spanning domain, and/or C-end anchors localization.According to the present invention, the immunogenic fragments that a kind of preferred viewpoint is BASB063 has comprised all extracellular domains of a peptide species basically, the sequence of this peptide species is with regard to the SEQ ID NO:10 of whole length, has 85% identity at least with SEQ ID NO:10, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, most preferably with its 97-99% identity or identical sequence at least.

The present invention further provides:

(a) a kind of isolating peptide section, this peptide section comprises an aminoacid sequence, this sequence has 85% identity with SEQ ID NO:12 at least, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, most preferably with its 97-99% identity or identical sequence at least.

(b) a kind of polypeptide of isolating polynucleotide encoding is arranged, this polynucleotide contain a nucleotide sequence, this sequence is with regard to the SEQ ID NO:11 of complete length, has 85% identity at least with SEQ IDNO:11, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, in addition more preferably with its 97-99% identity or identical sequence at least.

(c) by a kind of polypeptide of isolating polynucleotide encoding, the polynucleotide of this polypeptide of encoding contain a polynucleotide sequence, this sequence encoding one peptide species, the aminoacid sequence of this polypeptide and SEQ IDNO:12 has 85% identity at least, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, in addition more preferably with its 97-99% identity or identical sequence at least.

The BASB065 polypeptide that provides among the SEQ ID NO:12 is from Neisseria meningitidis strains A TCC13090.

In addition, the present invention also provides the immunogenic fragments of BASB065 polypeptide, also is the sequential portion of BASB065 polypeptide, this fragment and the identical or essentially identical immunogenicity of the polypeptide of the aminoacid sequence that contains SEQ ID NO:12.That is to say that (when with a kind of carrier when coupled, if necessary) this fragment just can cause the immune response of identification BASB065 polypeptide.Such fragment can comprise, for example lacks the BASB065 polypeptide of the terminal leader sequence of N-, and/or membrane-spanning domain, and/or C-end anchors localization.According to the present invention, the immunogenic fragments that a kind of preferred viewpoint is BASB065 has comprised all extracellular domains of a peptide species basically, the sequence of this peptide species has 85% identity at least with the SEQ ID NO:12 of whole length, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, most preferably with its 97-99% identity or identical sequence at least.

The present invention further provides:

(a) a kind of isolating peptide section, this peptide section comprises an aminoacid sequence, this sequence has 85% identity with SEQ ID NO:14 at least, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, most preferably with its 97-99% identity or identical sequence at least.

(b) a kind of polypeptide of isolating polynucleotide encoding is arranged, this polynucleotide contain a nucleotide sequence, this sequence is with regard to the SEQ ID NO:13 of complete length, has 85% identity at least with SEQ IDNO:13, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, in addition more preferably with its 97-99% identity or identical sequence at least.

(c) by a kind of polypeptide of isolating polynucleotide encoding, the polynucleotide of this polypeptide of encoding contain a polynucleotide sequence, this sequence encoding one peptide species, the aminoacid sequence of this polypeptide and SEQ IDNO:14 has 85% identity at least, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, in addition more preferably with its 97-99% identity or identical sequence at least.

The BASB066 polypeptide that provides among the SEQ ID NO:14 is from Neisseria meningitidis strains A TCC13090.

In addition, the present invention also provides the immunogenic fragments of BASB066 polypeptide, also is the sequential portion of BASB066 polypeptide, this fragment and the identical or essentially identical immunogenicity of the polypeptide of the aminoacid sequence that contains SEQ ID NO:14.That is to say that (when with a kind of carrier when coupled, if necessary) this fragment just can strengthen the immune response of discerning the BASB066 polypeptide.Such fragment can comprise, for example lacks the BASB066 polypeptide of the terminal leader sequence of N-, and/or membrane-spanning domain, and/or C-end anchors localization.According to the present invention, the immunogenic fragments that a kind of preferred viewpoint is BASB066 has comprised all extracellular domains of a peptide species basically, the sequence of this peptide species is with regard to the SEQID NO:14 of whole length, has 85% identity at least with SEQ ID NO:14, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, most preferably with its 97-99% identity or identical sequence at least.

The present invention further provides:

(a) a kind of isolating peptide section, this peptide section comprises an aminoacid sequence, this sequence has 85% identity with SEQ ID NO:16 at least, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, most preferably with its 97-99% identity or identical sequence at least.

(b) a kind of polypeptide of isolating polynucleotide encoding is arranged, this polynucleotide contain a nucleotide sequence, this sequence is with regard to the SEQ ID NO:15 of complete length, has 85% identity at least with SEQ IDNO:15, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, in addition more preferably with its 97-99% identity or identical sequence at least.

(c) by a kind of polypeptide of isolating polynucleotide encoding, the polynucleotide of this polypeptide of encoding contain a polynucleotide sequence, this sequence encoding one peptide species, the aminoacid sequence of this polypeptide and SEQ IDNO:16 has 85% identity at least, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, in addition more preferably with its 97-99% identity or identical sequence at least.

BASB07 1 polypeptide that provides among the SEQ ID NO:16 is from Neisseria meningitidis strains A TCC13090.

In addition, the present invention also provides the immunogenic fragments of BASB071 polypeptide, also is the sequential portion of BASB071 polypeptide, this fragment and the identical or essentially identical immunogenicity of the polypeptide of the aminoacid sequence that contains SEQ ID NO:16.That is to say that (when with a kind of carrier when coupled, if necessary) this fragment just can cause the immune response of identification BASB071 polypeptide.Such fragment can comprise, for example lacks the BASB071 polypeptide of the terminal leader sequence of N-, and/or membrane-spanning domain, and/or C-end anchors localization.According to the present invention, the immunogenic fragments that a kind of preferred viewpoint is BASB071 has comprised all extracellular domains of a peptide species basically, the sequence of this peptide species is with regard to the SEQ ID NO:16 of whole length, has 85% identity at least with SEQ ID NO:16, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, most preferably with its 97-99% identity or identical sequence at least.

These fragments are some polypeptide, and the partial amino-acid series of polypeptide is identical among the aminoacid sequence that these polypeptide contain and the present invention, rather than with the present invention in all aminoacid sequences of polypeptide identical.When containing BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 and BASB071 polypeptide, these fragments are " unbound states ", or be included in and form among a part or the regional long peptide section, most preferably in isolating long peptide section as isolating successive zone.

Preferred peptide section comprises, as contains SEQ ID NO:2,4,6,8,10,12,14,16 or polypeptide partial amino-acid series, that shorten of its variant, for example contain the successive series residue of the aminoacid sequence of a kind of amino and/or C-terminal.The polypeptide of the degraded form among the present invention who is present in the host cell or is produced by host cell also is preferred.Some fragments further preferably, they are characterised in that the characteristic of 26S Proteasome Structure and Function aspect, and for example some fragments comprise zone, hydrophilic area, hydrophobic region, α-amphipathic molecule district, β-amphipathic molecule district, flex region, surface formation district, substrate land and the high antigen index area that zone, coiled coil and the coiled coil of zone, corner and corner formation that zone, beta sheet and the beta sheet of a-spiral and alpha-helix formation form form.

Further preferred fragment comprises a kind of isolated peptides section, and it contains SEQ IDNO:2,4 at least, 6,8,10,15,20,30,40,50 or 100 in 12,14,16 the aminoacid sequence in abutting connection with amino acid, or a kind of isolated peptides section, it contains the IDNO:2 to SEQ, 4 at least, 6,8,10,15,20,30,40,50 or 100 contiguous amino acid of 12,14,16 sequential amino acid deletion or shortening.

Polypeptide fragment among the present invention can be used for by the synthetic polypeptide of producing its corresponding total length of peptide section; Therefore, these peptide sections can be used as the intermediate of the polypeptide of whole length among production the present invention.

Especially preferred is some variants, and several, 5-10, the 1-5 among them, 1-3,1-2 or 1 amino acid substitute in any combination, disappearance or add.

Polypeptide among the present invention or immunogenic fragments can " maturation " albumen forms or are existed with the form of large protein such as precursor or fusion rotein part.It is useful often to comprise some additional aminoacid sequences, and these aminoacid sequences comprise the sequence of secretion sequence or leader sequence, presequence, aided purification such as polyhistidine residue or the appended sequence relevant with stability in recombinant production.In addition, also consider with the immunogenic potential that increases final molecule adding allogenic polypeptide or lipid tail or polynucleotide sequence.

On the one hand, the present invention relates to by gene engineering method synthetic soluble fusion protein, these albumen comprise polypeptide or its fragment among the present invention, and various immunoglobulin subclass (IgG, IgM, IgA, heavy chain IgE) or the different piece of constant region of light chain.As immunoglobulin (Ig), preferably human IgG, the especially constant region of IgG1 heavy chain are carried out and fusion reaction is a hinge area at IgG1.In a specific scheme,, and just this can be cut the sequence excision by blooc coagulation factor Xa only by just the Fc district being got rid of in conjunction with a kind of cutting sequence.

In addition, the present invention relates to prepare the method for these fusion roteins by genetically engineered, with and application in medicine examination, diagnosis and treatment.The further aspect of the present invention also relates to polynucleotide encoding such as fusion rotein.The visible international patent application no of the example of fusion rotein technology is in the patent of No.WO94/29458 and WO94/22914.

Compare with non-fusion rotein, these albumen can be to exist in chemically combined mode in expression system, or are expressed as the recombination fusion protein form of improving the standard in expression system.Fusion partner can be assisted provides T to assist epitope (Immune Fusion mating partner), preferably can be assisted epitope by the T that the people discerns; Perhaps the more original recombinant protein of auxiliary expression has the albumen (expression enhancement factor) of higher output yield.Preferred fusion partner is not only the Immune Fusion mating partner, and is to express to strengthen mating partner.

Fusion partner comprises the protein D of Haemophilus influemae and the Nonstructural Protein of influenza virus NS1 (erythrocyte agglutination element).Another kind of fusion rotein is called LytA.Advantageous applications be the C-terminal portions of this molecule.LytA is derived from streptococcus pneumoniae, this bacterium is synthesized a kind of N-acetyl-L-ala amide enzyme, be Ntn hydrolase LytA (by the LytA gene Gene, 43 (1986) page 265-272}) coding, but this kind of enzyme is the autolysin of particular key in a kind of degrade specifically peptidoglycan skeleton.The proteic C-stub area of LytA is responsible for combining with choline or with cholinomimetic such as DEAE.This character has been applied to the growth of intestinal bacteria C-LytA expression plasmid, and this plasmid is useful in Expression of Fusion Protein.Comprise existing { Biotechnology:10, (1992) the page 795-798} of describing of the proteic purification process of the segmental hybrid of C-LytA at N-terminal.Might utilize the repeating part of the LytA molecule of C-end, these tumor-necrosis factor glycoproteinss are initial from 178 residues, for example the 188-305 residue.

The present invention also comprises aforementioned variant polypeptides, also promptly is different from the polypeptide of its indicator by conservative amino acid replacement, and displacement herein is meant that the amino acid with a kind of identical characteristics substitutes another kind of amino acid.Typical displacement occurs in Ala, and Val is between Leu and lle, between Ser and Thr, between acidic residues Asp and Glu, between Asn and Gln, between alkaline residue Lys and Arg or between aromatic residues Phe and Tyr.

Polypeptide among the present invention can prepare with arbitrary suitable method.Such polypeptide comprises naturally occurring isolated polypeptide, by the polypeptide of reorganization preparation, by the polypeptide of synthetic method preparation or the polypeptide for preparing by the combination that utilizes aforesaid method.In the art, to the existing very dark understanding of the preparation method of these polypeptide.

Polypeptide among the present invention is most preferably taken from Neisseria meningitidis, yet also can preferably obtain from its other biological body of the same race.Polypeptide among the present invention also can from, obtain in for example identical section and the purpose organism.Polynucleotide

A target of the present invention provides the polynucleotide of coding BASB051 polypeptide, the polynucleotide of the BASB051 polypeptide of indication herein of especially encoding.

In a kind of especially preferred embodiment of the present invention, these polynucleotide comprise the district of coding BASB051 polypeptide or its variant, this coding region comprises a kind of sequence among the SEQ ID NO:1, and this sequence comprises gene or its mutation of whole length of coding BASB051 polypeptide.

The BASB051 polynucleotide that provide among the SEQ ID NO:1 are Neisseria meningitidis strains A TCC13090.

Further, the invention provides coding and/or express BASB051 polypeptide and polynucleotide, especially the isolated nucleic acid molecule of Neisseria meningitidis BASB051 polypeptide and polynucleotide, these nucleic acid molecule comprise, as unprocessed RNA, ribozyme rna, mRNA, cDNA, genomic dna, B-and Z-DNA.Further embodiment comprises biologically among the present invention, diagnosis is gone up, useful polynucleotide and polypeptide and variant thereof in the prevention, clinically or on treating, comprise the composition of these materials.

Another aspect of the present invention relates to isolating polynucleotide, the polynucleotide and the variant thereof of tight association is arranged with it, and these isolating polynucleotide comprise gene a kind of whole length, coding BASB051 polypeptide at least, and this polypeptide contains the deduced amino acid from SEQ ID NO:2.

Polypeptide in another preferred embodiment of the present invention is BASB051 polypeptide or its variant, this BASB051 polypeptide is taken from Neisseria meningitidis, and it comprises a kind of aminoacid sequence of SEQ ID NO:2 or is made up of a kind of aminoacid sequence of SEQ ID NO:2.

Utilize information provided herein, as the polynucleotide sequence among the SEQ ID NO:1, the polynucleotide of a kind of BASB051 polypeptide of encoding among the present invention just can be by the clone and the screening method of standard, is that the material that sets out is cloned the bacterial chromosomal dna fragment and checked order and then obtain whole length clone's method as those with the Neisseria meningitidis cell, obtains.For example, seek out the polynucleotide sequence among the present invention, as given polynucleotide sequence among the SEQ ID NO:1, representative is the chromosomal DNA clone library of detecting the Neisseria meningitidis of intestinal bacteria or other suitable host, and probe is to be 17-mer or longer radio-labeling oligonucleotide derived from a kind of partial sequence, preferred length.Utilize strict hybridization conditions just the clone who carries with the probe same DNA can be distinguished.By each clone who is identified by hybridization being checked order with the sequencing primer that designs by original polypeptide or polynucleotide, these polynucleotide might be extended from both direction, thus the gene order of definite whole length.More convenient is that such order-checking can be passed through, and as utilizing from the denatured double stranded dna of plasmid clone preparation, carries out.Suitable technology is at Manistis, T., Fritsch, the MOLECULAR CLONING.A LABORATORYMAMIAL of E.F. and Sambrook etc., 2nd Ed.; ColdSpring Harbor Labocatory pnss, Cold Spring Harbor, New York has narration in (1989). and (seeing in particular.Screening By Hybridization1.90 and Sequencing Denatured Double-Stranded DNA Templates13.70).Directly the genomic dna order-checking also can be undertaken by the gene order of obtaining a kind of whole length.In the illustrative example of the present invention, each polynucleotide among the SEQ ID NO:1 all can be found in being derived from the DNA library of Neisseria meningitidis.

In addition, dna sequence dna in the Neisseria meningitidis contains a kind of open reading frame, a kind of protein of this open reading frame codified, the amino acid number that this protein contains amino acid about and among the SEQ ID NO:2 is identical, and its molecular weight can calculate by the amino-acid residue molecular weight Evaluation Method that those of skill in the art know.

Among the SEQ ID NO:1, at the codon at first Nucleotide place and since the polynucleotide between the end of a period codon of the 802nd Nucleotide, the polypeptide of coding SEQ ID NO:2.

Advance on the one hand, the invention provides the separation polynucleotide of forming or comprise following sequence by following sequence.

(a) a kind of polynucleotide sequence, this sequence has 85% identity with SEQ ID NO:1 at least with regard to the whole length of SEQ ID NO:1, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, even more preferably with its 97-99% identity or identical sequence at least, or

(b) a kind of polynucleotide sequence, this sequence has 85% identity with SEQ ID NO:2 at least with regard to the whole length of SEQ ID NO:2, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, in addition more preferably with its 97-99% identity or identical sequence at least.

A kind of polynucleotide of the polypeptide in the code book invention comprise homologous gene and orthologous gene in its kind outside Neisseria meningitidis, can be made by a kind of method.This method (for example is included under the strict hybridization conditions, temperature required between 45-60 ℃, SDS concentration is 0.1-1%), utilization contains SEQ ID NO:1 sequence or its fragment or screens a kind of suitable library by the probe that SEQ IDNO:1 sequence or its fragment are formed, and separates steps such as the gene of the whole length that comprise this polynucleotide sequence or genomic clone.

The invention provides a kind of with regard to its total length, with the identical polynucleotide of encoding sequence (open reading frame) among the SEQ ID NO:1.The present invention also provides a kind of mature polypeptide or its segmental encoding sequence, exist with a kind of maturation protein in this polynucleotide sequence self and the reading frame or the form of its segmental encoding sequence, this reading frame contains another kind of encoding sequence again, the sequence of for example encode a kind of guiding or secretion sequence, preceding-protein sequence, former protein sequence, preproprotein sequence.Polynucleotide among the present invention contain a kind of non-coding sequence at least; for example these non-coding sequences comprise as; but be not limited at least a 5 ' and 3 ' non-coding sequence, for example transcribe but sequence, intron and the poly-adenylylation signal of non-translated sequence, termination signal (as relying on ρ-factor and not relying on the termination signal of ρ-factor), ribosome bind site, Kozak sequence, stable mRNA.These polynucleotide sequences also can comprise the additional amino acid whose appended sequence of coding.For example, codified can promote the marker sequence to the purifying of fusion polypeptide.In a certain particular of the present invention, the standard substance sequence is a kind of six histidine peptide sections, as pQE carrier (Qiagen, Inc.) provide, at Gentz et al., Proc.Natl.Acad Sci. has six histidine peptide sections of description or a kind of HA peptide segment mark (Wilson et al. among the USA 86:821-824 (1989), Cell 3～: 767 (1984), these two kinds of sequences are useful when separating with peptide sequence that they merge mutually.Polynucleotide among the present invention also comprise, but are not limited to, and contain the sequence that is associated naturally that structure gene and its controlling gene are expressed.

The sequence of the BASB051 polypeptide of coding SEQ ID NO:2 can be identical with the polypeptid coding sequence of 1-801 the Nucleotide that is included in SEQ ID NO:1.Another kind of situation, as the result of genes encoding redundancy (degeneration), also the encode polypeptide of SEQ ID NO:2 of this polynucleotide sequence." polynucleotide encoding polypeptide " herein comprises those and comprises polypeptide, especially bacterial peptide in the code book invention, more particularly contains the polynucleotide of the Neisseria meningitidis BASB051 polypeptide of SEQ ID NO:2 aminoacid sequence." polynucleotide encoding polypeptide " this noun also comprise those polypeptide that comprise that coding links to each other with additional zone (as be integrated insertion sequence, the integration of polynucleotide that phage is interrupted, integration carrier sequence, integration the transposon sequence or cause rna editing or the sequence of genomic dna reorganization) the continuum or the polynucleotide of locus of discontinuity, and additional zone also can comprise coding and/or non-coding sequence.

The invention further relates to the variant of polynucleotide described herein, these polynucleotide variant codings contain the variant polypeptides of the derivation aminoacid sequence of SEQ ID NO:2.Polynucleotide passage among the present invention can be used for, as the polynucleotide of whole length among synthetic the present invention.

Further preferred embodiment is the polynucleotide of coding BASB051 variant, these BASB051 variants have the BASB051 amino acid sequence of polypeptide of SEQ ID NO:2, and in its sequence, several, some, 5-10,1-5,1-3, the 2nd, the 1st amino-acid residue or 0 amino-acid residue with substitute, modify, disappearance and/or the arbitrary combination mode of adding handled.Wherein especially preferred is reticent substituting, add or disappearance, because they do not change the activity or the characteristic of BASB057 polypeptide.

Further preferred embodiment of the present invention is with regard to its total length and the polynucleotide of BASB051 polypeptide at least 85% identity that contains SEQID NO:2 aminoacid sequence of encoding, and polynucleotide complementary polynucleotide therewith.In this, the polynucleotide that have 90% to 100% identity at least are especially preferred, and in these preferred polynucleotide, those at least 95% identity be especially preferred.Further, in the polynucleotide that have 95% identity at least, those at least 97% identity be very preferred, at least 98% and 99% identity be especially very preferably, at least 99% identity be more very preferably.

Embodiment preferred is that those codings remain with the biological activity identical with the coded mature polypeptide of the DNA of SEQ ID NO:1 or the polynucleotide of function basically.

Corresponding to the particular preferred embodiment among the present invention, the invention provides polynucleotide with BASB051, as those polynucleotide of SEQ ID NO:1, especially under the condition of strictness, the polynucleotide of hybridizing.

Polynucleotide have been the present invention further provides with polynucleotide sequence provided herein hybridization.In this, the invention particularly relates to those under stringent condition with the polynucleotide of multi-nucleotide hybrid described herein.Noun used herein " strict condition " and " strict hybridization conditions " refer to have only when at least 95%, preferably at least 97% identity is arranged between sequence and just can hybridize.An example of stringent hybridization condition is exactly in a kind of solution 42 ℃ of cultivations and spends the night, subsequently at 65 ℃ of hybridization upholders that wash down among 0.1 * SSC, solution herein contains: 50% formaldehyde, 5 * SSC (150mM NaCI, the 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5 * Denhardt ' s solution, the denatured DNA of the salmon sperm that 10% sulfuric acid dextran and 20 micrograms/ml have sheared.Hybridization and wash conditions are well-known, and at the Molecular Cloning:A LaboratoryManual of Sambrook etc., Second Edition, Cold Spring Harbor, N.Y. in (1989), especially has example in its Chapter 11.Hybridization solution also can be shared with polynucleotide sequence provided by the invention.

The present invention also provides a kind of polynucleotide of being formed or comprised this polynucleotide sequence by a polynucleotide sequence, this polynucleotide sequence is under the hybridization conditions of strictness, with this polynucleotide sequence or its fragment that contain among the SEQ ID NO:1 is probe, screening comprises the suitable library of complete genome of a kind of polynucleotide sequence among the SEQ IDNO:1, and this polynucleotide sequence is carried out isolating method obtains.Comprise probe and primer that other parts for example of the present invention are described in detail to obtaining the useful fragment of these polynucleotide.

Detect about polynucleotide, other parts of the present invention have discussion, for example the polynucleotide among the present invention can be used as the probe of RNA, cDNA and genomic dna, separate the cDNAs and the genomic clone of whole length of the BASB051 that encodes with this, and also can this separate and the high identity of BASB051 the cDNA of other genes of especially high sequence identity and genomic clone.In general, such probe contains 15 nucleotide residues or base pair at least.The preferred probe that contains the probe of 30 nucleotide residues or base pair at least or contain 50 nucleotide residues or base pair at least.Especially preferred is to contain 20 nucleotide residues or base pair at least but the probe that is less than 30 nucleotide residues or base pair is arranged.

Come the synthetic oligonucleotide probe to carry out method for screening by the dna sequence dna that utilizes SEQ ID NO:1 to provide, can be separated to the coding region of BASB051 gene.Then, utilize contain with the present invention in the labeled oligonucleotide screening cDNA of gene order complementary sequence, genomic dna or library mRNA, and then determine and the member in the library that probe is hybridized with this.

A target of the present invention provides the polynucleotide of coding BASB057 polypeptide, the polynucleotide of the BASB057 polypeptide of indication herein of especially encoding.

In a kind of especially preferred embodiment of the present invention, these polynucleotide comprise the district of coding BASB057 polypeptide or its variant, this coding region comprises a kind of sequence among the SEQ ID NO:3, and this sequence comprises gene or its variant of whole length of coding BASB057 polypeptide.

The BASB057 polynucleotide that provide among the SEQ ID NO:3 are Neisseria meningitidis strains A TCC13090.

Further, the invention provides coding and/or express BASB057 polypeptide and polynucleotide, especially the isolated nucleic acid molecule of Neisseria meningitidis BASB057 polypeptide and polynucleotide, these nucleic acid molecule comprise, as unprocessed RNA, ribozyme rna, mRNA, cDNA, genomic dna, B-and Z-DNA.Further embodiment comprises biologically among the present invention, diagnosis is gone up, useful polynucleotide and polypeptide and variant thereof in the prevention, clinically or on treating, comprise the composition of these materials.

Another aspect of the present invention relates to isolating polynucleotide, the polynucleotide and the variant thereof of tight association is arranged with it, and these isolating polynucleotide comprise gene a kind of whole length, coding BASB057 polypeptide at least, and this polypeptide contains the deduced amino acid from SEQ ID NO:4.

Of the present invention another especially the polypeptide in the preferred embodiment be BASB057 polypeptide or its variant, this BASB057 polypeptide is taken from and is comprised Neisseria meningitidis, and it comprises a kind of aminoacid sequence of SEQ IDNO:4 or is made up of a kind of aminoacid sequence of SEQ ID NO:4.

Utilize information provided herein, as the polynucleotide sequence among the SEQ ID NO:3, the polynucleotide of a kind of BASB057 polypeptide of encoding among the present invention just can be by the clone and the screening method of standard, be the material that sets out as those with the Neisseria meningitidis cell, the bacterial chromosomal dna fragment is cloned and checked order and then obtain whole length clone's method, obtain.For example, seek out the polynucleotide sequence among the present invention, as given polynucleotide sequence among the SEQ ID NO:3, representative is the chromosomal DNA clone library of detecting the Neisseria meningitidis of intestinal bacteria or other appropriate host, and probe is to be 17-mer or longer radio-labeling oligonucleotide derived from a kind of partial sequence, preferred length.Utilize strict hybridization conditions just the clone who carries with the probe same DNA can be distinguished.By each clone who is identified by hybridization being checked order with the sequencing primer that designs by original polypeptide or polynucleotide, these polynucleotide might be extended from both direction, thus the gene order of definite whole length.More convenient is that such order-checking can be passed through, and as utilizing from the denatured double stranded dna of plasmid clone preparation, carries out.Suitable technology is at Manistis, T., Fritsch, the MOLECULAR CLONING.A LABORATORYMAMIAL of E.F. and Sambrook etc., 2nd Ed.; Cold Spring Harbor Labocatory pnss, Cold Spring Harbor, NewYork has narration in (1989).(seeing in particular.Screening ByHybridization 1.90 and Sequencing Denatured Double-StrandedDNA Templates 13.70).Directly the genomic dna order-checking also can be undertaken by the gene order of obtaining a kind of whole length.In the illustrative example of the present invention, each polynucleotide among the SEQ ID NO:3 all can be found in being derived from the DNA library of Neisseria meningitidis.

In addition, the dna sequence dna of Neisseria meningitidis contains a kind of open reading frame, a kind of protein of this open reading frame codified, the amino acid number that this protein contains amino acid about and among the SEQ ID NO:4 is identical, and its molecular weight can calculate by the amino-acid residue molecular weight Evaluation Method that those of skill in the art know.

In SEQ ID NO:3, the codon at first Nucleotide place and since the polypeptide of the polynucleotide encoding SEQ ID NO:4 between the end of a period codon of the 1402nd Nucleotide.

Advance on the one hand, the invention provides the separation polynucleotide of forming or comprise following sequence by following sequence:

(a) a kind of polynucleotide sequence, this sequence is with regard to SEQ ID NO:3 total length, at least with SEQ ID NO:3 85% identity is arranged, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, even more preferably with its 97-99% identity or identical sequence at least, or

(b) a kind of polynucleotide sequence, this sequence is with regard to SEQ ID NO:4 total length, at least with SEQ ID NO:4 85% identity is arranged, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, in addition more preferably with its 97-99% identity or identical sequence at least.

A kind of polynucleotide of the polypeptide in the code book invention comprise homologous gene and orthologous gene in its kind outside Neisseria meningitidis, can be made by a kind of method.This method (for example is included under the strict hybridization conditions, temperature required between 45-60 ℃, SDS concentration is 0.1-1%), utilization contains the sequence of SEQ ID NO:3 or its fragment or screens a kind of suitable library by the probe that SEQID NO:3 sequence or its fragment are formed, and separates steps such as the gene of the whole length that comprise this polynucleotide sequence or genomic clone.

The invention provides a kind of with regard to total length, with a kind of and whole identical polynucleotide of encoding sequence (open reading frame) of length among the SEQ ID NO:3.The present invention also provides a kind of mature polypeptide or its segmental encoding sequence, exist with a kind of maturation protein in this polynucleotide sequence self and the reading frame or the form of its segmental encoding sequence, this reading frame contains another kind of encoding sequence again, the sequence of for example encode a kind of guiding or secretion sequence, preceding protein sequence, former protein sequence, preproprotein sequence.Polynucleotide among the present invention contain a kind of non-coding sequence at least; for example these non-coding sequences comprise as; but be not limited at least a 5 ' and 3 ' non-coding sequence, for example transcribe but sequence, intron and the poly-adenylylation signal of non-translated sequence, termination signal (as relying on ρ-factor and not relying on the termination signal of ρ-factor), ribosome bind site, Kozak sequence, stable mRNA.These polynucleotide sequences also can comprise the additional amino acid whose appended sequence of coding.For example, codified can promote the marker sequence to the purifying of fusion polypeptide.In a certain particular of the present invention, the standard substance sequence is a kind of six histidine peptide sections, as pQE carrier (Qiagen, Inc.) provide, at Gentz etc., Proc.Natl.Acad Sci. has six histidine peptide sections of description or a kind of HA peptide segment mark (Wilson et al. among the USA 86:821-824 (1989), Cell 3～: 767 (1984), these two kinds of sequences are useful when separating with peptide sequence that they merge mutually.Polynucleotide among the present invention also comprise, but are not limited to, contain that structure gene and its controlling gene are expressed, with its sequence that is associated naturally.

The sequence of the BASB057 polypeptide of coding SEQ ID NO:4 can be identical with the polypeptid coding sequence of 1-1401 the Nucleotide that is included in SEQ ID NO:3.Another kind of situation, as the result of genes encoding redundancy (degeneration), also the encode polypeptide of SEQ ID NO:4 of this polynucleotide sequence." polynucleotide encoding polypeptide " herein comprises those and comprises polypeptide, especially bacterial peptide in the code book invention, more particularly contains the polynucleotide of the Neisseria meningitidis BASB057 polypeptide of SEQ ID NO:4 aminoacid sequence." polynucleotide encoding polypeptide " this noun also comprise those polypeptide that comprise that coding links to each other with additional zone (as be integrated insertion sequence, the integration of polynucleotide that phage is interrupted, integration carrier sequence, integration the transposon sequence or cause rna editing or the sequence of genomic dna reorganization) the continuum or the polynucleotide of locus of discontinuity, and additional zone also can comprise coding and/or non-coding sequence.

The invention further relates to the variant of polynucleotide described herein, these polynucleotide variant codings contain the polypeptide variants of the derivation aminoacid sequence of SEQ ID NO:4.Polynucleotide passage among the present invention can be used for, as the polynucleotide of whole length among synthetic the present invention.

Further especially preferred embodiment is the polynucleotide of coding BASB057 variant, these BASB057 variants have the BASB057 amino acid sequence of polypeptide of SEQ ID NO:4, and in its sequence, several, some, 5-10,1-5,1-3, the 2nd, the 1st amino-acid residue or 0 amino-acid residue with substitute, modify, disappearance and/or add and their arbitrary combination mode was handled.Wherein especially preferred is reticent substituting, add or disappearance, because they do not change the activity or the characteristic of BASB057 polypeptide.

Further preferred embodiment of the present invention is with regard to its total length, and the BASB057 polypeptide that contains the aminoacid sequence of SEQID NO:4 with coding has 85% identical polynucleotide at least, and polynucleotide complementary polynucleotide therewith.In this, the polynucleotide that have 90% to 100% identity at least are especially preferred, and in these preferred polynucleotide, those at least 95% identity be especially preferred.Further, in the polynucleotide that have 95% identity at least, those at least 97% identity be very preferred, at least 98% and 99% identity be especially very preferably, at least 99% identity be more very preferably.

Embodiment preferred is that those codings maintain the biological activity identical with the coded mature polypeptide of the DNA of SEQ ID NO:3 or the polynucleotide of function basically.

According to the particular preferred embodiment among the present invention, the invention provides polynucleotide with BASB057, as those polynucleotide of SEQ ID NO:3, especially under the condition of strictness, the polynucleotide of hybridizing.

Polynucleotide have been the present invention further provides with polynucleotide sequence provided herein hybridization.In this, the invention particularly relates to the polynucleotide that those are hybridized with polynucleotide described herein under stringent condition.Noun used herein " strict condition " and " strict hybridization conditions " just can be hybridized when referring to 95%, preferably at least 97% identity is only arranged between sequence at least.An example of stringent hybridization condition is exactly in a kind of solution 42 ℃ of cultivations and spends the night, subsequently at 65 ℃ of hybridization upholders that wash down among 0.1 * SSC, solution herein contains: 50% formaldehyde, 5 * SSC (150mM NaCI, the 15mM trisodium citrate), 50 mM sodium phosphates (pH7.6), 5 * Denhardt ' s solution, the denatured DNA of the salmon sperm that 10% sulfuric acid dextran and 20 micrograms/ml have sheared.Hybridization and wash conditions are well-known in addition, and at the Molecular Cloning:ALaboratory Manual of Sambrook etc., Second Edition, Cold Spring Harbor, N.Y., in (1989), especially in its Chapter 11 example is arranged.Hybridization solution also can be shared with polynucleotide sequence provided by the invention.

The present invention also provides a kind of polynucleotide of being formed or comprised this polynucleotide sequence by a kind of polynucleotide sequence.This polynucleotide sequence is under the hybridization conditions of strictness, with this polynucleotide sequence or its fragment that contain among the SEQ ID NO:3 is probe, screening comprises the suitable library of complete genome of a kind of polynucleotide sequence among the SEQ IDNO:3, and this polynucleotide sequence is separated this serial of methods obtains.Be of value to the fragment that obtains these polynucleotide and comprise, for example the probe and the primer that are described in detail of other parts of the present invention.

Detection about polynucleotide, other parts of the present invention have discussion, for example the polynucleotide among the present invention can be used as the probe of RNA, cDNA and genomic dna, separate the cDNA and the genomic clone of whole length of the BASB057 that encodes with this, and also can this separation have high identity, the cDNA of other genes of especially high sequence identity and genomic clone with BASB057.In general, such probe contains 15 nucleotide residues or base pair at least.Preferably contain the probe of 30 nucleotide residues or base pair at least or contain the probe of 50 nucleotide residues or base pair at least.Especially preferred is to contain 20 nucleotide residues or base pair at least but the probe that is less than 30 nucleotide residues or base pair.

By the screening method that the dna sequence dna synthetic oligonucleotide probe that utilizes SEQ ID NO:3 to provide carries out, can be separated to the coding region of BASB057 gene.Then, utilize contain with the present invention in labeled oligonucleotide screening cDNA, genomic dna or the mRNA library of gene order complementary sequence, and then determine the member in the library of hybridizing with probe.

A target of the present invention provides the polynucleotide of coding BASB060 polypeptide, the polynucleotide of the BASB060 polypeptide of indication herein of especially encoding.

In a kind of especially preferred embodiment of the present invention, these polynucleotide comprise the district of coding BASB060 polypeptide or its variant, this coding region comprises the sequence among the SEQ ID NO:5, and this sequence comprises gene or its variant of whole length of coding BASB060 polypeptide.

The BASB060 polynucleotide that provide among the SEQ ID NO:5 are Neisseria meningitidis strains A TCC13090.

Further, the invention provides coding and/or express BASB060 polypeptide and polynucleotide, especially the isolated nucleic acid molecule of Neisseria meningitidis BASB060 polypeptide and polynucleotide, these nucleic acid molecule comprise, as unprocessed RNA, ribozyme rna, mRNA, cDNA, genomic dna, B-and Z-DNA.Further embodiment comprises biologically among the present invention, diagnosis is gone up, useful polynucleotide and polypeptide and variant thereof in the prevention, clinically or on treating, comprise the composition of these materials.

Another aspect of the present invention relates to isolating polynucleotide, the polynucleotide and the variant thereof of tight association is arranged with it, and these isolating polynucleotide comprise gene a kind of whole length, coding BASB060 polypeptide at least, and this polypeptide contains the deduced amino acid from SEQ ID NO:6.

Of the present invention another especially the polypeptide in the preferred embodiment be BASB060 polypeptide or its variant, this BASB060 polypeptide is taken from Neisseria meningitidis, and it comprises a kind of aminoacid sequence of SEQ ID NO:6 or is made up of a kind of aminoacid sequence of SEQ ID NO:6.

Utilize information provided herein, as the polynucleotide sequence among the SEQ ID NO:5, the polynucleotide of a kind of BASB060 polypeptide of encoding among the present invention just can be by the clone and the screening method of standard, be the material that sets out as those with the Neisseria meningitidis cell, the bacterial chromosomal dna fragment is cloned and checked order and then obtain whole length clone's method, obtain.For example, seek out the polynucleotide sequence among the present invention, as given polynucleotide sequence among the SEQ ID NO:5, representative is the chromosomal DNA clone library of detecting the Neisseria meningitidis of intestinal bacteria or other appropriate host, and probe is to be 17-mer or longer radio-labeling oligonucleotide derived from a kind of partial sequence, preferred length.Utilize strict hybridization conditions just the clone who carries with the probe same DNA can be distinguished.By each clone who is identified by hybridization being checked order with the sequencing primer that designs by original polypeptide or polynucleotide, these polynucleotide might be extended from both direction, thus the gene order of definite whole length.More convenient is that such order-checking can be passed through, and as utilizing from the denatured double stranded dna of plasmid clone preparation, carries out.Suitable technology is at Manistis, T., Fritsch, the MOLECULAR CLONING.A LABORATORYMAMIAL of E.F. and Sambrook etc., 2nd Ed.; Cold Spring Harbor Labocatory pnss, Cold Spring Harbor has narration (seeing in particular.Screening ByHybridization 1.90 and Sequencing Denatured Double-StrandedDNA Templates 13.70) among the NewYork (1989).Directly the genomic dna order-checking also can be undertaken by the gene order of obtaining a kind of whole length.In the illustrative example of the present invention, each polynucleotide among the SEQ ID NO:5 all can be found in being derived from the DNA library of Neisseria meningitidis.

In addition, dna sequence dna in the Neisseria meningitidis contains a kind of open reading frame, a kind of protein of this open reading frame codified, the amino acid number that this protein contains amino acid about and among the SEQ ID NO:6 is identical, and its molecular weight can calculate by the amino-acid residue molecular weight Evaluation Method that those of skill in the art know.

In SEQ ID NO:5, the codon at first Nucleotide place and since the polypeptide of the polynucleotide encoding SEQ ID NO:6 between the end of a period codon of the 418th Nucleotide.

Advance on the one hand, the invention provides the separation polynucleotide of forming or comprise following sequence by following sequence:

(a) a kind of polynucleotide sequence, this sequence is with regard to SEQ ID NO:5 total length, at least with SEQ ID NO:5 85% identity is arranged, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, even more preferably with its 97-99% identity or identical sequence at least, or

(b) a kind of polynucleotide sequence, this sequence is with regard to SEQ ID NO:6 total length, at least with SEQ ID NO:6 85% identity is arranged, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, in addition more preferably with its 97-99% identity or identical sequence at least.

A kind of polynucleotide of the polypeptide in the code book invention comprise homologous gene and orthologous gene in its kind outside Neisseria meningitidis, can be made by a kind of method.This method (for example is included under the strict hybridization conditions, temperature required between 45-60 ℃, SDS concentration is 0.1-1%), utilization contains the sequence of SEQ ID NO:5 or its fragment or screens a kind of suitable library by the probe that SEQID NO:5 sequence or its fragment are formed, and separates steps such as the gene of the whole length that comprise this polynucleotide sequence or genomic clone.

The invention provides a kind of with regard to its total length, with the identical polynucleotide of encoding sequence (open reading frame) among the SEQ ID NO:5.The present invention also provides a kind of mature polypeptide or its segmental encoding sequence, exist with a kind of maturation protein in this polynucleotide sequence self and the reading frame or the form of its segmental encoding sequence, this reading frame contains another kind of encoding sequence again, the sequence of for example encode a kind of guiding or secretion sequence, preceding protein sequence, former protein sequence, preproprotein sequence.Polynucleotide among the present invention contain a kind of non-coding sequence at least; for example these non-coding sequences comprise as; but be not limited at least a 5 ' and 3 ' non-coding sequence, for example transcribe but sequence, intron and the poly-adenylylation signal of non-translated sequence, termination signal (as relying on ρ-factor and not relying on the termination signal of the p-factor), ribosome bind site, Kozak sequence, stable mRNA.These polynucleotide sequences also can comprise the additional amino acid whose appended sequence of coding.For example, codified can promote the marker sequence to the purifying of fusion polypeptide.In a certain particular of the present invention, the standard substance sequence is a kind of six histidine peptide sections, as pQE carrier (Qiagen, Inc.) provide, at Gentz etc., Proc.Natl.Acad Sci. has six histidine peptide sections of description or a kind of HA peptide segment mark (Wilson et al. among the USA 86:821-824 (1989), Cell 3～: 767 (1984), these two kinds of sequences are useful when separating with peptide sequence that they merge mutually.Polynucleotide among the present invention also comprise, but are not limited to, contain that structure gene and its controlling gene are expressed, with its sequence that is associated naturally.

The sequence of the BASB060 polypeptide of coding SEQ ID NO:6 can be identical with the polypeptid coding sequence of 1-417 the Nucleotide that is included in SEQ ID NO:6.Another kind of situation, as the result of genes encoding redundancy (degeneration), also the encode polypeptide of SEQ ID NO:6 of this polynucleotide sequence." polynucleotide encoding polypeptide " herein comprises those and comprises polypeptide, especially bacterial peptide in the code book invention, more particularly contains the polynucleotide of the Neisseria meningitidis BASB060 polypeptide of SEQ ID NO:6 aminoacid sequence." polynucleotide encoding polypeptide " this noun also comprise those polypeptide that comprise that coding links to each other with additional zone (as be integrated insertion sequence, the integration of polynucleotide that phage is interrupted, integration carrier sequence, integration the transposon sequence or cause rna editing or the sequence of genomic dna reorganization) the continuum or the polynucleotide of locus of discontinuity, and additional zone also can comprise coding and/or non-coding sequence.

The invention further relates to the variant of polynucleotide described herein, these polynucleotide variant codings contain the polypeptide variants of the derivation aminoacid sequence of SEQ ID NO:6.Polynucleotide passage among the present invention can be used for, as the polynucleotide of whole length among synthetic the present invention.

Further especially preferred embodiment is the polynucleotide of coding BASB060 variant, these BASB060 variants have the BASB060 amino acid sequence of polypeptide of SEQ ID NO:6, and in its sequence, several, some, 5-10,1-5,1-3, the 2nd, the 1st amino-acid residue or 0 amino-acid residue with substitute, modify, disappearance and/or add and their arbitrary combination mode was handled.Wherein especially preferred is reticent substituting, add or disappearance, because they do not change the activity or the characteristic of BASB060 polypeptide.

Further preferred embodiment of the present invention is to have 85% identical polynucleotide at least with the BASB060 polypeptide of the aminoacid sequence that contains SEQID NO:6 of encoding with regard to its total length, and polynucleotide complementary polynucleotide therewith.In this, the polynucleotide that have 90% to 100% identity at least are especially preferred, and in these preferred polynucleotide, those at least 95% identity be especially preferred.Further, in the polynucleotide that have 95% identity at least, those at least 97% identity be very preferred, at least 98% and 99% identity be especially very preferably, at least 99% identity be more very preferably.

Embodiment preferred is that those codings maintain and the biological activity of the coded mature polypeptide identity of the DNA of SEQ ID NO:5 or the polynucleotide of function basically.

According to the particular preferred embodiment among the present invention, the invention provides polynucleotide with BASB060, as those polynucleotide of SEQ ID NO:5, especially under the condition of strictness, the polynucleotide of hybridizing.

Polynucleotide have been the present invention further provides with polynucleotide sequence provided herein hybridization.In this, the invention particularly relates to the polynucleotide that those are hybridized with polynucleotide described herein under stringent condition.Noun used herein " strict condition " and " strict hybridization conditions " just can be hybridized when referring to 95%, preferably at least 97% identity is only arranged between sequence at least.An example of stringent hybridization condition is exactly in a kind of solution 42 ℃ of cultivations and spends the night, subsequently at 65 ℃ of hybridization upholders that wash down among 0.1 * SSC, solution herein contains: 50% formaldehyde, 5 * SSC (150mM NaCI, the 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5 * Denhardt ' s solution, the denatured DNA of the salmon sperm that 10% sulfuric acid dextran and 20 micrograms/ml have sheared.Hybridization and wash conditions are well-known in addition, and at the Molecular Cloning:ALaboratory Manual of Sambrook etc., Second Edition, Cold Spring Harbor, N.Y., in (1989), especially in its Chapter 11 example is arranged.Hybridization solution also can be shared with polynucleotide sequence provided by the invention.

The present invention also provides a kind of polynucleotide of being formed or comprised this polynucleotide sequence by a kind of polynucleotide sequence.This polynucleotide sequence is under the hybridization conditions of strictness, with this polynucleotide sequence or its fragment that contain among the SEQ ID NO:5 is probe, screening comprises the suitable library of complete genome of a kind of polynucleotide sequence among the SEQ IDNO:5, and this polynucleotide sequence is separated this serial of methods obtains.Be of value to the fragment that obtains these polynucleotide and comprise, for example the probe and the primer that are described in detail of other parts of the present invention.

Detection about polynucleotide, other parts of the present invention have discussion, for example the polynucleotide among the present invention can be used as the probe of RNA, cDNA and genomic dna, separate the cDNA and the genomic clone of whole length of the BASB060 that encodes with this, and also can this separation have high identity, the cDNA of other genes of especially high sequence identity and genomic clone with BASB060.In general, such probe contains 15 nucleotide residues or base pair at least.Preferably contain the probe of 30 nucleotide residues or base pair at least or contain the probe of 50 nucleotide residues or base pair at least.Especially preferred is to contain 20 nucleotide residues or base pair at least but the probe that is less than 30 nucleotide residues or base pair.

By the screening method that the dna sequence dna synthetic oligonucleotide probe that utilizes SEQ ID NO:5 to provide carries out, can be separated to the coding region of BASB060 gene.Then, utilize contain with the present invention in labeled oligonucleotide screening cDNA, genomic dna or the mRNA library of gene order complementary sequence, and then determine the member in the library of hybridizing with probe.

A target of the present invention provides the polynucleotide of coding BASB061 polypeptide, the polynucleotide of the BASB061 polypeptide of indication herein of especially encoding.

In a kind of especially preferred embodiment of the present invention, these polynucleotide comprise the district of coding BASB061 polypeptide or its variant, this coding region comprises the sequence among the SEQ ID NO:7, and this sequence comprises gene or its variant of whole length of coding BASB061 polypeptide.

The BASB061 polynucleotide that provide among the SEQ ID NO:7 are Neisseria meningitidis strains A TCC13090.

Further, the invention provides coding and/or express BASB061 polypeptide and polynucleotide, especially the isolated nucleic acid molecule of Neisseria meningitidis BASB061 polypeptide and polynucleotide, these nucleic acid molecule comprise, as unprocessed RNA, ribozyme rna, mRNA, cDNA, genomic dna, B-and Z-DNA.Further embodiment comprises biologically among the present invention, diagnosis is gone up, useful polynucleotide and polypeptide and variant thereof in the prevention, clinically or on treating, comprise the composition of these materials.

Another aspect of the present invention relates to isolating polynucleotide, the polynucleotide and the variant thereof of tight association is arranged with it, and these isolating polynucleotide comprise gene a kind of whole length, coding BASB061 polypeptide at least, and this polypeptide contains the deduced amino acid from SEQ ID NO:8.

Of the present invention another especially the polypeptide in the preferred embodiment be BASB061 polypeptide or its variant, this BASB061 polypeptide is taken from Neisseria meningitidis, and it comprises a kind of aminoacid sequence of SEQ ID NO:8 or is made up of a kind of aminoacid sequence of SEQ ID NO:8.

Utilize information provided herein, as the polynucleotide sequence among the SEQ ID NO:7, the polynucleotide of a kind of BASB061 polypeptide of encoding among the present invention just can be by the clone and the screening method of standard, be the material that sets out as those with the Neisseria meningitidis cell, the bacterial chromosomal dna fragment is cloned and checked order and then obtain whole length clone's method, obtain.For example, seek out the polynucleotide sequence among the present invention, as given polynucleotide sequence among the SEQ ID NO:7, representative is the chromosomal DNA clone library of detecting the Neisseria meningitidis of intestinal bacteria or other appropriate host, and probe is to be 17-mer or longer radio-labeling oligonucleotide derived from a kind of partial sequence, preferred length.Utilize strict hybridization conditions just the clone who carries with the probe same DNA can be distinguished.By each clone who is identified by hybridization being checked order with the sequencing primer that designs by original polypeptide or polynucleotide, these polynucleotide might be extended from both direction, thus the gene order of definite whole length.More convenient is that such order-checking can be passed through, and as utilizing from the denatured double stranded dna of plasmid clone preparation, carries out.Suitable technology is at Manistis, T., Fritsch, the MOLECULAR CLONING.A LABORATORYMAMIAL of E.F. and Sambrook etc., 2nd Ed.; Cold Spring Harbor Labocatory pnss, Cold Spring Harbor has narration (seeing in particular.Screening ByHybridization 1.90 and Sequencing Denatured Double-StrandedDNA Templates 13.70) among the NewYork (1989).Directly the genomic dna order-checking also can be undertaken by the gene order of obtaining a kind of whole length.In the illustrative example of the present invention, each polynucleotide among the SEQ ID NO:7 all can be found in being derived from the DNA library of Neisseria meningitidis.

In addition, dna sequence dna in the Neisseria meningitidis contains a kind of open reading frame, a kind of protein of this open reading frame codified, the amino acid number that this protein contains amino acid about and among the SEQ ID NO:8 is identical, and its molecular weight can calculate by the amino-acid residue molecular weight Evaluation Method that those of skill in the art know.

In SEQ ID NO:7, the codon at first Nucleotide place and since the polypeptide of the polynucleotide encoding SEQ ID NO:8 between the end of a period codon of the 514th Nucleotide.

Advance on the one hand, the invention provides the separation polynucleotide of forming or comprise following sequence by following sequence:

(a) a kind of polynucleotide sequence, this sequence is with regard to SEQ ID NO:7, at least with SEQ ID NO:7 85% identity is arranged, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, even more preferably with its 97-99% identity or identical sequence at least, or

(b) a kind of polynucleotide sequence, this sequence is with regard to SEQ ID NO:8 total length, at least the SEQ ID NO:8 with whole length has 85% identity, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, in addition more preferably with its 97-99% identity or identical sequence at least.

A kind of polynucleotide of the polypeptide in the code book invention comprise homologous gene and orthologous gene in its kind outside Neisseria meningitidis, can be made by a kind of method.This method (for example is included under the strict hybridization conditions, temperature required between 45-60 ℃, SDS concentration is 0.1-1%), utilization contains the sequence of SEQ ID NO:7 or its fragment or screens a kind of suitable library by the probe that SEQID NO:7 sequence or its fragment are formed, and separates steps such as the gene of the whole length that comprise this polynucleotide sequence or genomic clone.

The invention provides a kind of with regard to its total length, with a kind of and whole identical polynucleotide of encoding sequence (open reading frame) of length among the SEQ ID NO:7.The present invention also provides a kind of mature polypeptide or its segmental encoding sequence, exist with a kind of maturation protein in this polynucleotide sequence self and the reading frame or the form of its segmental encoding sequence, this reading frame contains another kind of encoding sequence again, the sequence of for example encode a kind of guiding or secretion sequence, preceding protein sequence, former protein sequence, preproprotein sequence.Polynucleotide among the present invention contain a kind of non-coding sequence at least; for example these non-coding sequences comprise as; but be not limited at least a 5 ' and 3 ' non-coding sequence, for example transcribe but sequence, intron and the poly-adenylylation signal of non-translated sequence, termination signal (as relying on ρ-factor and not relying on the termination signal of ρ-factor), ribosome bind site, Kozak sequence, stable mRNA.These polynucleotide sequences also can comprise the additional amino acid whose appended sequence of coding.For example, codified can promote the marker sequence to the purifying of fusion polypeptide.In a certain particular of the present invention, the standard substance sequence is a kind of six histidine peptide sections, as pQE carrier (Qiagen, Inc.) provide, at Gentz etc., Proc.Natl.Acad Sci. has six histidine peptide sections of description or a kind of HA peptide segment mark (Wilson et al. among the USA 86:821-824 (1989), Cell 3～: 767 (1984), these two kinds of sequences are useful when separating with peptide sequence that they merge mutually.Polynucleotide among the present invention also comprise, but are not limited to, contain that structure gene and its controlling gene are expressed, with its sequence that is associated naturally.

The sequence of the BASB061 polypeptide of coding SEQ ID NO:8 can be identical with the polypeptid coding sequence of 1-513 the Nucleotide that is included in SEQ ID NO:8.Another kind of situation, as the result of genes encoding redundancy (degeneration), also the encode polypeptide of SEQ ID NO:8 of this polynucleotide sequence." polynucleotide encoding polypeptide " herein comprises those and comprises polypeptide, especially bacterial peptide in the code book invention, more particularly contains the polynucleotide of the Neisseriameningitides BASB061 polypeptide of SEQ ID NO:8 aminoacid sequence." polynucleotide encoding polypeptide " this noun also comprise those polypeptide that comprise that coding links to each other with additional zone (as be integrated insertion sequence, the integration of polynucleotide that phage is interrupted, integration carrier sequence, integration the transposon sequence or cause rna editing or the sequence of genomic dna reorganization) the continuum or the polynucleotide of locus of discontinuity, and additional zone also can comprise coding and/or non-coding sequence.

The invention further relates to the variant of polynucleotide described herein, these polynucleotide variant codings contain the polypeptide variants of the derivation aminoacid sequence of SEQ ID NO:8.Polynucleotide passage among the present invention can be used for, as the polynucleotide of whole length among synthetic the present invention.

Further especially preferred embodiment is the polynucleotide of coding BASB060 variant, these BASB061 variants have the BASB061 amino acid sequence of polypeptide of SEQ ID NO:8, and in its sequence, several, some, 5-10,1-5,1-3, the 2nd, the 1st amino-acid residue or 0 amino-acid residue with substitute, modify, disappearance and/or add and their arbitrary combination mode was handled.Wherein especially preferred is reticent substituting, add or disappearance, because they do not change the activity or the characteristic of BASB061 polypeptide.

Further preferred embodiment of the present invention is with regard to its total length, and the BASB061 polypeptide that contains the aminoacid sequence of SEQID NO:8 with coding has 85% identical polynucleotide at least, and polynucleotide complementary polynucleotide therewith.In this, the polynucleotide that have 90% to 100% identity at least are especially preferred, and in these preferred polynucleotide, those at least 95% identity be especially preferred.Further, in the polynucleotide that have 95% identity at least, those at least 97% identity be very preferred, at least 98% and 99% identity be especially very preferably, at least 99% identity be more very preferably.

Embodiment preferred is that those codings maintain the biological activity identical with the coded mature polypeptide of the DNA of SEQ ID NO:7 or the polynucleotide of function basically.

According to the particular preferred embodiment among the present invention, the invention provides polynucleotide with BASB061, as those polynucleotide of SEQ ID NO:7, especially under the condition of strictness, the polynucleotide of hybridizing.

Polynucleotide have been the present invention further provides with polynucleotide sequence provided herein hybridization.In this, the invention particularly relates to the polynucleotide that those are hybridized with polynucleotide described herein under stringent condition.Noun used herein " strict condition " and " strict hybridization conditions " just can be hybridized when referring to 95%, preferably at least 97% identity is only arranged between sequence at least.An example of stringent hybridization condition is exactly in a kind of solution 42 ℃ of cultivations and spends the night, subsequently at 65 ℃ of hybridization upholders that wash down among 0.1 * SSC, solution herein contains: 50% formaldehyde, 5 * SSC (150mM NaCI, the 15mM trisodium citrate), 50 mM sodium phosphates (pH7.6), 5 * Denhardt ' s solution, the denatured DNA of the salmon sperm that 10% sulfuric acid dextran and 20 micrograms/ml have sheared.Hybridization and wash conditions are well-known in addition, and at the Molecular Cloning:ALaboratory Manual of Sambrook etc., Second Edition, Cold Spring Harbor, N.Y., in (1989), especially in its Chapter 11 example is arranged.Hybridization solution also can be shared with polynucleotide sequence provided by the invention.

The present invention also provides a kind of polynucleotide of being formed or comprised this polynucleotide sequence by a kind of polynucleotide sequence.This polynucleotide sequence is under the hybridization conditions of strictness, with this polynucleotide sequence or its fragment that contain among the SEQ ID NO:7 is probe, screening comprises the suitable library of complete genome of a kind of polynucleotide sequence among the SEQ IDNO:7, and this polynucleotide sequence is separated this serial of methods obtains.Be of value to the fragment that obtains these polynucleotide and comprise, for example the probe and the primer that are described in detail of other parts of the present invention.

Detection about polynucleotide, other parts of the present invention have discussion, for example the polynucleotide among the present invention can be used as the probe of RNA, cDNA and genomic dna, separate the cDNA and the genomic clone of whole length of the BASB061 that encodes with this, and also can this separation have high identity, the cDNA of other genes of especially high sequence identity and genomic clone with BASB061.In general, such probe contains 15 nucleotide residues or base pair at least.Preferably contain the probe of 30 nucleotide residues or base pair at least or contain the probe of 50 nucleotide residues or base pair at least.Especially preferred is to contain 20 nucleotide residues or base pair at least but the probe that is less than 30 nucleotide residues or base pair.

By the screening method that the dna sequence dna synthetic oligonucleotide probe that utilizes SEQ ID NO:7 to provide carries out, can be separated to the coding region of BASB061 gene.Then, utilize contain with the present invention in labeled oligonucleotide screening cDNA, genomic dna or the mRNA library of gene order complementary sequence, and then determine the member in the library of hybridizing with probe.

A target of the present invention provides the polynucleotide of coding BASB063 polypeptide, the polynucleotide of the BASB063 polypeptide of indication herein of especially encoding.

In a kind of especially preferred embodiment of the present invention, these polynucleotide comprise the district of coding BASB063 polypeptide or its variant, this coding region comprises the sequence among the SEQ ID NO:9, and this sequence comprises gene or its variant of whole length of coding BASB063 polypeptide.

The BASB063 polynucleotide that provide among the SEQ ID NO:9 are Neisseria meningitidis strains A TCC13090.

Further, the invention provides coding and/or express BASB063 polypeptide and polynucleotide, especially the isolated nucleic acid molecule of Neisseria meningitidis BASB063 polypeptide and polynucleotide, these nucleic acid molecule comprise, as unprocessed RNA, ribozyme rna, mRNA, cDNA, genomic dna, B-and Z-DNA.Further embodiment comprises biologically among the present invention, diagnosis is gone up, useful polynucleotide and polypeptide and variant thereof in the prevention, clinically or on treating, comprise the composition of these materials.

Another aspect of the present invention relates to isolating polynucleotide, the polynucleotide and the variant thereof of tight association is arranged with it, and these isolating polynucleotide comprise gene a kind of whole length, coding BASB063 polypeptide at least, and this polypeptide contains the deduced amino acid from SEQ ID NO:10.

Of the present invention another especially the polypeptide in the preferred embodiment be BASB063 polypeptide or its variant, this BASB063 polypeptide is taken from Neisseria meningitidis, and it comprises a kind of aminoacid sequence of SEQ ID NO:10 or is made up of a kind of aminoacid sequence of SEQ ID NO:10.

Utilize information provided herein, as the polynucleotide sequence among the SEQ ID NO:9, the polynucleotide of a kind of BASB063 polypeptide of encoding among the present invention just can be by the clone and the screening method of standard, be the material that sets out as those with the Neisseria meningitidis cell, the bacterial chromosomal dna fragment is cloned and checked order and then obtain whole length clone's method, obtain.For example, seek out the polynucleotide sequence among the present invention, as given polynucleotide sequence among the SEQ ID NO:9, representative is the chromosomal DNA clone library of detecting the Neisseria meningitidis of intestinal bacteria or other appropriate host, and is 17-mer or longer radio-labeling oligonucleotide derived from a kind of partial sequence, preferred length.Utilize strict hybridization conditions just the clone who carries with the probe same DNA can be distinguished.By each being checked order by the clone that hybridization obtains with the sequencing primer that designs by original polypeptide or polynucleotide, these polynucleotide might be extended from both direction, thus the gene order of definite whole length.More convenient is that such order-checking can be passed through, and as utilizing from the denatured double stranded dna of plasmid clone preparation, carries out.Suitable technology is at Manistis, T., Fritsch, the MOLECULAR CLONING.A LABORATORYMAMIAL of E.F. and Sambrook etc., 2nd Ed.; Cold Spring Harbor Labocatory pnss, Cold Spring Harbor has narration (seeing in particular.Screening ByHybridization 1.90 and Sequencing Denatured Double-StrandedDNA Templates 13.70) among the NewYork (1989).Directly the genomic dna order-checking also can be undertaken by the gene order of obtaining a kind of whole length.In the illustrative example of the present invention, each polynucleotide among the SEQ ID NO:9 all can be found in being derived from the DNA library of Neisseria meningitidis.

In addition, dna sequence dna in the Neisseria meningitidis contains a kind of open reading frame, a kind of protein of this open reading frame codified, the amino acid number that this protein contains amino acid about and among the SEQ ID NO:10 is identical, and its molecular weight can calculate by the amino-acid residue molecular weight Evaluation Method that those of skill in the art know.

In SEQ ID NO:9, the codon at first Nucleotide place and since the polypeptide of the polynucleotide encoding SEQ ID NO:10 between the end of a period codon of the 814th Nucleotide.

Advance on the one hand, the invention provides the separation polynucleotide of forming or comprise following sequence by following sequence:

(a) a kind of polynucleotide sequence, this sequence is with regard to SEQ ID NO:9 total length, at least with SEQ ID NO:9 85% identity is arranged, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, even more preferably with its 97-99% identity or identical sequence at least, or

(b) a kind of polynucleotide sequence, this sequence is with regard to SEQ ID NO:10 total length, at least with SEQ ID NO:10 85% identity is arranged, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, in addition more preferably with its 97-99% identity or identical sequence at least.

A kind of polynucleotide of the polypeptide in the code book invention comprise homologous gene and orthologous gene in its kind outside Neisseria meningitidis, can be made by a kind of method.This method (for example is included under the strict hybridization conditions, temperature required between 45-60 ℃, SDS concentration is 0.1-1%), utilization contains the sequence of SEQ ID NO:9 or its fragment or screens a kind of suitable library by the probe that SEQID NO:9 sequence or its fragment are formed, and separates steps such as the gene of the whole length that comprise this polynucleotide sequence or genomic clone.

The invention provides a kind of with regard to its total length, with a kind of and encoding sequence (open reading frame) among the SEQ ID NO:9 polynucleotide of identity fully.The present invention also provides a kind of mature polypeptide or its segmental encoding sequence, and the form of a kind of maturation protein in this polynucleotide sequence self and the reading frame or its segmental encoding sequence exists, this reading frame contains another kind of encoding sequence again, the sequence of for example encode a kind of guiding or secretion sequence, preceding protein sequence, former protein sequence, preproprotein sequence.Polynucleotide among the present invention contain a kind of non-coding sequence at least; for example these non-coding sequences comprise as; but be not limited at least a 5 ' and 3 ' non-coding sequence, for example transcribe but sequence, intron and the poly-adenylylation signal of non-translated sequence, termination signal (as relying on ρ-factor and not relying on the termination signal of ρ-factor), ribosome bind site, Kozak sequence, stable mRNA.These polynucleotide sequences also can comprise the additional amino acid whose appended sequence of coding.For example, codified can promote the marker sequence to the purifying of fusion polypeptide.In a certain particular of the present invention, the standard substance sequence is a kind of six histidine peptide sections, as pQE carrier (Qiagen, Inc.) provide, at Gentz etc., Proc.Natl.Acad Sci. has six histidine peptide sections of description or a kind of HA peptide segment mark (Wilson et al. among the USA 86:821-824 (1989), Cell 3～: 767 (1984), these two kinds of sequences are useful when separating with peptide sequence that they merge mutually.Polynucleotide among the present invention also comprise, but are not limited to, contain that structure gene and its controlling gene are expressed, with its sequence that is associated naturally.

The sequence of the BASB063 polypeptide of coding SEQ ID NO:10 can with the polypeptid coding sequence identity of 1-813 the Nucleotide that is included in SEQ IDNO:10.Another kind of situation, as the result of genes encoding redundancy (degeneration), also the encode polypeptide of SEQ ID NO:10 of this polynucleotide sequence." polynucleotide encoding polypeptide " herein comprises those and comprises polypeptide, especially bacterial peptide in the code book invention, more particularly contains the polynucleotide of the Neisseria meningitides BASB063 polypeptide of SEQ ID NO:10 aminoacid sequence." polynucleotide encoding polypeptide " this noun also comprise those polypeptide that comprise that coding links to each other with additional zone (as be integrated insertion sequence, the integration of polynucleotide that phage is interrupted, integration carrier sequence, integration the transposon sequence or cause rna editing or the sequence of genomic dna reorganization) the continuum or the polynucleotide of locus of discontinuity, and additional zone also can comprise coding and/or non-coding sequence.

The invention further relates to the variant of polynucleotide described herein, these polynucleotide variant codings contain the polypeptide variants of the derivation aminoacid sequence of SEQ ID NO:10.Polynucleotide passage among the present invention can be used for, as the polynucleotide of whole length among synthetic the present invention.

Further especially preferred embodiment is the polynucleotide of coding BASB063 variant, these BASB063 variants have the BASB063 amino acid sequence of polypeptide of SEQ ID NO:10, and in its sequence, several, some, 5-10,1-5,1-3, the 2nd, the 1st amino-acid residue or 0 amino-acid residue with substitute, modify, disappearance and/or add and their arbitrary combination mode was handled.Wherein especially preferred is reticent substituting, add or disappearance, because they do not change the activity or the characteristic of BASB063 polypeptide.

Further preferred embodiment of the present invention is with regard to its total length and the BASB063 polypeptide of the aminoacid sequence that contains SEQID NO:10 of encoding has the polynucleotide of 85% identity at least, and polynucleotide complementary polynucleotide therewith.In this, the polynucleotide that have 90% to 100% identity at least are especially preferred, and in these preferred polynucleotide, those at least 95% identity be especially preferred.Further, in the polynucleotide that have 95% identity at least, those at least 97% identity be very preferred, at least 98% and 99% identity be especially very preferably, at least 99% identity be more very preferably.

Embodiment preferred is that those codings maintain the biological activity identical with the coded mature polypeptide of the DNA of SEQ ID NO:9 or the polynucleotide of function basically.

According to the particular preferred embodiment among the present invention, the invention provides polynucleotide with BASB063, as those polynucleotide of SEQ ID NO:9, especially under the condition of strictness, the polynucleotide of hybridizing.

Polynucleotide have been the present invention further provides with polynucleotide sequence provided herein hybridization.In this, the invention particularly relates to the polynucleotide that those are hybridized with polynucleotide described herein under stringent condition.Noun used herein " strict condition " and " strict hybridization conditions " just can be hybridized when referring to 95%, preferably at least 97% identity is only arranged between sequence at least.An example of stringent hybridization condition is exactly in a kind of solution 42 ℃ of cultivations and spends the night, subsequently at 65 ℃ of hybridization upholders that wash down among 0.1 * SSC, solution herein contains: 50% formaldehyde, 5 * SSC (150mM NaCI, the 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5 * Denhardt ' s solution, the denatured DNA of the salmon sperm that 10% sulfuric acid dextran and 20 micrograms/ml have pulverized.Hybridization and wash conditions are well-known in addition, and at the Molecular Cloning:ALaboratory Manual of Sambrook etc., Second Edition, Cold Spring Harbor, N.Y., in (1989), especially in its Chapter 11 example is arranged.Hybridization solution also can be shared with polynucleotide sequence provided by the invention.

The present invention also provides a kind of polynucleotide of being formed or comprised this polynucleotide sequence by a kind of polynucleotide sequence.This polynucleotide sequence is under the hybridization conditions of strictness, with this polynucleotide sequence or its fragment that contain among the SEQ ID NO:9 is probe, screening comprises the suitable library of complete genome of a kind of polynucleotide sequence among the SEQ IDNO:9, and this polynucleotide sequence is separated this serial of methods obtains.Be of value to the fragment that obtains these polynucleotide and comprise, for example the probe and the primer that are described in detail of other parts of the present invention.

Detection about polynucleotide, other parts of the present invention have discussion, for example the polynucleotide among the present invention can be used as the probe of RNA, cDNA and genomic dna, separate the cDNA and the genomic clone of whole length of the BASB063 that encodes with this, and also can this separation have high identity, the cDNA of other genes of especially high sequence identity and genomic clone with BASB063.In general, such probe contains 15 nucleotide residues or base pair at least.Preferably contain the probe of 30 nucleotide residues or base pair at least or contain the probe of 50 nucleotide residues or base pair at least.Especially preferred is to contain 20 nucleotide residues or base pair at least but the probe that is less than 30 nucleotide residues or base pair.

By the screening method that the dna sequence dna synthetic oligonucleotide probe that utilizes SEQ ID NO:9 to provide carries out, can be separated to the coding region of BASB063 gene.Then, utilize contain with the present invention in labeled oligonucleotide screening cDNA, genomic dna or the mRNA library of gene order complementary sequence, and then determine the member in the library of hybridizing with probe.

A target of the present invention provides the polynucleotide of coding BASB065 polypeptide, the polynucleotide of the BASB065 polypeptide of indication herein of especially encoding.

In a kind of especially preferred embodiment of the present invention, these polynucleotide comprise the district of coding BASB065 polypeptide or its variant, this coding region comprises the sequence among the SEQ ID NO:11, and this sequence comprises gene or its variant of whole length of coding BASB065 polypeptide.

The BASB065 polynucleotide that provide among the SEQ ID NO:11 are Neisseria meningitidis strains A TCC13090.

Further, the invention provides coding and/or express BASB065 polypeptide and polynucleotide, especially the isolated nucleic acid molecule of Neisseria meningitidis BASB065 polypeptide and polynucleotide, these nucleic acid molecule comprise, as unprocessed RNA, ribozyme rna, mRNA, cDNA, genomic dna, B-and Z-DNA.Further embodiment comprises biologically among the present invention, diagnosis is gone up, useful polynucleotide and polypeptide and variant thereof in the prevention, clinically or on treating, comprise the composition of these materials.

Another aspect of the present invention relates to isolating polynucleotide, the polynucleotide and the variant thereof of tight association is arranged with it, and these isolating polynucleotide comprise gene a kind of whole length, coding BASB065 polypeptide at least, and this polypeptide contains the deduced amino acid from SEQ ID NO:12.

Of the present invention another especially the polypeptide in the preferred embodiment be BASB065 polypeptide or its variant, this BASB065 polypeptide is taken from Neisseria meningitiilis, and it comprises a kind of aminoacid sequence of SEQ ID NO:12 or is made up of a kind of aminoacid sequence of SEQ ID NO:12.

Utilize information provided herein, as the polynucleotide sequence among the SEQ ID NO:11, the polynucleotide of a kind of BASB065 polypeptide of encoding among the present invention just can be by the clone and the screening method of standard, be the material that sets out as those with the Neisseria meningitidis cell, the bacterial chromosomal dna fragment is cloned and checked order and then obtain whole length clone's method, obtain.For example, seek out the polynucleotide sequence among the present invention, as given polynucleotide sequence among the SEQ ID NO:11, representative is the chromosomal DNA clone library of detecting the Neisseria meningitidis of intestinal bacteria or other appropriate host, and probe is to be 17-mer or longer radio-labeling oligonucleotide derived from a kind of partial sequence, preferred length.Utilize strict hybridization conditions just the clone who carries with the probe same DNA can be distinguished.By each clone who is identified by hybridization being checked order with the sequencing primer that designs by original polypeptide or polynucleotide, these polynucleotide might be extended from both direction, thus the gene order of definite whole length.More convenient is that such order-checking can be passed through, and as utilizing from the denatured double stranded dna of plasmid clone preparation, carries out.Suitable technology is at Manistis, T., Fritsch, the MOLECULAR CLONING.A LABORATORYMAMIAL of E.F. and Sambrook etc., 2nd Ed.; Cold Spring Harbor Labocatory pnss, Cold Spring Harbor has narration (seeing in particular.Screening ByHybridization 1.90 and Sequencing Denatured Double-StrandedDNA Templates 13.70) among the NewYork (1989).Directly the genomic dna order-checking also can be undertaken by the gene order of obtaining a kind of whole length.In the illustrative example of the present invention, each polynucleotide among the SEQ ID NO:11 all can be found in being derived from the DNA library of Neisseria meningitidis.

In addition, dna sequence dna in the Neisseria meningitidis contains a kind of open reading frame, a kind of protein of this open reading frame codified, the amino acid number that this protein contains amino acid about and among the SEQ ID NO:12 is identical, and its molecular weight can calculate by the amino-acid residue molecular weight Evaluation Method that those of skill in the art know.

In SEQ ID NO:11, the codon at first Nucleotide place and since the polypeptide of the polynucleotide encoding SEQ ID NO:12 between the end of a period codon of the 715th Nucleotide.

Advance on the one hand, the invention provides the separation polynucleotide of forming or comprise following sequence by following sequence:

(a) a kind of polynucleotide sequence, this sequence is with regard to SEQ ID NO:11 total length, at least with SEQ ID NO:11 85% identity is arranged, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, even more preferably with its 97-99% identity or identical sequence at least, or

(b) a kind of polynucleotide sequence, this sequence is with regard to SEQ ID NO:12 total length, at least with SEQ ID NO:12 85% identity is arranged, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, in addition more preferably with its 97-99% identity or identical sequence at least.

A kind of polynucleotide of the polypeptide in the code book invention comprise homologous gene and orthologous gene in its kind outside Neisseria meningitidis, can be made by a kind of method.This method (for example is included under the strict hybridization conditions, temperature required between 45-60 ℃, SDS concentration is 0.1-1%), utilization contains the sequence of SEQ ID NO:11 or its fragment or the probe be made up of SEQ ID NO:11 sequence or its fragment screens a kind of suitable library, and separates steps such as the gene of the whole length that comprise this polynucleotide sequence or genomic clone.

The invention provides a kind of with regard to its total length, with a kind of and whole identical polynucleotide of encoding sequence (open reading frame) of length among the SEQ ID NO:11.The present invention also provides a kind of mature polypeptide or its segmental encoding sequence, exist with a kind of maturation protein in this polynucleotide sequence self and the reading frame or its segmental encoding sequence form, this reading frame contains another kind of encoding sequence again, the sequence of for example encode a kind of guiding or secretion sequence, preceding protein sequence, former protein sequence, preproprotein sequence.Polynucleotide among the present invention contain a kind of non-coding sequence at least; for example these non-coding sequences comprise as; but be not limited at least a 5 ' and 3 ' non-coding sequence, for example transcribe but sequence, intron and the poly-adenylylation signal of non-translated sequence, termination signal (as relying on ρ-factor and not relying on the termination signal of ρ-factor), ribosome bind site, Kozak sequence, stable mRNA.These polynucleotide sequences also can comprise the additional amino acid whose appended sequence of coding.For example, codified can promote the marker sequence to the purifying of fusion polypeptide.In a certain particular of the present invention, the standard substance sequence is a kind of six histidine peptide sections, as pQE carrier (Qiagen, Inc.) provide, at Gentz etc., Proc.Natl.Acad Sci. has six histidine peptide sections of description or a kind of HA peptide segment mark (Wilson et al. among the USA 86:821-824 (1989), Cell 3～: 767 (1984), these two kinds of sequences are useful when separating with peptide sequence that they merge mutually.Polynucleotide among the present invention also comprise, but are not limited to, contain that structure gene and its controlling gene are expressed, with its sequence that is associated naturally.

The sequence of the BASB065 polypeptide of coding SEQ ID NO:12 can be contained in the polypeptid coding sequence of 1-714 Nucleotide of SEQ IDNO:12 identical with encoded packets.Another kind of situation, as the result of genes encoding redundancy (degeneration), also the encode polypeptide of SEQ ID NO:12 of this polynucleotide sequence." polynucleotide encoding polypeptide " herein comprises those and comprises polypeptide, especially bacterial peptide in the code book invention, more particularly contains the polynucleotide of the Neisseria meningitidis BASB065 polypeptide of SEQ ID NO:12 aminoacid sequence." polynucleotide encoding polypeptide " this noun also comprise those polypeptide that comprise that coding links to each other with additional zone (as be integrated insertion sequence, the integration of polynucleotide that phage is interrupted, integration carrier sequence, integration the transposon sequence or cause rna editing or the sequence of genomic dna reorganization) the continuum or the polynucleotide of locus of discontinuity, and additional zone also can comprise coding and/or non-coding sequence.

The invention further relates to the variant of polynucleotide described herein, these polynucleotide variant codings contain the polypeptide variants of the derivation aminoacid sequence of SEQ ID NO:12.Polynucleotide passage among the present invention can be used for, as the polynucleotide of whole length among synthetic the present invention.

Further especially preferred embodiment is the polynucleotide of coding BASB065 variant, these BASB065 variants have the BASB065 amino acid sequence of polypeptide of SEQ ID NO:12, and in its sequence, several, some, 5-10,1-5,1-3, the 2nd, the 1st amino-acid residue or 0 amino-acid residue with substitute, modify, disappearance and/or add and their arbitrary combination mode was handled.Wherein especially preferred is reticent substituting, add or disappearance, because they do not change the activity or the characteristic of BASB065 polypeptide.

Further preferred embodiment of the present invention is with regard to its total length, and the BASB065 polypeptide that contains the aminoacid sequence of SEQID NO:12 with coding has 85% identical polynucleotide at least, and polynucleotide complementary polynucleotide therewith.In this, the polynucleotide that have 90% to 100% identity at least are especially preferred, and in these preferred polynucleotide, those at least 95% identity be especially preferred.Further, in the polynucleotide that have 95% identity at least, those at least 97% identity be very preferred, at least 98% and 99% identity be especially very preferably, at least 99% identity be more very preferably.

Embodiment preferred is that those codings maintain the biological activity identical with the coded mature polypeptide of the DNA of SEQ ID NO:11 or the polynucleotide of function basically.

According to the particular preferred embodiment among the present invention, the invention provides polynucleotide with BASB065, as those polynucleotide of SEQ ID NO:11, especially under the condition of strictness, the polynucleotide of hybridizing.

Polynucleotide have been the present invention further provides with polynucleotide sequence provided herein hybridization.In this, the invention particularly relates to the polynucleotide that those are hybridized with polynucleotide described herein under stringent condition.Noun used herein " strict condition " and " strict hybridization conditions " just can be hybridized when referring to 95%, preferably at least 97% identity is only arranged between sequence at least.An example of stringent hybridization condition is exactly in a kind of solution 42 ℃ of cultivations and spends the night, subsequently at 65 ℃ of hybridization upholders that wash down among 0.1 * SSC, solution herein contains: 50% formaldehyde, 5 * SSC (150mM NaCI, the 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5 * Denhardt ' s solution, 10% sulfuric acid dextran and 20 micrograms/ml have sheared the denatured DNA of salmon sperm.Hybridization and wash conditions are well-known in addition, and at the Molecular Cloning:ALaboratory Manual of Sambrook etc., Second Edition, Cold Spring Harbor, N.Y., in (1989), especially in its Chapter 11 example is arranged.Hybridization solution also can be shared with polynucleotide sequence provided by the invention.

The present invention also provides a kind of polynucleotide of being formed or comprised this polynucleotide sequence by a kind of polynucleotide sequence.This polynucleotide sequence is under the hybridization conditions of strictness, with this polynucleotide sequence or its fragment that contain among the SEQ ID NO:11 is probe, screening comprises the suitable library of complete genome of a kind of sequence of polynucleotide among the SEQ IDNO:11, and this polynucleotide sequence is separated this serial of methods obtains.Be of value to the fragment that obtains these polynucleotide and comprise, for example the probe and the primer that are described in detail of other parts of the present invention.

Detection about polynucleotide, other parts of the present invention have discussion, for example the polynucleotide among the present invention can be used as the probe of RNA, cDNA and genomic dna, separate the cDNA and the genomic clone of whole length of the BASB065 that encodes with this, and also can this separation have high identity, the cDNA of other genes of especially high sequence identity and genomic clone with BASB065.In general, such probe contains 15 nucleotide residues or base pair at least.Preferably contain the probe of 30 nucleotide residues or base pair at least or contain the probe of 50 nucleotide residues or base pair at least.Especially preferred is to contain 20 nucleotide residues or base pair at least but the probe that is less than 30 nucleotide residues or base pair.

By the screening method that the dna sequence dna synthetic oligonucleotide probe that utilizes SEQ ID NO:11 to provide carries out, can be separated to the coding region of BASB065 gene.Then, utilize contain with the present invention in labeled oligonucleotide screening cDNA, genomic dna or the mRNA library of gene order complementary sequence, and then determine the composition in the library of hybridizing with probe.

A target of the present invention provides the polynucleotide of coding BASB066 polypeptide, the polynucleotide of the BASB066 polypeptide of indication herein of especially encoding.

In a kind of especially preferred embodiment of the present invention, these polynucleotide comprise the district of coding BASB066 polypeptide or its variant, this coding region comprises the sequence among the SEQ ID NO:13, and this sequence comprises gene or its variant of whole length of coding BASB066 polypeptide.

The BASB066 polynucleotide that provide among the SEQ ID NO:13 are Neisseria meningitidis strains A TCC13090.

Further, the invention provides coding and/or express BASB066 polypeptide and polynucleotide, especially the isolated nucleic acid molecule of Neisseria meningitidis BASB066 polypeptide and polynucleotide, these nucleic acid molecule comprise, as unprocessed RNA, ribozyme rna, mRNA, cDNA, genomic dna, B-and Z-DNA.Further embodiment comprises biologically among the present invention, diagnosis is gone up, useful polynucleotide and polypeptide and variant thereof in the prevention, clinically or on treating, comprise the composition of these materials.

Another aspect of the present invention relates to isolating polynucleotide, the polynucleotide and the variant thereof of tight association is arranged with it, and these isolating polynucleotide comprise gene a kind of whole length, coding BASB066 polypeptide at least, and this polypeptide contains the deduced amino acid from SEQ ID NO:14.

Of the present invention another especially the polypeptide in the preferred embodiment be BASB066 polypeptide or its variant, this BASB066 polypeptide is taken from Neisseria meningitidis, and it comprises a kind of aminoacid sequence of SEQ ID NO:14 or is made up of a kind of aminoacid sequence of SEQ ID NO:14.

Utilize information provided herein, as the polynucleotide sequence among the SEQ ID NO:13, the polynucleotide of a kind of BASB066 polypeptide of encoding among the present invention just can be by the clone and the screening method of standard, be the material that sets out as those with the Neisseria meningitidis cell, the bacterial chromosomal dna fragment is cloned and checked order and then obtain whole length clone's method, obtain.For example, seek out the polynucleotide sequence among the present invention, as given polynucleotide sequence among the SEQ ID NO:13, representative is the chromosomal DNA clone library of detecting the Neisseria meningitidis of intestinal bacteria or other appropriate host, and probe is to be 17-mer or longer radio-labeling oligonucleotide derived from a kind of partial sequence, preferred length.Utilize strict hybridization conditions just the clone who carries with the probe same DNA can be distinguished.By each being identified that by hybridization the clone checks order with the sequencing primer that designs by original polypeptide or polynucleotide, these polynucleotide might be extended from both direction, thus the gene order of definite whole length.More convenient is that such order-checking can be passed through, and as utilizing from the denatured double stranded dna of plasmid clone preparation, carries out.Suitable technology is at Manistis, T., Fritsch, the MOLECULAR CLONING.A LABORATORYMAMIAL of E.F. and Sambrook etc., 2nd Ed.; Cold Spring Harbor Labocatory pnss, Cold Spring Harbor has narration (seeing in particular.Screening ByHybridization 1.90 and Sequencing Denatured Double-StrandedDNA Templates 13.70) among the NewYork (1989).Directly the genomic dna order-checking also can be undertaken by the gene order of obtaining a kind of whole length.In the illustrative example of the present invention, each polynucleotide among the SEQ ID NO:13 all can be found in being derived from the DNA library of Neisseria meningitidis.

In addition, dna sequence dna in the Neisseria meningitidis contains a kind of open reading frame, a kind of protein of this open reading frame codified, the amino acid number that this protein contains amino acid about and among the SEQ ID NO:14 is identical, and its molecular weight can calculate by the amino-acid residue molecular weight Evaluation Method that those of skill in the art know.

In SEQ ID NO:13, the codon at first Nucleotide place and since the polypeptide of the polynucleotide encoding SEQ ID NO:14 between the end of a period codon of the 1174th Nucleotide.

Advance on the one hand, the invention provides the separation polynucleotide of forming or comprise following sequence by following sequence:

(a) a kind of polynucleotide sequence, this sequence is with regard to SEQ ID NO:13 total length, at least with SEQ ID NO:13 85% identity is arranged, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, even more preferably with its 97-99% identity or identical sequence at least, or

(b) a kind of polynucleotide sequence, this sequence is with regard to SEQ ID NO:14 total length, at least with SEQ ID NO:14 85% identity is arranged, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, in addition more preferably with its 97-99% identity or identical sequence at least.

A kind of polynucleotide of the polypeptide in the code book invention comprise homologous gene and orthologous gene in its kind outside Neisseria meningitidis, can be made by a kind of method.This method (for example is included under the strict hybridization conditions, temperature required between 45-60 ℃, SDS concentration is 0.1-1%), utilization contains the sequence of SEQ ID NO:13 or its fragment or the probe be made up of SEQ ID NO:13 sequence or its fragment screens a kind of suitable library, and separates steps such as the gene of the whole length that comprise this polynucleotide sequence or genomic clone.

The invention provides a kind of with regard to its total length, with a kind of and identical polynucleotide of encoding sequence (open reading frame) among the SEQ ID NO:13.The present invention also provides a kind of mature polypeptide or its segmental encoding sequence, exist with a kind of maturation protein in this polynucleotide sequence self and the reading frame or the form of its segmental encoding sequence, this reading frame contains another kind of encoding sequence again, the sequence of for example encode a kind of guiding or secretion sequence, preceding protein sequence, former protein sequence, preproprotein sequence.Polynucleotide among the present invention contain a kind of non-coding sequence at least; for example these non-coding sequences comprise as; but be not limited at least a 5 ' and 3 ' non-coding sequence, for example transcribe but sequence, intron and the poly-adenylylation signal of non-translated sequence, termination signal (as relying on ρ-factor and not relying on the termination signal of ρ-factor), ribosome bind site, Kozak sequence, stable mRNA.These polynucleotide sequences also can comprise the additional amino acid whose appended sequence of coding.For example, codified can promote the marker sequence to the purifying of fusion polypeptide.In a certain particular of the present invention, the standard substance sequence is a kind of six histidine peptide sections, as pQE carrier (Qiagen, Inc.) provide, at Gentz etc., Proc.Natl.Acad Sci. has six histidine peptide sections of description or a kind of HA peptide segment mark (Wilson et al. among the USA 86:821-824 (1989), Cell 3～: 767 (1984), these two kinds of sequences are useful when separating with peptide sequence that they merge mutually.Polynucleotide among the present invention also comprise, but are not limited to, contain that structure gene and its controlling gene are expressed, with its sequence that is associated naturally.

The sequence of the BASB060 polypeptide of coding SEQ ID NO:14 can be identical with the polypeptid coding sequence of 1-1173 the Nucleotide that comprises SEQ ID NO:14.Another kind of situation, as the result of genes encoding redundancy (degeneration), also the encode polypeptide of SEQ ID NO:14 of this polynucleotide sequence." polynucleotide encoding polypeptide " herein comprises those and comprises polypeptide, especially bacterial peptide in the code book invention, more particularly contains the polynucleotide of the Neisseria meningitides BASB066 polypeptide of SEQ ID NO:14 aminoacid sequence." polynucleotide encoding polypeptide " this noun also comprise those polypeptide that comprise that coding links to each other with additional zone (as be integrated insertion sequence, the integration of polynucleotide that phage is interrupted, integration carrier sequence, integration the transposon sequence or cause rna editing or the sequence of genomic dna reorganization) the continuum or the polynucleotide of locus of discontinuity, and additional zone also can comprise coding and/or non-coding sequence.

The invention further relates to the variant of polynucleotide described herein, these polynucleotide variant codings contain the polypeptide variants of the derivation aminoacid sequence of SEQ ID NO:14.Polynucleotide passage among the present invention can be used for, as the polynucleotide of whole length among synthetic the present invention.

Further especially preferred embodiment is the polynucleotide of coding BASB066 variant, these BASB066 variants have the BASB066 amino acid sequence of polypeptide of SEQ ID NO:14, and in its sequence, several, some, 5-10,1-5,1-3, the 2nd, the 1st amino-acid residue or 0 amino-acid residue with substitute, modify, disappearance and/or add and their arbitrary combination mode was handled.Wherein especially preferred is reticent substituting, add or disappearance, because they do not change the activity or the characteristic of BASB066 polypeptide.

Further preferred embodiment of the present invention is with regard to its total length, and the BASB066 polypeptide that contains the aminoacid sequence of SEQID NO:14 with coding has the identical polynucleotide of 85% whole length at least, and polynucleotide complementary polynucleotide therewith.In this, the polynucleotide that have 90% to 100% identity at least are especially preferred, and in these preferred polynucleotide, those at least 95% identity be especially preferred.Further, in the polynucleotide that have 95% identity at least, those at least 97% identity be very preferred, at least 98% and 99% identity be especially very preferably, at least 99% identity be more very preferably.

Embodiment preferred is that those codings maintain the biological activity identical with the coded mature polypeptide of the DNA of SEQ ID NO:13 or the polynucleotide of function basically.

According to the particular preferred embodiment among the present invention, the invention provides polynucleotide with BASB066, as those polynucleotide of SEQ ID NO:13, especially under the condition of strictness, the polynucleotide of hybridizing.

Polynucleotide have been the present invention further provides with polynucleotide sequence provided herein hybridization.In this, the invention particularly relates to the polynucleotide that those are hybridized with polynucleotide described herein under stringent condition.Noun used herein " strict condition " and " strict hybridization conditions " just can be hybridized when referring to 95%, preferably at least 97% identity is only arranged between sequence at least.An example of stringent hybridization condition is exactly in a kind of solution 42 ℃ of cultivations and spends the night, subsequently at 65 ℃ of hybridization upholders that wash down among 0.1 * SSC, solution herein contains: 50% formaldehyde, 5 * SSC (150mM NaCI, the 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5 * Denhardt ' s solution, 10% sulfuric acid dextran and 20 micrograms/ml have sheared the denatured DNA of salmon sperm.Hybridization and wash conditions are well-known in addition, and at the Molecular Cloning:ALaboratory Manual of Sambrook etc., Second Edition, Cold Spring Harbor, N.Y., in (1989), especially in its Chapter 11 example is arranged.Hybridization solution also can be shared with polynucleotide sequence provided by the invention.

The present invention also provides a kind of polynucleotide of being formed or comprised this polynucleotide sequence by a kind of polynucleotide sequence.This polynucleotide sequence is under the hybridization conditions of strictness, with this polynucleotide sequence or its fragment that contain among the SEQ ID NO:13 is probe, screening comprises the suitable library of complete genome of a kind of polynucleotide sequence among the SEQ IDNO:13, and this polynucleotide sequence is separated this serial of methods obtains.Be of value to the fragment that obtains these polynucleotide and comprise, for example the probe and the primer that are described in detail of other parts of the present invention.

Detection about polynucleotide, other parts of the present invention have discussion, for example the polynucleotide among the present invention can be used as the probe of RNA, cDNA and genomic dna, separate the cDNA and the genomic clone of whole length of the BASB066 that encodes with this, and also can this separation have high identity, the cDNA of other genes of especially high sequence identity and genomic clone with BASB066.In general, such probe contains 15 nucleotide residues or base pair at least.Preferably contain the probe of 30 nucleotide residues or base pair at least or contain the probe of 50 nucleotide residues or base pair at least.Especially preferred is to contain 20 nucleotide residues or base pair at least but the probe that is less than 30 nucleotide residues or base pair.

By the screening method that the dna sequence dna synthetic oligonucleotide probe that utilizes SEQ ID NO:13 to provide carries out, can be separated to the coding region of BASB066 gene.Then, utilize contain with the present invention in labeled oligonucleotide screening cDNA, genomic dna or the mRNA library of gene order complementary sequence, and then determine the member in the library of hybridizing with probe.

A target of the present invention provides the polynucleotide of coding BASB071 polypeptide, the polynucleotide of the BASB071 polypeptide of indication herein of especially encoding.

In a kind of especially preferred embodiment of the present invention, these polynucleotide comprise the district of coding BASB071 polypeptide or its variant, this coding region comprises the sequence among the SEQ ID NO:15, and this sequence comprises gene or its variant of whole length of coding BASB071 polypeptide.

The BASB071 polynucleotide that provide among the SEQ ID NO:15 are Neisseria meningitidis strains A TCC13090.

Further, the invention provides coding and/or express BASB071 polypeptide and polynucleotide, especially the isolated nucleic acid molecule of Neisseria meningitidis BASB071 polypeptide and polynucleotide, these nucleic acid molecule comprise, as unprocessed RNA, ribozyme rna, mRNA, cDNA, genomic dna, B-and Z-DNA.Further embodiment comprises biologically among the present invention, diagnosis is gone up, useful polynucleotide and polypeptide and variant thereof in the prevention, clinically or on treating, comprise the composition of these materials.

Another aspect of the present invention relates to isolating polynucleotide, the polynucleotide and the variant thereof of tight association is arranged with it, and these isolating polynucleotide comprise gene a kind of whole length, coding BASB060 polypeptide at least, and this polypeptide contains the deduced amino acid from SEQ ID NO:16.

Of the present invention another especially the polypeptide in the preferred embodiment be BASB071 polypeptide or its variant, this BASB071 polypeptide is taken from Neisseria meningitidis, and it comprises a kind of aminoacid sequence of SEQ ID NO:16 or is made up of a kind of aminoacid sequence of SEQ ID NO:16.

Utilize information provided herein, as the polynucleotide sequence among the SEQ ID NO:15, the polynucleotide of a kind of BASB071 polypeptide of encoding among the present invention just can be by the clone and the screening method of standard, be the material that sets out as those with the Neisseria meningitidis cell, the bacterial chromosomal dna fragment is cloned and checked order and then obtain whole length clone's method, obtain.For example, seek out the polynucleotide sequence among the present invention, as given polynucleotide sequence among the SEQ ID NO:15, representative is the chromosomal DNA clone library of detecting the Neisseria meningitidis of intestinal bacteria or other appropriate host, and probe is to be 17-mer or longer radio-labeling oligonucleotide derived from a kind of partial sequence, preferred length.Utilize strict hybridization conditions just the clone who carries with the probe same DNA can be distinguished.By each clone who is identified by hybridization being checked order with the sequencing primer that designs by original polypeptide or polynucleotide, these polynucleotide might be extended from both direction, thus the gene order of definite whole length.More convenient is that such order-checking can be passed through, and as utilizing from the denatured double stranded dna of plasmid clone preparation, carries out.Suitable technology is at Manistis, T., Fritsch, the MOLECULAR CLONING.A LABORATORYMAMIAL of E.F. and Sambrook etc., 2nd Ed.; Cold Spring Harbor Labocatory pnss, Cold Spring Harbor has narration (seeing in particular.Screening ByHybridization 1.90 and Sequencing Denatured Double-StrandedDNA Templates 13.70) among the NewYork (1989).Directly the genomic dna order-checking also can be undertaken by the gene order of obtaining a kind of whole length.In the illustrative example of the present invention, each polynucleotide among the SEQ ID NO:15 all can be found in being derived from the DNA library of Neisseria meningitidis.

In addition, dna sequence dna in the Neisseria meningitidis contains a kind of open reading frame, a kind of protein of this open reading frame codified, the amino acid number that this protein contains amino acid about and among the SEQ ID NO:16 is identical, and its molecular weight can calculate by the amino-acid residue molecular weight Evaluation Method that those of skill in the art know.

In SEQ ID NO:15, the codon at first Nucleotide place and since the polypeptide of the polynucleotide encoding SEQ ID NO:16 between the end of a period codon of the 805th Nucleotide.

Advance on the one hand, the invention provides the separation polynucleotide of forming or comprise following sequence by following sequence:

(a) a kind of polynucleotide sequence, this sequence is with regard to SEQ ID NO:15 total length, at least with SEQ ID NO:15 85% identity is arranged, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, even more preferably with its 97-99% identity or identical sequence at least, or

(b) a kind of polynucleotide sequence, this sequence is with regard to SEQ ID NO:16 total length, at least with SEQ ID NO:16 85% identity is arranged, the sequence that preferably has 90% identity at least with it, yet more preferably with the sequence of its at least 95% identity, in addition more preferably with its 97-99% identity or identical sequence at least.

A kind of polynucleotide of the polypeptide in the code book invention comprise homologous gene and orthologous gene in its kind outside Neisseria meningitidis, can be made by a kind of method.This method (for example is included under the strict hybridization conditions, temperature required between 45-60 ℃, SDS concentration is 0.1-1%), utilization contains the sequence of SEQ ID NO:15 or its fragment or the probe be made up of SEQ ID NO:15 sequence or its fragment screens a kind of suitable library, and separates steps such as the gene of the whole length that comprise this polynucleotide sequence or genomic clone.

The invention provides a kind of with regard to its total length, with the identical polynucleotide of encoding sequence (open reading frame) among the SEQ ID NO:15.The present invention also provides a kind of mature polypeptide or its segmental encoding sequence, exist with a kind of maturation protein in this polynucleotide sequence self and the reading frame or the form of its segmental encoding sequence, this reading frame contains another kind of encoding sequence again, the sequence of for example encode a kind of guiding or secretion sequence, preceding protein sequence, former protein sequence, preproprotein sequence.Polynucleotide among the present invention contain a kind of non-coding sequence at least; for example these non-coding sequences comprise as; but be not limited at least a 5 ' and 3 ' non-coding sequence, for example transcribe but sequence, intron and the poly-adenylylation signal of non-translated sequence, termination signal (as relying on ρ-factor and not relying on the termination signal of ρ-factor), ribosome bind site, Kozak sequence, stable mRNA.These polynucleotide sequences also can comprise the additional amino acid whose appended sequence of coding.For example, codified can promote the marker sequence to the purifying of fusion polypeptide.In a certain particular of the present invention, the standard substance sequence is a kind of six histidine peptide sections, as pQE carrier (Qiagen, Inc.) provide, at Gentz etc., Proc.Natl.Acad Sci. has six histidine peptide sections of description or a kind of HA peptide segment mark (Wilson et al. among the USA 86:821-824 (1989), Cell 3～: 767 (1984), these two kinds of sequences are useful when separating with peptide sequence that they merge mutually.Polynucleotide among the present invention also comprise, but are not limited to, contain that structure gene and its controlling gene are expressed, with its sequence that is associated naturally.

The sequence of the BASB071 polypeptide of coding SEQ ID NO:16 can be identical with the polypeptid coding sequence of 1-804 the Nucleotide that is included in SEQ IDNO:16.Another kind of situation, as the result of genes encoding redundancy (degeneration), also the encode polypeptide of SEQ ID NO:16 of this polynucleotide sequence." polynucleotide encoding polypeptide " herein comprises those and comprises polypeptide, especially bacterial peptide in the code book invention, more particularly contains the polynucleotide of the Neisseria meningitides BASB071 polypeptide of SEQ ID NO:16 aminoacid sequence." polynucleotide encoding polypeptide " this noun also comprise those polypeptide that comprise that coding links to each other with additional zone (as be integrated insertion sequence, the integration of polynucleotide that phage is interrupted, integration carrier sequence, integration the transposon sequence or cause rna editing or the sequence of genomic dna reorganization) the continuum or the polynucleotide of locus of discontinuity, and additional zone also can comprise coding and/or non-coding sequence.

The invention further relates to the variant of polynucleotide described herein, these polynucleotide variant codings contain the polypeptide variants of the derivation aminoacid sequence of SEQ ID NO:16.Polynucleotide passage among the present invention can be used for, as the polynucleotide of whole length among synthetic the present invention.

Further especially preferred embodiment is the polynucleotide of coding BASB071 variant, these BASB071 variants have the BASB071 amino acid sequence of polypeptide of SEQ ID NO:16, and in its sequence, several, some, 5-10,1-5,1-3, the 2nd, the 1st amino-acid residue or 0 amino-acid residue with substitute, modify, disappearance and/or add and their arbitrary combination mode was handled.Wherein especially preferred is reticent substituting, add or disappearance, because they do not change the activity or the characteristic of BASB071 polypeptide.

Further preferred embodiment of the present invention is with regard to its total length, and the BASB071 polypeptide that contains the aminoacid sequence of SEQID NO:16 with coding has 85% identical polynucleotide at least, and polynucleotide complementary polynucleotide therewith.In this, the polynucleotide that have 90% to 100% identity at least are especially preferred, and in these preferred polynucleotide, those at least 95% identity be especially preferred.Further, in the polynucleotide that have 95% identity at least, those at least 97% identity be very preferred, at least 98% and 99% identity be especially very preferably, at least 99% identity be more very preferably.

Embodiment preferred is that those codings maintain the biological activity identical with the coded mature polypeptide of the DNA of SEQ ID NO:15 or the polynucleotide of function basically.

According to the particular preferred embodiment among the present invention, the invention provides polynucleotide with BASB071, as those polynucleotide of SEQ ID NO:15, especially under the condition of strictness, the polynucleotide of hybridizing.

Polynucleotide have been the present invention further provides with polynucleotide sequence provided herein hybridization.In this, the invention particularly relates to the polynucleotide that those are hybridized with polynucleotide described herein under stringent condition.Noun used herein " strict condition " and " strict hybridization conditions " just can be hybridized when referring to 95%, preferably at least 97% identity is only arranged between sequence at least.An example of stringent hybridization condition is exactly in a kind of solution 42 ℃ of cultivations and spends the night, subsequently at 65 ℃ of hybridization upholders that wash down among 0.1 * SSC, solution herein contains: 50% formaldehyde, 5 * SSC (150mM NaCI, the 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5 * Denhardt ' s solution, the denatured DNA of the salmon sperm that 10% sulfuric acid dextran and 20 micrograms/ml have sheared.Hybridization and wash conditions are well-known in addition, and at the Molecular Cloning:ALaboratory Manual of Sambrook etc., Second Edition, Cold Spring Harbor, N.Y., in (1989), especially in its Chapter 11 example is arranged.Hybridization solution also can be shared with polynucleotide sequence provided by the invention.

The present invention also provides a kind of polynucleotide of being formed or comprised this polynucleotide sequence by a kind of polynucleotide sequence.This polynucleotide sequence is under the hybridization conditions of strictness, with this polynucleotide sequence or its fragment that contain among the SEQ ID NO:15 is probe, screening comprises the suitable library of complete genome of a kind of polynucleotide sequence among the SEQ IDNO:15, and this polynucleotide sequence is separated this serial of methods obtains.Be of value to the fragment that obtains these polynucleotide and comprise, for example the probe and the primer that are described in detail of other parts of the present invention.

Detection about polynucleotide, other parts of the present invention have discussion, for example the polynucleotide among the present invention can be used as the probe of RNA, cDNA and genomic dna, separate the cDNA and the genomic clone of whole length of the BASB060 that encodes with this, and also can this separation have high identity, the cDNA of other genes of especially high sequence identity and genomic clone with BASB071.In general, such probe contains 15 nucleotide residues or base pair at least.Preferably contain the probe of 30 nucleotide residues or base pair at least or contain the probe of 50 nucleotide residues or base pair at least.Especially preferred is to contain 20 nucleotide residues or base pair at least but the probe that is less than 30 nucleotide residues or base pair.

By the screening method that the dna sequence dna synthetic oligonucleotide probe that utilizes SEQ ID NO:15 to provide carries out, can be separated to the coding region of BASB071 gene.Then, utilize contain with the present invention in labeled oligonucleotide screening cDNA, genomic dna or the mRNA library of gene order complementary sequence, and then determine the member in the library of hybridizing with probe.

The method that can utilize multiple those of skill in the art to know (is seen, as Frohman based on terminal rapid amplifying (RACE) method of cDNA as those, et al., PNAS IlSA85:8998-9002,1988) method on obtains the DNA of total length or extends the DNA of short chain.Nearest modification to these technology is as Marathon ^TMThe example that technology did (Clontech Laboratories Inc.) has significantly been simplified the searching to long-chain DNA.At Marathon ^TMBe from the tissue of selecting, to extract RNA among the technology, connect ' receptor ' sequence then at two ends respectively, thus preparation cDNA.In conjunction with the Oligonucleolide primers special that utilizes gene specific, come 5 ' end of DNA amplification disappearance by nucleic acid amplification (PCR) technology with conjugant.Utilize nested primer then, i.e. design comes amplified production annealed primer (be typically the special primer of 3 ' terminal annealed, the conjugant of promoting the conjugant sequence and promote gene order 5 ' terminal annealed of selecting, the primer of gene specific) come this pcr amplification reaction of repetition.By dna sequencing this reaction product is analyzed, and this full length DNA is by product being directly connected among the existing DNA producing a kind of sufficient sequence, or the new sequence information that is used to design 5 ' primer separates, and the method for total length PCR makes up.

Polynucleotide and polypeptide among the present invention can be used as, and as studying with reagent and the material that carries out the discovery of medical diagnosis on disease and methods of treatment, these diseases especially refer to Human diseases, and these have further discussion in the part that relates to the polynucleotide detection.

Polynucleotide among the present invention are the oligonucleotide derived from the sequence of SEQ ID NO:1-16, the method that they can be used for having described, but PCR preferably, thereby determine whether the polynucleotide that this place is differentiated have carried out part or all of transcribing in the bacterium of infected tissue.Empirical tests, these peptide sections also will be applied in the diagnosis of the diagnosis of infective stage and the infection type that pathogenic agent causes.

The present invention also provides the polynucleotide of some coded polypeptides, and these polypeptide are maturation proteins of the amino acid (for example when this polypeptide mature form contains more than a kind of polypeptide chain) that includes additional amino or C-terminal amino acid or mature polypeptide inside.Such sequence may play an important role in the processing of albumen from the precursor to the mature form, can allow to send in other materials, can prolong or shorten proteic half life maybe can promote operations such as test for protein or production.In vivo, utilize cellular enzymes from maturation protein, to dispose with adding amino acid.

To each and all polynucleotide among the present invention, the present invention also provides and its complementary polynucleotide.Preferably these complementary polynucleotide and each polynucleotide complementation of complementary with it.

Precursor protein can be the inactive form of polypeptide, contains the mature form polypeptide that is fused on one or more presequences.But after presequence was got rid of, the precursor of these inactivations generally can become its activity form.Some or all of presequences can be got rid of before activation.In general, these precursors are called as preceding albumen (proprotein).

Outside A, the G of the representative nucleosides of standard, C, T/U, " N " also can be used as a certain polynucleotide of describing among the present invention.N is that in four kinds of DNA or the RNA nucleosides any all can appear at the specified location of this DNA or RNA, as long as combined with the adjacent nucleotide position, when reading in correct reading frame, preferred N is not the Nucleotide that produces the precocious terminator codon in this reading frame.

Generally speaking, the ripe albumen of polynucleotide codified among the present invention, have leader sequence maturation protein (can think preceding albumen), contain one or more non-before the presequence maturation protein precursor or the preproprotein of protein leader, and preproprotein is preceding proteic precursor, he contains a kind of leader sequence and one or more presequence, and these presequences have generally just been got rid of in processing the process that active and sophisticated polypeptide form are arranged with acquisition.

According to one aspect of the present invention, the polynucleotide that the invention provides among the present invention are being treated or prevention the especially application of genetic immunization aspect.

Polynucleotide of the present invention are the suitable delivering method of advantageous applications in the application on the genetic immunization, direct injection plasmid DNA (Wolff et al. in muscle for example, Hum Mol Genet (1992) 1:363, Manthorpe et al., Hum.Gene Ther. (1983) 4:419) send, with special protein carrier compound DNA (Wu et al., J Biol Chem. (1989) 364:16985), with coprecipitation of calcium phosphate (Benvenisty ﹠amp; Reshef, PNAS USA, (1986) 83:9551), the DNA of the various ways of being undertaken by liposome bag is by (Kanedaet al., Science (1989) 243:375), particle bombardment (Tang et al., Nature (1992) 356:152, Eisenbraun et al., DNA Cell Biol (1993) 12:791) and in the body that carries out of the retrovirus vector by the clone infect (Seeger et al., PNAS USA (1984) 81:5849).Carrier, host cell, expression system

The carrier that the present invention relates to comprises one or more polynucleotide among the present invention, and the host cell that relates to utilizes recombinant technology that carrier of the present invention and polypeptide product of the present invention have been carried out the genetically engineered design.Application source can prepare such protein from RNA, the cell free translation system of DNA construction of the present invention.

The technology of utilizing those of skill in the art to know, the recombinant polypeptide among the present invention can prepare from genetically engineered host cell design, that contain expression system.Further, the present invention relates to the expression system that comprises one or more polynucleotide among the present invention accordingly, also relate to contain such expression system, carry out the host cell that genetically engineered designed, also relate among the present invention product by the recombinant technology preparation.

For the polypeptide among recombinant production the present invention, will carry out genetic engineering modifiedly to host cell, make its a part or polynucleotide of the present invention in conjunction with expression system or expression system.Utilize the BASIC METHODS IN of the laboratory manual of many standards such as Davis etc.

MOLECULAR BIOLOGY, (1986) and the MOLECULARCLONING:A LABORATORY MANUAL of Sambrook etc., 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. method of describing in (1989), introduce and infection as transfection, electroporation, transduction, scratch loading, impact type that calcium phosphate transfection, the transfection of DEAE-dextran mediation, transposition, microinjection, cation lipid mediate, polynucleotide are effectively introduced host cell.

Suitable representative host comprises bacterial cell such as suis cell, staphylococcus, enterococcus bacteria, Bacillus coli cells, streptomyces cell, cyanobacteria, Bacillus subtilus, Moraxella catarrhalis (Moraxella catarrhalis), hemophilus influenzae and Neisseria meningitidis; Fungal cell such as yeast cell, Kluyveromyces sp, yeast belong cell (Saccharomyces), club fungi cell, Candida albicans and Aspergillus cell; Noctuid Sf9 is coveted on insect cell such as Drosophila S2 cell and meadow; Zooblast such as CHO, COS, HeLa, C127,3T3, BHK, 293, CV-1 and Bowes melanoma cells; And vegetable cell gymnosperm cell or angiosperm cell.

There is multiple expression system to can be used for preparing polypeptide among the present invention.In other expression system, these carriers comprise the karyomit(e) origin, free origin, carrier with viral origin, for example derived from virus particle, bacteriophage, transposon, the yeast episome, insertion element, the carrier of yeast chromosomal element, reach derived from virus as baculovirus, papovavirus such as SV40, vaccinia virus, adenovirus, fowl avipoxvirus, pseudorabies virus, little rrna nucleic acid virus, the carrier of retrovirus and alphavirus virus, and derived from these viral combinations as combination derived from plasmid and bacteriophage gene element, carrier as the combination of clay and phagemid.The formation of expression system comprises regulates and produces the control region of expressing.In this, in general, any system that is suitable for keeping in the host, breed or expresses multiple polynucleotide and/or a kind of polynucleotide can be used for expressing the polypeptide among the present invention.Utilize any technology in multiple well-known, the procedural technology, the MOLECULAR CLONING.A LABORATORY MANUAL (seeing above) as Sambrook etc. just can be inserted into suitable dna sequence dna in the expression system.

In eukaryotic recombinant expression system, for protein excretion that will translation in endoplasmic reticulum inner chamber, periplasmic space or born of the same parents' external environment, just need will be suitable secretion signal introducing polypeptide expressed in.These signals can be the endogenous or outer source signals of polypeptide.

Utilization comprises ammonium sulfate or well-known methods such as ethanol sedimentation, acid extraction, positively charged ion or anion-exchange chromatography, phosphorylated cotton chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite and Sugar receptors chromatography, can from the reconstitution cell culture, reclaim and purifying the present invention in polypeptide.Most preferably utilize ionic metal chromatography (IMAC) to come purified polypeptide.When polypeptide is synthetic in cell, in the process of separation and/or purifying during sex change, well-known refolding proteins technology can be used for producing again its activity form.

The living microorganism that expression system is also recombinated is as virus or bacterium.Useful gene can be inserted in the genome of live body recombinant virus or bacterium.Infect this live body carrier in inoculation or the body and will cause expressing in vivo this antigen and induction of immunity reaction.The virus and the bacterium that are used for this operation comprise as poxvirus (as cowpox, tame fowl pox, canary pox), alphavirus virus (Sindbis virus, Semliki Forest virus, Venezuelian EquineEncephalitis virus), adenovirus, adeno-associated virus, picornavirus (poliovirus, rhinovirus), simplexvirus (varicella zoster virus etc.), listeria, Salmonella, Shigella, neisseria, BCG.These viruses and bacterium can be fatal, also can be by the several different methods attenuation with preparation live body vaccine.These live body vaccines also are the parts among the present invention.Diagnostic detection, prediction detects, and serotype detects and sudden change detects

The present invention also relates to BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 and BASB071 polynucleotide and polypeptide in the application aspect diagnostic reagent.To eukaryote, particularly the detection of Mammals, especially people's BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 and BASB071 polynucleotide and/or polypeptide will provide a kind of medical diagnosis on disease, disease stage divide or at microbial infection to the diagnostic method aspect the reaction of medicine.Eukaryote, Mammals particularly, especially people, particularly those have infected or have been easy to infect the people of containing BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 and BASB071 gene or proteic organism, can utilize multiple technology well-known and provided by the invention to check on nucleic acid or amino acid levels.

Be used for preventing, diagnose or the polynucleotide and the polypeptide of other analyses can infect by supposition/and/or the body material of the individuality that infected obtain.The polynucleotide in this source, especially DNA or RNA can be directly used in detection, also can be used for carrying out enzymatic amplification by PCR or other amplification techniques before the analysis.RNA, especially mRNA, cDNA and genomic dna also can have identical application.

Utilize amplification technique,, just can obtain these infectious or the kind of resident organism and characteristics of strain by analyzing the genotype of polynucleotide selected from organism.The change of the length by amplified production, with the related organisms of selecting, preferably after the biology not of the same race of same genus or the genotype contrast, just can detect insertion and deficient phenomena with a kind of reference sequences of different strains.The DNA of amplification and BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 and the hybridization of BASB071 polynucleotide sequence of mark just can be detected point mutation.By utilizing Dnase or Rnase that DNA or RNA are digested respectively, or by detecting the difference of melting temperature (Tm) or reannealing kinetics, just can be perfectly or remarkable sequence and the duplex faulty or mispairing more that mates distinguish.With situation that the electrophoretic mobility of reference sequences is compared under, the electrophoretic mobility by polynucleotide passage in the gel changes the difference that just can detect between polynucleotide sequence.This detection is having denaturing agent to exist or is not having under the situation of denaturing agent and can both carry out.Also can detect difference between polynucleotide by direct DNA or RNA order-checking.See as Myers etc., Science, 130:1242 (1985).By ribozyme protection detection method such as RNase, V1 and S1 protects detection method or also can detect the sequence variation of specific site by the chemical chop method.See as Cotton etc., Proc.Natl.Acad Sci., USA, 85:4397-4401 (1985).

In another embodiment, can make up and comprise BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 and BASB071 nucleotide sequence or its segmental a series of oligonucleotide probes, thereby effectively screen, as classification on transgenation, serotype, the taxonomy or discriminating.This a series of technological method is well-known, and they have general suitability, they can be used for solving the various problems that molecular gene comprises genetic expression, gene interlocking and genetic variability in learning ((see as Chee etc., Science, 17～: 610 (1996)).

Therefore, on the other hand, the diagnostic kit that the present invention relates to comprises:

(a) polynucleotide among the present invention, SEQ ID NO:1 preferably, 3,5,7,9,11,13,15 nucleotide sequence or its fragment;

(b) with (a) complementary polynucleotide sequence;

(c) polypeptide among the present invention, SEQ ID NO:2 preferably, 4,6,8,10,12,14,16 nucleotide sequence or its fragment; Or

(d) antibody of the polypeptide among the present invention, SEQ ID NO:2 preferably, 4,6,8,10, the antibody of 12,14,16 polypeptide.

Will be understood that the (a) and (b), (c) of arbitrary test kit or (d) all contain basic component.In the mentioned reagent box, these test kits will be used for medical diagnosis on disease or to the diagnosis of the susceptibility of disease.

The present invention also relates to the application of the polynucleotide among the present invention as diagnostic reagent.To the mutant form of polynucleotide of the present invention, preferably with disease or the pathogenic SEQID NO:1 that is associated, 3,5,7,9,11,13,15 polynucleotide, detection, a kind of diagnostic tool will be provided, he can assist or define the determining or the infectivity of disease of prediction, disease stage of diagnostic method, the disease process of disease, and these diseases are because the expression deficiency of polynucleotide, overexpression or express changes and produce.Utilize multiple technologies, as the technology that other parts among the present invention are described, then can be with organism, the organism that has infected especially, the sudden change of these entrained polynucleotide detects on the polynucleotide level.

By multiple technologies, as take into account by serotype and classify, on the polynucleotide level, can detect carrying the polynucleotide among the present invention and/or the biological cell of sudden change on the polypeptide or polymorphism (allelic variation).For example, RT-PCR can be used for detecting the sudden change of RNA.Especially preferred is to utilize and automatic detection architecture such as GeneScan, the RT-PCR that combines.RNA, cDNA or genomic dna also can be used for same purpose-PCR.For example, the polynucleotide complementary PCR primer with coding BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 and BASB071 polypeptide can be used for differentiating and analyzing some sudden changes.

The present invention further provides some from 5 ' and/or 3 ' end remove the primer of 1,2,3 or 4 Nucleotide.These primers can be used for increasing BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 DNA and/or RNA, these DNA and/or RNA are from being derived from an individuality, separate in the sample as the material of health.The polynucleotide that primer can be used for increasing and is separated to from infected individuals utilize multiple technologies to explain this polynucleotide then.So just can detect the sudden change in the polynucleotide sequence, also this sudden change can be applied to diagnose and/or predict the stage of disease infection or infection or the process of infection, or be used for determining easy infection thing serotype and/or it is classified.

The present invention further provides and diagnosed the illness, infectation of bacteria preferably, the more preferably method of the infection that causes by Neisseria meningitidis, these methods comprise determines to contain SEQID NO:1,3,5,7,9, the polynucleotide enhanced of 11,13,15 sequences is expressed, and these polynucleotide are from being derived from an individuality, separate in the sample as the material of health.Utilize that any one those of skill in the art know, to the polynucleotide quantitative methods; for example the amplification, PCR;, RT-PCR, RNase the protection; the Northern marking, spectrometry and other hybridizing method are measured enhancing or minimizing that BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polynucleotide are expressed.

In addition, according to the present invention, thereby by comparing and can the overexpression of BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptide be detected with normal control tissue sample, this diagnostic method also can be used for detecting, as whether thoughts are dyed existence.To those of skill in the art, these can be used for determining that the detection method of BA5B051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptide level is well-known, these BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptide are from being derived from a host, separate in the sample as the material of health.Such detection method comprises that radioimmunoassay, competitive binding assay, the analysis of the Westem marking, antibody three-ply wood mensuration, antibody test and ELISA measure.

Polynucleotide among the present invention can be used as the polynucleotide array, preferably the component of high-density polynucleotide array (array) or polynucleotide grid (grid).These high density arrays are especially useful to the diagnosis and the prediction of disease.For example, one group of point, each all comprises a kind of different gene, and contain a kind of polynucleotide or multiple polynucleotide among the present invention, can be used for detecting, this detecting is to utilize by hybridization or nucleic acid amplification, utilization deutero-or utilize the probe that obtains from the body sample to determine that the method for the existence of special polynucleotide sequence in the individuality or correlated series carries out from the body sample.The existence of special polynucleotide sequence like this shows germ, the especially existence of Neisseria meningitidis, and can effectively be used for the diagnosis and/or the prevention of disease or lysis.Contain a large amount of SEQ IDNO:1,3,5,7,9,11,13,15 polynucleotide sequence variant grid is preferred.Contain a large amount of coding SEQ ID NO:2,4,6,8,10, the polynucleotide sequence variant grid of 12,14,16 peptide sequence also is preferred.Antibody

Polypeptide among the present invention and polynucleotide or its variant or its express cell can be used as immunogen, utilize this immunogen can produce respectively antibody to polypeptide or polynucleotide specific immunity.

The antibody of anti-BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptide or polynucleotide is provided in some preferred embodiment of the present invention.

According to conventional rules, with the polypeptide among the present invention and/or polynucleotide or the two or one of them the fragment that contains antigenic determinant, the two or one of them resemblance, express the two or one of them cell to animal, preferred inhuman animals administer just can obtain the antibody of anti-polypeptide of the present invention or polynucleotide.Arbitrary method that those of skill in the art know, that prepare antibody with the cause continuous cell line all can be used for preparing monoclonal antibody.Such example comprises multiple technologies, as at Kohler, G. and Milstein, C., among the Nature 156:495-497 (1975), among the Immunology Today 4:72 (1983) of Kozbor etc., Cole etc., pg 77-96 in MONOCLONAL ANTIBODIESAND CANCERTHERAPY, Alan R.Liss, the technology among the Inc. (1985).

The technology (U.S.Patent No.4,946,778) of preparation single-chain antibody can be used for preparing the single-chain antibody that the polynucleotide among the present invention or polypeptide is had resistance.In addition, transgenic mice or other biological body or animal, as the Mammals except that mouse, can be used for expressing to polypeptide among the present invention or polynucleotide tool specific immunity, humanized antibody.

On the other hand, utilize display technique of bacteriophage, can be from lymphocyte v-gene PCR amplified material or the library that is used to first to test (McCafferty, et al., (1990), Nature 348,552-554; Marks, et al., (1992) Biotechnology 10, selecting 779-783) has in conjunction with active antibody gene polypeptide of the present invention, and these lymphocytes v gene PCR amplified material is to obtain from the people who handles screening by anti--BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071.By also can improve the affinity of these antibody as chain reorganization (Clackson et al., (1991) Nature 352:628).

Above-mentioned antibody can be used for separating and identifying the clone that can express polypeptide of the present invention or polynucleotide, and then by come these polypeptide of purifying or polynucleotide as affinity chromatography.

Therefore, the antibody of anti-BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptide or polynucleotide can be used for treatment to be infected, especially infectation of bacteria.

The variant that comprises antigen variant of equal value, antigenic determinant variant of equal value or immunological equivalence variant also is the special one side of the present invention.

Preferably modified mistake, individuality is produced less immunogenic antibody or its variant.For example, if this individuality is the people, then this antibody is " humanized ", complementarity-determining region or hybridoma deutero-antibody district are implanted in the human monoclonal antibodies herein, see (1986) such as Jones, Nature 321,522-525 or Tempest et al., (1991) Biotechnology 9, the description of 266-273..Antagonist and agonist detect and various molecule

Polypeptide of the present invention or polynucleotide also can be assessed small molecules substrate and aglucon as the combination in cell, cell-free preparation, pharmaceutical chemicals storehouse and the natural product mixture.These substrates and aglucon can be natural substrate and aglucon or its structure or functional analogue.See as Coligan etc., Current Protocols in Immunology I (2): Chapter 5 (1991).

Utilize the marker that directly or indirectly links to each other with the candidate compound, then screening method just can easy to doly detect candidate compound and polypeptide or polynucleotide or with the cell that contains polypeptide or polynucleotide or film or with the combining of the fusion rotein of polypeptide.In addition, this screening method comprises with the competition thing of mark and competing mutually.Further, utilize the detection architecture of the cell be suitable for containing polypeptide or polynucleotide, can these screening methods can make the candidate compound produce signal to the activation of polypeptide or polynucleotide or restraining effect and detect.Generally be that the inhibitor to activation detects in the presence of known agonist, and under the situation that the candidate compound exists, just can be observed the influence that agonist produces activation.Under the condition that does not have agonist or antagonist to exist, whether the activation of polypeptide or polynucleotide is produced and suppress by detecting the candidate compound, the polypeptide expressed of the active polypeptide of composition and/or composition and polynucleotide just can be used for reversing in the screening method of effect of agonist or antagonist.And the candidate compound is might produce the activation of polypeptide or polynucleotide to suppress.Further, these screening methods comprise with candidate compound and the solution that contains polypeptide of the present invention or polynucleotide mix, form a kind of mixture, detect the active of BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptide and/or polynucleotide in the mixture and with the BASB051 in the mixture, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptide and or the activity of polynucleotide and standard step such as compare.Fusion rotein, as fusion rotein from the aforesaid Fc part of this paper and BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or the preparation of BASB071 polypeptide, also can be used for the high throughput testing method, identify the antagonist of the polypeptide among the present invention with this, also can be used to identify with its polypeptide relevant on system's generation and/or function and (see D.Bennett er al., J MolRecognition, 8:52-58 (1995); And K.Johanson er al., J BiolChem, 270 (16): 9459-9471 (1995)).

Be connected with polypeptide among the present invention or polynucleotide interactional with it, polypeptide and antibody also can be used for forming new screening method, detect the mRNA in the compound pair cell of adding and/or the influence of polypeptide output with this.For example, by the standard method of knowing in this area, just can utilize monoclonal antibody and polyclonal antibody to make up a kind of ELISA detection method that is intended to detect the polypeptide level that is associated with secretion or cell.This method can be used for finding those inhibition or strengthen from produce the material (also being called antagonist or inhibitor accordingly) of polypeptide through the cell or tissue of suitable operation.

The present invention also provides a kind of SCREENED COMPOUND, identify the compound that those can strengthen (agonist) or suppress (antagonist) BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptide or polynucleotide effect, and especially those can sterilizations or the method for bacteriostatic compound.These screening methods can comprise high-throughout technology.For example, in order to screen agonist or antagonist, to a kind of synthetic mixture, a kind of cellular compartment, as contain BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, the labeled substrate of BASB066 or BASB071 polypeptide and these polypeptide or the film of part, epicyte or cell walls, or their arbitrary preparation, cultivate having the candidate molecule or do not exist under the condition of candidate molecule, these candidate molecules can contain BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 antagonist or agonist.These candidate molecules strengthen or the ability of antagonism BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptide is reflected in that the tagged ligand bonded reduces or from the reduction of substrate products therefrom output.Those carry out voluntary combination, and also promptly not inducing the bonded molecule of BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or the effect of BASB071 polypeptide most possibly is good antagonist.Depending on circumstances, those are in conjunction with good, and productive rate, increase signal transduction or the active molecule of increase chemical access that can increase the product that obtains from substrate are agonists.Depending on circumstances, by utilizing a kind of system of reporting to increase detection to active speed of product, signal transduction or the chemical access that obtains from substrate or level.In this, the report system is useful, he comprises, but be not limited to substrate conversion colorimetric, mark is product and binding assay as known in the art, and these products are that BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polynucleotide or polypeptide active are changed the reporter gene that responds.

BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, another example of BASB066 or BASB071 agonist detection method is a competitive assay, competitive assay is meant under the conditions suitable that competitive inhibition detects, with BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 and a kind of potential agonist and BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 binding molecule, natural substrate or part, or substrate or ligand mimics combine.Can be to BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 carry out mark, the mark that is undertaken by radioactivity or colorimetric compound for example, therefore can determine the BASB051 that combines with binding molecule accurately, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 molecule or be converted into the BASB051 of product, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 molecule number, thus the effectiveness of this potential antagonist is assessed.

Potential antagonist comprises that those are attached on the polynucleotide and/or polypeptide among the present invention, thus the little organic molecule, peptide section, polypeptide and the antibody that suppress or remove their active fully or express.Potential antagonist also can be little organic molecule, the peptide section, the protein of polypeptide as being closely related, or the antibody in identical combination site arranged on binding molecule, these binding molecules for example: do not bring out BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or the active binding molecule of BASB071 inductive, by getting rid of BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, the combination of BASB066 or BASB071 polypeptide and/or polynucleotide, thus they just can prevent BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, the effect of BASB066 or BASB071 polypeptide and/or polynucleotide or expression.

Potential antagonist comprises some small molecules, these small molecules in conjunction with and occupy the binding site of polypeptide, thereby prevent that them from combining with the cell binding molecule, thereby also just prevented normal biological activity.Micromolecular example comprises, but is not limited to little organic molecule, peptide section or polypeptide resemblance molecule.Other potential antagonist comprises that antisense molecule (sees 0kano, J.Neurochem.16:560 (1991); OLIGODEOXYNUCLEOTIDESASANTISENSEINHIB1TORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL (1988), the description that these molecules are done).Preferred potential antagonist comprises the compound that is associated with BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 and the variant of BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071.

Further, the present invention relates to contain a peptide species of the present invention or it is segmental, by the soluble proteins of genetically engineered design, and the immunoglobulin (Ig) of multiple subclass (IgG, IgM, IgA, the different piece of constant region in heavy chain IgE) or the light chain.To immunoglobulin (Ig), preferably human IgG, the especially constant region of IgG1 heavy chain, and fusion reaction just occurs in the hinge area of IgG.In a special embodiment, only just Fc partly can be cut away, and this cutting sequence can cut away by blooc coagulation factor Xa by introducing the cutting sequence.In addition, the present invention also relates to utilize genetically engineered to prepare the method for those fusion roteins, and these methods can be used for drug screening, medical diagnosis on disease and treatment.The present invention further designs the polynucleotide of these fusion roteins of coding.The example of fusion protein technology can be found in the international patent application sequence number is the patent application of WO94/29458 and WO94/22914.

Each polynucleotide sequence provided by the invention can be used for finding or the exploitation antimicrobial compounds.When expressing, proteins encoded can be used as the target material of antibacterials screening.In addition, these polynucleotide sequences can be used for making up the antisense sequences that the interested encoding sequence of control is expressed, and these polynucleotide sequences be can fgs encoder albumen N-terminal district or the translation of Shine-Delgarno or other corresponding RNA promote sequence.

The present invention also provides the polypeptide among the present invention, polynucleotide, agonist or antagonist interfering one or more pathogenic bacteria and bearing the eucaryon host that infects sequela, the preferably application aspect the initial physiological response between mammalian hosts.Molecule among the present invention especially can be used for: prevent bacterium, especially Gram-positive and/or gram negative bacterium and bury eukaryote on the equipment, the adhesion between the extracellular matrix protein of preferred mammal, or and the wound extracellular matrix protein between adhesion; And/or the inhibition eukaryote, the adhesion between bacterium BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or the BASB071 albumen of preferred mammal extracellular matrix protein and mediation tissue injury; The positive regular incidence of the infection that inhibition is caused by buried implant equipment or other surgical operations.

According to an aspect of the present invention, the invention provides BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 agonist and antagonist, preferred antibacterial or germ-resistant agonist and antagonist.

Antagonist and agonist among the present invention can be used for, as prevent, suppress and/or treat disease.

Advance on the one hand, the present invention relates to the plan position (mimotope) of the polypeptide among the present invention.Intending the position is exactly a kind of peptide section sequence, he and native peptides section closely similar (on sequence composition or structure), and this peptide section sequence to be the antibody that can be able to be discerned the native peptides section discern, perhaps when this peptide section and suitable carriers mutually during coupling, he just can produce the antibody that can discern the native peptides section.

By the amino acid of interpolation, disappearance or alternative, just the peptide section can be intended the position and be designed for specific purpose.Therefore, just the peptide section can be done some and modify, thereby make the keying action of itself and protein carrier be easy to carry out.For example, it is desirable to comprise some Chemical bond methods of terminal aminothiopropionic acid.In addition, the peptide section combines with protein carrier, and comprising hydrophobic end with the far-end in conjunction with terminal at peptide also is ideal, so peptide section free does not still link to each other with the carrier proteins surface in conjunction with end.Thereby with this peptide section be in to whole native peptides segment molecule environment under the most similar conformational state of peptide section.For example, peptide shed repair decorations can be become the peptide section that contains terminal aminothiopropionic acid of N-and the terminal hydrophobic acid amides tail of C-.On the other hand, just can produce useful derivative, for example can improve the derivative of peptide section stability by adding or substituting one or more amino acid whose D-steric isomers.

On the other hand, utilize some technology such as display technique of bacteriophage (EP 0 552 267 B1), just can identify peptide section plan position, because these antibody itself just can combine with the polypeptide among the present invention by antibody.Utilize this technology to produce a large amount of peptide section sequences, the similar of these sequences and native peptides section, thereby they can be attached on the anti-natural peptide section antibody, but they itself not necessarily contain and the most of sequence of native peptides section homologous.Vaccine

Another aspect of the present invention relates to induces individuality; especially Mammals; the preferred human method that produces immunne response; these methods comprise the individuality inoculation with sufficient dosage, BASB051, the BASB057 that can produce antibody and/or T-cellullar immunologic response, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polynucleotide and/or polypeptide; or its fragment or variant; and these immunne responses can be protected this individuality to exempt to be infected; especially infectation of bacteria, the most especially Neisseria meningitidis infect.Certain methods also is provided, can have slowed down duplicating of bacterium by means of immunne response wherein.

Another aspect of the present invention relates to the method for induce immune response; these methods comprise transports nucleic acid carrier in the body; sequence or ribozyme to these individualities to instruct BASB051; BASB057; BASB060; BASB061; BASB063; BASB065; BASB066 or BASB071 polynucleotide and/or polypeptide expression; because these BASB051; BASB057; BASB060; BASB061; BASB063; BASB065; BASB066 or BASB071 polynucleotide and/or polypeptide; or its fragment or variant expression in vivo just can be induced the generation immunne response; as produce antibody and/or T cellullar immunologic response; these T cells comprise generation cytokine T cell or cytotoxin T cell; they can protect this individuality; preferred human individual; avoid disease, no matter whether this disease exists in vivo.To an example of this gene drug delivery is by its acceleration is entered in the purpose cell, and this gene just can be as the dressing of particle or other materials.Such nucleic acid carrier comprises nucleic acid, DNA/RNA hybrid, DNA-protein complexes or the RNA-protein complexes of DNA, RNA, ribozyme, modified.

Of the present inventionly advance to relate in one aspect to immune composition, when being introduced, these compositions can induce a certain individuality that produces immunne response in vivo, preferred individual human, they just can induce generation to BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, the immunne response of the polypeptide of BASB066 or BASB071 polynucleotide and/or these polynucleotide encodings, compositions herein comprises reorganization BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, the polypeptide of BASB066 or BASB071 polynucleotide and/or these polynucleotide encodings, and/or coding and express this BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polynucleotide, reach antigen by the polypeptide of these polynucleotide encodings, or other polypeptide among the present invention.These immunne responses can be used for the prevention or the treatment of disease, and they also can antibody mediated immunity and/or cellular immunization, exist as the form of the cellular immunization that caused by CTL or CD4+T cell.

BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptide or its fragment can merge mutually with accessory protein or chemical halfbody; itself can maybe can not produce antibody these accessory proteins or chemical halfbody; but they can stablize first albumen; and generation is that merge or albumen modified, that have antigenic or immunogenic characteristic, especially protection feature.Therefore, further the recombinant protein that has preferably merged preferably comprises the antigen accessory protein, for example, lipoprotein D, glutathione-S-transferase (GST) or the beta-galactosidase enzymes that obtains from hemophilus influenzae or those relatively large, can solubilized protein and quicken the albumen of its preparation and purifying.In addition, these albumen all can make the immunity system of the organism of accepting them produce general stimulation, and on this point, accessory protein might be as adjuvant.Accessory protein can be attached to first proteic amino or the C-terminal.

In vaccine composition of the present invention, BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptide or polynucleotide or its fragment or its intend the position or its variant can be present in the carrier, for example above-mentioned live body recombinant vectors is as the live bacteria carrier.

Other carriers that are suitable for BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptide are non-living body carriers, for example bacterial outer membrane vesicle or " vesicle (blebs) ".The OM vesicle is to obtain from the adventitia of gram negative bacterium duplicature, and verified they also are present in and comprise C.trachomatis and C.psittaci ((Zhou, L et al.1998.FEMS Microbiol.Lett.163:223-228) in interior many gram negative bacteriums.One reported but and the not exhaustive bacterial pathogens list that can produce vesicle comprise: bordetella pertussis, B. burgdorferi, Malta Bacillus brucellae, sheep Bacillus brucellae, intestinal bacteria, hemophilus influenzae, have a liking for lung Legionnella, Diplococcus gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa and Yersinia enterocolitica.

The advantage of vesicle is that outer membrane protein can be provided in the native conformation, thereby it is particularly useful for vaccine.Bacterium is carried out some designs, change the expression of one or more outer membrane molecules, just can do some improvement the vesicle that is used for vaccine.Like this, incremental adjustments (for example by changing promotor) just can be introduced or be subjected to for example desired adventitia immunogenic protein as BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptide expression.Method as an alternative or additional method, the expression of incoherent outer membrane molecule (for example non-protective antigen or immundominance but variable albumen) or deleterious outer membrane molecule (the potential inductor of replying as poisonous molecule such as LPS or autoimmunity) is subjected to negative the adjusting.These methods have more detailed discussion hereinafter.

The non-coding flanking region of BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 gene contains regulatory element more important in genetic expression.These be adjusted in transcribe with translation skill on carry out.Just can obtain the sequence of the upstream or the catchment of gene open reading frame by dna sequencing.Potential adjusting motif is determined in these sequence informations permissions, as different promoter elements, terminator sequence, derivable sequential element, repressor, the element of being responsible for stage variation, shine-dalgarno sequence and those districts of containing the potential secondary structure relevant with adjusting and being correlated with other multiple adjusting motifs or sequence.

These sequence informations allow the adjusting of expressing naturally of BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 gene.Just can just regulate by modification promotor, shine-dalgamo sequence, potential repressor or operator gene element or other relevant elements genetic expression.Equally, just can finish negative adjusting by carrying out similar modification to them.On the other hand, just expression of gene can be placed under the control of stage variation sequence, it is no longer regulated by changing the stage variation sequence.Utilize another kind of method, expression of gene can be placed one or more to allow inducing under the element of the expression regulated.These adjustings comprise, induce, add and induce substrate as the carbohydrate selected or their derivative, trace elements, VITAMIN, cofactor, metal ion etc. but be not limited to temperature variation.

Above-mentioned modification can be introduced by different ways.By random mutagenesis, can finish modification in vivo to the sequence that relates to genetic expression, select the phenotype that needs subsequently.Other method comprises separates useful district, and by random mutagenesis, site-directed mutagenesis, insertion mutagenesis or deletion mutagenesis he is modified.Then, just the district of modified can be introduced in the bacterial genomes again, and also can assess their influence genetic expression by homologous recombination.In other method, the sequence information in these useful districts can be used for substituting or lacking the natural adjusting sequence of all or part.Just the fixed regulatory region of target is separated since it is so and modify, make its regulatory element that contains combination, synthetic regulatory region or other regulatory regions, or make its disappearance wild-type regulate the selected part of sequence from another gene, heterogeneic regulatory element.Via homologous recombination the sequence of modified is introduced in the bacterial genomes again then.Preferred those lists that can be used for the up-regulated promotor of genetic expression comprise: the porA of Neisseria meningitidis or gonorrhoea naphthalene plucked instrument Salmonella, porB, lbpB, tbpB, pl10, lst, hpuAB; OmpCD, copB, lbpB, ompE, UspA1; UspA2; The TbpB of M.Catarrhalis; The p1 of hemophilus influenzae H.influenzae, p2, p4, p5, p6, lpD, tbpB, D15, Hia, Hmw1, Hmw2.

In an example, by the promotor of gene is exchanged the (upstream sequence that separates this gene with a stronger promotor, then this sequence is modified, subsequently by homologous recombination with in this sequence quiding gene group again) come the expression of regulatory gene.From these two kinds of bacteriums, can obtain to be subjected to up-regulated expression, also can from adventitia vesicle that prepare by bacterium or, obtain by bacterium release.

In another example, utilize aforesaid method can produce the recombinant bacteria bacterial strain, these bacterial strains have the characteristic that has improved, can be used as vaccine.These bacterial strains can comprise, but are not limited to attenuated strain, can increase and express the antigenic bacterial strain selected, rejected the gene of interfering immunne response and maybe can reduce the bacterial strain of the genetic expression of interfering immunne response, the bacterial strain with expression that immundominance albumen regulates, the bacterial strain with adventitia vesicle adjustment release.

Thereby, the present invention also provides the upstream of BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or the modified mistake of BASB071 gene, the upstream of this modified contains a kind of allos regulatory element, and this regulatory element can change the proteic expression level of BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 that is positioned at adventitia.According to the present invention, this upstream comprises the upstream sequence of BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 gene.This upstream further extends to apart from the ATG initiator codon and is no more than the 1000bp place from the upstream that BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 gene are right after.Be positioned at this gene under the situation of polycistron (operon) sequence, upstream can be initial foremost or initial before first gene of operon from being right after this gene.According to the present invention, preferably a kind of upstream of modified, this upstream contains the exogenous promoter at 500-700bp place, ATG upstream.

Thereby, the invention provides BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polypeptide in the bacterium vesicle that is present in modified mistake.The present invention further provides the host cell of modified, these host cells can produce the vesicle carrier based on non-living body film base.The present invention further provides the nucleic acid carrier that contains BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 gene, these genes contain and comprise the upstream external source regulatory element, modified.

The present invention further provides preparation among the present invention host cell and the method for bacterium vesicle.

The present invention also provides some compositions, vaccine composition especially, and comprise polypeptide among the present invention and/or polynucleotide, at Sato, the method for the immunostimulating dna sequence dna described in the Y.et al.Science 273:352 (1996).

The present invention also provides certain methods, these methods are utilized those above-mentioned polynucleotide that proved codified bacterium surface albumen constant region or its special fragments, and these polynucleotide or its special fragment are present in the polynucleotide construction, and these constructions are used in the genetic immunization experiment of carrying out in the animal infection modal of Neisseria meningitidis.These immunization experiments are particularly useful to differentiating those proteic antigenic determinants that can cause that prevention or therapeutic immunization are replied.We believe, this method makes those preparations with monoclonal antibody formulation of special value subsequently become possibility, these monoclonal antibodies come from those for developing Mammals, especially people's infection, especially the prevention infected of Neisseria meningitidis or the medicine of treatment, thereby successfully opposing or remove and infect necessary organ.

The present invention also comprises a kind of vaccine preparation, comprises and suitable carriers, and as acceptable carrier on the medicament, the immunogenicity recombinant polypeptide of the present invention or the polynucleotide that combine.Because polypeptide and polynucleotide can be degraded under one's belt, thereby they must carry out administered parenterally, comprise, as subcutaneous administration, muscle administration, intravenously administrable or intradermal administration.The formulation that is suitable for administered parenterally comprises and contains antioxidant, damping fluid, bacteriostatic compound and make preparation and the water-based or the non-aqueous aseptic injectable solution of the solution that the preferred blood of body fluid etc. is opened; And the hydration or the non-hydrated suspended substance that can contain the suspension factor or the thickening factor.These formulations can be sub-packed in unitary dose or the multi-dose container, in the ampoule or bottle as sealing, also can be stored under the cryodesiccated condition, only need add sterile liquid carrier before use to this.

Vaccine formulation among the present invention also can comprise the immunogenic adjuvant system that is used to improve formulation.Preferred adjuvant system can cause preferably that the TH1 type replys.

But a kind of immunne response generalized is divided into two kinds of final types, i.e. body fluid or cell-mediated immune responses (they are characterised in that it is respectively the cytological effect thing mechanism of antibody and protection traditionally).These two kinds of type of immune response are called TH1 type immunne response (cell-mediated replys) and TH2 type immunne response (replying of body fluid mediation) again

Extreme TH1 type immunne response feature is in the lymphocytic generation of cytotoxic T of the generation of its antigen-specific, haplotype restriction, and the natural killer cell generation of replying.Mouse TH1 type is replied feature and usually is IgG2a hypotype production of antibodies, and is to produce IgGI type antibody accordingly in human body.TH2 type immunne response feature comprises mouse IgGI, IgA and IgM in producing numerous homotypic immunity sphaeroprotein.

Can think that the motivating force of these two kinds of immunne responses of development is cytokines.High-caliber TH1 cytokines tends to induce given antigenic cell-mediated immune responses, and high-caliber TH2 cytokines tends to induce the immunne response of given antigenic body fluid mediation.

The difference of TH1 type and TH2 type immunne response is not absolute.In fact, body will be supported a kind of immunne response one by one, wherein be that TH1 type or TH2 type are preponderated.Yet, advantageously according to Mosmann and Coffman at the cytokine (Mosmann that considers each family described in the mouse CD4+ve T cell clone, T.R.and Coffman, R.L. (1989) TH1 and TH2 cells:differernt patterns of lymphokine secretionlead to different funcitional properties.Annual Review ofImmunology, 7, p145-173).On the tradition, TH1 type immunne response is to interrelate with INF-γ that produces by the T lymphocyte and IL-2 cytokine.It is not by the T cell that other induction phases direct and TH1 type immunne response are got in touch cytokine, produces as IL-12.On the contrary, TH2 type immunne response is that secretion with IL-4, IL-5, IL-6 and IL-13 interrelates.

Known some specific vaccine adjuvant is particularly suited for stimulating replying of generation TH1 or TH2 cytokines.In vaccine inoculation or infecting, after the best indicator of TH1:TH2 balance of immunne response is included in and remises sharp antigen traditionally, by the output of T lymphocyte TH1 or TH2 in the direct mensuration body, and/or measure the IgGI:IgG2a ratio that antigen-specific antibodies reacts.

Thereby, TH1 type auxiliary agent is can preferably stimulate when swashing antigen isolating T cell mass producing high-caliber TH1 cytokines when remising in the body, and the CD8+ cytotoxic T cell that interrelates of promotion and homotype TH1 type and the material of antigen specific immune globulin reaction.

Those can preferably stimulate the auxiliary agent of TH1 cell response in international patent application NO.WO94/00153 and WO95/17209 description to be arranged.

3-deoxidation-acyl group monophosphoryl lipid A (3D-MPL) is exactly a kind of such auxiliary agent.This can be by knowing among the GB 2220211 (Ribi).From chemistry, it is the mixture that contains the 3-deoxidation-acyl group monophosphoryl lipid A of 3,4,5 or 6 acidylate chains, is by RibiImmunochem, the Montana preparation.A kind of preferred form of 3-deoxidation-acyl group monophosphoryl lipid A is open in European patent 0 689 454 B1 (SmithKline Beecham BiologicalsSA).

Preferably enough little, 3D-MPL particle (european patent number 0 689 454) that can 0.22 micron filter membrane of aseptic filtration.The amount of 3D-MPL is 10 μ g-100 μ g in each preparation, preferred 25-50 μ g, and antigen wherein is typically 2-50 μ g/ agent.

Another kind of preferred auxiliary agent comprises QS21, and it is a kind of QuillajaSaponaria of coming from Molina bark, through the nontoxic fragment of high-efficient liquid phase chromatogram purification.It is welcome mixes with 3-deoxidation-acyl group monophosphoryl lipid A (3D-MPL), also welcomely mixes with carrier.

The preparation method of QS21 is at United States Patent (USP) NO.5, disclosed narration arranged in 057,540.

Contain the non-reactionogenicity auxiliary agent formulation existing narration (WO96/33739) in front of QS21.When becoming preparation with a kind of antigen co-production, the preparation that contains QS21 and cholesterol has proved effective TH1 stimulating additive.

The auxiliary agent that further can be used as the preferred stimulator of TH1 cell response comprises immunity modulation oligonucleotide, for example disclosed unmethylated CpG sequence among the WO 96/02555.

The binding substances of different TH1 stimulating additives, as the binding substances of above-mentioned auxiliary agent, also being considered to provide auxiliary agent, and this auxiliary agent is the preferred stimulator of TH1 cell response.For example, QS21 can plant the preparation preparation jointly with 3D-MPL.The typical proportion of QS21 and 3D-MPL is 1: 10-10: 1, preferred 1: 5-5: 1, often be essentially 1: 1.The preferable range of synergy is 3D-MPL: QS21 is 2.5; 1-1: 1.

According to the present invention, preferred a kind of carrier also is present in the vaccine composition.This carrier can be an oil-in-water emulsion, or aluminium salt, as aluminum phosphate or aluminium hydroxide.

A kind of preferred oil-in-water emulsion comprises a kind of metabolizable oil, as shark alkene, alpha-tocopherol, and tween 80.According to the present invention, one preferred aspect, in the vaccine composition antigen be in such emulsion with 3D-MPL and QS21 bonded.In addition, oil-in-water emulsion can contain span 85 and/or Yelkin TTS and/or tricaprylin.

Typically, the QS21 and the measures range of 3D-MPL in vaccine that can be used for people's administration be 1 μ g-200 μ g, as 10-100 μ g, preferred 10 μ g-50 μ g/ agent.Typical oil-in-water emulsion comprises 2-10% shark alkene, 2-10% alpha-tocopherol and 0.3-3% tween 80.Preferred shark alkene: the alpha-tocopherol ratio is equal to or less than 1, because can provide more stable emulsion like this.Span 85 also can be 1% level.In some cases, further to contain stablizer will be useful to the vaccine among the present invention.

Atoxic oil-in-water emulsion preferably contains non-toxicity oil, as is present in shark alkane (squalane) and shark alkene, emulsifying agent such as tween 80 in the aqueous carrier.These aqueous carriers can be, as phosphate buffered saline(PBS).

A kind of especially effectively auxiliary agent formulation comprises QS21,3D-MPL and the tocopherol in the oil-in-water emulsion described in the WO95/17210.

The present invention also provides a kind of multivalent vaccine composition, and he contains among the present invention and other antigen, is particularly useful for effectively treating the vaccine preparation that cancer, autoimmune disease and relative disease combine.Such multivalent vaccine composition can comprise, as the aforementioned TH1 inducibility auxiliary agent.Though the present invention is to specific BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 and BASB071 polypeptide and the existing description of polynucleotide, we are construed as the fragment that he has comprised the polypeptide or the polynucleotide of natural generation, and do not influence interpolation, the disappearance of the immunogen characteristic of recombinant polypeptide or polynucleotide basically or substitute similar polypeptide or the polynucleotide of operating.

Antigen also can transmit with form and the segmental form of ubcellular of whole bacterium (dead or live), and these possible delivery forms also comprise Neisseria meningitidis itself really.Composition, test kit and administration

Of the present inventionly advance to provide some compositions, these compositions to contain on the one hand to can be used for the pair cell administration or to BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or BASB071 polynucleotide and/or BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 or the BASB071 polypeptide of multicellular organisms administration.

The present invention also relates to discussed herein, that contain polynucleotide or polypeptide and their agonist and antagonist composition.Polypeptide among the present invention and polynucleotide can with non-sterile or sterile carrier or pair cell, tissue or organism useful carrier, as be suitable for pharmaceutical carrier, in conjunction with using to individual administration.Such composition comprises, for example among media additive or the present invention, the polypeptide and/or the polynucleotide of effective dose in the treatment, and medicine acceptable carrier or vehicle.These carriers can comprise, but be not limited to salt, buffering salt, dextrose, water, glycerine, ethanol and their binding substances.Formulation should be suitable mutually with administering mode.The invention further relates to diagnosis and pharmaceutical pack and test kit, they contain be equipped with that one or more is aforesaid, one or more container of the composition components among the present invention.Polypeptide among the present invention, polynucleotide and the separable application of other compounds, or with other compounds, as therapeutic compound, be used in combination.

Curative composition can effectively any and mode administration easily, and these modes comprise, for example by part, mouth, anus, vagina, intravenously, intraperitoneal, muscle, subcutaneous, nose interior or intradermal administration etc.

In treatment or prevention, the form that active agents can injectable composition is to individual administration, and for example aseptic hydration disperses thing, preferably waits the aseptic hydration of opening to disperse thing.

Advance on the one hand, the invention provides some pharmaceutical compositions, they comprise the medicine acceptable carrier or combine with vehicle, the polypeptide and/or the polynucleotide of effective dose in the treatment, for example soluble form of polypeptide among the present invention and/or polynucleotide, the agonist or antagonist peptide section or the micromolecular compound that combine with medicine acceptable carrier or vehicle.These carriers can comprise, but be not limited to salt solution, buffer saline, dextrose, water, glycerine, ethanol and their composition.The invention further relates to diagnosis and pharmaceutical pack and test kit, they contain be equipped with that one or more is aforesaid, one or more container of the composition components among the present invention.Polypeptide among the present invention, polynucleotide and the separable application of other compounds, or with other compounds, be used in combination as therapeutic compound.

These compositions will be suitable for route of administration, as passing through system or oral administration.The preferred systems administering mode comprises injection, is typically by intravenous injection.Other injecting pathway also can as subcutaneous injection, intramuscular injection or peritoneal injection.The mode of being administered systemically in addition comprises utilizes permeate agent, strides mucous membrane or endermic administration as what cholate or fusidinic acid or other stain removers carried out.In addition, if the polypeptide among the present invention or other compounds can be prepared into enteric or capsule preparations, just may carry out oral administration.Also partial and/or localization to the administration of these compounds, its form can be ointment, paste, gel, solution, powder or other.

To Mammals, especially to people's administration, the active medicine dosage level of expection is 0.01mg/kg-10mg/kg, generally is about 1mg/kg.The doctor can determine actual dose under particular case, these dosage will with individual fit, and change with the reaction of age, body weight and particular individual is different.Above-mentioned dosage is example generally speaking.Certainly, the individuality that has can administration with higher or lower dosage range, this is also within the scope of the invention.

The dosage range that requires depends on selection, route of administration, the preparation characteristic of peptide section, the person's that is subjected to the medicine type of situation and doctor's diagnosis.Yet to the person that is subjected to the medicine, proper dosage is the 0.1-100 μ g/kg person's body weight that is subjected to the medicine.

Vaccine composition is easily with injectable forms.Traditional auxiliary agent can be used for improving immunne response.The suitable dose of vaccine inoculation is 0.5-5 microgram/kg antigen, and such dosage wants preferred administration 1-3 time, is spaced apart 1-3 week.In given dosage range, do not observe compound among the present invention and produce side effect on the toxicology, and the generation of the side effect on the toxicology will get rid of him can be to the administration of suitable individuality.

Yet, consider the type of compounds that can get and the efficient difference of different way of administration, it is in expectancy that the broad of required dosage changes.For example, orally just require the dosage more higher than intravenous injection.The variation of dosage level can utilize the standard empirical method that is used to optimize to adjust, and this inflation method is existing in the art well to be understood.Sequence library, sequence and operational method in media (tangible medium) as can be known

Polypeptide and polynucleotide sequence have formed valuable information resources, just can determine that by these information their 2-or 3-tie up structure, and the sequence that can further differentiate similar homologue.The way of the easiest these methods of promotion is that sequence is stored in the computer-readable media, utilizes the information that has stored then or utilize well-known research tool in known macromolecular structure, comes the search sequence database as the GCG routine package.

The present invention also provides the method for analytical characteristic sequence or sequence string, especially gene order or their encoded protein sequence.Preferred sequence analysis method comprises, sequence homology thing analytical procedure for example is as identity and similarity analysis; DNA, RNA and structural analysis of protein; Sequence assembling; Branch analyzes; Sequence motif is analyzed; Open reading frame is determined; Nucleic acid base is checked; The codon operational analysis; Nucleic acid base is modified; And the sequence chromatographic peak is analyzed.

Provide a kind of computer-based method to come homology is differentiated.This method may further comprise the steps: first polynucleotide sequence that the polynucleotide sequence that contains among the present invention is provided in computer readable medium; This first polynucleotide sequence and at least a second polynucleotide or peptide sequence are compared, to differentiate homology.

The present invention also provides a kind of computer-based method to come homologue is differentiated.This method may further comprise the steps: first peptide sequence that the peptide sequence that contains among the present invention is provided in computer readable medium; This first peptide sequence and at least a second polynucleotide or peptide sequence are compared, to differentiate homology.

All publications and reference include but are not limited to: patent and patent application, all quote in this manual, point out that as clear and definite, discrete each separates publication or reference is all quoted in this article with for referencial use.The patent application of any right of priority as present patent application also quotes in full herein with for referencial use, and mode is the same.Definition

As known in the art, " identity " be two the multiple polypeptides sequence or two or multiple polynucleotide sequence between relation, can compare to determine relation between them to sequence.In the art, " identity " also can refer to the degree of correlation between polypeptide or polynucleotide sequence, can be by the sequence string being compared to determine the degree of correlation that they are original.Identity can be calculated easily by existent method, these methods comprise, but are not limited to those in (Computational Molecular Biology, Lesk, A.M., ed., OxfordUniversity Press, New York, 1988; Biocomputing:Informaticsand Genome Projects, Smith, D.W., ed., Academic Press, NewYork, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffn, H.G., eds., Humana press, NewJergey, 1994; Sequence Analysis in Molecular Biology, vonHeine, G., Academic Press, 1987; And Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M Stockton Press, NewYork, 1991; And Cacillo, H., and Lipman, D., SIAM J.AppliedMath has the method for description among the .48:1073 (1988).Be used for determining that the method for identity is the method that design provides maximum match between sequence to be measured.In addition, but the method compilation of determining identity in the computer program of open purchase.The computer algorithm of determining identity between two sequences comprises, but be not limited to the GAP program (Devereux in the GCG routine package, J., et al., 387 (1984)), BLASTP, BLASTN (Altschul Nucleic Acids Research 12 (1):, and FAFSTA (Pearson and Lipman Proc.Natl.Acad.Sci.USA 85 S.F.et al., J.Mol.Biol.215:403-410 (1990)); 2444-2448 (1988)).BLAST family program can from NCBI or other channel open purchases (BLAST Manual, Alt schul, S., et al., NCBI NLM NIHBethesda, MD 20894; Altschul, S., et al., J.Mol.Biol.215:406-410 (1990)).Well-known Smith Waterman operational method also can be used for determining identity.

The correlated parameter of peptide sequence comprises:

Operational method: Needleman and Wunsch, J.Mol Biol.48:443-453 (1970)

Contrast media: BLOSSUM62 from Henikoff and Henikoff, Proc.Natl.Acad.Sci.USA.89:10915-10919 (1992)

Breach penalties (penalty): 8

Notch length penalties (penalty): 2

The effective procedure that has these parameters can arrive by open purchase, and as from GeneticsComputer Group, Madison WI can buy at the place " breach " program.Aforementioned parameters is the correlated default parameters of peptide section (terminal breach does not have penalties (penalty)).

The correlated parameter of polynucleotide sequence comprises:

Operational method: Needleman and Wunsch, J.Mol Biol.48:443-453 (1970)

The contrast penalties: coupling=+ 10, unmatched=0

Breach penalties: 50

Notch length loss: 3

From: from Genetics Computer Group, " breach " program that Madison WI buys.These parameters are the correlated default parameters of nucleic acid (terminal breach do not have penalties).

Depending on circumstances, to polynucleotide and polypeptide, " identity " preferred meaning as seen following (1) and (2).

(1) the polynucleotide embodiment further comprises isolating polynucleotide, he contains the polynucleotide sequence that has 50,60,70,80,85,90,95,97 or 100% identity with SEQ ID NO:1 reference sequences at least, and wherein this polynucleotide sequence is can be with SEQ ID NO:1 reference sequences identical or contain the polynucleotide sequence that has a specific integer Nucleotide to change with the reference sequences contrast; Wherein this change be from by at least a nucleotide deletion, substitute, comprise conversion and transversion or insert and select in the group of forming; And wherein this change can occur in 5 of reference nucleotide sequence ' or 3 ' end, or occur in any position between these two ends, they can be dispersed in the Nucleotide of reference sequences by discrete, or with the population dispersal of one or more adjacency in reference sequences; And wherein this nucleosides change number is to multiply by integer divided by 100 definition per-cent identity by the Nucleotide sum with SEQID NO:1, deducts from the Nucleotide sum of this SEQ ID NO:1 then that this product obtains, or

Nn≤Xn-(Xny), wherein, Nn is a Nucleotide change number; Xn is the Nucleotide sum of SEQ ID NO:1; 0.50 of y refers to that 50%, 0.60 refers to that 60%, 0.70 refers to that 70%, 0.80 refers to that 80%, 0.85 refers to that 85%, 0.90 refers to that 90%, 0.95 refers to that 95%, 0.97 refers to that 97%, 1.00 refer to 100%; Represent multiplication sign; Wherein non-integral Xn and y just were approximately the most close integer with it before deducting from Xn.The change of the polynucleotide sequence of the polypeptide of coding SEQ ID NO:2 can produce nonsense mutation, missense mutation or phase shift mutation in this encoding sequence, thereby changes the polypeptide through the polynucleotide encoding after these changes.

By example as can be known, polynucleotide sequence among the present invention can be identical with the reference sequences of SEQ ID NO:1, be 100% identity also, perhaps he can comprise that the nucleic acid of comparing specific integer with reference sequences changes, thereby identity percentage ratio just is lower than 100%.Wherein this change be from by at least a nucleotide deletion, substitute, comprise conversion and transversion or insert and select in the group of forming; And wherein this change can occur in 5 of reference nucleotide sequence ' or 3 ' end, or occurs in any position between these two ends, and they can discrete, or with the population dispersal of one or more adjacency in the Nucleotide of reference sequences, or scatter; And wherein this Nucleotide change number is to multiply by integer divided by 100 definition per-cent identity by the Nucleotide sum with SEQ ID NO:1, deducts from the Nucleotide sum of this SEQ ID NO:1 then that this product obtains, or

Nn≤Xn-(Xny), wherein, Nn is a nucleosides change number; Xn is the nucleosides sum of SEQ ID NO:1; 0.50 of y refers to that 50%, 0.60 refers to that 60%, 0.70 refers to that 70%, 0.80 refers to that 80%, 0.85 refers to that 85%, 0.90 refers to that 90%, 0.95 refers to that 95%, 0.97 refers to that 97%, 1.00 refer to 100%; Represent multiplication sign; Wherein non-integral Xn and y just were approximately the most close integer with it before deducting from Xn.

(2) the polynucleotide embodiment further comprises isolated polypeptide, he contains the peptide sequence that has 50,60,70,80,85,90,95,97 or 100% identity with SEQ IDNO:2 reference sequences at least, and wherein this peptide sequence is can be with SEQ ID NO:2 reference sequences identical or contain the peptide sequence that has a specific integer amino acid to change with the reference sequences contrast; Wherein this change be from by at least a aminoacid deletion, substitute, comprise conservative substitute and non-conservative substitute or the group of compositions such as insertion in select; And wherein this change can occur in the amino or the C-terminal of reference sequences, or occurs in any position between these two ends, they can discrete or with the population dispersal of one or more adjacency in the amino acid of reference sequences; And wherein this amino acid change number is to multiply by integer divided by 100 definition per-cent identity by the amino acid sum with SEQ ID NO:2, deducts from the amino acid sum of this SEQ ID NO:2 then that this product obtains, or

Na≤Xa-(Xay), wherein, Na is an amino acid change number; Xa is the amino acid sum of SEQ ID NO:2; 0.50 of y refers to that 50%, 0.60 refers to that 60%, 0.70 refers to that 70%, 0.80 refers to that 80%, 0.85 refers to that 85%, 0.90 refers to that 90%, 0.95 refers to that 95%, 0.97 refers to that 97%, 1.00 refer to 100%; Represent multiplication sign; Wherein non-integral Xa and y just were approximately the most close integer with it before deducting from Xa.

As an example, peptide sequence among the present invention can be identical with the reference sequences of SEQ ID NO:2, be 100% identity also, perhaps he can comprise that the amino acid of comparing specific integer with reference sequences changes, thereby identity percentage ratio just is lower than 100%.Wherein this change be from by at least a aminoacid deletion, substitute, comprise conservative replace and the group of compositions such as non-conservative replacement or insertion in select; And wherein this change can occur in the amino or the C-terminal of reference polypeptide sequence, or occurs in any position between these two ends, and they can discrete, or with the population dispersal of one or more adjacency in the amino acid of reference sequences; And wherein the amino acid of this specific identity % change number is to multiply by integer divided by 100 definition per-cent identity by the amino acid sum with SEQID NO:2, deduct from the amino acid sum of this SEQ ID NO:2 then that this product obtains, or

Na≤Xa-(Xay), wherein, Na is an amino acid change number; Xa is the amino acid sum of SEQ ID NO:2; 0.50 of y refers to that 50%, 0.60 refers to that 60%, 0.70 refers to that 70%, 0.80 refers to that 80%, 0.85 refers to that 85%, 0.90 refers to that 90%, 0.95 refers to that 95%, 0.97 refers to that 97%, 1.00 refer to 100%; Represent multiplication sign; Wherein non-integral Xa and y just were approximately the most close integer with it before deducting from Xa.

" individuality " refers to a kind of organism when mentioning in the present invention, meaning is the many cells eukaryotes, includes but are not limited to: metazoan, Mammals, OVID, bovid, ape, primate and people.

" isolating " mean from virgin state and changed through " staff ", if also promptly he appears at nature, he has changed from its initial environment or irrelevant with initial environment, or the two has concurrently.For example, the polynucleotide or the polypeptide that are present in naturally in the organism alive are not " isolating ", and the identical polynucleotide or the polypeptide that are separated to the concurrent under its native state are exactly " isolating ", and this noun just refers to this meaning herein.In addition, the polynucleotide or the polypeptide that import organisms by transformation, genetic manipulation or other any recombination methods are " isolating ", if even they still be present in this organism, and this organism can be or or non-living body.

" polynucleotide " refer generally to polybribonucleotide or polydeoxyribonucleotide, and these polybribonucleotides or polydeoxyribonucleotide can be not modified RNA or DNA or pass through the RNA that modifies or comprise strand and the DNA of double stranded region.

" variant " refer to different with reference to polynucleotide or polypeptide, but remain with the polynucleotide or the polypeptide of fundamental characteristics.The nucleotide sequence of the typical variant of polynucleotide and another variant and different with reference to the nucleotide sequence of polynucleotide.The change of variant nucleotide sequence might change or not change the aminoacid sequence of reference sequences encoded polypeptides.Nucleotide changes the amino acid that can cause the reference sequences encoded polypeptides to be taken place to substitute, add, lacks, merges and shortens, and hereinafter this is had discussion.The aminoacid sequence of the typical variant of polypeptide is different with the aminoacid sequence of another variant and reference polypeptide.In general, difference is limited, so reference polypeptide and variant sequence are closely similar on the whole, and some district is identical.Variant and reference sequences can be owing to one or more any bonded substitution effects, interpolation effect, disappearance effect and are different on aminoacid sequence.The amino-acid residue of alternate or insertion can be by gene codon coding or can't help the gene codon coding.The polynucleotide variant can be naturally occurring, as allele variant, also can be the variant of natural the unknown.Polynucleotide that non-natural takes place or polypeptide variants can be made or directly be passed through to synthesize and prepare by the variation generation technique.

" disease " refer to that infectation of bacteria causes or with the infectation of bacteria diseases associated, comprise, as upper respiratory tract infection, invasive bacterial disease such as microbemia and meningitis.Example

Following example is carried out according to standard method, for those of skill in the art be well-known, stylize, yet those be described in detail except.These examples are illustrative, but they do not limit the present invention.BASB051 gene among the example 1 Neisseria meningitidis strains A TCC 13090

BASB051 gene among the Neisseria meningitidis strains A TCC 13090 has in SEQ IDNO:1 and shows.Sequence is seen shown in the SEQ ID NO:2 after the translation of BASB051 polynucleotide sequence, and he shows that this BASB051 polypeptide and Diplococcus gonorrhoeae ComL lipoprotein have significant similarity.The BASB051 polypeptide contains a kind of leader sequence, it is characterized in that a lipoprotein signal sequence.BASB057 gene among the example 2 Neisseria meningitidis strains A TCC 13090

BASB057 gene among the Neisseria meningitidis strains A TCC 13090 has in SEQ IDNO:3 and shows.Sequence is seen shown in the SEQ ID NO:4 after the translation of BASB057 polynucleotide sequence, and he shows that this BASB057 polypeptide and gonococcus MtrE outer membrane lipoprotein have significant similarity.The BASB057 polypeptide contains a kind of leader sequence, it is characterized in that a lipoprotein signal sequence.BASB060 gene among the example 3 Neisseria meningitidis strains A TCC 13090

BASB060 gene among the Neisseria meningitidis strains A TCC 13090 has in SEQ IDNO:5 and shows.Sequence is seen shown in the SEQ ID NO:6 after the translation of BASB060 polynucleotide sequence, and he shows that this BASB060 polypeptide and known protein do not have significant similarity.The BASB060 polypeptide contains a kind of leader sequence, it is characterized in that a lipoprotein signal sequence, and contains the characteristics of outer membrane lipoprotein.BASB061 gene among the example 4 Neisseria meningitidis strains A TCC 13090

BASB061 gene among the Neisseria meningitidis strains A TCC 13090 has in SEQ IDNO:7 and shows.Sequence is seen shown in the SEQ ID NO:8 after the translation of BASB061 polynucleotide sequence, and he shows that the product of this BASB061 polypeptide and Neisseria meningitidis mlp gene has significant similarity.The BASB061 polypeptide contains a kind of leader sequence, it is characterized in that a lipoprotein signal sequence, and contains the characteristics of outer membrane lipoprotein.BASB063 gene among the example 5 Neisseria meningitidis strains A TCC 13090

BASB063 gene among the Neisseria meningitidis strains A TCC 13090 has in SEQ IDNO:9 and shows.Sequence is seen shown in the SEQ ID NO:10 after the translation of BASB063 polynucleotide sequence, and he shows that this BASB063 polypeptide and known protein have significant similarity.The BASB063 polypeptide contains a kind of leader sequence, it is characterized in that a lipoprotein signal sequence, and contains the characteristics of outer membrane lipoprotein.BASB065 gene among the example 6 Neisseria meningitidis strains A TCC 13090

BASB065 gene among the Neisseria meningitidis strains A TCC 13090 has in SEQ IDNO:11 and shows.Sequence is seen shown in the SEQ ID NO:12 after the translation of BASB065 polynucleotide sequence, and he shows that this BASB065 polypeptide and known protein have significant similarity.The BASB065 polypeptide contains a kind of leader sequence, it is characterized in that a lipoprotein signal sequence, and contains the characteristics of outer membrane lipoprotein.BASB066 gene among the example 7 Neisseria meningitidis strains A TCC 13090

BASB066 gene among the Neisseria meningitidis strains A TCC 13090 has in SEQ IDNO:13 and shows.Sequence is seen shown in the SEQ ID NO:14 after the translation of BASB066 polynucleotide sequence, and he shows that this BASB066 polypeptide and Neisseria meningitidis CtrA albumen have significant similarity.The BASB066 polypeptide contains a kind of leader sequence, it is characterized in that a lipoprotein signal sequence, and contains the proteic characteristics that are positioned on the adventitia.BASB071 gene among the example 8 Neisseria meningitidis strains A TCC 13090

BASB071 gene among the Neisseria meningitidis strains A TCC 13090 has in SEQ IDNO:15 and shows.Sequence is seen shown in the SEQ ID NO:16 after the translation of BASB071 polynucleotide sequence, and he shows that this BASB071 polypeptide and gonococcus HisJ albumen have significant similarity.The BASB071 polypeptide contains a kind of leader sequence, it is characterized in that a lipoprotein signal sequence, and contains the characteristics of outer membrane lipoprotein.

Polynucleotide and polypeptide sequences SEQ ID NO: 1 Neisseria meningitidis strains ATCC13090 of BASB051 polynucleotide sequences ATGAAAAAAATTCTTTTAACGGTTTCATTAGGTTTGGCACTGAGTGCCTGTGCCACTCAA GGTACGGTCGATAAAGATGCTCAGATTACCCAAGATTGGAGTGTGGAGAAGCTCTATGCC GAAGCCCAGGACGAATTGAACAGCAGCAATTATACGCGGGCTGTCAAGTTATACGAAATC TTGGAATCGCGCTTCCCCACCAGCCGCCATGCCCGGCAATCCCAACTGGATACCGCATAC GCCTATTATAAAGACGATGAAAAAGACAAGGCTCTGGCGGCAATCGAACGCTTCCGCCGC CTCCATCCGCAGCATCCGAATATGGATTACGCGCTGTATCTGCGCGGCTTGGTGCTGTTC AACGAAGACCAGTCCTTCTTGAACAAACTGGCCTCGCAAGACTGGTCCCACCGCGACCCG AAAGCCAACCGCGAAGTAACCCAGGCGTTTGCGGAACTCGTCCAACGCTTCCCCAACAGC AAATACGCCGCCGATGCGACCGCACGCATGGTCAAACTGGTCGATGCACTGGGCGGCAAT GAAATGTCGGTGGCGCGCTACTACATGAAACGCGGCGCATATATCGCCGCCGCCAACCGC GCCCAAAAAATTATCGGCAGCTACCAAAATACACGCTATGTCGAAGAATCGCTCGCCATC TTGGAACTTGCCTACCAAAAACTCGGCAAACCACAGCTTGCCGCCGATACGCGCCGCGTG TTGGAAACCAACTTCCCGAAAAGCCCGTTTTTGACGCACGCTTGGCAGCCCGACGATATG CCTTGGTGGCGTTACTGGCATTAA SEQ ID NO: 2 Neisseria meningitidis strains ATCC13090, and from SEQ ID NO: 1 polynucleotide sequence BASB051 polypeptide sequence deduced column MKKILLTVSLGLALSACATQGTVDKDAQITQDWSVEKLYAEAQDELNSSNYTRAVKLYEI LESRFPTSRHARQSQLDTAYAYYKDDEKDKALAAIERFRRLHPQHPNMDYALYLRGLVLF NEDQSFLNKLASQDWSDRDPKANREVTQAFAELVQRFPNSKYAADATARMVKLVDALGGN EMSVARYYMKRGAYIAAANRAQKIIGSYQNTRYVEESLAILELAYQKLGKPQLAADTRRV LETNFPKSPFLTHAWQPDDMPWWRYWH SEQ ID NO: 3 Neisseria meningitidis strains ATCC13090 of BASB057 polynucleotide sequences ATGGATACTACATTGAAAACCACCTTGACTTCTGTTGCAGCAGCCTTCGCATTATCCGCC TGCACCATGATTCCCCAATACGAGCAGCCCAAAGTCGAAGTTGCCGAAACGTTTAAAAAC GATACCGCCGACAGCGGCATCCGTGCGGTCGATTTAGGTTGGCATGACTATTTTGCCGAC CCGCGCCTGCAAAAGCTGATCGACATCGCACTCGAGCGCAATACCAGTTTGCGTACCGCC GTATTGAACAGCGAAATCTACCGCAAACAATACATGATTGAGCGCAACAACCTCCTGCCC ACGCTTGCCGCCAATGCGAACGGCTCGCGCCAAGGCAGCTTGAGCGGCGGCAATGTCAGC AGCAGCTACAATGTCGGACTGGGTGCGGCATCTTACGAACTCGACCTGTTCGGACGCGTC CGCAGCAGCAGCGAAGCAGCACTGCAAGGCTATTTTGCAAGTGTCGCCAACCGCGATGCG GCACATTTGAGCCTGATTGCCACCGTTGCCAAAGCCTATTTCAACGAACGTTATGCCGAA GAAGCGATGTCTTTGGCGCAGCGTGTTTTGAAAACGCGCGAGGAAACCTACAAGCTGTCC GAATTACGTTACAAGGCAGGCGTGATTTCCGCCGTCGCCCTACGTCAGCAGGAAGCCCTG ATCGAATCTGCCAAAGCCGATTATGCCCATGCCGCGCGCAGCCGCGAACAGGCGCGCAAT GCCTTGGCAACCTTGATTAACCAACCGATACCCGAAGACCTGCCTGCCGGTTTGCCGCTG GACAAGCAGTTTTTTGTTGAAAAACTGCCGGCCGGTTTGAGTTCCGAAGTATTGCTCGAC CGTCCCGATATCCGTGCTGCCGAACACGCGCTCAAACAGGCAAACGCCAATATCGGTGCG GCACGCGCCGCCTTTTTCCCATCCATCCGCCTGACCGGAACCGTCGGTACGGGTTCTGCC GAATTGGGTGGGTTGTTCAAAAGCGGCACGGGCGTTTGGTCGTTCGCGCCGTCTATTACC CTGCCGATTTTTACCTGGGGTACGAACAAAGCCAACCTTGATGTAGCCAAGCTGCGCCAA CAGGCACAAATCGTTGCCTATGAAGCCGCCGTCCAATCCGCATTTCAAGACGTGGCAAAC GCATTGGCGGCGCGCGAGCAGCTGGATAAAGCCTATGACGCTTTAAGCAAACAAAGCCGC GCCTCTAAAGAGGCGTTGCGCTTGGTCGGCCTGCGTTACAAGCACGGCGTATCCGGCGCG CTCGACTTGCTCGATGCGGAACGCAGCAGCTATGCGGCGGAGGGTGCGGCTTTGTCGGCA CAACTGACCCGCGCCGAAAACCTTGCCGATTTGTACAAGGCACTCGGCGGCGGATTGAAA CGGGATACCCAAACCGACAAATAA SEQ ID NO: 4 From SEQ ID NO: 3 polynucleotide sequence deduced strain of Neisseria meningitidis BASB057 Polypeptide sequence MDTTLKTTLTSVAAAFALSACTMIPQYEQPKVEVAETFKNDTADSGIRAVDLGWHDYFAD PRLQKLIDIALERNTSLRTAVLNSEIYRKQYMIERNNLLPTLAANANGSRQGSLSGGNVS SSYNVGLGAASYELDLFGRVRSSSEAALQGYFASVANRDAAHLSLIATVAKAYFNERYAE EAMSLAQRVLKTREETYKLSELRYKAGVISAVALRQQEALIESAKADYAHAARSREQARN ALATLINQPIPEDLPAGLPLDKQFFVEKLPAGLSSEVLLDRPDIRAAEHALKOANANIGA ARAAFFPSIRLTGTVGTGSAELGGLFKSGTGVWSFAPSITLPIFTWGTNKANLDVAKLRQ QAQIVAYEAAVQSAFQDVANALAAREQLDKAYDALSKQSRASKEALRLVGLRYKHGVSGA LDLLDAERSSYAAEGAALSAQLTRAENLADLYKALGGGLKRDTQTDK SEQ ID NO: 5 Neisseria meningitidis strains ATCC13090 of BASB060 polynucleotide sequences ATGAAAAAACTTCTAATGATAACCCTCACCGGTATGCTTGCAGCTTGTGCAACAGGTGTC AATGTCGGCCGGTTGATGGTTGAAATGCCGCAGGGAGAACGTTCTGTCGTTGTGCAGGTT CCCGCGACAAATAACCCGCTTTCCGATACGGTAGCTGTCGGAATGATTAAAACATCCGGT TCGCCTTCGGCATCAAATATGATTGAAATGCTCGGCGCGGACAATATCAACGTCGGCGTG GTGGGAAGCAGCCAAATGCTTAATAAGGCGACCGCACTTTATTCCTTAAACCATGCAAAG AAAGTCGGAAATAATGTCAGTGTTTATATGATGGGCGACAGCGAAAGTGACAAGGCCGAT TTGGAAAACGCGGCAAATGCCAAAAATATCAAATTGCATTATTTCTTTAACCAAAAATAA SEQ ID NO: 6 From SEQ ID NO: 5 polynucleotide sequence deduced Neisseria meningitidis strains BASB060 Polypeptide sequence MKKLLMITLTGMLAACATGVNVGRLMVEMPQGERSVVVQVPATNNPLSDTVAVGMIKTSG SPSASNMIEMLGADNINVGVVGSSQMLNKATALYSLNHAKKVGNNVSVYMMGDSESDKAD LENAANAKNIKLHYFFNQK SEQ ID NO: 7 Neisseria meningitidis strains ATCC13090 of BASB061 polynucleotide sequences ATGAAAATCAAACAAATCGTCAAACCGGGCTTGGCAGTATTGGCGGCGGGCGTTCTGTCT GCCTGCGCAACCAAAAGCAACGTCAAAGCCGACGGAACGACCGACAATCCGGTTTTCCCG AAACCCTATTCCGTAACGCTCGACAACAATCGCGGTACATTCCCGACCTATGACGAATTG GACTTGATGCGTCCCGGTCTGACCAAAGACGACATCTACAAAATCCTGGGTCGTCCGCAT TACGACGAAGGTATGTACGGCGTGCGCGAATGGGATTATCTGTTCCACTTCCACACCCCG GGCGTAGGCATCGACCCTGAAAACACTTCCGGCGTAGAAGGCATTACCACCTGTCAATAC AAAATTATTTTCGATAAAGACAAATTTGCCCGCAGCTTCTACTGGAACCCCGTCTTCCCG AAAGATGCCGCCTGTCCGCCGCCCGCACCCAAAGCCGAGCCGCAAgTCATCATCCGCGAA ATCGTGCCCGCCAAAcCCAAACGCATCCGCCAATAA SEQ ID NO: 8 From SEQ ID NO: 7 polynucleotide sequence deduced strain of Neisseria meningitidis BASB061 Polypeptide sequence MKIKQIVKPGLAVLAAGVLSACATKSNVKADGTTDNPVFPKPYSVTLDNNRGTFPTYDEL DLMRPGLTKDDIYKILGRPHYDEGMYGVREWDYLFHFHTPGVGIDPENTSGVEGITTCQY KIIFDKDKFARSFYWNPVFPKDAACPPPAPKAEPQVIIREIVPAKPKRIRQ SEQ ID NO: 9 Neisseria meningitidis strains ATCC13090 of BASB063 polynucleotide sequences ATGAGACCATATGCTACTACCATTTATCAACTTTTTATTTTGTTTATTGGGAGTGTTTTT ACTATGACCTCATGTGAACCTGTGAATGAAAAGACAGATCAAAAAGCAGTAAGTGCGCAA CAGGCTAAAGAACAAACCAGTTTCAACAATCCCGAGCCAATGACAGGATTTGAACATACG GTTACATTTGATTTTCAGGGCACCAAAATGGTTATCCCCTATGGCTATCTTGCACGGTAT ACGCAAGACAATGCCACAAAATGGCTTTCCGACACGCCCGGGCAGGATGCTTACTCCATT AATTTGATAGAGATTAGCGTCTATTACAAAAAAACCGACCAAGGCTGGGTTCTTGAGCCA TACAACCAGCAAAACAAAGCACACTTTATCCAATTTCTACGCGACGGTTTGGATAGCGTG GACGATATTGTTATCCGAAAAGATGCGTGTAGTTTAAGTACGACTATGGGAGAAAGATTG CTTACTTACGGGGTTAAAAAAATGCCATCTGCCTATCCTGAATACGAGGCTTATGAAGAT AAAAGACATATTCCTGAAAATCCATATTTTCATGAATTTTACTATATTAAAAAAGGAGAA AATCCGGCGATTATTACTCATCGGAATAATCGAATAAACCAAACTGAAGAAGATAGTTAT AGCACTAGCGTAGGTTCCTGTATTAACGGTTTCACGGTACAGTATTACCCGTTTATTCGG GAAAAGCAGCAGCTCACACAGCAGGAGTTGGTAGGTTATCACCAACAAGTAGAGCAATTG GTACAGAGTTTTGTAAACAATTCAAATAAAAAATAA SEQ ID NO: 10 From SEQ ID NO: 9 polynucleotide sequence deduced strain of Neisseria meningitidis BASB063 Polypeptide sequence MRPYATTIYQLFILFIGSVFTMTSCEPVNEKTDQKAVSAQQAKEQTSFNNPEPMTGFEHT VTFDFQGTKMVIPYGYLARYTQDNATKWLSDTPGQDAYSINLIEISVYYKKTDQGWVLEP YNQQNKAHFIQFLRDGLDSVDDIVIRKDACSLSTTMGERLLTYGVKKMPSAYPEYEAYED KRHIPENPYFHEFTYIKKGENPAIITHRNNRINQTEEDSYSTSVGSCINGFTVQYYPFIR EKQQLTQQELVGYHQQVEQLVQSFVNNSNKK SEQ ID NO: 11 Neisseria meningitidis strains ATCC13090 of BASB065 polynucleotide sequences ATGAAGACCAAATTACCGCTTTTTATCATTTGGCTGTCCGTATCCGCCGCCTGTTCTTCC CCTGTTTCCCGCAATATTCAGGATATGCGGCCCGAACCGCAGGCAGAGGCAGGTAGTTCG GACGCTATTCCCTATCCCGTTCCCACTCTGCAAGACCGTTTGGATTATCTGGAAGGCACA CTCGTCCGCCTGTCGAACGAAGTGGAAACCTTAAACGGCAAAGTCAAAGCACTGGAGCAT GCGAAAACACACCCTTCCGGTAGGGCATACGTCCAAAAACTCGACGACCGCAAGTTGAAA GAGCATTACCTCAATACCGAAGGCGGCAGCGCATCCGCACATACCGTCGAAACCGCACAA AACCTCTACAATCAGGCACTCAAACACTATAAAAGCGGCAGGTTTTCTGCCGCAGCCGCC CTGTTGAAAGGCGCGGACGGAGGCGACGGCGGCAGCATCGCGCAACGCAGTATGTACCTG TTGCTGCAAAGCAGGGCGCGTATGGGCAACTGCGAATCCGTCATCGAAATCGGAGGGCGT TACGCCAACCGTTTCAAAGACAGCCCAACCGCGCCCGAAGCCATGTTCAAAATCGGCGAA TGCCAATACAGGTTGCAGCAGAAAGACATTGCAAGGGCAACTTGGCGCAGCCTGATACAG GCTTACCCGAGCAGCCCGGCGGCAAAACGCGCCGCCGCAGCCGTACGCAAACGATAG SEQ ID NO: 12 From SEQ ID NO: 11 polynucleotide sequence deduced strain of Neisseria meningitidis BASB065 Polypeptide sequence MKTKLPLFIIWLSVSAACSSPVSRNIQDMRPEPQAEASSSDAIPYPVPTLQDRLDYLEGT LVRLSNEVETLNGKVKALEHAKTHPSGRAYVQKLDDRKLKEHYLNTEGGSASAHTVETAQ NLYNQALKHYKSGRFSAAAALLKGADGGDGGSIAQRSMYLLLQSRARMGNCESVIEIGGR YANRFKDSPTAPEAMFKIGECQYRLQQKDIARATWRSLIQAYPSSPAAKRAAAAVRKR SEQ ID NO: 13 Neisseria meningitidis strains ATCC13090 of BASB066 polynucleotide sequences GTGTTTAAAGTGAAATTTTATATTCGTCACGCAGTATTATTATTGTGTGGAAGTTTAATT GTAGGATGCTCTGCGATTCCTTCATCAGGCCCCAGCGCAAAAAAAATTGTCTCTTTAGGG CAACAATCTGAAGTTCAAATTCCTGAAGTGGAGCTGATTGATGTGAATCATACGGTTGCT CAGTTATTATATAAGGCTCAGATAAATCAGTCATTCACTCAGTTTGGCGATGGTTATGCT TCGGCTGGTACGCTAAATATTGGTGATGTATTGGATATTATGATTTGGGAAGCGCCGCCG GCAGTATTGTTTGGTGGTGGCCTTTCTTCGATGGGCTCGGGTAGTGCGCATCAAACTAAG TTGCCAGAGCAGTTGGTCACGGCACGTGGTACGGTTTCTGTGCCGTTTGTTGGCGATATT TCGGTGGTCGGTAAAACGCCTGGTCAGGTTCAGGAAATTATTAAAGGCCGCCTGAAAAAA ATGGCCAATCAGCCACAAGTGATGGTGCGTTTGGTGCAGAATAATGCGGCGAATGTGTCG GTGATTCGTGCTGGGAATAGTGTGCGTATGCCGCTGACGGCAGCCGGTGAGCGTGTGTTG GATGCGGTGGCTGCGGTAGGTGGTTCAACGGCAAATGTGCAGGATACGAATGTGCAGCTG ACACGTGGCAATGTAGTACGAACTGTTGCCTTGGAAGATTTAGTTGCAAATCCGCGACAA AATATTTTGCTGCGTCGCGGTGATGTGGTTACCATGATTACCAATCCCTATACCTTTACG TCTATGGGTGCGGTGGGGAGAACACAAGAAATCGGTTTTTCAGCCAGAGGCTTATCGCTT TCTGAAGCCATTGGCCGTATGGGCGGTTTGCAAGATCGCCGTTCTGATGCGCGTGGTGTG TTTGTGTTCCGCTATACGCCATTGGTGGAATTGCCGGCAGAACGTCAGGATAAATGGATT GCTCAAGGTTATGGCAGTGAGGCAGAGATTCCAACGGTATATCGTGTGAATATGGCTGAT GCGCATTCGCTATTTTCTATGCAGCGCTTTCCTGTGAAGAATAAAGATGTATTGTATGTG TCGAATGCGCCGTTGGCTGAAGTGCAGAAATTCTTGTCGTTTGTGTTCTCGCCGGTTACC AGTGGCGCGAACAGTATTAATAATTTAACTAATTAA SEQ ID NO: 14 From SEQ ID NO: 13 polynucleotide sequences derived strains of Neisseria meningitidis BASB066 Polypeptide sequence MFKVKFYIRHAVLLLCGSLIVGCSAIPSSGPSAKKIVSLGQQSEVQIPEVELIDVNHTVA QLLYKAQINQSFTQFGDGYASAGTLNIGDVLDIMIWEAPPAVLFGGGLSSMGSGSAHQTK LPEQLVTARGTVSVPFVGDISVVGKTPGQVQEIIKGRLKKMANQPQVMVRLVQNNAANVS VIRAGNSVRMPLTAAGERVLDAVAAVGGSTANVQDTNVQLTRGNVVRTVALEDLVANPRQ NILLRRGDVVTMITNPYTFTSMGAVGRTQEIGFSARGLSLSEAIGRMGGLQDRRSDARGV FVFRYTPLVELPAERQDKWIAQGYGSEAEIPTVYRVNMADAHSLFSMQRFPVKNKDVLYV SNAPLAEVQKFLSFVFSPVTSGANSINNLTN SEQ ID NO: 15 Neisseria meningitidis strains ATCC13090 of BASB071 polynucleotide sequences ATGAATATGAAAAAATGGATTGCCGCCGCCCTTGCCTGTTCCGCGCTCGCGCTCTCTGCC TGCGGCGGTCAGGGCAAAGATGCCGCCGCGCCCGCCGCAAACCCCGACAAAGTGTACCGC GTGGCTTCCAACGCCGAGTTTGCCCCCTTTGAATCTTTAGACTCGAAAGGCAATGTTGAA GGTTTCGATGTGGATTTGATGAACGCGATGGCGAAGGCGGGCAATTTTAAAATCGAATTC AAACACCAGCCGTGGGACAGCCTTTTCCCCGCCTTGAACAACGGCGATGCGGACGTTGTG ATGTCGGGCGTAACCATTACCGACGACCGCAAACAGTCTATGGACTTCAGCGACCCGTAT TTTGAAATCACCCAAGTCGTCCTCGTTCCGAAAGGCAAAAAAATATCTTCTTCCGAAGAT TTGAAAAACATGAACAAAGTCGGCGTGGTAACCGGCTACACGGGCGATTTCTCCGTATCC AAACTCTTGGGCAACGACAACCCGAAAATCGCGCGCTTTGAAAACGTTCCCCTGATTATC AAAGAACTGGAAAACGGCGGCTTGGATTCCGTGGTCAGCGACAGCGCAGTCATCGCCAAT TATGTGAAAAACAATCCGACCAAAGGGATGGACTTCGTTACCCTGCCCGACTTCACCACC GAACACTACGGCATCGCGGTACGCAAAGGCGACGAAGCAACCGTCAAAATGCTGAACGAT GCGTTGAAAAAAGTACGCGAAAGCGGCGAATACGACAAAATCTACGCCAAATATTTTGCA AAAGAAGACGGACAGGCCGCAAAATAA SEQ ID NO: 16 From SEQ ID NO: 15 polynucleotide sequence deduced strain of Neisseria meningitidis BASB071 Polypeptide sequence MNMKKWIAAALACSALALSACGGQGKDAAAPAANPDKVYRVASNAEFAPFESLDSKGNVE GFDVDLMNAMAKAGNFKIEFKHQPWDSLFPALNNGDADVVMSGVTITDDRKQSMDFSDPY FEITQVVLVPKGKKISSSEDLKNMNKVGVVTGYTGDFSVSKLLGNDNPKIARFENVPLII KELENGGLDSVVSDSAVIANYVKNNPTKGMDFVTLPDFTTEHYGIAVRKGDEATVKMLND ALKKVRESGEYDKIYAKYFAKEDGQAAK Deposited material ...

The B serological type strain that contains Neisseria meningitidis on June 22nd, 1997 in American Type Culture Collection (also being " ATCC ") storage, storage number is 13090.The storage thing is to be registered as Neisseria meningitidis (Albrecht and Ghon), and he is through lyophilize, the long insertion library of 1.5-2.9kb, and he makes up from the Neisseria meningitidis isolate.This storage thing in Int.Bull.Bacteriol.Nomencl.Taxon.8:1-15 (1958). in description is arranged.

Neisseria meningitidis bacterial strain storage thing refer to herein " storage bacterial strain " or " DNA of storage bacterial strain ".

The storage bacterial strain contains BASB051, BASB057, BASB060, BASB061, BASB063, BASB065, BASB066 and the BASB071 gene of whole length.If take place and the afoul situation of sequence described herein, based on the polynucleotide sequence that contains in the storage bacterial strain and the aminoacid sequence of encoded polypeptides thereof.

The storage of this storage bacterial strain is according to clause patented procedure, international endorsement, microorganism storage carries out about being applied among the Budapest Treaty.When patent one issue, this bacterial strain can not be recalled, and can not add the inflow specific crowd of restriction and supplementary condition.The storage bacterial strain only is for those of skill in the art provide convenience, and is not the requirement of vest right institute, as the requirement of 35U.S.C. § 112, carry out bacterial strain storage.

Sequence table

<110>SmithKline?Beecham?Biologicals?S.A.

＜120〉new compound

<130>BM45348

<160>16

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

<210>1

<211>804

<212>DNA

<213>Neisseria?meningitidis

＜214〉Neisseria meningitidis

<400>1atgaaaaaaa?ttcttttaac?ggtttcatta?ggtttggcac?tgagtgcctg?tgccactcaa?????60ggtacggtcg?ataaagatgc?tcagattacc?caagattgga?gtgtggagaa?gctctatgcc????120gaagcccagg?acgaattgaa?cagcagcaat?tatacgcggg?ctgtcaagtt?atacgaaatc????180ttggaatcgc?gcttccccac?cagccgccat?gcccggcaat?cccaactgga?taccgcatac????240gcctattata?aagacgatga?aaaagacaag?gctctggcgg?caatcgaacg?cttccgccgc????300ctccatccgc?agcatccgaa?tatggattac?gcgctgtatc?tgcgcggctt?ggtgctgttc????360aacgaagacc?agtccttctt?gaacaaactg?gcctcgcaag?actggtccga?ccgcgacccg????420aaagccaacc?gcgaagtaac?ccaggcgttt?gcggaactcg?tccaacgctt?ccccaacagc????480aaatacgccg?ccgatgcgac?cgcacgcatg?gtcaaactgg?tcgatgcact?gggcggcaat????540gaaatgtcgg?tggcgcgcta?ctacatgaaa?cgcggcgcat?atatcgccgc?cgccaaccgc????600gcccaaaaaa?ttatcggcag?ctaccaaaat?acacgctatg?tcgaagaatc?gctcgccatc????660ttggaacttg?cctaccaaaa?actcggcaaa?ccacagcttg?ccgccgatac?gcgccgcgtg????720ttggaaacca?acttcccgaa?aagcccgttt?ttgacgcacg?cttggcagcc?cgacgatatg????780ccttggtggc?gttactggca?ttaa???????????????????????????????????????????804

<210>2

<211>267

<212>PRT

<213>Neisseria?meningitidis

＜214〉Neisseria meningitidis

<400>2Met?Lys?Lys?Ile?Leu?Leu?Thr?Val?Ser?Leu?Gly?Leu?Ala?Leu?Ser?Ala?1???????????????5??????????????????10??????????????????15Cys?Ala?Thr?Gln?Gly?Thr?Val?Asp?Lys?Asp?Ala?Gln?Ile?Thr?Gln?Asp

20??????????????????25??????????????????30Trp?Ser?Val?Glu?Lys?Leu?Tyr?Ala?Glu?Ala?Gln?Asp?Glu?Leu?Asn?Ser

35??????????????????40??????????????????45Ser?Asn?Tyr?Thr?Arg?Ala?Val?Lys?Leu?Tyr?Glu?Ile?Leu?Glu?Ser?Arg

50??????????????????55??????????????????60Phe?Pro?Thr?Ser?Arg?His?Ala?Arg?Gln?Ser?Gln?Leu?Asp?Thr?Ala?Tyr65??????????????????70??????????????????75??????????????????80Ala?Tyr?Tyr?Lys?Asp?Asp?Glu?Lys?Asp?Lys?Ala?Leu?Ala?Ala?Ile?Glu

85??????????????????90??????????????????95Arg?Phe?Arg?Arg?Leu?His?Pro?Gln?His?Pro?Asn?Met?Asp?Tyr?Ala?Leu

100?????????????????105?????????????????110Tyr?Leu?Arg?Gly?Leu?Val?Leu?Phe?Asn?Glu?Asp?Gln?Ser?Phe?Leu?Asn

115?????????????????120?????????????????125Lys?Leu?Ala?Ser?Gln?Asp?Trp?Ser?Asp?Arg?Asp?Pro?Lys?Ala?Asn?Arg

130?????????????????135?????????????????140Glu?Val?Thr?Gln?Ala?Phe?Ala?Glu?Leu?Val?Gln?Arg?Phe?Pro?Asn?Ser145?????????????????150?????????????????155?????????????????160Lys?Tyr?Ala?Ala?Asp?Ala?Thr?Ala?Arg?Met?Val?Lys?Leu?Val?Asp?Ala

165?????????????????170?????????????????175Leu?Gly?Gly?Asn?Glu?Met?Ser?Val?Ala?Arg?Tyr?Tyr?Met?Lys?Arg?Gly

180?????????????????185?????????????????190Ala?Tyr?Ile?Ala?Ala?Ala?Asn?Arg?Ala?Gln?Lys?Ile?Ile?Gly?Ser?Tyr

195?????????????????200?????????????????205Gln?Asn?Thr?Arg?Tyr?Val?Glu?Glu?Ser?Leu?Ala?Ile?Leu?Glu?Leu?Ala

210?????????????????215?????????????????220Tyr?Gln?Lys?Leu?Gly?Lys?Pro?Gln?Leu?Ala?Ala?Asp?Thr?Arg?Arg?Val225?????????????????230?????????????????235?????????????????240Leu?Glu?Thr?Asn?Phe?Pro?Lys?Ser?Pro?Phe?Leu?Thr?His?Ala?Trp?Gln

245?????????????????250?????????????????255Pro?Asp?Asp?Met?Pro?Trp?Trp?Arg?Tyr?Trp?His

260?????????????????265

<210>3

<211>1404

<212>DNA

<213>Neisseria?meningitidis

＜214〉Neisseria meningitidis

<400>3atggatacta?cattgaaaac?caccttgact?tctgttgcag?cagccttcgc?attatccgcc?????60tgcaccatga?ttccccaata?cgagcagccc?aaagtcgaag?ttgccgaaac?gtttaaaaac????120gataccgccg?acagcggcat?ccgtgcggtc?gatttaggtt?ggcatgacta?ttttgccgac????180ccgcgcctgc?aaaagctgat?cgacatcgca?ctcgagcgca?ataccagttt?gcgtaccgcc????240gtattgaaca?gcgaaatcta?ccgcaaacaa?tacatgattg?agcgcaacaa?cctcctgccc????300acgcttgccg?ccaatgcgaa?cggctcgcgc?caaggcagct?tgagcggcgg?caatgtcagc????360agcagctaca?atgtcggact?gggtgcggca?tcttacgaac?tcgacctgtt?cggacgcgtc????420cgcagcagca?gcgaagcagc?actgcaaggc?tattttgcaa?gtgtcgccaa?ccgcgatgcg????480gcacatttga?gcctgattgc?caccgttgcc?aaagcctatt?tcaacgaacg?ttatgccgaa????540gaagcgatgt?ctttggcgca?gcgtgttttg?aaaacgcgcg?aggaaaccta?caagctgtcc????600gaattacgtt?acaaggcagg?cgtgatttcc?gccgtcgccc?tacgtcagca?ggaagccctg????660atcgaatctg?ccaaagccga?ttatgcccat?gccgcgcgca?gccgcgaaca?ggcgcgcaat?????720gccttggcaa?ccttgattaa?ccaaccgata?cccgaagacc?tgcctgccgg?tttgccgctg?????780gacaagcagt?tttttgttga?aaaactgccg?gccggtttga?gttccgaagt?attgctcgac?????840cgtcccgata?tccgtgctgc?cgaacacgcg?ctcaaacagg?caaacgccaa?tatcggtgcg?????900gcacgcgccg?cctttttccc?atccatccgc?ctgaccggaa?ccgtcggtac?gggttctgcc?????960gaattgggtg?ggttgttcaa?aagcggcacg?ggcgtttggt?cgttcgcgcc?gtctattacc????1020ctgccgattt?ttacctgggg?tacgaacaaa?gccaaccttg?atgtagccaa?gctgcgccaa????1080caggcacaaa?tcgttgccta?tgaagccgcc?gtccaatccg?catttcaaga?cgtggcaaac????1140gcattggcgg?cgcgcgagca?gctggataaa?gcctatgacg?ctttaagcaa?acaaagccgc????1200gcctctaaag?aggcgttgcg?cttggtcggc?ctgcgttaca?agcacggcgt?atccggcgcg????1260ctcgacttgc?tcgatgcgga?acgcagcagc?tatgcggcgg?agggtgcggc?tttgtcggca????1320caactgaccc?gcgccgaaaa?ccttgccgat?ttgtacaagg?cactcggcgg?cggattgaaa????1380cgggataccc?aaaccgacaa?ataa???????????????????????????????????????????1404

<210>4

<211>467

<212>PRT

<213>Neisseria?meningitidis

＜214〉Neisseria meningitidis

<400>4Met?Asp?Thr?Thr?Leu?Lys?Thr?Thr?Leu?Thr?Ser?Val?Ala?Ala?Ala?Phe?1???????????????5??????????????????10??????????????????15Ala?Leu?Ser?Ala?Cys?Thr?Met?Ile?Pro?Gln?Tyr?Glu?Gln?Pro?Lys?Val

20??????????????????25??????????????????30Glu?Val?Ala?Glu?Thr?Phe?Lys?Asn?Asp?Thr?Ala?Asp?Ser?Gly?Ile?Arg

35??????????????????40??????????????????45Ala?Val?Asp?Leu?Gly?Trp?His?Asp?Tyr?Phe?Ala?Asp?Pro?Arg?Leu?Gln

50??????????????????55??????????????????60Lys?Leu?Ile?Asp?Ile?Ala?Leu?Glu?Arg?Asn?Thr?Ser?Leu?Arg?Thr?Ala65??????????????????70??????????????????75??????????????????80Val?Leu?Asn?Ser?Glu?Ile?Tyr?Arg?Lys?Gln?Tyr?Met?Ile?Glu?Arg?Asn

85??????????????????90??????????????????95Asn?Leu?Leu?Pro?Thr?Leu?Ala?Ala?Asn?Ala?Asn?Gly?Ser?Arg?Gln?Gly

100?????????????????105?????????????????110Ser?Leu?Ser?Gly?Gly?Asn?Val?Ser?Ser?Ser?Tyr?Asn?Val?Gly?Leu?Gly

115?????????????????120?????????????????125Ala?Ala?Ser?Tyr?Glu?Leu?Asp?Leu?Phe?Gly?Arg?Val?Arg?Ser?Ser?Ser

130?????????????????135?????????????????140Glu?Ala?Ala?Leu?Gln?Gly?Tyr?Phe?Ala?Ser?Val?Ala?Asn?Arg?Asp?Ala145?????????????????150?????????????????155?????????????????160Ala?His?Leu?Ser?Leu?Ile?Ala?Thr?Val?Ala?Lys?Ala?Tyr?Phe?Asn?Glu

165?????????????????170?????????????????175Arg?Tyr?Ala?Glu?Glu?Ala?Met?Ser?Leu?Ala?Gln?Arg?Val?Leu?Lys?Thr

180?????????????????185?????????????????190Arg?Glu?Glu?Thr?Tyr?Lys?Leu?Ser?Glu?Leu?Arg?Tyr?Lys?Ala?Gly?Val

195?????????????????200?????????????????205Ile?Ser?Ala?Val?Ala?Leu?Arg?Gln?Gln?Glu?Ala?Leu?Ile?Glu?Ser?Ala

210?????????????????215?????????????????220Lys?Ala?Asp?Tyr?Ala?His?Ala?Ala?Arg?Ser?Arg?Glu?Gln?Ala?Arg?Asn225?????????????????230?????????????????235?????????????????240Ala?Leu?Ala?Thr?Leu?Ile?Asn?Gln?Pro?Ile?Pro?Glu?Asp?Leu?Pro?Ala

245?????????????????250?????????????????255Gly?Leu?Pro?Leu?Asp?Lys?Gln?Phe?Phe?Val?Glu?Lys?Leu?Pro?Ala?Gly

260?????????????????265?????????????????270Leu?Ser?Ser?Glu?Val?Leu?Leu?Asp?Arg?Pro?Asp?Ile?Arg?Ala?Ala?Glu

275?????????????????280?????????????????285His?Ala?Leu?Lys?Gln?Ala?Asn?Ala?Asn?Ile?Gly?Ala?Ala?Arg?Ala?Ala

290?????????????????295?????????????????300phe?Phe?Pro?Ser?Ile?Arg?Leu?Thr?Gly?Thr?Val?Gly?Thr?Gly?Ser?Ala305?????????????????310?????????????????315?????????????????320Glu?Leu?Gly?Gly?Leu?Phe?Lys?Ser?Gly?Thr?Gly?Val?Trp?Ser?Phe?Ala

325?????????????????330?????????????????335Pro?Ser?Ile?Thr?Leu?Pro?Ile?phe?Thr?Trp?Gly?Thr?Asn?Lys?Ala?Asn

340?????????????????345?????????????????350Leu?Asp?Val?Ala?Lys?Leu?Arg?Gln?Gln?Ala?Gln?Ile?Val?Ala?Tyr?Glu

355?????????????????360?????????????????365Ala?Ala?Val?Gln?Ser?Ala?Phe?Gln?Asp?Val?Ala?Asn?Ala?Leu?Ala?Ala

370?????????????????375?????????????????380Arg?Glu?Gln?Leu?Asp?Lys?Ala?Tyr?Asp?Ala?Leu?Ser?Lys?Gln?Ser?Arg385?????????????????390?????????????????395?????????????????400Ala?Ser?Lys?Glu?Ala?Leu?Arg?Leu?Val?Gly?Leu?Arg?Tyr?Lys?His?Gly

405?????????????????410?????????????????415Val?Ser?Gly?Ala?Leu?Asp?Leu?Leu?Asp?Ala?Glu?Arg?Ser?Ser?Tyr?Ala

420?????????????????425?????????????????430Ala?Glu?Gly?Ala?Ala?Leu?Ser?Ala?Gln?Leu?Thr?Arg?Ala?Glu?Asn?Leu

435?????????????????440?????????????????445Ala?Asp?Leu?Tyr?Lys?Ala?Leu?Gly?Gly?Gly?Leu?Lys?Arg?Asp?Thr?Gln

450?????????????????455?????????????????460Thr?Asp?Lys465

<210>5

<211>420

<212>DNA

<213>Neisseria?meningitidis

＜214〉Neisseria meningitidis

<400>5atgaaaaaac?ttctaatgat?aaccctcacc?ggtatgcttg?cagcttgtgc?aacaggtgtc?????60aatgtcggcc?ggttgatggt?tgaaatgccg?cagggagaac?gttctgtcgt?tgtgcaggtt????120cccgcgacaa?ataacccgct?ttccgatacg?gtagctgtcg?gaatgattaa?aacatccggt????180tcgccttcgg?catcaaatat?gattgaaatg?ctcggcgcgg?acaatatcaa?cgtcggcgtg????240gtgggaagca?gccaaatgct?taataaggcg?accgcacttt?attccttaaa?ccatgcaaag????300aaagtcggaa?ataatgtcag?tgtttatatg?atgggcgaca?gcgaaagtga?caaggccgat????360ttggaaaacg?cggcaaatgc?caaaaatatc?aaattgcatt?atttctttaa?ccaaaaataa????420

<210>6

<211>139

<212>PRT

<213>Neisseria?meningitidis

＜214〉Neisseria meningitidis

<400>6Met?Lys?Lys?Leu?Leu?Met?Ile?Thr?Leu?Thr?Gly?Met?Leu?Ala?Ala?Cys?1???????????????5??????????????????10??????????????????15Ala?Thr?Gly?Val?Asn?Val?Gly?Arg?Leu?Met?Val?Glu?Met?Pro?Gln?Gly

20??????????????????25??????????????????30Glu?Arg?Ser?Val?Val?Val?Gln?Val?Pro?Ala?Thr?Asn?Asn?Pro?Leu?Ser

35??????????????????40??????????????????45Asp?Thr?Val?Ala?Val?Gly?Met?Ile?Lys?Thr?Ser?Gly?Ser?Pro?Ser?Ala

50??????????????????55??????????????????60Ser?Asn?Met?Ile?Glu?Met?Leu?Gly?Ala?Asp?Asn?Ile?Asn?Val?Gly?Val65??????????????????70??????????????????75??????????????????80Val?Gly?Ser?Ser?Gln?Met?Leu?Asn?Lys?Ala?Thr?Ala?Leu?Tyr?Ser?Leu

85??????????????????90??????????????????95Asn?His?Ala?Lys?Lys?Val?Gly?Asn?Asn?Val?Ser?Val?Tyr?Met?Met?Gly

100?????????????????105?????????????????110Asp?Ser?Glu?Ser?Asp?Lys?Ala?Asp?Leu?Glu?Asn?Ala?Ala?Asn?Ala?Lys

115?????????????????120?????????????????125Asn?Ile?Lys?Leu?His?Tyr?Phe?Phe?Asn?Gln?Lys

130?????????????????135

<210>7

<211>516

<212>DNA

<213>Neisseria?meningitidis

＜214〉Neisseria meningitidis

<400>7atgaaaatca?aacaaatcgt?caaaccgggc?ttggcagtat?tggcggcggg?cgttctgtct?????60gcctgcgcaa?ccaaaagcaa?cgtcaaagcc?gacggaacga?ccgacaatcc?ggttttcccg????120aaaccctatt?ccgtaacgct?cgacaacaat?cgcggtacat?tcccgaccta?tgacgaattg????180gacttgatgc?gtcccggtct?gaccaaagac?gacatctaca?aaatcctggg?tcgtccgcat????240tacgacgaag?gtatgtacgg?cgtgcgcgaa?tgggattatc?tgttccactt?ccacaccccg????300ggcgtaggca?tcgaccctga?aaacacttcc?ggcgtagaag?gcattaccac?ctgtcaatac????360aaaattattt?tcgataaaga?caaatttgcc?cgcagcttct?actggaaccc?cgtcttcccg????420aaagatgccg?cctgtccgcc?gcccgcaccc?aaagccgagc?cgcaagtcat?catccgcgaa????480atcgtgcccg?ccaaacccaa?acgcatccgc?caataa??????????????????????????????516

<210>8

<21l>17L

<212>PRT

<213>Neisseria?meningitidis

＜214〉Neisseria meningitidis

<400>8Met?Lys?Ile?Lys?Gln?Ile?Val?Lys?Pro?Gly?Leu?Ala?Val?Leu?Ala?Ala?1???????????????5??????????????????10??????????????????15Gly?Val?Leu?Ser?Ala?Cys?Ala?Thr?Lys?Ser?Asn?Val?Lys?Ala?Asp?Gly

20??????????????????25??????????????????30Thr?Thr?Asp?Asn?Pro?Val?Phe?Pro?Lys?Pro?Tyr?Ser?Val?Thr?Leu?Asp

35??????????????????40??????????????????45Asn?Asn?Arg?Gly?Thr?Phe?Pro?Thr?Tyr?Asp?Glu?Leu?Asp?Leu?Met?Arg

50??????????????????55??????????????????60Pro?Gly?Leu?Thr?Lys?Asp?Asp?Ile?Tyr?Lys?Ile?Leu?Gly?Arg?Pro?His65??????????????????70??????????????????75??????????????????80Tyr?Asp?Glu?Gly?Met?Tyr?Gly?Val?Arg?Glu?Trp?Asp?Tyr?Leu?Phe?His

85??????????????????90??????????????????95Phe?His?Thr?Pro?Gly?Val?Gly?Ile?Asp?Pro?Glu?Asn?Thr?Ser?Gly?Val

100?????????????????105?????????????????110Glu?Gly?Ile?Thr?Thr?Cys?Gln?Tyr?Lys?Ile?Ile?Phe?Asp?Lys?Asp?Lys

115?????????????????120?????????????????125Phe?Ala?Arg?Ser?Phe?Tyr?Trp?Asn?Pro?Val?Phe?Pro?Lys?Asp?Ala?Ala

130?????????????????135?????????????????140Cys?Pro?Pro?Pro?Ala?Pro?Lys?Ala?Glu?Pro?Gln?Val?Ile?Ile?Arg?Glu145?????????????????150?????????????????155?????????????????160Ile?Val?Pro?Ala?Lys?Pro?Lys?Arg?Ile?Arg?Gln

165?????????????????170

<210>9

<211>816

<212>DNA

<213>Neisseria?meningitidis

＜214〉Neisseria meningitidis

<400>9atgagaccat?atgctactac?catttatcaa?ctttttattt?tgtttattgg?gagtgttttt?????60actatgacct?catgtgaacc?tgtgaatgaa?aagacagatc?aaaaagcagt?aagtgcgcaa????120caggctaaag?aacaaaccag?tttcaacaat?cccgagccaa?tgacaggatt?tgaacatacg????180gttacatttg?attttcaggg?caccaaaatg?gttatcccct?atggctatct?tgcacggtat????240acgcaagaca?atgccacaaa?atggctttcc?gacacgcccg?ggcaggatgc?ttactccatt????300aatttgatag?agattagcgt?ctattacaaa?aaaaccgacc?aaggctgggt?tcttgagcca????360tacaaccagc?aaaacaaagc?acactttatc?caatttctac?gcgacggttt?ggatagcgtg????420gacgatattg?ttatccgaaa?agatgcgtgt?agtttaagta?cgactatggg?agaaagattg????480cttacttacg?gggttaaaaa?aatgccatct?gcctatcctg?aatacgaggc?ttatgaagat????540aaaagacata?ttcctgaaaa?cccatatttt?catgaatttt?actatattaa?aaaaggagaa????600aatccggcga?ttattactca?tcggaataat?cgaataaacc?aaactgaaga?agatagttat????660agcactagcg?taggttcctg?tattaacggt?ttcacggtac?agtattaccc?gtttattcgg????720gaaaagcagc?agctcacaca?gcaggagttg?gtaggttatc?accaacaagt?agagcaattg????780gtacagagtt?ttgtaaacaa?ttcaaataaa?aaataa??????????????????????????????816

<210>10

<211>271

<212>PRT

<213>Neisseria?meningitidis

＜214〉Neisseria meningitidis

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

20??????????????????25??????????????????30Asp?Gln?Lys?Ala?Val?Ser?Ala?Gln?Gln?Ala?Lys?Glu?Gln?Thr?Ser?Phe

35??????????????????40??????????????????45Asn?Asn?Pro?Glu?Pro?Met?Thr?Gly?Phe?Glu?His?Thr?Val?Thr?Phe?Asp

50??????????????????55??????????????????60Phe?Gln?Gly?Thr?Lys?Met?Val?Ile?Pro?Tyr?Gly?Tyr?Leu?Ala?Arg?Tyr65??????????????????70??????????????????75??????????????????80Thr?Gln?Asp?Asn?Ala?Thr?Lys?Trp?Leu?Ser?Asp?Thr?Pro?Gly?Gln?Asp

85??????????????????90??????????????????95Ala?Tyr?Ser?Ile?Asn?Leu?Ile?Glu?Ile?Ser?Val?Tyr?Tyr?Lys?Lys?Thr

100?????????????????105?????????????????110Asp?Gln?Gly?Trp?Val?Leu?Glu?Pro?Tyr?Asn?Gln?Gln?Asn?Lys?Ala?His

115?????????????????120?????????????????125Phe?Ile?Gln?Phe?Leu?Arg?Asp?Gly?Leu?Asp?Ser?Val?Asp?Asp?Ile?Val

130?????????????????135?????????????????140Ile?Arg?Lys?Asp?Ala?Cys?Ser?Leu?Ser?Thr?Thr?Met?Gly?Glu?Arg?Leu145?????????????????150?????????????????155?????????????????160Leu?Thr?Tyr?Gly?Val?Lys?Lys?Met?Pro?Ser?Ala?Tyr?Pro?Glu?Tyr?Glu

165?????????????????170?????????????????175Ala?Tyr?Glu?Asp?Lys?Arg?His?Ile?Pro?Glu?Asn?Pro?Tyr?Phe?His?Glu

180?????????????????185?????????????????190Phe?Tyr?Tyr?Ile?Lys?Lys?Gly?Glu?Asn?Pro?Ala?Ile?Ile?Thr?His?Arg

195?????????????????200?????????????????205Asn?Asn?Arg?Ile?Asn?Gln?Thr?Glu?Glu?Asp?Ser?Tyr?Ser?Thr?Ser?Val

210?????????????????215?????????????????220Gly?Ser?Cys?Ile?Asn?Gly?Phe?Thr?Val?Gln?Tyr?Tyr?Pro?Phe?Ile?Arg225?????????????????230?????????????????235?????????????????240Glu?Lys?Gln?Gln?Leu?Thr?Gln?Gln?Glu?Leu?Val?Gly?Tyr?His?Gln?Gln

245?????????????????250?????????????????255Val?Glu?Gln?Leu?Val?Gln?Ser?Phe?Val?Asn?Asn?Ser?Asn?Lys?Lys

260?????????????????265?????????????????270

<210>11

<211>717

<212>DNA

<213>Neisseria?meningitidis

＜214〉Neisseria meningitidis

<400>11atgaagacca?aattaccgct?ttttatcatt?tggctgtccg?tatccgccgc?ctgttcttcc?????60cctgtttccc?gcaatattca?ggatatgcgg?cccgaaccgc?aggcagaggc?aggtagttcg????120gacgctattc?cctatcccgt?tcccactctg?caagaccgtt?tggattatct?ggaaggcaca????180ctcgtccgcc?tgtcgaacga?agtggaaacc?ttaaacggca?aagtcaaagc?actggagcat????240gcgaaaacac?acccttccgg?tagggcatac?gtccaaaaac?tcgacgaccg?caagttgaaa????300gagcattacc?tcaataccga?aggcggcagc?gcatccgcac?ataccgtcga?aaccgcacaa????360aacctctaca?atcaggcact?caaacactat?aaaagcggca?ggttttctgc?cgcagccgcc????420ctgttgaaag?gcgcggacgg?aggcgacggc?ggcagcatcg?cgcaacgcag?tatgtacctg????480ttgctgcaaa?gcagggcgcg?tatgggcaac?tgcgaatccg?tcatcgaaat?cggagggcgt????540tacgccaacc?gtttcaaaga?cagcccaacc?gcgcccgaag?ccatgttcaa?aatcggcgaa????600tgccaataca?ggttgcagca?gaaagacatt?gcaagggcaa?cttggcgcag?cctgatacag????660gcttacccga?gcagcccggc?ggcaaaacgc?gccgccgcag?ccgtacgcaa?acgatag???????717

<210>12

<211>238

<212>PRT

<213>Neisseria?meningitidis

＜214〉Neisseria meningitidis

<400>12Met?Lys?Thr?Lys?Leu?Pro?Leu?Phe?Ile?Ile?Trp?Leu?Ser?Val?Ser?Ala?1???????????????5??????????????????10??????????????????15Ala?Cys?Ser?Ser?Pro?Val?Ser?Arg?Asn?Ile?Gln?Asp?Met?Arg?Pro?Glu

20??????????????????25??????????????????30Pro?Gln?Ala?Glu?Ala?Gly?Ser?Ser?Asp?Ala?Ile?Pro?Tyr?Pro?Val?Pro

35??????????????????40??????????????????45Thr?Leu?Gln?Asp?Arg?Leu?Asp?Tyr?Leu?Glu?Gly?Thr?Leu?Val?Arg?Leu

50??????????????????55??????????????????60Ser?Asn?Glu?Val?Glu?Thr?Leu?Asn?Gly?Lys?Val?Lys?Ala?Leu?Glu?His65??????????????????70??????????????????75??????????????????80Ala?Lys?Thr?His?Pro?Ser?Gly?Arg?Ala?Tyr?Val?Gln?Lys?Leu?Asp?Asp

85??????????????????90??????????????????95Arg?Lys?Leu?Lys?Glu?His?Tyr?Leu?Asn?Thr?Glu?Gly?Gly?Ser?Ala?Ser

100?????????????????105?????????????????110Ala?His?Thr?Val?Glu?Thr?Ala?Gln?Asn?Leu?Tyr?Asn?Gln?Ala?Leu?Lys

115?????????????????120?????????????????125His?Tyr?Lys?Ser?Gly?Arg?Phe?Ser?Ala?Ala?Ala?Ala?Leu?Leu?Lys?Gly

130?????????????????135?????????????????140Ala?Asp?Gly?Gly?Asp?Gly?Gly?Ser?Ile?Ala?Gln?Arg?Ser?Met?Tyr?Leu145?????????????????150?????????????????155?????????????????160Leu?Leu?Gln?Ser?Arg?Ala?Arg?Met?Gly?Asn?Cys?Glu?Ser?Val?Ile?Glu

165?????????????????170?????????????????175Ile?Gly?Gly?Arg?Tyr?Ala?Asn?Arg?Phe?Lys?Asp?Ser?Pro?Thr?Ala?Pro

180?????????????????185?????????????????190Glu?Ala?Met?Phe?Lys?Ile?Gly?Glu?Cys?Gln?Tyr?Arg?Leu?Gln?Gln?Lys

195?????????????????200?????????????????205Asp?Ile?Ala?Arg?Ala?Thr?Trp?Arg?Ser?Leu?Ile?Gln?Ala?Tyr?Pro?Ser

210?????????????????215?????????????????220Ser?Pro?Ala?Ala?Lys?Arg?Ala?Ala?Ala?Ala?Val?Arg?Lys?Arg225?????????????????230?????????????????235

<210>13

<211>1176

<212>DNA

<213>Neisseria?meningitidis

＜214〉Neisseria meningitidis

<400>13gtgtttaaag?tgaaatttta?tattcgtcac?gcagtattat?tattgtgtgg?aagtttaatt??????60gtaggatgct?ctgcgattcc?ttcatcaggc?cccagcgcaa?aaaaaattgt?ctctttaggg?????120caacaatctg?aagttcaaat?tcctgaagtg?gagctgattg?atgtgaatca?tacggttgct?????180cagttattat?ataaggctca?gataaatcag?tcattcactc?agtttggcga?tggttatgct?????240tcggctggta?cgctaaatat?tggtgatgta?ttggatatta?tgatttggga?agcgccgccg?????300gcagtattgt?ttggtggtgg?cctttcttcg?atgggctcgg?gtagtgcgca?tcaaactaag?????360ttgccagagc?agttggtcac?ggcacgtggt?acggtttctg?tgccgtttgt?tggcgatatt?????420tcggtggtcg?gtaaaacgcc?tggtcaggtt?caggaaatta?ttaaaggccg?cctgaaaaaa?????480atggccaatc?agccacaagt?gatggtgcgt?ttggtgcaga?ataatgcggc?gaatgtgtcg?????540gtgattcgtg?ctgggaatag?tgtgcgtatg?ccgctgacgg?cagccggtga?gcgtgtgttg?????600gatgcggtgg?ctgcggtagg?tggttcaacg?gcaaatgtgc?aggatacgaa?tgtgcagctg?????660acacgtggca?atgtagtacg?aactgttgcc?ttggaagatt?tagttgcaaa?tccgcgacaa?????720aatattttgc?tgcgtcgcgg?tgatgtggtt?accatgatta?ccaatcccta?tacctttacg?????780tctatgggtg?cggtggggag?aacacaagaa?atcggttttt?cagccagagg?cttatcgctt?????840tctgaagcca?ttggccgtat?gggcggtttg?caagatcgcc?gttctgatgc?gcgtggtgtg?????900tttgtgttcc?gctatacgcc?attggtggaa?ttgccggcag?aacgtcagga?taaatggatt?????960gctcaaggtt?atggcagtga?ggcagagatt?ccaacggtat?atcgtgtgaa?tatggctgat????1020gcgcattcgc?tattttctat?gcagcgcttt?cctgtgaaga?ataaagatgt?attgtatgtg????1080tcgaatgcgc?cgttggctga?agtgcagaaa?ttcttgtcgt?ttgtgttctc?gccggttacc????1140agtggcgcga?acagtattaa?taatttaact?aattaa??????????????????????????????1176

<210>14

<211>391

<212>PRT

<213>Neisseria?meningitidis

＜214〉Neisseria meningitidis

<400>14Met?Phe?Lys?Val?Lys?Phe?Tyr?Ile?Arg?His?Ala?Val?Leu?Leu?Leu?Cys?1???????????????5??????????????????10??????????????????15Gly?Ser?Leu?Ile?Val?Gly?Cys?Ser?Ala?Ile?Pro?Ser?Ser?Gly?Pro?Ser

20??????????????????25??????????????????30Ala?Lys?Lys?Ile?Val?Ser?Leu?Gly?Gln?Gln?Ser?Glu?Val?Gln?Ile?Pro

35??????????????????40??????????????????45Glu?Val?Glu?Leu?Ile?Asp?Val?Asn?His?Thr?Val?Ala?Gln?Leu?Leu?Tyr

50??????????????????55??????????????????60Lys?Ala?Gln?Ile?Asn?Gln?Ser?Phe?Thr?Gln?Phe?Gly?Asp?Gly?Tyr?Ala65??????????????????70??????????????????75??????????????????80Ser?Ala?Gly?Thr?Leu?Asn?Ile?Gly?Asp?Val?Leu?Asp?Ile?Met?Ile?Trp

85??????????????????90??????????????????95Glu?Ala?Pro?Pro?Ala?Val?Leu?Phe?Gly?Gly?Gly?Leu?Ser?Ser?Met?Gly

100?????????????????105?????????????????110Ser?Gly?Ser?Ala?His?Gln?Thr?Lys?Leu?Pro?Glu?Gln?Leu?Val?Thr?Ala

115?????????????????120?????????????????125Arg?Gly?Thr?Val?Ser?Val?Pro?Phe?Val?Gly?Asp?Ile?Ser?Val?Val?Gly

130?????????????????135?????????????????140Lys?Thr?Pro?Gly?Gln?Val?Gln?Glu?Ile?Ile?Lys?Gly?Arg?Leu?Lys?Lys145?????????????????150?????????????????155?????????????????160Met?Ala?Asn?Gln?Pro?Gln?Val?Met?Val?Arg?Leu?Val?Gln?Asn?Asn?Ala

165?????????????????170?????????????????175Ala?Asn?Val?Ser?Val?Ile?Arg?Ala?Gly?Asn?Ser?Val?Arg?Met?Pro?Leu

180?????????????????185?????????????????190Thr?Ala?Ala?Gly?Glu?Arg?Val?Leu?Asp?Ala?Val?Ala?Ala?Val?Gly?Gly

195?????????????????200?????????????????205Ser?Thr?Ala?Asn?Val?Gln?Asp?Thr?Asn?Val?Gln?Leu?Thr?Arg?Gly?Asn

210?????????????????215?????????????????220Val?Val?Arg?Thr?Val?Ala?Leu?Glu?Asp?Leu?Val?Ala?Asn?Pro?Arg?Gln225?????????????????230?????????????????235?????????????????240Asn?Ile?Leu?Leu?Arg?Arg?Gly?Asp?Val?Val?Thr?Met?Ile?Thr?Asn?Pro

245?????????????????250?????????????????255Tyr?Thr?Phe?Thr?Ser?Met?Gly?Ala?Val?Gly?Arg?Thr?Gln?Glu?Ile?Gly

260?????????????????265?????????????????270Phe?Ser?Ala?Arg?Gly?Leu?Ser?Leu?Ser?Glu?Ala?Ile?Gly?Arg?Met?Gly

275?????????????????280?????????????????285Gly?Leu?Gln?Asp?Arg?Arg?Ser?Asp?Ala?Arg?Gly?Val?Phe?Val?Phe?Arg

290?????????????????295?????????????????300Tyr?Thr?Pro?Leu?Val?Glu?Leu?Pro?Ala?Glu?Arg?Gln?Asp?Lys?Trp?Ile305?????????????????310?????????????????315?????????????????320Ala?Gln?Gly?Tyr?Gly?Ser?Glu?Ala?Glu?Ile?Pro?Thr?Val?Tyr?Arg?Val

325?????????????????330?????????????????335Asn?Met?Ala?Asp?Ala?His?Ser?Leu?Phe?Ser?Met?Gln?Arg?Phe?Pro?Val

340?????????????????345?????????????????350Lys?Asn?Lys?Asp?Val?Leu?Tyr?Val?Ser?Asn?Ala?Pro?Leu?Ala?Glu?Val

355?????????????????360?????????????????365Gln?Lys?Phe?Leu?Ser?Phe?Val?Phe?Ser?Pro?Val?Thr?Ser?Gly?Ala?Asn

370?????????????????375?????????????????380Ser?Ile?Asn?Asn?Leu?Thr?Asn385?????????????????390

<210>15

<211>807

<212>DNA

<213>Neisseria?meningitidis

＜214〉Neisseria meningitidis

<400>15atgaatatga?aaaaatggat?tgccgccgcc?cttgcctgtt?ccgcgctcgc?gctgtctgcc?????60tgcggcggtc?agggcaaaga?tgccgccgcg?cccgccgcaa?accccgacaa?agtgtaccgc????120gtggcttcca?acgccgagtt?tgcccccttt?gaatctttag?actcgaaagg?caatgttgaa????180ggcctcgatg?tggatttgat?gaacgcgatg?gcgaaggcgg?gcaattttaa?aatcgaattc????240aaacaccagc?cgtgggacag?ccttttcccc?gccttgaaca?acggcgatgc?ggacgttgtg????300atgtcgggcg?taaccattac?cgacgaccgc?aaacagtcta?tggacttcag?cgacccgtat????360tttgaaatca?cccaagtcgt?cctcgttccg?aaaggcaaaa?aaatatcttc?ttccgaagat????420ttgaaaaaca?tgaacaaagt?cggcgtggta?accggctaca?cgggcgattt?ctccgtatcc????480aaactcttgg?gcaacgacaa?cccgaaaatc?gcgcgctttg?aaaacgttcc?cctgattatc????540aaagaactgg?aaaacggcgg?cttggattcc?gtggtcagcg?acagcgcagt?catcgccaat????600tatgtgaaaa?acaatccgac?caaagggatg?gacttcgtta?ccctgcccga?cttcaccacc????660gaacactacg?gcatcgcggt?acgcaaaggc?gacgaagcaa?ccgtcaaaat?gctgaacgat????720gcgttgaaaa?aagtacgcga?aagcggcgaa?tacgacaaaa?tctacgccaa?atattttgca????780aaagaagacg?gacaggccgc?aaaataa????????????????????????????????????????807

<210>16

<211>268

<212>PRT

<213>Neisseria?meningitidis

＜214〉Neisseria meningitidis

<400>16Met?Asn?Met?Lys?Lys?Trp?Ile?Ala?Ala?Ala?Leu?Ala?Cys?Ser?Ala?Leu?1???????????????5??????????????????10??????????????????15Ala?Leu?Ser?Ala?Cys?Gly?Gly?Gln?Gly?Lys?Asp?Ala?Ala?Ala?Pro?Ala

20??????????????????25??????????????????30Ala?Asn?Pro?Asp?Lys?Val?Tyr?Arg?Val?Ala?Ser?Asn?Ala?Glu?Phe?Ala

35??????????????????40??????????????????45Pro?Phe?Glu?Ser?Leu?Asp?Ser?Lys?Gly?Asn?Val?Glu?Gly?Phe?Asp?Val

50??????????????????55??????????????????60Asp?Leu?Met?Asn?Ala?Met?Ala?Lys?Ala?Gly?Asn?Phe?Lys?Ile?Glu?Phe65??????????????????70??????????????????75??????????????????80Lys?His?Gln?Pro?Trp?Asp?Ser?Leu?Phe?Pro?Ala?Leu?Asn?Asn?Gly?Asp

85??????????????????90??????????????????95Ala?Asp?Val?Val?Met?Ser?Gly?Val?Thr?Ile?Thr?Asp?Asp?Arg?Lys?Gln

100?????????????????????105?????????????????110Ser?Met?Asp?Phe?Ser?Asp?Pro?Tyr?Phe?Glu?Ile?Thr?Gln?Val?Val?Leu

115?????????????????120?????????????????125Val?Pro?Lys?Gly?Lys?Lys?Ile?Ser?Ser?Ser?Glu?Asp?Leu?Lys?Asn?Met

130?????????????????135?????????????????140Asn?Lys?Val?Gly?Val?Val?Thr?Gly?Tyr?Thr?Gly?Asp?Phe?Ser?Val?Ser145?????????????????150?????????????????155?????????????????160Lys?Leu?Leu?Gly?Asn?Asp?Asn?Pro?Lys?Ile?Ala?Arg?Phe?Glu?Asn?Val

165?????????????????170?????????????????175Pro?Leu?Ile?Ile?Lys?Glu?Leu?Glu?Asn?Gly?Gly?Leu?Asp?Ser?Val?Val

180?????????????????185?????????????????190Ser?Asp?Ser?Ala?Val?Ile?Ala?Asn?Tyr?Val?Lys?Asn?Asn?Pro?Thr?Lys

195?????????????????????200?????????????????205Gly?Met?Asp?Phe?Val?Thr?Leu?Pro?Asp?Phe?Thr?Thr?Glu?His?Tyr?Gly

210?????????????????215?????????????????220Ile?Ala?Val?Arg?Lys?Gly?Asp?Glu?Ala?Thr?Val?Lys?Met?Leu?Asn?Asp225?????????????????230?????????????????235?????????????????240Ala?Leu?Lys?Lys?Val?Arg?Glu?Ser?Gly?Glu?Tyr?Asp?Lys?Ile?Tyr?Ala

245?????????????????250?????????????????255Lys?Tyr?Phe?Ala?Lys?Glu?Asp?Gly?Gln?Ala?Ala?Lys

260?????????????????265

Claims (24)

1. the isolated polypeptide that contains a kind of aminoacid sequence, this aminoacid sequence has 85% identity at least with the aminoacid sequence that is selected from the sequence set of being made up of SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16.

2. the isolated polypeptide in the claim 1, its aminoacid sequence has 95% identity at least with the aminoacid sequence that is selected from the sequence set of being made up of SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16.

3. the polypeptide in the claim 1, it contains the aminoacid sequence that is selected from the sequence set of being made up of SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16.

4.SEQ the isolated polypeptide of ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16.

5. the immunogenic fragments of polypeptide among the claim 1-4, the immunogen activity of this immunogenic fragments polypeptide with SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 basically is identical.

6. the separation polynucleotide that contain the nucleotide sequence of the peptide species of encoding, this nucleotide sequence coded polypeptide is with regard to the SEQ ID NO:2,4,6,8,10,12,14 or 16 of whole length, with SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, aminoacid sequence have 85% identity at least, or separate polynucleotide sequence complementary nucleotide sequence with this.

7. the separation polynucleotide that contain a kind of nucleotide sequence, this nucleotide sequence and coding SEQ ID NO:2,4,6,8,10,12,14 or 16 polypeptide have at least 85% identity in whole coding regions of length; Or separate polynucleotide complementary polynucleotide with this.

8. the separation polynucleotide that contain a kind of nucleotide sequence, this nucleotide sequence are with regard to the SEQ ID NO:1 of total length, and 3,5,7,9,11,13 have 85% identity at least with SEQ ID NO:1,3,5,7,9,11,13 or 15; Or with this polynucleotide sequence complementary polynucleotide sequence.

9. the polynucleotide that separate that have 95% identity among the claim 6-8 with SEQ ID NO:1,3,5,7,9,11,13 or 15 at least.

10. the separation polynucleotide of nucleotide sequence that contain the polypeptide of coding SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14 or SEQ ID NO:16.

11. contain the separation polynucleotide of the polynucleotide of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13 or SEQ ID NO:15.

12. contain coding SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, the separation polynucleotide of the nucleotide sequence of the polypeptide of SEQ ID NO:14 or SEQ ID NO:16, he can contain SEQ ID NO:1 by utilization, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13 or SEQ ID NO:15 sequence or its segmental probe, under the hybridization conditions of strictness, obtaining by screening suitable library.

13. contain expression vector or reorganization living microorganism body corresponding to the separation polynucleotide of claim 6-12.

14. contain the host cell of expression vector of claim 13 or a kind of film of ubcellular fragment or this host cell, can express a kind of isolated polypeptide, the aminoacid sequence of selecting in the aminoacid sequence that this polypeptide contains and the group of being made up of SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, EQ ID NO:16 has 85% identity at least.

15. preparation contains a kind of method of polypeptide of aminoacid sequence, the aminoacid sequence of selecting in this aminoacid sequence and the group of being made up of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQID NO:10, SEQ ID NO:12, SEQ ID NO:14 or SEQ ID NO:16 has 85% identity at least, this method is also included within the host cell of cultivating claim 14 under the condition that can fully prepare this polypeptide, and reclaims this polypeptide from substratum.

16. express the method for the polynucleotide among the claim 6-12, comprise and utilize the carrier transformed host cell that contains at least a these polynucleotide, and under the condition that can give full expression to these polynucleotide, cultivate this host cell.

17. contain the vaccine composition of polypeptide among the claim 1-5, effective dose and pharmaceutically acceptable carrier.

18. contain the vaccine composition of polypeptide among the claim 6-12, effective dose and pharmaceutically acceptable carrier.

19. the vaccine composition of claim 17 or 18, wherein said composition contains at least a other I (Neisseria meningitidis) antigens.

20. the antibody that the polypeptide among the claim 1-5 or immunology fragment are had immunologic opsonin.

21. the method that the diagnosis of meningitis Neisseria gonorrhoeae infects is included in from suspecting and infects the polypeptide of differentiating in the biological samples of obtaining this infected animals among the claim 1-5, or the existence of the antibody of this polypeptid specificity immunity.

22. contain the composition of polypeptide among the claim 1-5 of effective dose on the immunology, be used for producing in animal body the application of the pharmaceutical preparation of immunne response in preparation.

23. contain the composition of Nucleotide among the claim 6-12 of effective dose on the immunology is used for producing in animal body the pharmaceutical preparation of immunne response in preparation application.

24. can effectively treat people Neisseria meningitidis treatment of diseases composition, said composition contains the antibody and the suitable carriers of the polypeptide among the direct anti-claim 1-5 at least.