CN101481409A - BASB027 proteins and genes from Moraxella catarrhalis, antigens, antibodies, and uses - Google Patents

Abstract

The invention provides BASB027 polypeptides and polynucleotides encoding BASB027 polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are diagnostic, prophylactic and therapeutic uses.

Description

Derive from BASB027 albumen and BASB027 gene, antigen, antibody and the application of morazella catarrhalis

The application is dividing an application of following application: May 31 1999 applying date, and application number 99809199.5 (PCT/EP99/03822), title " derives from BASB027 albumen and BASB027 gene, antigen, antibody and the application of morazella catarrhalis ".

Technical field

The present invention relates to polynucleotide (this paper is called " BASB027 polynucleotide "), by the polypeptide (this paper is called " BASB027 " or " BASB027 polypeptide ") of described polynucleotide encoding, produce their recombined material and method.On the other hand, the present invention relates to use the method for such polypeptide and polynucleotide, comprise vaccine bacterial-infection resisting.More on the one hand, the present invention relates to detect the diagnostic test of some pathogenic infection.

Background technology

Morazella catarrhalis (Moraxella catarrhalis) (being also referred to as catarrh branhamella (Branhamella catarrhalis)) is the gram negative bacterium that often is separated to from the human upper respiratory tract.It causes several symptom, mainly contains the otitis media among baby and the children and the pneumonia of aging philtrum.It also causes sinusitis paranasal sinusitis, hospital infection, and causes affecting conditions infrequently.

From case quantity and potential sequela, otitis media is a kind of important childhood disease.Record every year in the U.S. and to surpass 3.5 hundred ten thousand cases, and estimate that 80% children experienced an otitis outbreak (Klein, JO (1994) Clin.Inf.Dis 19:823) in the past at least reaching 3 years old.When this disease does not obtain medical treatment or becomes when chronic, may cause temporary transient hearing loss (at liquid under the situation of middle ear accumulation) or permanent hearing loss (if auditory nerve sustains damage).In the baby, such hearing loss may cause that language learning postpones.

Mainly isolate three kinds of bacterial species from the children's that suffer from otitis media middle ear: streptococcus pneumoniae (Streptococcus pneumoniae), (non typeable) hemophilus influenzae (Haemophilus influenzae) that can't finalize the design are (NTHi) and morazella catarrhalis.They are present in 60% to 90% the case.The summary of research shows that streptococcus pneumoniae and NTHi have represented about 30% otitis media case separately recently, and morazella catarrhalis has been represented about 15% otitis media case (Murphy, TF (1996) Microbiol.Rev.60:267).Also can be separated to other bacterium (Type B hemophilus influenzae, streptococcus pyogenes (S.pyogenes) etc.) from middle ear, but frequency much lower (2% case or still less).

Epidemic data shows: for the pathogenic agent of finding in middle ear, building group (colonization) in the upper respiratory tract is the sine qua non who develops into otitis; Yet also need other prerequisite with cause disease (Dickinson, people such as DP (1988) J.Infect.Dis.158:205, Faden, HL etc. (1991) Ann.Otorhinol.Laryngol, 100:612).These for trigger bacterium by pharyngotympanic tube move to middle ear, initial inflammatory process is important subsequently.These factors are unknown so far.Suppose, for example, behind virus infection immune of short duration may cause unusually being controlled at respiratory tract build group (Faden, HL etc. (1994) J.Infect.Dis.169:1312).Another explanation is, is exposed to environmental factor and builds the group with making some child's greater amounts, and these children are because the lasting existence of middle ear pathology substance and become subsequently and be easy to develop otitis media (Murphy, TF (1996) Microbiol.Rev.60:267).

Immunne response at morazella catarrhalis does not obtain good characterized as yet.The analysis of the bacterial strain that order the baby's that followed the tracks of from 0 to 2 years old the nasopharynx is separated to is pointed out: they obtain and remove new bacterial strain continually.This points out, has set up effective immunne response (Faden, HL etc. (1994) J.Infect.Dis.169:1312) of resisting this bacterium in the children that build the group.

In the adult that great majority were tested, bacteriocidin (Chapman, AJ etc. (1985) J.Infect.Dis.151:878) have extremely been identified.There is variation in the bacterial strain of morazella catarrhalis on the ability of their opposing serum bactericidal activity: in general, the isolate that derives from diseased individuals has more resistance (Hol than the isolate that derives from the individuality of only having built the group, C etc. (1993) Lancet341:1281, Jordan, KL etc. (1990) Am.J.Med.88 (supplementary issue 5A): 28S).Therefore the virulence factor that seroresistance can be regarded as described bacterium.The children's that recover from otitis media serum, observed opsonic activity.

Except OMP B1 (the 84kDa albumen that a kind of expression is regulated by iron, it is suffered from the patient's of pneumonia serum identification (Sethi, and UspA1 and UspA2 (Chen D. etc. (1999) (1995) Infect.Immun.63:1516 such as S.)), Infect.Immun.67:1310) outside, also do not identify in human body antigen by these different immunne responses institute targets.

Several membranins that other is present in the morazella catarrhalis surface have used biochemical method to carry out characterized; or their potential associations (summary is seen Murphy, TF (1996) Microbiol.Rev.60:267) in inducing protective immunity have been identified.In mouse pneumonia model, (UspA, CopB) existence of the antibody that produces promotes to remove quickly pulmonary infection to resist some described albumen.Another kind of polypeptide (OMP CD) is conservative at morazella catarrhalis bacterial strain camber, and there is homology with the porin of Pseudomonas aeruginosa (Pseudomonas aeruginosa), shown that this porin effectively resists described bacterium in animal model.

In the past few decades, the frequency of morazella catarrhalis infection sharply rises.This increases owing to the crowd of appearance with multiple strains and immunity system weakening.The morazella catarrhalis bacterial strain that is separated to the some or all of standard antibiotic of opposing no longer has been uncommon thing.This phenomenon has been created needs of medical treatment and the demand of demanding urgently satisfying at this biological novel anti-microbial agent, vaccine, drug screening method and diagnostic test.

Summary of the invention

The present invention relates to BASB027, especially BASB027 polypeptide and BASB027 polynucleotide are produced their recombined material and method.On the other hand, the present invention relates to use the method for such polypeptide and polynucleotide, comprising prevention and treatment microbial diseases.More on the one hand, the present invention relates to be used to detect the diagnostic test with infected by microbes diseases associated and the illness relevant with such infection, for example detect BASB027 polynucleotide or polypeptide expression or active test.

By reading the other parts of the following description and the reading disclosure of invention, various changes in the spirit and scope of disclosed invention and modification will be very tangible for those skilled in the art.

Description of drawings

Fig. 1: the photo of the running gel that in the rflp analysis of BASB027, obtains.

Fig. 2: the sequence contrast of BASB027 polynucleotide sequence.Point out with point with the identity of SeqID No:1.

Fig. 3: the sequence contrast of BASB peptide sequence.Point out with point with the identity of SeqID No:2.

Fig. 4: the SDS-PAGE after the proteic coomassie dyeing of the BASB027 of purifying.

Fig. 5: the proteic western blotting of BASB027 that uses the tera-His purifying antibody.

Fig. 6: the proteic western blotting of BASB027 that uses the purifying of corresponding anti-recombinant protein serum.Plate A: preimmune serum.Plate B: immune serum.

Fig. 7: the western blotting of the full cell lysate of the 16 strain morazella catarrhalis that the anti-BASB027 albumen serum (serum dilutes with 1:2000) that use compiles carries out.

Fig. 8: the western blotting that uses the reorganization BASB027 of the purifying that corresponding anti-peptide serum carries out.2 roads and 3 roads are NISs.1 road and 4 roads are immune serums.

Embodiment

The present invention relates to as hereinafter BASB027 polypeptide and polynucleotide in greater detail.The invention particularly relates to polypeptide and the polynucleotide of the BASB027 of morazella catarrhalis, the BASB027 of described morazella catarrhalis is relevant on amino acid sequence homology with Neisseria meningitidis (Neisseriameningitidis) OMP85 outer membrane protein.The present invention be more particularly directed to have SEQ ID NO:1 or 3 and SEQ ID NO:2 or 4 nucleotide sequence of stating respectively and the BASB027 of aminoacid sequence.Be appreciated that, the sequence that is enumerated as " DNA " below in the sequence table has been represented the example of one embodiment of the invention, because those skilled in the art will recognize such sequence and can usefully generally be used in polynucleotide, comprise ribose polynucleotide (ribopolynucleotide).

Polypeptide

In one aspect of the invention, the polypeptide of the morazella catarrhalis that this paper is called " BASB027 " and " BASB027 polypeptide " is provided and biologically, in the diagnosis, in the prevention, clinically or in the treatment useful varient, and the composition that comprises aforementioned polypeptides, its varient.

The present invention also provides:

(a) isolated polypeptide, described polypeptide comprise aminoacid sequence with SEQ ID NO:2 or 4 at least 85% identity, preferred at least 90% identity, more preferably at least 95% identity, the most preferably 97-99% or the aminoacid sequence of identity at least fully;

(b) by the polypeptide of isolating polynucleotide encoding, described isolating polynucleotide comprise respectively on the total length of SEQ ID NO:1 or 3 with SEQ ID NO:1 or 3 at least 85% identity, preferred at least 90% identity, more preferably at least 95% identity even the more preferably 97-99% or the polynucleotide sequence of identity at least fully; Or

(c) by the polypeptide of isolating polynucleotide encoding, described isolating polynucleotide comprise coding and SEQ ID NO:2 or 4 aminoacid sequence at least 85% identity, preferred at least 90% identity, more preferably at least 95% identity, the most preferably 97-99% or the polynucleotide sequence of the polypeptide of identity at least fully.

The BASB027 polypeptide that provides among the SEQ ID NO:2 or 4 is the BASB027 polypeptide that derives from morazella catarrhalis bacterial strain Mc2931 (ATCC 43617).

The present invention also provides the immunogenic fragments of BASB027 polypeptide, promptly has the sequential portion of identical or basic identical immunogenic BASB027 polypeptide with the polypeptide of the aminoacid sequence that comprises SEQID NO:2 or 4; In other words, described fragment (being coupled on the carrier if desired) can cause the immunne response of the described BASB027 polypeptide of identification.A kind of like this immunogenic fragments can comprise the BASB027 polypeptide that for example lacks N end leader sequence and/or membrane spaning domain and/or C end anchoring structure territory.Aspect preferred, comprise basic all ectodomains of polypeptide according to the immunogenic fragments of BASB027 of the present invention, wherein said polypeptide has at least 85% identity, preferably at least 90% identity, more preferably at least 95% identity, 97-99% identity at least most preferably with SEQ ID NO:2 or 4 on the total length of SEQ ID NO:2.

A kind of fragment is the polypeptide with such aminoacid sequence: any aminoacid sequence part of described aminoacid sequence and any polypeptide of the present invention but be not all identical.With regard to the BASB027 polypeptide, fragment can be " freestanding ", or is contained in the bigger polypeptide, and they form a part or zone therein, and described fragment most preferably is as the single successive zone in the single bigger polypeptide.

Preferred fragment comprises the aminoacid sequence that for example has part SEQ ID NO:2 or 4 or the brachymemma polypeptide of its varient, for example comprises the successive series of residues of amino terminal amino acid sequence and/or C-terminal aminoacid sequence.The degraded form of the described polypeptide of the present invention that also preferably produces or in host cell, produce by host cell.More preferably be characterised in that the fragment of structure or functional attributes, for example comprise alpha-helix and form district, the spiral (coil) of district, corner and formation corner of district, beta sheet and formation beta sheet of alpha-helix and the amphipathic district of district, hydrophilic area, hydrophobic region, α, the amphipathic district of β, flex region (flexible region), the surface that form spiral form distinguish, the fragment of substrate land and high antigen indicator.

Other preferred fragment comprises isolated polypeptide, described isolated polypeptide comprises the aminoacid sequence that has from least 15,20,30,40,50 or 100 continuous amino acids of the aminoacid sequence of SEQ ID NO:2 or 4, and perhaps described isolated polypeptide comprises the aminoacid sequence of having clipped or lacked at least 15,20,30,40,50 or 100 continuous amino acids from the aminoacid sequence of SEQ ID NO:2 or 4.

Can use the fragment of polypeptide of the present invention to synthesize the full-length polypeptide that produces correspondence by peptide; Therefore, can use these fragments as the intermediate that produces full-length polypeptide of the present invention.

Especially preferred be wherein with any combination replace, lack or add several, 5-10,1-5,1-3,1-2 or 1 amino acid whose varient.

Polypeptide of the present invention or immunogenic fragments can be " maturation " proteic forms, maybe can be the parts of bigger albumen (as precursor protein or fusion rotein).The aminoacid sequence that includes interpolation usually is favourable, and described sequence comprises secretion sequence or leader sequence, former sequence (pro-sequence), helps the sequence (as the polyhistidine residue) of purifying or helps interpolation sequence stable in the recombinant production process.In addition, interpolation allogenic polypeptide or lipid afterbody or polynucleotide sequence have also been considered, to increase the immunogenicity potentiality of final molecule.

On the one hand, the present invention relates to genetically engineered soluble fusion protein, described fusion rotein comprises polypeptide of the present invention or its fragment, and the different piece of the constant region of the heavy chain of different immunoglobulin subclass (IgG, IgM, IgA, IgE) or light chain.As the immunoglobulin (Ig) constant portion of the heavy chain of IgG (especially IgG1) preferably, wherein merge and occur in hinge region.In a specific embodiment, can remove the Fc part with the cutting sequence of factor Xa cutting by inserting one simply.

In addition, the present invention relates to prepare the method for these fusion roteins by genetic engineering, with and be used for the application of drug screening, diagnosis and treatment.The polynucleotide that also relate to the such fusion rotein of coding more on the one hand of the present invention.The example of fusion protein technology can find in international patent application WO94/29458 number and WO94/22914 number.

Described albumen can be chemically conjugated or be expressed as recombination fusion protein, so that compare with non-fusion rotein, the level that produces in expression system increases.Fusion partner (partner) can help to provide t helper cell epi-position (Immune Fusion mating partner), the t helper cell epi-position of being discerned by the people preferably, perhaps fusion partner can help to express described albumen (expression reinforce) with the output that is higher than natural recombinant protein.Described fusion partner preferably is the Immune Fusion mating partner simultaneously and expresses the enhancing mating partner.

Fusion partner comprises from the protein D of hemophilus influenzae with from the non-structural protein NS 1 (hemagglutinin) of influenza virus.Another kind of mating partner is the albumen that is called LytA.Preferably use the C end parts of described molecule.Lyta derives from streptococcus pneumoniae, and synthetic N-acetyl-L-ala amide enzyme-Ntn hydrolase LytA (by lytA genes encoding { Gene, 43 (1986) 265-272 pages or leaves }), this enzyme are the autolysins of some key in the specificity degraded peptidoglycan skeleton.The affinity to choline or some cholinomimetics (as DEAE) is responsible in the proteic C end structure of LytA territory.Utilized this specific character development to can be used for the intestinal bacteria C-LytA expression plasmid of expressed fusion protein.Described at N-terminal and comprised the proteic purifying of the segmental hybrid of C-LytA { Biotechnology:10, (1992) 795-798 pages or leaves }.Might use the repeating part of the LytA molecule of finding at C-terminal that originates in residue 178, as residue 188-305.

The present invention also comprises the varient of polypeptide noted earlier, and promptly because conservative amino-acid substitution and the polypeptide different with polypeptide noted earlier, another residue that residue is had similar features in conservative amino-acid substitution replaces.Typical such replacement occurs between Ala, Val, Leu and the Ile; Between Ser and the Thr; Between acidic residues Asp and the Glu; Between Asn and the Gln; Between alkaline residue Lys and the Arg; Or between aromatic residue Phe and the Tyr.

Can prepare polypeptide of the present invention with any appropriate method.Such polypeptide comprises the polypeptide of the polypeptide of isolating natural appearance, the polypeptide that reorganization produces, synthetic generation or passes through the polypeptide of the combination results of these methods.The method for preparing such polypeptide is well-known in this area.

Polypeptide most preferably of the present invention derives from morazella catarrhalis, yet described polypeptide preferably derives from other biology that same taxonomy belongs to.For example, polypeptide of the present invention also can derive from same classification subject or purpose biology.

Polynucleotide

A target of the present invention provides the polynucleotide of coding BASB027 polypeptide, and this paper that especially encodes is called the polynucleotide of the polypeptide of BASB027.

In a particularly preferred embodiment according to the invention, described polynucleotide comprise the district of coding BASB027 polypeptide, and described district comprises the sequence of statement in SEQ ID NO:1 or 3, and described sequence comprises gene or its varient of total length.

The BASB027 polynucleotide that provide in SEQ ID NO:1 or 3 are the BASB027 polynucleotide that derive from morazella catarrhalis bacterial strain Mc2931 (ATCC 43617).

As one side more of the present invention, coding is provided and/or has expressed the isolated nucleic acid molecule of BASB027 polypeptide and polynucleotide, especially morazella catarrhalis BASB027 polypeptide and polynucleotide, comprised for example unprocessed RNA, ribozyme rna, mRNA, cDNA, genomic dna, b form dna and Z-DNA.Other embodiments of the present invention are included in biologically, in the diagnosis, in the prevention, clinically or treatment go up useful polynucleotide and polypeptide and their varient, and the composition that comprises described polynucleotide and polypeptide.

Another aspect of the present invention relates to the varient of the isolating polynucleotide that comprise at least one full-length gene, polynucleotide closely-related with it and described isolating polynucleotide, and wherein said isolating polynucleotide encoding has the BASB027 polypeptide of SEQ ID NO:2 or 4 deduced amino acid.

In another particularly preferred embodiment of the present invention, the BASB027 polypeptide or its varient that derive from morazella catarrhalis are arranged, and described BASB027 polypeptide comprises the aminoacid sequence of SEQ ID NO:2 or 4 or is made up of the aminoacid sequence of SEQ ID NO:2 or 4.

Use information provided herein, polynucleotide sequence as statement in SEQ ID NO:1 or 3, can use the clone of standard and the polynucleotide of the present invention that screening method obtains coding BASB027 polypeptide, described method is as using morazella catarrhalis Catlin cell as parent material, from bacterial clone and chromosomal dna fragment and to its order-checking, obtain the clone's of total length method subsequently.For example, for obtaining nucleotide sequence of the present invention, as the polynucleotide sequence that in SEQ ID NO:1 or 3, provides, usually survey the clone of morazella catarrhalis Caltin chromosomal DNA in intestinal bacteria or some other suitable host library with radiolabeled oligonucleotide, wherein said oligonucleotide derives from partial sequence, preferably 17 aggressiveness (17-mer) or longer.Can use strict hybridization conditions then, pick out the clone who has with the probe same DNA.Identify the clone by using design obtains according to original polypeptide or polynucleotide sequence sequencing primer to hybridize,, then might extend described polynucleotide sequence to determine the gene order of total length at both direction to each cloning and sequencing of evaluation like this.Be for example to use denatured double stranded dna to carry out such order-checking easily by the plasmid clone preparation.Maniatis, T., Fritsch, E.F. and Sambrook etc., MOLECULAR CLONING, ALABORATORY MANUAL, second edition; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) have described suitable technique (especially seeing " screening by hybridization 1.90 " and " order-checking 13.70 of denatured double stranded dna template ").Also can carry out direct genomic dna order-checking to obtain the gene order of total length.As explanation of the present invention, each polynucleotide of statement are all deriving from the DNA library of morazella catarrhalis and are finding in SEQ ID NO:1 or 3.

In addition, each dna sequence dna of statement all comprises an open reading-frame (ORF) in SEQ ID NO:1 or 3, described open reading-frame (ORF) coding has the albumen of the described amino-acid residue of statement number in about SEQ ID NO:2 or 4, and the molecular weight that described albumen is inferred can use the known amino-acid residue molecular weight values of those skilled in that art to calculate.

The polynucleotide of SEQ ID NO:1 are in the initiator codon of the Nucleotide of SEQ ID NO:1 number 1 with start between the terminator codon of Nucleotide numbers 2440 coding SEQ ID NO:2 polypeptide.

The polynucleotide of SEQ ID NO:3 are in the initiator codon of the Nucleotide of SEQ ID NO:3 number 1 with start between the terminator codon of Nucleotide numbers 2440 coding SEQ ID NO:4 polypeptide.

On the other hand, the invention provides the isolating polynucleotide that comprise following sequence or form by following sequence:

(a) polynucleotide sequence has at least 85% identity, preferably at least 90% identity, more preferably at least 95% identity even more preferably 97-99% or fully identity at least with SEQ IDNO:1 or 3 respectively on the total length of SEQ ID NO:1 or 3; Or

(b) polynucleotide sequence of coded polypeptide, described polypeptide have at least 85% identity, preferably at least 90% identity, more preferably at least 95% identity even more preferably 97-99% or 100% complete identity at least with the aminoacid sequence of SEQ ID NO:2 or 4 respectively on the total length of SEQ ID NO:2 or 4.

Can obtain the polynucleotide of code book invention polypeptide by a kind of method, comprise the homologue and the ortholog that derive from the species except morazella catarrhalis, described method (for example comprises the following steps: under stringent condition, use interior temperature of 45-65 ℃ of scope and from 0.1% to 1% SDS concentration), but with comprising or screening suitable library by mark or detection probes that the sequence or their fragment of SEQ ID NO:1 or 3 are formed; And separation comprises the full-length gene and/or the genomic clone of described polynucleotide sequence.

The invention provides on total length with SEQ ID NO:1 or 3 in the identical polynucleotide sequence of encoding sequence (open reading-frame (ORF)).The present invention also provides mature polypeptide or its segmental encoding sequence itself, and meeting common mature polypeptide or its segmental encoding sequence that exists in frame ground with another encoding sequence, described another encoding sequence is as the sequence of coding leader sequence or secretion sequence, preceding albumen (pre-protein) sequence, former albumen (pro-protein) sequence or preceding former albumen (prepro-protein) sequence.Polynucleotide of the present invention can also comprise at least one non-coding sequence, described non-coding sequence comprises such as but not limited at least one non-coding 5 ' and 3 ' sequence, as transcribes but the sequence do not translated, termination signal (as depend on the termination signal of rho and do not rely on the termination signal of rho), ribosome bind site, Kozak sequence, the sequence of stablizing mRnA, intron and polyadenylation signal.Described polynucleotide sequence also can comprise the encoding sequence of the amino acid whose interpolation of coding interpolation.For example, can encode and make things convenient for the flag sequence of purified fusion protein.In certain embodiments of the invention, described flag sequence is at pQE carrier (Qiagen, Inc.) provide in and at Gentz etc., Proc.Natl.Acad.Sci., the six Histidine peptides of describing among the USA 86:821-824 (1989), or the peptide-labeled thing of HA (Wilson etc., Cell37:767 (1984), these two kinds of flags sequence may be used to the peptide sequence that merge purifying and their.Polynucleotide of the present invention also include but not limited to: comprise structure gene and the polynucleotide of the sequence expressed with its natural controlling gene of linking to each other.

The nucleotide sequence of coding SEQ ID NO:2 or 4 BASB027 polypeptide can be identical with the polypeptid coding sequence among the Nucleotide 1-2439 that is included in SEQ ID NO:1 or 3 respectively.Perhaps it can be such sequence: as the result of the Feng Yuxing (degeneracy) of genetic code, the polypeptide of also encode SEQ ID NO:2 or 4.

Term " polynucleotide of coded polypeptide " comprises such polynucleotide as used herein: described polynucleotide comprise the sequence of code book invention polypeptide, wherein said polypeptide of the present invention is bacterial peptide especially, more particularly has the polypeptide of the morazella catarrhalis BASB027 of SEQ ID NO:2 or 4 aminoacid sequences of stating.This term (for example also comprises the single continuum that comprises coding said polypeptide or discontinuous a plurality of district, the polynucleotide that the carrier sequence of the phage that is integrated, the insertion sequence of integration, integration, the transposon sequence of integration interrupt, or owing to rna editing or genomic dna are organized the polynucleotide that interrupt again) polynucleotide and the district of interpolation, the district of described interpolation also can comprise encoding sequence and/or non-coding sequence.

The invention still further relates to the varient of polynucleotide as herein described, described varient coding has the varient of polypeptide of the deduced amino acid of SEQ ID NO:2 or 4.Polynucleotide passage of the present invention can be used for for example synthetic total length polynucleotide of the present invention.

More particularly preferred embodiment is the polynucleotide of coding BASB027 varient, described BASB027 varient has the BASB027 amino acid sequence of polypeptide of SEQ ID NO:2 or 4, in described aminoacid sequence several, several, 5 to 10,1 to 5,1 to 3,2,1 or 0 amino-acid residue be substituted, modify, lack and/or add with arbitrary combination.Particularly preferably be the character and active reticent replacement, interpolation and the disappearance that do not change the BASB027 polypeptide therein.

Other preferred embodiment of the present invention be such polynucleotide and with such polynucleotide complementary polynucleotide: to have polynucleotide at least 85% of BASB027 polypeptide of aminoacid sequences of SEQ IDNO:2 or 4 statements identical with coding on total length for described polynucleotide.Perhaps, more it is highly preferred that the polynucleotide in the polynucleotide that comprise with coding BASB027 polypeptide at least 90% identical district on total length, and with its complementary polynucleotide.At this on the one hand, especially preferably with above-mentioned polynucleotide at least 95% identical polynucleotide on total length.In addition, at least 97% identical polynucleotide are highly preferred in those at least 95% identical polynucleotide, more preferred at least 98% polynucleotide identical therein with at least 99%, and at least 99% identical polynucleotide are most preferred.

Embodiment preferred is the polynucleotide of the such polypeptide of coding: described polypeptide has kept same biological function or the activity of mature polypeptide with the dna encoding of SEQ ID NO:1 or 3 substantially.

According to some preferred embodiment of the present invention, provide and BASB027 polynucleotide sequence hybridization, the especially polynucleotide of hybridize under stringent condition, as those polynucleotide in SEQ ID NO:1 or 3.

The invention still further relates to polynucleotide sequence with polynucleotide sequence hybridization provided herein.At this on the one hand, The present invention be more particularly directed under stringent condition polynucleotide with multi-nucleotide hybrid as herein described.As used herein, term " stringent condition " and " stringent hybridization condition " are meant only at least 95% between sequence, just hybridize during best 97% identity.An object lesson of stringent hybridization condition is to spend the night in 42 ℃ of cultivations in comprising the solution of following material: 50% methane amide, 5x SSC (150mM NaCl, the 15mM trisodium citrate), 50mM sodium phosphate (pH 7.6), 5x Denhardt ' s solution, 10% dextran sulfate and 20 micrograms/ml sex change, the salmon sperm dna of shearing: in 0.1x SSC, wash supports for hybridization down subsequently in about 65 ℃.The condition of hybridization and washing is well-known, and at Sambrook etc., Molecular Cloning:A Laboratory Manual, second edition, Cold Spring Harbor, N.Y., in (1989), especially the Chapter 11 of this book illustrates.Also can use polynucleotide sequence provided by the invention to carry out solution hybridization.

The present invention also provides the polynucleotide that comprise or be made up of such polynucleotide sequence, described polynucleotide sequence is by under stringent hybridization condition, screen the polynucleotide sequences of SEQ ID NO:1 or 3 statements and obtain in comprising the suitable library of described complete genome with probe, wherein said probe has in SEQ ID NO:1 or 3 described polynucleotide sequence or its fragments sequence of statement; Separate described polynucleotide sequence then.The fragment that can be used for obtaining a kind of like this polynucleotide for example comprises probe and the primer that other places are in this article described comprehensively.

Discuss about polynucleotide mensuration of the present invention as this paper other places, polynucleotide of the present invention for example can be used as the hybridization probe of RNA, cDNA and genomic dna, separating full-length cDNA and the genomic clone of coding BASB027, and separation and BASB027 gene have height identity, the especially highly cDNA and the genomic clone of other gene of sequence identity.Such probe generally will comprise at least 15 nucleotide residues or base pair.Such probe preferably has at least 30 nucleotide residues or base pair, can have at least 50 nucleotide residues or base pair.Especially preferred probe will have at least 20 nucleotide residues or base pair, and will have and be less than 30 nucleotide residues or base pair.

The dna sequence dna synthetic oligonucleotide probe that uses SEQ ID NO:1 or 3 to provide can be by the coding region of screening and separating BASB027 gene.With labeled oligonucleotide screening cDNA library, genome dna library or mRNA library, determine described probe hybridization to which member in library then, wherein said probe has the sequence complementary sequence with gene of the present invention.

Available several method obtains full length DNA or extends short dna, and this several method is that those skilled in the art are known, for example based on the method for those methods of the terminal rapid amplifying of cDNA (RACE) (for example seeing Frohman etc., PNAS USA 85:8998-9002,1988).The up-to-date improvement of this technology is for example with Marathon ^TMTechnology (Clonetech LaboratoriesInc.) is an example, has significantly simplified and has sought longer cDNA.At Marathon ^TMIn the technology, prepare cDNA and connect " connector " sequence at each end by mRNA from selected tissue extraction.Use the combination of gene specific and joint specific oligonucleotide primer then, carry out nucleic acid amplification (PCR) and " lose " 5 ' end with the described DNA that increases.Use " nested " primer to repeat the PCR reaction then, described " nested " primer promptly be design in amplified production the annealed primer (generally be annealed combination in the connector sequence 3 ' and the connector Auele Specific Primer in downstream and annealed combination in described selected genes 5 ' and the gene-specific primer of upstream).Can analyze the product of this reaction then by dna sequencing, and or by described product being directly connected to existing DNA upward to produce complete sequence or to design 5 ' primer to carry out another total length PCR by the information of using new sequence, make up full length DNA.

Measure institute as this paper about polynucleotide and further discuss, polynucleotide of the present invention and polypeptide can be as for example finding disease, especially human disease treatment and Studies on Diagnosis reagent and material.

Can be used for method as described herein as polynucleotide of the present invention from the sequence deutero-oligonucleotide of SEQ ID NO:1 or 3, but be preferably used among the PCR, whether intactly or partly transcribe in the bacterium in infected tissue to determine identified polynucleotides of the present invention.Have realized that such sequence also can be used to infective stage and the infection type of diagnosing pathogenic agent to reach.

The present invention also provides the polynucleotide of coded polypeptide, described polypeptide is amino terminal amino acid or the amino acid whose maturation protein of C-terminal that also has interpolation, or added amino acid whose maturation protein (for example, when described mature form has more than one polypeptide chain) in described mature polypeptide inside.Wherein such sequence can work in the processing of albumen from the precursor to the mature form, can prolong or shorten the proteic transformation period so that can carry out protein transport, or can convenient protein determination or the operation of production.Owing to generally be situation in vivo, therefore can remove the amino acid of described interpolation from described maturation protein processing by cellular enzymes.

For each and all polynucleotide of the present invention, complementary polynucleotide are with it all provided.Preferred these complementary polynucleotide and each their complementary polynucleotide complementation fully with it.

Precursor protein may be the inactive form of described polypeptide, in mature form and one or more former sequence (prosequence) fusion of polypeptide described in the described precursor protein.When removing former sequence, such inactive precursor is activated usually.Before activation, can remove part or all of former sequence.Such precursor is commonly referred to as former albumen.

Except that A, G, C, T/U with standard represent the Nucleotide, also use letter " N " to describe some polynucleotide of the present invention.On any such specified location that all can appear in DNA or the RNA sequence in four kinds of DNA of " N " expression or the RNA Nucleotide, but preferred N is not such nucleic acid: when with the adjacent nucleotide position grouping, when reading with correct open reading-frame (ORF), have the effect of the too early terminator codon of in such open reading-frame (ORF) generation.

In a word, the maturation protein (can be referred to as preceding albumen) that polynucleotide of the present invention can encoding mature albumen, coding adds leader sequence, coding have be not before former albumen before the precursor of maturation protein of one or more former sequences of proteic leader sequence or the coding, the former proteic precursor that promptly has leader sequence and one or more former sequences, wherein said leader sequence and former sequence are generally removed in producing the procedure of processing that active and sophisticated form are arranged of described polypeptide.

According to one aspect of the present invention, provide polynucleotide of the present invention to be used for the treatment of or to prevent the purposes of purpose, especially genetic immunization.

In genetic immunization, use polynucleotide of the present invention, suitable transfer approach will preferably be used, advance muscle (Wolff etc. as the direct injection plasmid DNA, HumMol Genet (1992) 1:363.Manthorpe etc., Hum.Gene Ther. (1983) 4:419), transmit and specific proteins carrier compound DNA (Wu etc., J Biol Chem. (1989) 264:16985), with coprecipitation of calcium phosphate DNA (Benvenisty and Reshef, PNAS USA, (1986) 83:9551), DNA is wrapped into (Kaneda etc. in the various forms of liposomes, Science (1989) 243:375), particle bombardment (Tang etc., Nature (1992) 356:152, Eisenbraun etc., DNA Cell Biol (1993) 12:791) and with the retroviral vector of cloning carry out infecting in the body (Seeger etc., PNAS USA (1984) 81:5849).

Carrier, host cell, expression system

The invention still further relates to the carrier that comprises one or more polynucleotide of the present invention, produce polypeptide of the present invention with the genetically engineered host cell of carrier of the present invention and by recombinant technology.Also can use the RNA that obtains by DNA structure of the present invention, produce such albumen with cell free translation system.

Can pass through the known method of those skilled in that art, prepare recombinant polypeptide of the present invention from the genetically engineered host cell that comprises expression system.Therefore, on the other hand, the present invention relates to comprise the expression system of one or more polynucleotide of the present invention, relate to, and relate to by recombinant technology and produce polypeptide of the present invention with the genetically engineered host cell of such expression system.

Be that reorganization produces polypeptide of the present invention, can genetically engineered host cell to introduce expression system of the present invention or its part or polynucleotide.Can adopt the method for in many standard laboratory handbooks, describing that polynucleotide are introduced described host cell, described laboratory manual such as Davis etc., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook etc., MOLECULAR CLONING:A LABORATORY MANUAL, second edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), described method such as calcium phosphate transfection, the transfection of DEAE-dextran mediation, transposition, microinjection, the transfection of cationic lipid mediation, electroporation, transduction, scrape loading, trajectory is introduced (ballistic introduction) and is infected.

The representational example of suitable host comprises bacterial cell, as the cell of suis (streptococci), staphylococcus (staphylococci), faecalis (enterococci), intestinal bacteria (E.coli), streptomyces (streptomyces), cyanobacteria (cyanobacteria), Bacillus subtilus (Bacillus subtilis), Neisseria meningitidis and morazella catarrhalis; The fungal cell is as the cell of yeast, kluyveromyces spp (Kluveromyces), Saccharomycodes (Saccharomyces), basidiomycetes (basidiomycete), Candida albicans (Candida albicans) and Aspergillus (Aspergillus); Insect cell is as the cell of Drosophila S2 (Drosophila S2) and greedy Noctua Sf9 (Spodoptera) Sf9; Zooblast is as CHO, COS, HeLa, Cl27,3T3, BHK, 293, CV-1 and Bowes melanoma cells; And vegetable cell, as gymnosperm or angiospermous cell.

Can use very multiple expression system to produce polypeptide of the present invention.Such carrier comprising, karyomit(e) deutero-carrier, episome deutero-carrier and viral deutero-carrier, for example, from bacterial plasmid deutero-carrier, from phage-derived carrier, from transposon deutero-carrier, from yeast episome deutero-carrier, from insertion element deutero-carrier, from yeast chromosomal element deutero-carrier, from viral deutero-carrier and from their combination deutero-carrier, as those from plasmid and phage genetic elements deutero-carrier (as clay and phagemid), wherein said virus such as baculovirus, papovavirus (as SV40), vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus, picornavirus, retrovirus and alphavirus (alphavirus).Described expression system structure can comprise the control region of regulating and causing expression.In general, at this on the one hand, can use any system that is suitable in the host, keeping, breed or express polynucleotide and/or express polypeptide or carrier to express being used for.Can suitable dna sequence dna be inserted in the described expression system by any in the multiple well-known routine techniques, described routine techniques such as those are at Sambrook etc., MOLECULARCLONING, A LABORATORY MANUAL, the technology of being stated in (seeing above).

In the recombinant expression system in eukaryotic cell, advance chamber, the secretion of endoplasmic reticulum for the protein excretion after will translating and advance periplasmic space or secrete born of the same parents' external environment, suitable secretion signal can be introduced expressed polypeptide.These signals can be endogenous for described polypeptide, and perhaps they can be the allos signals.

Can be by well-known method, reclaim and purifying polypeptide of the present invention from the reconstitution cell culture, described method comprises ammonium sulfate precipitation or ethanol sedimentation, acid extraction, anion-exchange chromatography or cation-exchange chromatography, phosphorylated cotton chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite and lectin chromatography.Most preferably use ionic metal affinity chromatography (IMAC) to carry out purifying.When described polypeptide is synthetic in cell, in the process of isolated or purified during sex change, can use the proteic technology regeneration activity of well-known refolding conformation.

Described expression system also can be the live microorganism of reorganization, as virus or bacterium.Target gene can be inserted in the genome of recombinant virus alive or bacterium.To cause antigenic expression in vivo and induce immune response with infecting in this live vector inoculation and the body.The virus and the bacterium that are used for this purpose have, and for example: poxvirus is (as vaccinia virus, fowlpox virus, canary pox virus), alphavirus (sindbis virus (Sindbis virus), Semliki Forest virus (Semliki ForestVirus), the auspicious Internal drawing horse encephalomyelitis virus (Venezuelian Equine EncephalitisVirus) of entrusting), adenovirus, adeno associated virus, picornavirus (poliovirus, rhinovirus), simplexvirus (varicella zoster virus etc.), Li Site Jun Shi belongs to (Listeria), salmonella (Salmonella), Shigella (Shigella), bacille Calmette-Guerin vaccine (BCG).Be to obtain the live body vaccine, these viruses and bacterium can be virulence arranged or attenuation in every way.Such live body vaccine also constitutes a part of the present invention.

Diagnostic assay, prognosis mensuration, serotype are measured and sudden change is measured

The invention still further relates to use BASB027 polynucleotide of the present invention and polypeptide purposes as diagnostic reagent.Detection BASB027 polynucleotide and/or polypeptide will provide for medical diagnosis on disease, staging or the infectivity biology diagnostic method to the reaction of medicine in eukaryotic cell, especially Mammals, the particularly mankind.Can be by multiple well-known technology, and by method provided herein, on nucleic acid or amino acid levels, detect eukaryote, especially Mammals, particularly human, wherein especially be subjected to or suspect the people who is subjected to comprising BASB027 gene or proteic biological infection.

Obtain to be used for the polypeptide and the polynucleotide of prognosis, diagnosis or other analysis the body material of the individuality that can be infected and/or infected from supposition.From any polynucleotide, especially DNA or RNA in these sources, can be directly used in detection or before analysis, pass through to use PCR or any other amplification technique enzymatic amplification.Also can be by using RNA (especially mRNA), cDNA and genomic dna with quadrat method.Use amplification, or the species of residence biological (resident organism) and the characterized of strain system biological to the infectivity that exists in the individuality can be finished by the genotype of analyzing the selected polynucleotide of this biology.Make comparisons with the genotype of the reference sequences of being selected by associated biomolecule, can detect disappearance or insert by the variation on the amplified production size, wherein said associated biomolecule is the different plant species of same genus or the not homophyletic system of same species preferably.By the DNA of amplification and the BASB027 polynucleotide sequence of mark are hybridized, can identify point mutation.Can be by DNA enzyme or RNA enzymic digestion (respectively for DNA or RNA) or by detecting the difference of melting temperature(Tm) or reannealing kinetics, with perfectly matched sequence or significantly matched sequence with not exclusively mispairing or more remarkable mispairing duplex make a distinction.Also can detect the difference of polynucleotide sequence with the variation of the electrophoretic mobility of polynucleotide passage on gel by comparing with reference sequences.This can use or not use sex change reagent to carry out.Also can detect polynucleotide difference by direct dna sequencing or RNA order-checking.See for example Myers etc., Science, 230:1242 (1985).Also can be disclosed in the sequence change of specific site by RNase protection analysis (analyzing) or chemical chop method as RNA enzyme, V1 and S1 protection.See for example Cotton etc., Proc.Natl.Acad.Sci., USA, 85:4397-4401 (1985).

In another embodiment, can make up the array that comprises BASB027 nucleotide sequence or its segmental oligonucleotide probe, to carry out for example effective screening, serotype, taxonomy classification or the evaluation of genetic mutation.The array technique method is well-known and has general applicability, and can be used for solving the various problems of molecular genetics, comprises genetic expression, genetic linkage and hereditary variability (seeing for example Chee etc., Science, 274:610 (1996)).

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

(a) polynucleotide of the present invention, preferably SEQ ID NO:1 or 3 nucleotide sequence or its fragment;

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

(c) polypeptide of the present invention, preferably SEQ ID NO:2 or 4 polypeptide or its fragment; Or

(d) at the antibody of polypeptide of the present invention, preferably at the antibody of the polypeptide of SEQ ID NO:2 or 4.

People will appreciate that, in any such test kit, and (a) and (b), (c) or (d) can comprise substantial composition.Such test kit will be especially diagnosing the illness or is diagnosing useful aspect the susceptibility of disease.

The invention still further relates to and use polynucleotide of the present invention as diagnostic reagent.The mutant form of detection and disease or pathogenic relevant polynucleotide of the present invention (preferably SEQ ID NO:1 or 3), can help providing or definite medical diagnosis on disease, predict disease course, determine disease stage or to the diagnostic tool of the susceptibility of disease, described disease comes from expression deficiency, the overexpression of described polynucleotide or expresses and changes.Can pass through multiple technologies on the polynucleotide level, as the technology that those this paper other places are described, detection has the biology of sudden change, especially infectivity biology on such polynucleotide.

Also can pass through multiple technologies, detecting the cell that has the biology of sudden change or polymorphism (allelic variation) on comfortable polynucleotide of the present invention and/or the polypeptide on polynucleotide or the polypeptide level, to carry out for example serotype.For example, can use RT-PCR to detect sudden change among the RNA.Especially preferably use RT-PCR in conjunction with automated detection system, for example GeneScan.RNA, cDNA or genomic dna also can be used for same purpose through PCR.For example, sudden change is identified and analyzed to can use and the encode polynucleotide complementary PCR primer of BASB027 polypeptide.

The present invention also provides the primer of removing 1,2,3 or 4 Nucleotide from 5 ' and/or 3 ' end.These primers can be used for, especially, and isolated BASB027 DNA of sample (as body material) and/or RNA that amplification is obtained by individuality.Described primer can be used for amplification from the isolating polynucleotide of infected individuals, so that subsequently can be by the described polynucleotide of various technical study to illustrate described polynucleotide sequence.Like this, can detect the sudden change in the described polynucleotide sequence, and be used for diagnosis and/or prediction infection or its stage or the course of disease, perhaps be used for described infectious agent is carried out serotype and/or classification.

The present invention also provide be used to diagnose the illness, preferably infectation of bacteria, be more preferably the method for the infection that morazella catarrhalis causes, comprise that from derive from individual sample (as body material) mensuration has the increase of expression level of polynucleotide of the sequence of SEQ ID NO:1 or 3.Can use the well-known method that is used for quantitative polynucleotide in any this area; measure increase or reduction that the BASB027 polynucleotide are expressed, described method is as for example amplification, PCR, RT-PCR, RNA enzyme protection, RNA blotting, spectrometry and other hybridizing method.

In addition, be used to detect the diagnostic assay of comparing BASB027 polypeptide overexpression with the normal control tissue sample, can be used for for example detecting the existence of infection according to of the present invention.Can be used for measuring the determination techniques of the level of BASB027 polypeptide from the sample (as body material) that the host obtains, know for those skilled in the art.Such analytical procedure comprises that radioimmunoassay, competitive binding assay, western blot analysis, antibody sandwich mensuration, antibody test and ELISA measure.

Polynucleotide of the present invention can be used as polynucleotide array, the composition of high density arrays or grid preferably.These high density arrays are particularly useful for diagnosis and prediction purpose.For example, one group of point is arranged, each point comprises a different gene, and comprise one or more polynucleotide of the present invention, this group point can be used for surveying, as obtaining or the deutero-probe, determine the existence of specific polynucleotide sequence in the individuality or correlated series by using hybridization or nucleic acid amplification, using from body sample.A kind of like this existence can be indicated the existence of pathogenic agent, especially morazella catarrhalis, and can be used to diagnose and/or predict the course of disease of disease or disease.Preferably include the grid of varient of the polynucleotide sequence of a large amount of SEQ ID NO:1 or 3.Also preferably include the grid of varient of polynucleotide of the peptide sequence of a large amount of coding SEQ ID NO:2 or 4.

Antibody

Polypeptide of the present invention and polynucleotide or its varient or express above-mentioned cell and can be used as immunogen are to produce respectively the antibody to such polypeptide or polynucleotide immune specificity.

In some embodiment preferred of the present invention, provide the antibody of antagonism BASB027 polypeptide or polynucleotide.

Can use ordinary method, by giving animal (preferably be not human) polypeptide of the present invention and/or polynucleotide or wherein one or both the fragment that has epi-position, one or both analogue or express in the two wherein one or both cell wherein in the two in the two, obtain the antibody that produces at polypeptide of the present invention or polynucleotide.For the preparation monoclonal antibody, can use any technology that the antibody that is produced by the continuous cell line culture is provided in this area.Example comprises various technology, as the technology in following document: Kohler, and G. and Milstein, C., Nature 256:495-497 (1975); Kozbor etc., Immunology Today 4:72 (1983); Cole etc., MONOCLONAL ANTIBODIES AND CANCER THERAPY, 77-96 page or leaf, Alan R.Liss, Inc. (1985).

Can revise the technology (United States Patent (USP) the 4th, 946, No. 778) that produces single-chain antibody, to produce single-chain antibody at polypeptide of the present invention or polynucleotide.In addition, can use the humanized antibody of transgenic mice or other biology or animal (as other Mammals) expression to polypeptide of the present invention or polynucleotide immune specificity.

On the other hand, can utilize display technique of bacteriophage, perhaps from according to the repertoire of the lymphocyte v gene pool that has the pcr amplification that people that anti-BSAB027 filters out obtains or from originally library (naive library), selection has in conjunction with active antibody gene (McCafferty etc. polypeptide of the present invention, (1990) .Nature 348,552-554; Marks etc., (1992) Biotechnology 10,779-783).Also can improve the avidity (Clackson etc., (1991) Nature 352:628) of these antibody by for example chain reorganization.

Can use above-mentioned antibody to separate or identify the clone who expresses polypeptide of the present invention or polynucleotide, with by for example described polypeptide of affinitive layer purification or polynucleotide.

Therefore, wherein can use the antibody of antagonism BASB027 polypeptide or BASB027 polynucleotide to treat infection, especially infectation of bacteria.

Polypeptide variants is included on the antigen, on the epi-position or the varient that is equal in the immunity, these constitute concrete aspects of the present invention.

Preferably modify described antibody and varient thereof so that its immunogenicity in individuality is lower.For example, if described individuality is the people, so best " humanization " described antibody, the one or more complementary determining regions of antibody of wherein hybridoma being derived are transplanted in the human monoclonal antibodies, for example in (1986) such as Jones, Nature 321,522-525 or Tempest etc., (1991) Biotechnology9 is described in the 266-273.

Antagonist and agonist-mensuration and molecule

Also can use polypeptide of the present invention and polynucleotide, assessment small molecules substrate and part combining in for example cell, acellular prepared product, chemical library and natural product mixture.These substrates and part can be natural substrate and part, perhaps can be structural simulation thing or functional analogue thing.See for example Coligan etc., Current Protocols in Immunology 1 (2): the 5th chapter (1991).

Use and the direct or indirect bonded mark of candidate compound, screening method can only be measured combining or described candidate compound and the cell of the fusion rotein that has described polypeptide or combining of film of combining of described candidate compound and described polypeptide or polynucleotide or described candidate compound and cell that has described polypeptide or polynucleotide or film.Perhaps, described screening method relates to the competition with the competition thing of mark.In addition, use for the suitable detection system of the cell that comprises described polypeptide or polynucleotide, these screening methods can be tested the signal whether described candidate compound causes activation or inhibition owing to described polypeptide or polynucleotide to produce.General in the presence of a kind of known agonist, measure the activated inhibition, and observe in the presence of described candidate compound described agonist the activated effect.Whether the polypeptide of activated polypeptide of composing type and/or constitutive expression and polynucleotide can be used for screening the screening method of reverse agonist or inhibitor: under the situation that lacks agonist or inhibition, test described candidate compound as the case may be and cause the activated of described polypeptide or polynucleotide is suppressed.In addition, described testing method can only may further comprise the steps: candidate compound is mixed with the solution that contains polypeptide of the present invention or polynucleotide, form mixture, measure BASB027 polypeptide and/or polynucleotide activity in the described mixture, and the BASB027 polypeptide and/or the active activity with standard of polynucleotide of described mixture are made comparisons.Also can use fusion rotein, those fusion roteins of making by Fc part and BASB027 polypeptide as mentioned before for example, carry out the high throughput screening assay, with the antagonist of identifying polypeptide of the present invention and system take place to go up and/or function on the antagonist of related polypeptide (see D.Bennett etc., J Mol Recognition, 8:52-58 (1995); With K.Johanson etc., J Biol Chem, 270 (16): 9459-9471 (1995)).

Also can use described polynucleotide, polypeptide and dispose screening method, the effect of the generation of mRNA and/or polypeptide in the compound pair cell that adds with test in conjunction with polypeptide of the present invention and/or with the interactional antibody of polypeptide of the present invention.For example,, use monoclonal antibody and polyclonal antibody, can make up ELISA and measure to measure the secretion level or the cell related levels of polypeptide by standard method known in the art.This can be used for finding to suppress or to strengthen the reagent (also being called antagonist or agonist) that polypeptide produces from the cell or tissue that proper handling is crossed.

The present invention also provides the method for SCREENED COMPOUND, with compound, especially those system bacterium and/or germ-resistant compounds of the effect of identifying those enhancings (agonist) or blocking-up (antagonist) BASB027 polypeptide or polynucleotide.Described screening method relates to the high throughput technology.For example, be screening agonist or antagonist, existing or not existing may be under the situation of candidate molecules of BASB027 agonist or antagonist, the synthesis reaction mixture, cellular compartment (as film, cell envelope or cell walls or any prepared product in them) that will comprise the BASB027 polypeptide with as described in the labeled substrate or the part incubation of polypeptide.The ability of candidate molecules excitement or antagonism BASB027 polypeptide is reflected in reducing in conjunction with minimizing or from such substrate production product of tagged ligand.Free bonded molecule, the molecule of promptly not inducing the effect of BASB027 polypeptide probably are good antagonists.In conjunction with fine and as the case may be, increase from substrate produce product speed, to increase signal transduction or increase the active molecule of chemical access be agonist.The operation report system can strengthen (as the case may be) to produced the detection of product, signal transduction or active speed of chemical access or level by substrate.Include but not limited to that in this reporting system that comes in handy on the one hand colorimetry, labeled substrate change into product, make the reporter gene of replying and known in the art in conjunction with measuring for the activity change of BASB027 polynucleotide or polypeptide.

Another example of measuring the BASB027 agonist is a competitive assay, soon BASB027 and possible agonist mix under conditions suitable with BASB027 binding molecule, recombinate BASB027 binding molecule, natural substrate or part or substrate or ligand mimics, with being at war with property inhibition analysis.Can be by coming mark BASB027 as radioactivity or color-developing compounds, so that can accurately measure the quantity of the BASB027 molecule that combines or change into product with binding molecule, to assess the validity of described possible antagonist.

Possible antagonist comprising, in conjunction with polynucleotide of the present invention and/or polypeptide and therefore suppress or eliminate its active or express little organic molecule, peptide, polypeptide and antibody.Possible antagonist can also be little organic molecule, peptide, polypeptide, as be combined in closely-related albumen or the antibody that binding molecule (as binding molecule) is gone up same loci, described albumen or antibody are not induced BASB027 institute inductive activity simultaneously, therefore make it can not be in conjunction with effect that prevents BASB027 polypeptide and/or polynucleotide or expression by getting rid of BASB027 polypeptide and/or polynucleotide.

Possible antagonist comprises such small molecules: described small molecules in conjunction with and occupy described polypeptide binding site, therefore prevent that it is attached to the cell binding molecule so that prevent normal biological activity.Micromolecular example includes but not limited to little organic molecule, peptide or peptide sample molecule.Other possible antagonist comprises that antisense molecule (sees Okano, J.Neurochem.56:560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENEEXPRESSION, CRC Press, Boca Raton, FL (1988) is to the description of these molecules).Preferably may comprise compound relevant and the varient of BASB027 by antagonist with BASB027.

More on the one hand, the present invention relates to genetically engineered soluble fusion protein, described albumen comprises polypeptide of the present invention or its fragment, and the different piece of the constant region of the heavy chain of different subclass immunoglobulin (Ig) (IgG, IgM, IgA, IgE) or light chain.As the constant portion of the heavy chain of the preferred IgG of immunoglobulin (Ig) (especially IgG1), wherein merge and occur in hinge region.In specific embodiment, can remove the Fc part simply with the cutting sequence of factor Xa cutting by inserting.In addition, the present invention relates to prepare the method for these fusion roteins, and described fusion rotein is used for the application of drug screening, diagnosis and treatment by genetic engineering.Another aspect of the present invention also relates to the polynucleotide of the such fusion rotein of coding.The example of fusion protein technology can be asked in international monopoly in WO94/29458 number and WO94/22914 number and be found.

Each polynucleotide sequence provided herein may be used to find and the development antibacterium compound.After expression, coded albumen can be as the target of screening anti-bacterial drug.In addition, corresponding mRNA goes up the polynucleotide sequence in the described proteins encoded N-terminal of coding district or the sequence of Shine-Delgamo or other convenient translation can be used to make up the antisense sequences that control purpose encoding sequence is expressed.

The present invention also provides and uses polypeptide of the present invention, polynucleotide, agonist or antagonist to disturb a kind of pathogenic agent or multiple pathogenic agent with eucaryon host, preferably cause infecting the initial physical interaction of sequela between the mammalian hosts.Molecule of the present invention especially can be used for: prevent that bacterium, especially Gram-positive and/or gram negative bacterium are attached to eukaryote on the indwelling equipment, mammiferous extracellular matrix protein preferably, or be attached to the extracellular matrix protein in the wound; The eukaryote of blocking-up mediation tissue injury, the bacterial adhesion between mammiferous extracellular matrix protein and bacterium BASB027 albumen preferably, and/or; Blocking-up is except normally carrying out owing to pathogenetic in the infection that causes implantation indwelling equipment or other surgical technic.

Provide BASB027 agonist and antagonist more on the one hand according to of the present invention, preferably made bacterium or germ-resistant agonist and antagonist.

Antagonist of the present invention and agonist also can be used for for example preventing, suppressing and/or treat disease.

On the other hand, the present invention relates to the mimotope of polypeptide of the present invention.Mimotope is the peptide sequence of enough similar to native peptides (on the sequence or on the structure), can be identified the antibody recognition of native peptides; Perhaps when being coupled to suitable carrier, can produce the antibody of identification native peptides.

Can by add, disappearance or replace selected amino acid and designed peptide mimotope is used for specific end use.Therefore, can modify described peptide in order to be easy to put together in protein carrier.For example, some chemically conjugated methods may need to comprise a terminal cysteine.In addition, the peptide of puting together with protein carrier may be puted together at described peptide and terminally comprise a hydrophobic end at a distance, so that described peptide free is not in conjunction with the terminal surface bonding that keeps with carrier proteins.Therefore make described peptide present such conformation: described conformation is the closest similar to the conformation of the described peptide of finding in complete natural molecule.For example, can change described peptide makes it have a N-terminal halfcystine and a C-terminal hydrophobic amide tail.Perhaps, can carry out the interpolation or the replacement of one or more amino acid whose D-steric isomers, to produce useful derivative, for example to strengthen the stability of described peptide.

Perhaps, use technology, can identify peptide mimotope in conjunction with the antibody of polypeptide of the present invention with itself as display technique of bacteriophage (EP 0 552 267 B1).This technology has produced the structure of a large amount of simulation native peptides and therefore can be in conjunction with the peptide sequence of anti-native peptides antibody, but described peptide sequence itself might not have significant sequence identity with described natural polypeptides.

Vaccine

Another aspect of the present invention relates in individual, particularly Mammals, the method for induce immune response among the mankind preferably; comprise with the BASB027 polynucleotide that are enough to produce antibody and/or T cellullar immunologic response and/or polypeptide or their fragment or varient and inoculate described individuality; avoid infecting to protect described individuality; especially infectation of bacteria, more especially morazella catarrhalis infects.Also provide such method: rely on this method, such immunne response the duplicating of bacterium of slowing down.And the method that relates in one aspect to induce immune response in individuality more of the present invention; comprise the transmission nucleic acid carrier; sequence or ribozyme are given such individuality; instruct BASB027 polynucleotide and/or polypeptide; or the expression of its fragment or varient; to express BASB027 polynucleotide and/or polypeptide in vivo; or its fragment or varient; with this induce immune response; as produce antibody and/or T cellullar immunologic response (comprising the T cell or the cytotoxic T cell that for example produce cytokine); protect described individuality (preferably human) to avoid disease, no matter and whether this disease is set up in described individuality.An example that gives described gene is that it is quickened to enter required cell on the contrary as particulate encrusting substance or phase.Such nucleic acid carrier can comprise nucleic acid, DNA/RNA hybrid, DNA-protein complex or the RNA-protein complex of DNA, RNA, ribozyme, modified.

The immune composition that relates in one aspect to again of the present invention, when described immune composition is introduced individual, in the time of preferably among the mankind, since described immune composition can be in described individuality induce immune response, therefore in described individuality, induce at the BASB027 polynucleotide and/or by the immunne response of its encoded polypeptides, wherein said composition comprises reorganization BASB027 polynucleotide and/or by its encoded polypeptides, and/or described composition comprises coding and expresses the antigenic DNA and/or the RNA of described BASB027 polynucleotide, by its encoded polypeptides, or other polypeptide of the present invention.Described immunne response can be used for the treatment of or be the prevention purpose, and adopts the form of antibody mediated immunity and/or cellular immunization, as the cellular immunization that is caused by CTL or CD4+ T cell.

BASB027 polypeptide or its fragment can merge with auxilliary albumen (co-protein) or chemical part; described chemical part itself may produce antibody or not produce antibody, but can stablize described first kind of albumen and produce have antigenicity and/or immunogenic characteristic, the preferably fusion or the modified protein of protection feature.Therefore, the recombinant protein that merges preferably also comprises the auxilliary albumen of a kind of antigenicity, or any other stablized described albumen and convenient its produced and the relative big auxilliary albumen of purifying, and wherein said antigenicity is assisted lipoprotein D, glutathione-S-transferase (GST) or the beta-galactosidase enzymes of albumen Tathagata from hemophilus influenzae.In addition, described auxilliary albumen can work as adjuvant providing on the meaning that the immune universality of accepting described proteic biology is stimulated.Described auxilliary albumen can be connected to first kind of proteic aminoterminal or carboxyl terminal.

The invention provides composition, especially vaccine composition, and the method that comprises polypeptide of the present invention and/or polynucleotide and immunostimulating dna sequence dna, described immunostimulating dna sequence dna such as those are at Sato, the sequence described in the Science 273:352 (1996) such as Y..

The present invention also provides described polynucleotide or its specific segmental method used, wherein be presented in the morazella catarrhalis animal models infected, in the polynucleotide structure that in such inherited immunity experiment, uses, the non-variable region of described polynucleotide or its specific fragment coding bacterial cell surface protein.Such experiment can cause that for identifying protein epitope preventative or that therapeutic immunization is replied is particularly useful.Believe that this method will make it possible to prepare subsequently useful especially monoclonal antibody, with prevention reagent or the treatment therapy of exploitation at the infectation of bacteria in Mammals, the especially mankind (especially morazella catarrhalis infection), wherein said monoclonal antibody derives from the essential organ that infected animals was resisted or removed in success.

The present invention also comprises vaccine preparation, and described vaccine preparation comprises immunogenicity recombinant polypeptide of the present invention and/or polynucleotide and suitable carriers, as pharmaceutically acceptable carrier.Because described polypeptide and polynucleotide may decompose under one's belt, so each the best parenteral in them gives, comprise for example subcutaneously give, intramuscular gives, intravenously gives or intracutaneous gives.Be suitable for the preparation that parenteral gives and comprise water-based and non-aqueous aseptic injectable solution, described solution can comprise antioxidant, buffer reagent, system bacterium compound and make described preparation and the individual isoosmotic solute of body fluid (preferably blood); And water-based and non-aqueous sterile suspensions, described suspension can comprise suspension agent or thickening material.Described preparation can be contained in unitary dose or the multi-dose container (as the ampoule that seals and phial), and can preserve under lyophilisation condition, is only required in to use the preceding sterile liquid carrier that adds.

Vaccine preparation of the present invention can comprise that also adjuvant system is to strengthen the immunogenicity of described preparation.Described adjuvant system preferably preferentially causes the reaction of TH1 type.

Immunne response can broadly be included into two classes the most basic: humoral immunoresponse(HI) or cell-mediated immune responses (coming characterizing definition with antibody protection mechanism or cytological effect thing protection mechanism respectively traditionally).Two classes of this of immunne response are named as the reaction of TH1 type (cell-mediated reaction) and TH2 type immunne response (humoral response).

The most basic TH1 type immunne response is characterised in that and produces antigen-specific haplotype restrictive cell toxicity T cell and natural killer cell is replied.In mouse, the reaction of TH-1 type is characterized as usually and produces IgG2a hypotype antibody, and in the people, these antibody are corresponding to IgG1 type antibody.TH-2 type immunne response is characterised in that the immunoglobulin (Ig) isotype that produces broad range, comprises IgG1, IgA and IgM at the isotype of immunoglobulin (Ig) described in the mouse.

Can think that producing impellent behind at these two kinds of type of immune response is cytokine.High-caliber TH-1 cytokines tends to promote to induce at given antigenic cell-mediated immune responses, and high-caliber TH-2 cytokines tends to promote to induce antigenic humoral immunoresponse(HI).

The difference of TH-1 and TH-2 type immunne response is not absolute.In fact certain know from experience to support to be described to mainly to be TH1 or mainly to be the immunne response of TH2.Yet, considering described in the mouse CD4+ve T cell clone that according to Mosmann and Coffman cytokine family usually is (Mosmann easily, T.R. and Coffman, R.L. (1989) TH1 and TH2 cell: different lymphokine secretion patterns causes different functional performance .Annual Reviewof Immunology, 7, the 145-173 pages or leaves).Traditionally, the reaction of TH-1 type is relevant with the IL-2 cytokine with T lymphocyte generation INF-γ.Other usually directly related with inducing TH1 type immunne response cytokine is not to be produced by the T cell, as IL-12.By contrast, the reaction of TH2 type is relevant with the secretion of IL-4, IL-5, IL-6 and IL-13.

Known some vaccine adjuvant is particularly suitable for stimulating TH1 or TH2 cytokines to reply.Traditionally, reply TH1:TH2 equilibrated optimal parameter in vaccination or premunition, being included in external use antigen stimulates the back directly to measure the generation of lymphocytic TH1 of T or TH2 cytokine again, and/or measures the IgG1:IgG2a ratio of antigen-specific antibodies reaction.

Therefore, TH1 type adjuvant is a kind of like this adjuvant: it preferentially stimulates isolating T cell colony producing high-level TH1 cytokines when external use antigen stimulates again, and promotes the generation of CD8+ cytotoxic T lymphocyte and the antigen specific immune globulin reaction relevant with TH1 type isotype.

The adjuvant that can preferably stimulate the TH1 cell response has been described in international patent application No.WO 94/00153 and WO 95/17209.

3-deoxidation acetyl monophosphoryl lipid A (3D-MPL) a kind of adjuvant that comes to this.This can learn from GB 2220211 (Ribi).Chemically it is the mixture that the 3-deoxidation acetyl monophosphoryl lipid A of 4,5 or 6 acetylize chains is arranged, and by Ribi Immunochem, Montana produces.A kind of preferred form of 3-deoxidation acetyl monophosphoryl lipid A is disclosed in European patent 0 689 454 B1 (SmithKline Beecham Biologicals SA).

The particle of 3D-MPL is preferably little that the film that is enough to by 0.22 micron carries out Sterile Filtration (described in European patent 0 689 454).The scope that 3D-MPL uses in every dosage is 10 μ g-100 μ g, 25-50 μ g preferably, the wherein scope used in every dosage of antigen 2-50 μ g normally.

Another kind of preferred adjuvants comprises QS21, a kind of nontoxic component of the Hplc purifying that obtains from the bark of Quillaia saponaria (Quillaja SaponariaMolina).This adjuvant can mix with 3-deoxidation acetyl monophosphoryl lipid A (3D-MPL) alternatively, wherein can also have a kind of carrier alternatively.

United States Patent (USP) the 5th, 057, the production method that discloses QS21 No. 540.

The reactionless originality adjuvant formulation (WO96/33739) that contains QS21 had been described in the past.When preparing with antigen, the preparation that has shown this QS21 of comprising and cholesterol is that successful TH1 stimulates adjuvant.

Other adjuvant for the preferential stimulator of TH1 cell response comprises the immunomodulatory oligonucleotide, for example disclosed not methylated CpG sequence among the WO 96/02555.

Different TH1 stimulate the combination of adjuvants (as mentioned above those) also should be considered to provide to be the adjuvant of the preferential stimulator of TH1 cell response.For example, QS21 can prepare with 3D-MPL.The ratio of QS21:3D-MPL is about 1:10 usually to 10:1; Preferably 1:5 is to 5:1, and in fact usually is 1:1.The excellent scope of synergy is that 2.5:1 is to 1:13D-MPL:QS21.

According to preferably also there being a kind of carrier in the vaccine composition of the present invention.Described carrier can be a water external emulsion, or a kind of aluminium salt, as aluminum phosphate or aluminium hydroxide.

Preferred water external emulsion comprises a kind of metabolizable oil, as squalene, alpha-tocopherol and Tween 80.One particularly preferred aspect, mixing with QS21 and 3D-MPL in such emulsion according to the antigen in the vaccine composition of the present invention.Described in addition water external emulsion can contain span 85 and/or Yelkin TTS and/or tricaprylin.

When administration of human, QS21 that uses in the vaccine and 3D-MPL usually in the scope of every dosage 1 μ g-200 μ g, as 10-100 μ g, 10 μ g-50 μ g preferably.And water external emulsion can contain the squalene of 2-10%, the alpha-tocopherol of 2-10% and the Tween80 of 0.3-3% usually.Squalene: the ratio of alpha-tocopherol preferably is equal to or less than 1, because this provides a kind of more stable milk sap.Can on 1% level, use Span 85.In some cases, to contain a kind of stablizer in addition be possible favourable to vaccine of the present invention.

Nontoxic water external emulsion is preferably in and contains a kind of nontoxic oil in a kind of aqueous carrier, as squalane or squalene, and a kind of emulsifying agent, as Tween 80.Described aqueous carrier can be a phosphate buffered saline(PBS) for example.

Described a kind of especially effectively adjuvant formulation among the WO 95/17210, said preparation relates to QS21,3D-MPL and the tocopherol in water external emulsion.

The present invention also provides multivalent vaccine composition, and described multivalent vaccine composition comprises the vaccine preparation of the present invention with other antigen combination, and described other antigen is particularly useful for treating the antigen of cancer, autoimmune disease and associated conditions.A kind of like this multivalent vaccine composition can comprise the adjuvant of inducing TH-1 as previously described.

Though describe the present invention according to some BASB027 polypeptide and polynucleotide, but be to be understood that the present invention covers the fragment of the polypeptide and the polynucleotide of natural appearance, and the similar polypeptide and the polynucleotide that have interpolation, disappearance or replace, wherein said interpolation, disappearance or replacement do not influence the immunogenicity characteristic of described recombinant polypeptide or polynucleotide substantially.

Composition, test kit and administration

In one side more of the present invention, provide the composition that comprises BASB027 polynucleotide and/or BASB027 polypeptide that is used to give individual cells or multicellular organism.

The invention still further relates to the composition that comprises polynucleotide discussed in this article and/or polypeptide or their agonist or antagonist.Polypeptide of the present invention and polynucleotide can be used in combination to be used for cell, tissue or biology with non-sterile or aseptic a kind of carrier or variety carrier, and described carrier is as being suitable for giving individual pharmaceutical carrier.Such composition comprises polypeptide of the present invention and/or polynucleotide and the pharmaceutically acceptable carrier or the vehicle of media additive (media additive) for example or treatment significant quantity.Such carrier can include but not limited to salt solution, buffer saline, glucose, water, glycerine, ethanol and their composition.Described preparation should cooperate mode of administration.The invention still further relates to diagnosis and use and medicinal bag and test kit, described bag and test kit comprise one or more containers, and one or more compositions of the described composition of the present invention of preamble are housed in the described container.

Polypeptide of the present invention, polynucleotide and other compound can use separately or be used in combination as the treatment compound with other compound.

Described medicinal compositions can with any effectively, mode gives easily, described mode is comprising approach or intradermal routes in for example surperficial approach, oral route, anus approach, vaginal approach, intravenous route, intraperitoneal approach, intramuscular approach, subcutaneous route, the nose.

In treatment or when the prophylactic agent, can give individuality as injectable composition with described active agents, for example as the sterile aqueous dispersion, described dispersion is preferably isoosmotic.

On the other hand, the invention provides medicinal compositions, described medicinal compositions comprises polypeptide and/or polynucleotide (as the polypeptide of the present invention and/or the polynucleotide of soluble form), agonist peptide or the antagonist peptide or the micromolecular compound of the treatment significant quantity that has made up pharmaceutically acceptable carrier or vehicle.Such carrier includes but not limited to salt solution, buffer saline, glucose, water, glycerine, ethanol and their composition.The invention still further relates to medicine bag and test kit, described medicine bag and test kit comprise one or more containers, and one or more compositions of the described composition of the present invention of preamble are housed in the described container.

Polypeptide of the present invention, polynucleotide and other compound can use separately or be used in combination as the treatment compound with other compound.

Can change described composition to adapt to route of administration, described route of administration such as systemic approach or oral route.The preferred form of systemic administration comprises injection, generally is by intravenous injection.Can use other injecting pathway, as subcutaneous injection, intramuscularly or peritoneal injection.Other optional method that is used for systemic administration comprises the use permeate agent through mucous membrane and percutaneous dosing, described permeate agent such as biliary salts or fusidinic acid or other stain remover.In addition, if polypeptide of the present invention or other compound can be formulated in Enteral formulations or the encapsulated preparation, oral administration also is possible so.These compounds also can be with form surface and/or topical administrations such as ointment, paste, gel, solution, pulvis.

For giving Mammals, especially human, expect that dosage level every day of described active agents will generally be about 1mg/kg from 0.01mg/kg to 10mg/kg.Under any circumstance, the doctor will determine to be suitable for individual actual dose most, and this dosage will be different along with age, body weight and the reaction of particular individual.Top dosage is the example of generalized case.Certainly, the indivedual examples that need higher or lower dosage range might be arranged, and these situations are in the scope of the present invention.

Required dosage range depends on selection, route of administration, the character of preparation, the character of patient's illness and attending doctor's the judgement of peptide.Yet proper dosage is in the scope of 0.1-100 μ g/kg weight in patients.

Vaccine composition adopts injectable form easily.Can use conventional adjuvant to reply with enhancing immunity.The appropriate units dosage that is used for vaccine is the antigen of 0.5-5 microgram/kg, and preferably gives such dosage 1-3 time with the interval in 1-3 week.Use the dosage range of indication, will can not observe prevention with compound of the present invention and give the disadvantageous toxicological effect of suitable individuality them.

Yet, consider the diversity of available compound and the different efficacies of various route of administration, be expected on the required dosage great changes have taken place.For example, expection is oral will be than give more high dosage by intravenous injection.Use the standard experience program of knowing in this area that is used to optimize, can adjust the variation on these dosage levels.

Sequence library, the sequence in tangible media and algorithm

Polynucleotide and peptide sequence form valuable information source, use such information source to determine their 2 peacekeepings, 3 dimension structures, and identify other sequence with similar identity.By sequence being stored in the computer readable medium and using the data of being stored subsequently in known macromolecular structure program, or use the data stored, by well-known research tool such as GCG routine package search sequence database, big convenience these methods.

The present invention also provides the analytical procedure of characteristic sequence or bit string (string), especially gene order or coded protein sequence.Preferred sequence analysis method comprises for example method of sequence homology analysis (as identity and similarity analysis), DNA, RNA and structural analysis of protein, sequence assembling, branch's analysis, sequence motifs analysis, determines that open reading-frame (ORF), nucleic acid base are called in (nucleic acid base calling), codon selects analysis, nucleic acid base finishing (nucleicacid base trimming) and sequence chromatographic peak to analyze.

Provide computer-based method for carrying out the homology evaluation.This method comprises the following steps: to provide first polynucleotide sequence of the sequence that comprises polynucleotide of the present invention in computer readable medium; Described first polynucleotide sequence and at least a second polynucleotide or peptide sequence are compared, identify homology.

Also provide computer-based method for carrying out homology analysis, described method comprises the following steps: to provide first peptide sequence of the sequence that comprises polypeptide of the present invention in computer readable medium; Described first peptide sequence and at least a second polynucleotide or peptide sequence are compared, identify homology.

All publications quoted in this manual and bibliography (including but not limited to patent and patent application) intactly are attached to herein by reference, are attached to herein by reference as pointing out individually also that specifically each discrete publication or bibliography have been stated fully.Aforesaid way with publication and bibliography intactly is attached to any patent application that the application requires to obtain right of priority herein by reference simultaneously.

Definition

" identity " as known in the art, refers to according to circumstances, definite two or more peptide sequence or the relation between two or more polynucleotide sequence by comparative sequences.In this area, " identity " also refer to according to circumstances, the sequence similarity degree by the bit string of coupling polypeptide or polynucleotide sequence between the sequence of definite polypeptide or polynucleotide.By currently known methods, can easily calculate " identity ", described method includes but not limited to the method that those are described in following document: Computaional MolecularBiology, Lesk, A.M. edits, Oxford University Press, New York, 1988; Biocomputing:Informatics and Genone Projects, Smith, D.W. edits, Academic Press, New York, 1993; Computer Analysis of Sequence Data, part 1, Griffin, A.M., and Griffin, H.G. edits, Humana Press, NewJersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; With Sequence Analysis Priner, Gribskov, M. and Devereux, J., editor, M Stockton Press, New York, 1991; And Carillo, H., and Lipman, D., SIAM J.Applied Math., 48:1073 (1988).The method design that is used to measure identity is used for providing the maximum match between institute's cycle tests.In addition, measure the method available computer program code of the public of identity.The computer program means that is used for measuring identity between two sequences includes but not limited to: the GAP program (Devereux of GCG routine package, J. etc., 387 (1984)), BLASTP, BLASTN (Altschul Nucleic Acids Research 12 (1):, J.Molec.Biol.215:403-410 (1990)) and FASTA (Pearson and Lipman Proc.Natl.Acad.Sci.USA 85:2444-2448 (1988) S.F. etc..The public can obtain publicly from NCBI and other source blast program series (BLASTManual, Altschul, S. etc., NCBI NLM NIH Bethesda, MD 20894; Altschul, S. etc., J.Mol.Biol.215:403-410 (1990).Also can use well-known SmithWaterman algorithm to measure identity.

The parameter that is used for the peptide sequence comparison comprises following:

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

Comparator matrix (Comparison matrix): the BLOSSUM62 that derives from Henikoff and Henikoff Proc.Natl.Acad.Sci.USA.89:10915-10919 (1992)

Compensation (Gap Penalty) at interval: 8

Gap length compensation: 2

Can be used as Genetics Computer Group according to the operable program of these parameters, " (gap) at interval " program of Madison WI obtains publicly.Above-mentioned parameter is to carry out the default parameters that peptide relatively (does not have compensation to space from end).

The parameter that is used for the polynucleotide comparison comprises following:

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

Comparator matrix: coupling=+ 10, mispairing=0

Compensation at interval: 50

Gap length compensation: 3

Can be used as Genetics Computer Group, " at interval " program of Madison WI obtains.These parameters are to carry out nucleic acid default parameters relatively.

According to circumstances, provide in preferred meaning (1) and (2) below for polynucleotide and polypeptide " identity ".

(1) the polynucleotide embodiment also comprises isolating polynucleotide, described isolating polynucleotide comprise that the reference sequences with SEQ ID NO:1 has at least 50,60,70,80,85,90,95, the polynucleotide sequence of 97 or 100% identity, wherein said polynucleotide sequence can be identical with the reference sequences of SEQ ID NO:1, perhaps compare with described reference sequences, the Nucleotide that comprises a certain integer of as many as changes, wherein said change is selected from following: at least one nucleotide deletion, replace (comprising base conversion and transversion) or insertion, and described change can occur between 5 ' or 3 ' terminal position of described reference nucleotide sequence or those terminal positions Anywhere, perhaps intersperse among in the Nucleotide of reference sequences separately separately, perhaps intersperse among in the reference sequences with one or more successive groups, the quantity that wherein said Nucleotide changes is following to be determined: will define the percentile integer division of identity with 100, multiply by the sum of Nucleotide among the SEQ ID NO:1 then, from described SEQ ID NO:1, deduct this product in the sum of Nucleotide subsequently, or:

n _n≤x _n-(x _n·y)

N wherein _nBe the number that Nucleotide changes, x _nIt is the sum of Nucleotide among the SEQ ID NO:1, y 0.50 represents 50%, 0.60 to represent 60%, 0.70 to represent 70%, 0.80 to represent 80%, 0.85 to represent 85%, 0.90 to represent 90%, 0.95 to represent 95%, 0.97 to represent 97% or 1.00 to represent 100%, but the symbol of multiplication operator, wherein with any x _nWith the non-integer product of y from x _nBefore deducting, this product is rounded to immediate integer.The change of the polynucleotide of the polypeptide of coding SEQID NO:2 may produce nonsense, missense or phase shift mutation in this encoding sequence, and therefore changes the polypeptide by described polynucleotide encoding after such change.

For instance, polynucleotide sequence of the present invention can be identical with the reference sequences of SEQ ID NO:1, and is promptly can 100% identical, perhaps with described reference sequences relatively, this sequence comprises that the nucleic acid of a certain integer of as many as changes, so that the identity percentage is less than 100% identity.Such change is selected from following: at least one nucleotide deletion, replacement (comprising base conversion and transversion) or insertion, wherein said change can occur in described with reference to polynucleotide sequence 5 ' or 3 ' terminal position or those terminal positions between Anywhere, perhaps intersperse among separately separately in the nucleic acid of reference sequences, perhaps intersperse among in the reference sequences with one or more successive groups.The following number of determining for given percentile nucleic acid change: will define the percentile integer division of identity with 100, multiply by the sum of SEQ ID NO:1 amplifying nucleic acid then, from the sum of described SEQID NO:1 amplifying nucleic acid, deduct this product subsequently, or:

n _n≤x _n-(x _n·y)

N wherein _nBe the number that nucleic acid changes, x _nBe the sum of SEQ ID NO:1 amplifying nucleic acid, y for example 0.70 represents 70%, 0.80 to represent 80%, 0.85 to represent 85% or the like, but the symbol of multiplication operator, wherein with any x _nWith the non-integer product of y from x _nBefore deducting, this product is rounded to immediate integer.

(2) the polypeptide embodiment also comprises isolated polypeptide, described isolated polypeptide comprises that the polypeptide reference sequences with SEQ ID NO:2 has at least 50,60,70,80,85,90,95, the polypeptide of 97 or 100% identity, wherein said peptide sequence can be identical with the reference sequences of SEQ ID NO:2, perhaps compare with described reference sequences, the amino acid change that comprises a certain integer of as many as, wherein said change is selected from following: at least one aminoacid deletion, replace (comprising conservative the replacement and non-conservative replacement) or insertion, and described change can occur between the N-terminal of described reference polypeptide sequence or C-terminal position or those terminal positions Anywhere, perhaps intersperse among in the amino acid of reference sequences separately separately, perhaps intersperse among in the reference sequences with one or more successive groups, the wherein following quantity of determining described amino acid change: will define the percentile integer division of identity with 100, multiply by amino acid whose sum among the SEQ ID NO:2 then, from described SEQ ID NO:2, deduct this product in the amino acid whose sum subsequently, or:

n _a≤x _a-(x _a·y)

N wherein _aBe the number of amino acid change, x _aIt is amino acid whose sum among the SEQ ID NO:2, y 0.50 represents 50%, 0.60 to represent 60%, 0.70 to represent 70%, 0.80 to represent 80%, 0.85 to represent 85%, 0.90 to represent 90%, 0.95 to represent 95%, 0.97 to represent 97% or 1.00 to represent 100%, but the symbol of multiplication operator, wherein with any x _aWith the non-integer product of y from x _aBefore deducting, this product is rounded to immediate integer.

For instance, peptide sequence of the present invention can be identical with the reference sequences of SEQ ID NO:2, and is promptly can 100% identical, perhaps with described reference sequences relatively, this sequence comprises the amino acid change of a certain integer of as many as, so that the identity percentage is less than 100% identity.Such change is selected from following: at least one aminoacid deletion, replacement (comprising conservative the replacement and non-conservative replacement) or insertion, and described change can occur between the N-terminal of described reference polypeptide sequence or C-terminal position or those terminal positions Anywhere, perhaps intersperse among separately in the amino acid of reference sequences, perhaps intersperse among in the reference sequences with one or more successive groups.The quantity of the amino acid change of given % identity is following to be determined: will define the percentile integer division of identity with 100, multiply by amino acid whose sum among the SEQ ID NO:2 then, from described SEQ ID NO:2, deduct this product in the amino acid whose sum subsequently, or:

n _a≤x _a-(x _a·y)

N wherein _aBe the number of amino acid change, x _aBe amino acid whose sum among the SEQ ID NO:2, y for example 0.70 represents 70%, 0.80 to represent 80%, 0.85 to represent 85% or the like, but the symbol of multiplication operator, wherein with any x _aWith the non-integer product of y from x _aBefore deducting, this product is rounded to immediate integer.

" individuality ", when in this paper in order to refer to the biochron, refer to the many cells eukaryote, include but not limited to metazoan, Mammals, caprid (ovid), bovid (bovid), ape, primate and the mankind.

" isolating " refers to that " by people's work " changes from its state of nature, if promptly there is occurring in nature in it, then its changes from primal environment or takes out, and perhaps changes and takes out.For example, natural polynucleotide or the polypeptide that is present in the living organism is not " isolating ", but same polynucleotide or the polypeptide separated from the material of its native state coexistence are " isolating ", as this term use in this article.In addition, be " isolating " by conversion, genetic manipulation or polynucleotide or the polypeptide introduced in biology by any other recombination method, even it still is present in the described biology, and described biology can be that live or non-work.

" polynucleotide " refer generally to any polyribonucleotide or polydeoxyribonucleotide, and described polyribonucleotide or polydeoxyribonucleotide can be to comprise the unmodified rna of strand district and double stranded region or the RNA or the DNA of DNA or modified.

" varient " refers to different but kept the polynucleotide or the polypeptide of fundamental characteristics with reference to polynucleotide or polypeptide.Typical polynucleotide varient lists promptly different with reference to polynucleotide with another polynucleotide at nucleotides sequence.The change of described varient nucleotide sequence may change or not change by described amino acid sequence of polypeptide with reference to polynucleotide encoding.Discuss as following, the Nucleotide variation may cause by the aminoacid replacement in the reference sequences encoded polypeptides, interpolation, disappearance, fusion and brachymemma.Typical polypeptide variants is that reference polypeptide is different with another polypeptide on aminoacid sequence.In general, difference is restricted, so that the sequence of reference polypeptide and varient is closely similar and identical in many districts on the whole.Varient and reference polypeptide can be on aminoacid sequence because with one or more replacements of any combination, interpolation, disappearance and different.The amino-acid residue that replaces or insert can yes or no by the residue of genetic code coding.The varient of polynucleotide or polypeptide can be natural appearance (as an allelic variant), perhaps can be the varient of going back unknown natural appearance.Can be by induced-mutation technique or directly synthesize the varient that the non-natural of making polynucleotide and polypeptide occurs.

" disease " refer to any by infectation of bacteria cause or with the infectation of bacteria diseases associated, comprise for example baby and children's otitis media, auld people's pneumonia, sinusitis paranasal sinusitis, hospital infection and affecting conditions, with the chronic otitis media of hearing loss, body fluid inflammation at middle ear accumulation, auditory nerve damage, language learning delay, upper respiratory tract infection and middle ear.

Embodiment

Use standard technique to implement the following examples, described technology is known and routine for those skilled in the art except that other detailed description.Embodiment is illustrative, and does not limit the present invention.

Embodiment 1

Discovery and authenticity dna sequencing from the BASB027 gene of morazella catarrhalis strains A TCC 43617

At first in the Incyte PathoSeq database of the uncompleted genomic dna sequence that comprises morazella catarrhalis strains A TCC 43617 (being also referred to as bacterial strain Mc2931), find the BASB027 gene of SEQ ID NO:1.Shown in SEQ ID NO:2, the translation of described BASB027 polynucleotide sequence shows and the OMP85 outer membrane protein of Neisseria meningitidis has remarkable similarity (in 817 amino acid overlaps 32% identity being arranged).

Further confirmed the sequence of BASB027 gene with experimental technique.For this purpose, use QIAGEN genome DNA extracting reagent kit (Qiagen Gmbh), from 10 ¹⁰Individual morazella catarrhalis cell (strains A TCC 43617) extracts genomic dna, and use primer E515515 (5 '-ACT-ATA-GGG-CAC-GCG-TG-3 ') [SEQ ID NO:5] and E515528:(5 '-CCT-GCG-TTT-GTT-TGA-TTG-AG-3 ') [SEQ ID NO:6], carry out the polymerase chain reaction DNA cloning with 1 this material of μ g.The described PCR product of purifying on Biorobot 9600 (Qiagen Gmbh) instrument, and carry out dna sequencing with Big Dye CycleSequencing test kit (Perkin-Elmer) and ABI 377/PRISM dna sequencing instrument.With Feng Yuxing (redundancy) is 2, carries out dna sequencing on two chains, and uses the SeqMan program assembling full length sequence of DNASTAR Lasergene software package.The dna sequence dna that obtains and the peptide sequence of derivation show as SEQ ID NO:3 and SEQ ID NO:4 respectively.The difference of four Nucleotide comes SEQ ID NO:3 and SEQ ID NO:1 difference.Use the MEGALIGN program of DNASTAR Lasergene software package, carry out the sequence contrast of the polynucleotide sequence of SEQID NO:1 and 3, this sequence contrast is shown in Fig. 2; It is calculated that their identity level is 99.8%.Use same program, carry out the sequence contrast of the peptide sequence of SEQ IDNO:2 and 4, this sequence contrast is shown in Fig. 3; It is calculated that their identity level is 99.8%.

Embodiment 2

The Variability Analysis of BASB027 gene in several morazella catarrhalis bacterial strains

2A: restriction fragment length is analyzed (RFLP)

The genomic dna that from 16 morazella catarrhalis bacterial strains (being shown in table 1), extracts as described below.Morazella catarrhalis rule on the BHI agar plate forms single bacterium colony, and 37 ℃ of overnight incubation.Three or four single bacterium colonies of picking are inoculated into about 1.5ml BHI (brain-heart immersion liquid) meat soup seed culture medium, descend about 300rpm overnight incubation in 37 ℃ on shaking table.Inoculate the 500ml Erlenmeyer flask that comprises about 150ml BHI meat soup with this inoculum, cultivated about 12 to 16 hours in 37 ℃ of following about 175rpm on the shaking table, produce the cellular material that is used for DNA isolation.In Sorvall GSA rotor at room temperature centrifugal 15 minutes with about 2000 * g, collecting cell.Remove supernatant liquor and cell precipitation is resuspended in about 5.0ml sterilized water.Add equal-volume lysis buffer (200mM NaCl, 20mM EDTA, 40mM Tris-HCl, pH8.0,0.5% (weight/volume) SDS, 0.5% (volume ratio) 2 mercapto ethanol and 250 μ g/ml Proteinase Ks) and by mild stirring with grind suspension cell.Then described cell suspension thing is cultivated about 12 hours with lysing cell and discharge chromosomal DNA at 50 ℃.By adding the saturated NaCl of 5.0ml (about 6.0M is in sterilized water) and at room temperature centrifugal with about 5,500 * g in Sorvall SS34 rotor, the material of precipitating proteins.By adding 100% ethanol of two volumes, from the supernatant liquor of clarifying, precipitate chromosomal DNA.Collect accumulative DNA, and in 70% ethanolic soln of small volume, use mild stirring and wash.Chromosomal DNA behind the purifying is resuspended in the sterilized water and under 4 ℃ and makes its dissolution by slight rocked overnight.Under 260nm, use the optical extinction coefficient of the about 50 μ g/ml of 1.0O.D. unit, by the concentration of spectrophotometry dissolving DNA.

Use MC-D15-BamF (5 '-AAG GGC CCA ATT ACG CAGAGG GGA TCC ACA GGA CTA CAG CGA GTG ACC ATT GAAAGC TTA C-3 ') [SEQ ID NO:7] and MC-D15-SalRC (AAG GGC CCAATT ACG CAG AGG GTC GAC TTA TTA AAA GAC ACT ACC AATCTG GAA CTG TAC CGT ATC G-3 ') [SEQ ID NO:8] oligonucleotide subsequently, this material is carried out pcr amplification.Use BASB027 gene amplification of restriction enzyme (AciI, HindIII, MaeIII, NlaIII, RsaI, Sau3AI) hydrolysis correspondence independently then, and use standard molecular biology program, by agarose gel electrophoresis or polyacrylamide gel electrophoresis separation limit product, described standard biological is learned program as describing in " Molecular Cloning; a LaboratoryManual; second edition; editor: Sambrook; Fritsch and Maniatis, Cold SpringHarbor press 1989 ".The photo of the running gel that obtains is shown in Fig. 1.Record also makes up the RFLP pattern of each bacterial strain corresponding to 6 kinds of restriction enzymes.Determine to have the group of the bacterial strain of same RFLP mode combinations then.Make in this way, the bacterial strain that will in this research, test be divided into 4 gene groups ( Group 1: Mc2906, Mc2908, Mc2912, Mc2926; The group 2: Mc2905, Mc2907, Mc2909, Mc2911, Mc2913, Mc2960, Mc2975; Group 3: Mc2910, Mc2912, Mc2956, Mc2969; Group 4: Mc2931).These data are supported this viewpoint: the morazella catarrhalis colony that uses in this research shows limited nucleotide sequence diversity on the BASB027 gene.

Table 1: the feature of the morazella catarrhalis bacterial strain that in this research, uses

Bacterial strain	Separate certainly	From
			Mc2904	The U.S.	Auripuncture tapping art
Mc2905	The U.S.	Auripuncture tapping art
			Mc2906	The U.S.	Auripuncture tapping art
Mc2907	The U.S.	Auripuncture tapping art
			Mc2908	The U.S.	Acute otitis auripuncture tapping art
Mc2909	The U.S.	Auripuncture tapping art
			Mc2910	The U.S.	Auripuncture tapping art
Mc2911	The U.S.	Acute otitis auripuncture tapping art
			Mc2912	The U.S.	Acute otitis auripuncture tapping art
Mc2913	The U.S.	Acute otitis auripuncture tapping art
			Mc2926	The U.S.	Auripuncture tapping art
Mc2931/ATCC	The U.S.	Transtracheal aspirate

43617		(transtracheal aspirate)
			Mc2956	Finland	MEF (middle ear fluid)
Mc2960	Finland	MEF
			Mc2969	Norway	Nasopharynx (pharyngitis-rhinitis)
Mc2975	Norway	Nasopharynx (rhinitis)

Embodiment 3

The plasmid of structure express recombinant BASB027

The clone of A:BASB027

With BamHI and SalI restriction site respectively through engineering approaches advance complementary amplimer ([SEQID NO:7]) of forward and reverse complemental amplimer ([SEQ ID NO:8]), the PCR product that makes it possible to the about 2500bp of directed cloning advances the commercial colibacillus expression plasmid pQE30 (QiaGen that gets, anti-penbritin), so that ripe BASB027 albumen can be used as the expressing fusion protein that comprises (His) 6 affinity chromatography marks at N-terminal.Use based on the column spinner (spin column) of silica gel (QiaGen), according to the guidance of producer, purifying BASB027PCR product from the amplified reaction thing.For producing clone required BamHI and SalI end, that the digestion that the PCR product of purifying uses BamHI and SalI restriction enzyme such as producer (Life Technologies) to be recommended in proper order is complete.After the restriction digestion for the first time, before the enzymic digestion second time, desalt to remove by the described PCR product of column spinner purifying as mentioned above, then wash-out in sterilized water.With before the pQE30 plasmid is connected, the dna fragmentation of using the column spinner purifying based on silica gel to be digested once more.

B: the generation of expression vector

For preparing expression plasmid pQE30 to be used for connection, it is similarly complete with BamHI and SalI digestion, use calf small intestine Phosphoric acid esterase (CIP, about 0.02 unit/picomole 5 ' end subsequently, Life Technologies) instructs processing according to producer, connect certainly with prevention.Carry out described ligation with Duo the described digestion fragment of about 5 times of moles than prepared carrier.Use T4DNA ligase enzyme (about 2.0 unit/reactions, Life Technologies),, carry out the ligation (about 16 ℃, about 16 hours) of about 20 μ l of standard with well-known method in this area.According to well-known method in this area, use an equal portions connector (about 5 μ l) to transform M15 (pREP4) cell of electroreception attitude (electro-competent).In about 1.0ml LB meat soup, after 37 ℃ of vegetative period of following about 2-3 hour, transformant is inoculated on the LB agar plate that contains kantlex (50 μ g/ml) and penbritin (100 μ g/ml).In selecting substratum, comprise this two kinds of microbiotic, to guarantee that all transformants all have pREP4 plasmid (KnR) and pQE30-BASB027 plasmid (ApR), wherein the pREP4 plasmid is loaded with and prevents the necessary lacIq gene of expression of going up the expression of IPTG inducible protein for pQE30.Spent the night about 16 hours at 37 ℃ of culture plates.With each KnR/ApR bacterium colony of aseptic toothpick picking and be used for the dull and stereotyped and about 1.0ml LB KnR/ApR broth culture of the fresh LB KnR/ApR of " patch " inoculation.Dull and stereotyped and the broth culture of Patch or in the type culture case (flat board) or in shaking bath 37 ℃ of following overnight incubation.

Use confirms that based on the pcr analysis of full cell transformant comprises described BASB027DNA and inserts fragment.At this, the LB KnR/ApR broth culture that about 1.0ml spends the night is transferred to the 1.5ml polypropylene tube, and centrifugal in the Beckmann Eppendorf centrifuge (about 3 minutes, room temperature, about 12,000 * g), collecting cell.Cell precipitation is suspended in about 200 μ l sterilized waters, and carries out the PCR reaction of about 50 μ l final volumes with about 10 μ l aliquot sample, described PCR reaction comprises BASB027 forward amplimer and reverse amplimer.The ultimate density of described PCR reacted constituent is basic as indication among the embodiment 2, but uses the Taq polysaccharase of about 5.0 units.95 ℃ of initial denaturing steps are increased to 3 minutes, to guarantee the broken described bacterial cell of heat and to discharge plasmid DNA.Use ABI 9700 type thermal cyclers and 32 hot amplification methods of three step of round-robin, promptly 95 ℃, 45 seconds; 55-58 ℃, 45 seconds, 72 ℃, 1 minute, amplification was from the BASB027 PCR fragment of cracked transformant sample.After the heat amplification, analyze the reactant of about 20 μ l equal portions by agarose gel electrophoresis (0.8% agarose in Tris-acetate-EDTA (TAE) damping fluid).Behind gel electrophoresis and ethidium bromide staining, make dna fragmentation as seen by uviolizing.Dna molecular size criteria of parallel electrophoresis with specimen (1Kb ladder (ladder), Life Technologies), and in order to estimate the size of PCR product.The transformant that produces about 2500bp PCR product of expection is accredited as and comprises the bacterial strain that BASB027 expresses structure.Subsequent analysis comprises the inducible expression of reorganization BASB027 of the bacterial strain of expression plasmid.

The expression analysis of C:PCR positive transformant

For each PCR positive transformant of identifying above, use the about 5.0ml LB meat soup that contains kantlex (50 μ g/ml) and penbritin (100 μ g/ml) from the cell inoculation of patch flat board, and spend the night in 37 ℃ of following shaking culture (about 250rpm).The inoculum that spends the night (about 1.0ml) inoculation of one equal portions is advanced to contain the 125ml Erlenmeyer flask of about 25ml LB Kn/Ap meat soup, and to reach O.D.600 in 37 ℃ of following shaking culture (about 250rpm) up to the turbidity of culture be 0.5, i.e. mid-log phase (about usually 1.5-2.0 hour).To make an appointment with the culture (about 12.5ml) of half to transfer to second 125ml Erlenmeyer flask this moment, and (1.0M of sterilized water preparation stores liquid, Sigma) reaches the final concentration of 1.0mM, induces the proteic expression of reorganization BASB027 to add IPTG.Continuation is induced and about 4 hours of inductive culture not at 37 ℃ of following shaking culture IPTG.After inductive phase, get and induce and the sample of inducing culture thing (about 1.0ml) not, and in an Eppendorf centrifuge under the room temperature centrifugal about 3 minutes, collecting cell.Each cell precipitation is suspended in about 50 μ l sterilized waters, mixes with isopyknic 2X Laemelli SDS-PAGE sample buffer that contains 2 mercapto ethanol then, and place about 3 minutes of boiling water bath with metaprotein.With equal-volume (about 15 μ l) IPTG inductive cell and not the inductive cell the duplicate sample on the ground of thick lysate to 12% Tris/ glycine polyacrylamide gel (minigel that 1mm is thick, Novex).Under normal condition, use standard SDS/Tris/ glycine electrophoretic buffer (BioRad), will induce and inductive lysate sample and the molecular weight marker that dyes in advance (SeeBlue, Novex) electrophoresis together not.Behind the electrophoresis, glue is with coomassie brilliant blue R250 (BioRad) dyeing, but with rear decoloring so that novel B ASB027 IPTG inducible protein as seen.Use BioRad Mini-Protean II trace instrument and Towbin ethanol (20%) transfering buffering liquid, (0.45 micron pore size Novex) went up electroblotting about 2 hours at pvdf membrane with second glue under 4 ℃.According to well-known method closing membrane in this area and carry out antibody and cultivate.Using monoclonal anti RGS (His) 3 antibody, is the second rabbit anti-mouse antibody (QiaGen) of having puted together HRP then, confirms described BASB027 Recombinant Protein Expression and identity.Use or the insoluble substrate of ABT or be furnished with the Hyperfilm of AmershamECL chemiluminescence system, the reaction pattern that makes anti-His antibody as seen.

D: sequence confirms

For further confirming that the derivable reorganization of expressed IPTG BASB027 albumen is in correct open reading-frame (ORF) and not being, measured the segmental dna sequence dna of insertion of cloning because the clone makes the molecule of the vacation that thing (artifact) (being phase shift mutation) produces.(ABI Prism Dye-Terminator Cycle Sequencing Perkin-Elmer), obtains the dna sequence dna of morazella catarrhalis BASB027 gene from a chain to use conventional asymmetric PCR cycle sequencing method.Use indigested expression plasmid DNA (about 0.5 μ g/rxn) as template, (about 3.5 picomole/rxn) carry out sequencing reaction with suitable pQE30 carrier specificity sequencing primer and ORF specificity sequencing primer.Except that described template and sequencing primer, also comprise ddNTP (being ddA, ddG, ddC and the ddT) terminator nucleotides of four kinds of different dNTP (being A, G, C and T) and four kinds of correspondences in each sequencing reaction (about 20 μ l); Every kind of terminator is in conjunction with one of four kinds of fluorescence dyes simultaneously, and described four kinds of fluorescence dyes are Joe, Tam, Rox or Fam.Owing to insert the ddNTP terminator of dye marker, strand order-checking extension products stops at random site along described template.Use the termination product of microcentrifugation size exclusion chromatography post (Princeton Genetics) purifying fluorochrome label, dry under vacuum, be suspended in template resuspending damping fluid (Template Resuspension Buffer) (Perkin-Elmer) in to carry out capillary electrophoresis, or be suspended in the deionized methane amide to carry out PAGE, about 5 minutes of 95 ℃ of sex change, and by the high resolving power capillary electrophoresis (ABI 310 automatization dna sequencing instrument, Perkin-Elmer) or high resolving power PAGE (ABI 377 automatization dna sequencing instrument) analyze by manufacturer's recommended.Collect the dna sequence data that each reaction produces, and on the PowerMac computer, use ABI sequence analysis software (Perkin-Elmer) automated analysis relative fluorescence peak intensity.Use AutoAssembler software (Perkin-Elmer) separately the dna sequence dna of automated analysis to be merged into total single stranded sequence " bit string " preceding, the described dna sequence dna of human-edited is in the hope of accurately.Order-checking has determined that described expression plasmid comprises correct sequence with correct open reading-frame (ORF).

Embodiment 4: the generation of reorganization BASB027

Bacterial isolates

Produce the cellular material that is used for purification of recombinant proteins with the recombinant strains intestinal bacteria M15 (pREP4) that comprises the plasmid (pQE30) of coding from the BASB027 of morazella catarrhalis.Culture expression bacterial strain on the LB agar plate that contains 50 μ g/ml kantlex (" Kn ") and 100 μ g/ml penbritins (" Ap ") is to guarantee that pREP4 lacIq control plasmid and pQE30-BASB027 express structure and be maintained.For at-80 ℃ of cryopreservations, the described bacterial strain of breeding in containing the antibiotic LB meat soup of same concentration mixes with the LB meat soup that equal-volume contains 30% (weight/volume) glycerine then.

Substratum

The fermention medium that is used to produce recombinant protein comprises the 2X YT meat soup (Difco) that contains 50 μ g/ml Kn and 100 μ g/mlAp.(Antifoam 204, Sigma) to add defoamer with 0.25ml/L at the substratum that is used for fermentor tank.For inducing the BASB027 Recombinant Protein Expression, in fermentor tank, add IPTG (sec.-propyl-β-D sulfo-galactopyranoside) (1mM, final concentration).

Fermentation

With the frozen culture of 0.3ml quick-thawing or come several colony inoculations of self-selectively agar plate culture to contain the 500-ml taper seed bottle of 50ml working volume, in shaking table, cultivated about 12 hours down in 37 ± 1 ℃ then with 150rpm (Innova 2100, New Brunswick Scientific).The fermentor tank that contains the antibiotic 5-L working volume of 2X YT meat soup and Kn and Ap then with this inoculum inoculation.Fermentor tank (Bioflo 3000, New BrunswickScientific) is with 37 ± 1 ℃, 0.2-0.4 VVM air spray, Rushton impeller 250rpm operation.In bottled inoculum or fermentor tank, do not control pH.During the fermentation, the pH in the fermentor tank is in 6.5 to 7.3 the scope.When culture reaches logarithmic growth mid-term (about 0.7O.D.600 unit), in fermentor tank, add IPTG (1.0M stores liquid, prepares) in sterilized water.Inducing cell 2-4 hour, use or 28RS Heraeus (Sepatech) or RC5C ultracentrifuge (Sorvall Instrument) passed through centrifugal cell harvesting then.The cell paste is stored in-20 ℃ until further processing.

Purifying

Pharmaceutical chemicals and material

The imidazoles of biotechnology level (biotechnology grade) or higher category, Guanidinium hydrochloride, Tris (methylol) and EDTA (ethylenediamine tetraacetic acid (EDTA)) derive from Ameresco Chemical, Solon, Ohio.Triton X-100 (uncle's octylphenoxy polyethoxyethanol), SODIUM PHOSPHATE, MONOBASIC and urea are SILVER REAGENT or higher category, derive from Sigma Chemical Company, St.Louis, Missouri.Glacial acetic acid and hydrochloric acid derive from Mallincrodt BakerInc., Phillipsburg, NewJersey.Methyl alcohol derives from Fisher Scientific, Fairlawn, New Jersey. SC (4-(2-aminoethyl)-benzene sulfonyl fluorine), adequate proteins enzyme inhibitors mixing tablet and PMSF (phenylmethylsulfonyl fluoride) derive from Roche Diagnostics Corporation, Indianapolis, Indiana.Bestatin, pepstatin A and E-64 proteinase inhibitor derive from Calbiochem, LaJolla, California.Dulbecco phosphate buffered saline (1X PBS) derives from QualityBiological, Inc., Gaithersburg, Maryland.Dulbecco phosphate buffered saline(PBS) (10XPBS) derives from BioWhittaker, Walkersville, Maryland.The five Histidine antibody of no BSA derive from QiaGen, Valencia, California.The mountain sheep anti-mouse igg of puting together the affinity purification of peroxidase derives from Jackson Immuno Research, West Grove, Penn.The single solution of AEC (single solution) derives from Zymed, South San Francisco, California.All other reagent all are SILVER REAGENT or higher category.

Ni-NTA Superflow resin derives from QiaGen Inc., Valencia, California.Ready-formed Tris-glycine 4-20% and 10-20% polyacrylamide gel, all electrophoretic buffers and solution, SeeBlue dye standard, MultiMark polychrome standard and PVDF transfer film in advance and derive from Novex, San Diego, California.SDS-PAGE silver transfection reagent box derives from Daiichi PureChemicals Company Limited, Tokyo, Japan.The coomassie staining fluid derives from Bio-RadLaboratories, Hercules, California.Acrodisc PF 0.2m syringe filter derives from Pall Gelman Science, Ann Arbor, Michigan.GD/X 25mm one-shot injector formula filter derives from Whatman Inc., Clifton, New Jersey.Dialysis tubing 8,000 MWCO derive from BioDesign Inc.Od New York, Carmal New York.BCA protein determination reagent and snakeskin (Snake Skin) dialysis tubing 3,500 MWCO derive from Pierce Chemical Co., Rockford, Illinois.

Extracting method

The cell that at room temperature thaws was stuck with paste 30 to 60 minutes.Take by weighing five to six gram materials, place the disposable centrifuge tube of 50ml.Add 5ml/ gram Guanidinium hydrochloride (Gu-HCl) damping fluid (10mM Tris and 0.05% Triton X-100, pH 8.0 for 6M Guanidinium hydrochloride, 100mM SODIUM PHOSPHATE, MONOBASIC) therein.Use PRO300D proscientific homogenizer 3/4 power one minute, the resuspension cell is stuck with paste.Then described extraction mixture was placed under the room temperature mild stirring 60 to 90 minutes.After 60 to 90 minutes, and centrifugal described extraction mixture 15 minutes under 15,800 * g (Sorvall RC5C whizzer, 11,500rpm).Pour out supernatant liquor (S1) and preservation to carry out further purifying.Keep precipitation (P1) to analyze.

BASR027 is in conjunction with nickel-NTA resin

In S1, add 3-4ml Ni-NTA resin.Be placed under the room temperature mild stirring one hour subsequently.After one hour S1/Ni-NTA loaded into XK 16 Pharmacia posts.Use 1M Gu-HCl damping fluid (10mM Tris and 0.05% Triton X-100, pH 8.0 for 1M Guanidinium hydrochloride, 100mM SODIUM PHOSPHATE, MONOBASIC) to wash post then.Use phosphoric acid buffer (pH 6.3 for 100mM SODIUM PHOSPHATE, MONOBASIC, 10mM Tris and 0.05% Triton X-100) to wash post then.Use 250mM imidazole buffer (monoatomic base, 10mM Tris and 0.05% Triton X-100, pH 5.9 for 250mM imidazoles, 100mM sodium phosphate) eluted protein from the post then.

Final preparation

To 0.1% Triton X-100 and 1x PBS, pH 7.4 dialysed overnight are also changed solution three times therein, remove remaining Guanidinium hydrochloride and imidazoles, with preparation BASB027.Identify purified proteic feature and as described belowly be used to produce antibody.

Biochemical character is identified

SDS-PAGE analyzes and western blot analysis

(Thebaine etc. 1979, Proc.Natl.Acad.Sci.USA 76:4350-4354) as mentioned previously, the recombinant protein of separation and purification in the 4-20% polyacrylamide gel, and under 100V 1 hour with its electrophoretic transfer to pvdf membrane.Then with the described pvdf membrane of 25ml Dulbecco phosphate buffered saline(PBS) pre-treatment that contains 5% spent meal breast.Use this pre-treatment damping fluid to carry out all cultivations subsequently.

1:500 dilution 25ml with preimmune serum or the anti-His immune serum of rabbit under the room temperature cultivated 1 hour with pvdf membrane.Use lavation buffer solution (20mM Tris damping fluid, pH 7.5, contain 150mM sodium-chlor and 0.05% Tween-20) to wash pvdf membrane twice then.(1:5000 dilution 25ml PA) cultivated 30 minutes with pvdf membrane the goat anti-rabbit igg of usefulness peroxidase labelling for Jackson ImmunoResearchLaboratories, West Grove under the room temperature.Wash pvdf membrane 4 times with lavation buffer solution then, (SanFrancisco, the 3-that CA) provides amino-9-ethyl carbazole and urea peroxide respectively developed the color 10 minutes to use Zymed then.

The result of SDS-PAGE (Fig. 4) shows the albumen of a kind of about 95kDa, this albumen in the western blotting (Fig. 5) of SDS-PAGE with anti--RGS (His) antibody response.

Protein sequencing

At the Hewlett-Packard G1000A type sequenator with 1090 type LC and having on the Hewlett-Packard 24l type sequenator of 1100 type LC, use the chemical process of fine foundation, carry out purified proteic amino terminal amino acid order-checking, to confirm to produce correct recombinant protein.

Embodiment 5: produce the antiserum(antisera) at reorganization BASB027

By two rabbits of reorganization BASB027 albumen inoculation with purifying, the proteic polyvalent antiserum of the BASB027 that creates antagonism.Give every animal and receive three intramusculars (i.m.) immunization altogether, the about 20 μ g BASB027 albumen of per injection (begin to use up the Quan Fushi adjuvant, Quan Fushi adjuvant subsequently toos many or too much for use), about 21 days at interval.Before immunity for the first time (" getting blood (pre-bleed) before the immunity ") and got blood from animal on the 35th and 57 day.

Use the reorganization BASB027 albumen (0.5 μ g/ hole) of purifying, measure anti-BASB027 albumen by ELISA and tire.Tire to be defined as and equal or than big 0.1 the high dilution that calculates by following formula: the average OD of two damping fluid specimen of average OD-of two antiserum(antisera) specimen.As described in top embodiment 4, in western blotting, identify described albumen as first antibody with described antiserum(antisera).Western blotting is presented in the serum of immunity back animal and has anti-BASB027 antibody (Fig. 6).

Embodiment 6: amynologic characteristic is identified

Western blot analysis

Among 5% CO2, on the Chocolate Agar flat board, cultivated several morazella catarrhalis bacterial strains 48 hours under 35 ℃.Use the 25ml Muller Hinton meat soup in several colony inoculation 250ml bottles.With the culture overnight incubation and by centrifugal collection.Cultivated described suspended substance 5 minutes, dissolved cell by the 30 μ g cells that suspend in 150 μ l PAGE sample buffers (360mM Tris damping fluid, pH 8.8, contain 4% sodium lauryl sulphate and 20% glycerine) and at 100 ℃ then.(Thebaine etc. 1979, Proc.Natl.Acad.Sci.USA 76:4350-4354) as mentioned previously separate institute's dissolved cell in the 4-20% polyacrylamide gel, and under 100V 1 hour, isolating protein electrophoresis is transferred on the pvdf membrane.Then with the described pvdf membrane of 25ml Dulbecco phosphate buffered saline(PBS) pre-treatment that contains 5% skimmed milk powder.Use this pre-treatment damping fluid to carry out all cultivations subsequently.

1:500 dilution 25ml with preimmune serum or rabbit immune serum under the room temperature cultivated pvdf membrane 1 hour.Use lavation buffer solution (20mM Tris damping fluid, pH 7.5, contain 150mM sodium-chlor and 0.05% Tween-20) to wash pvdf membrane twice then.Use goat anti-rabbit igg (Jackson ImmunoResearch Laboratories, WestGrove, 1:5000 dilution 25ml cultivation pvdf membrane PA) 30 minutes of peroxidase labelling under the room temperature.Wash pvdf membrane 4 times with lavation buffer solution then, and (San Francisco, the 3-that CA) provides amino-9-ethyl carbazole and urea peroxide developed the color respectively 10 minutes with Zymed.

In all catarrhalis bacterial strains, detect the albumen (Fig. 7) of about 95kDa (corresponding to the expection molecular weight of BASB027) of a kind of and described antiserum(antisera) reaction.

Fungicidal activity

Detect the cytotoxic activity of the complement-mediated of anti-BASB027 antibody, to determine the vaccine potentiality (potential) of BASB027 polypeptide.As above prepare antiserum(antisera)." serum sterilizing test " that use Zollinger etc. describes is (to the immunne response of Neisseria meningitidis, at Manual of Clinical Laboratory Immunology, the third edition, in the 347-349 page or leaf), detect preimmune serum and anti-BASB027 serum and kill activity in the morazella catarrhalis at the mediation complement, but use the cell of morazella catarrhalis bacterial strain or kind, rather than Neisseria meningitidis cell.

The sterilization of rabbit anti-serum tire (killing 50% homologous strain) be＜1:8 (preimmune serum) and 1:128 (immune serum).

Embodiment 7: have the antibody at BASB027 in human convalescent phase serum

As carrying out the western blot analysis of the reorganization BASB027 of purifying as described in top embodiment 4 and 6, but use from the human Serum Bank that is subjected to the children that morazella catarrhalis infects as the first antibody prepared product.The result shows from the antiserum(antisera) of natural infection individuality and purified recombinant protein reaction.

Embodiment 8:BASB027 peptide, sero-fast generation and their reactivity

Use well-known method, produce two short amino acid BASB027 specific peptides in the laboratory, described peptide has the sequence of CYAKPLNKKQNDQTDT (SEQ ID NO:9) and YLTARRGQQTTLGEVVC (SEQ ID NO:10).In the female rabbit of 12 week specified-pathogens free New Zealand in age, produce antibody with these peptides that are coupled to KLH.Rabbit is accepted 4 injections with about 3 weekly intervals, and per injection is at Fu Shi adjuvant (injection for the first time) fully or incomplete 200 μ g peptide-KLH in the Fu Shi adjuvant (second, third, the 4th injection).Before immunity for the first time, got blood from animal in back one month with the 4th injection.

Use free peptide, measure the mid point of anti-peptide by ELISA and tire.Back one month of the 4th immunity, the mid point of anti-peptide is tired greater than 15000.Described in embodiment 4 and 6, use anti-peptide antibody as first antibody, the western blotting of the reorganization BASB027 of preparation purifying.The results are shown in Fig. 8.

Preserved material

The preservation thing that comprises morazella catarrhalis Caltin bacterial strain is deposited in American type culture collection (this paper is called " ATCC ") on June 21st, 1997, and preserving number is 43617.Described preservation thing is described to catarrh branhamella (Frosch and Kolle) and is freeze-dried products.1.5-2.9kb insert the morazella catarrhalis isolate that the fragment library construction obtains in a miner's who suffers with chronic bronchitis transtracheal aspirates thing.This preservation thing is described in Antimicrob.Agents Chemother.21:506-508 (1982).Morazella catarrhalis bacterial strain preservation thing is called " described preservation strain " or " DNA of described preservation strain " in this paper.

Described preservation strain comprises the BASB027 gene of total length.

The preservation thing that comprises the carrier pMC-D15 that inserts the morazella catarrhalis DNA in the pQE30 is deposited in American type culture collection (ATCC) on February 12nd, 1999, and preserving number is 207105.

Just in case have with any description of this paper and to conflict, then the sequence that modulation (controlling) is included in the polynucleotide in described preservation strain/clone with and any amino acid sequence of polypeptide of encoding.

Described preservation strain is according to the clause preservation of Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.In case after the granted patent, then described preservation strain will be irrevocable ground and unrestrictedly or unconditionally provide and give the public.Provide described preservation strain only to make things convenient for those skilled in the art, rather than admit that preservation is that realization is required, as desired according to 35 U.S.C. § 112.

<110>SmithKline Beecham Biologicals

＜120〉novel cpd

<130>BM45324

<160>10

＜170〉FastSEQ for version of window 3.0

<210>1

<211>2442

<212>DNA

＜213〉bacterium

<400>1

atgcgtaatt catattttaa aggtttccag gtcagtgcaa tgacaatggc tgtcacgatg 60

gcaatgtcaa ctcatgcaca agcggcggac tttatggcaa atgacattac catcacagga 120

ctacagcgag tgaccattga aagcttacaa agcgtgctgc cgtttcgctt gggtcaagtg 180

gtgagcgaaa accagttggc tgatggtgtc aaagcacttt atgcaacagg caattttcca 240

gatgtgcaag tctatcatca agaagggcgt accatctatc aggtaaccga aaggccgtta 300

accgctgaga ccaattttga gggcaatcgc ttaactccaa aagaaggtct acaagaaggg 360

ctaaaaaatg ctggcttagc tgtgggtcaa ccactaaaac aagccacagt acagatgatc 420

gaaaccgagc ttaccaatca atatatatca caaggctatt ataataccga aactactgtc 480

aaacagacga tgcttgatgg taatcgtgtt aagcttgata tgacctttgc tgaaggtaaa 540

cctgcacggg tggttgatat taatatcatt ggcaatcagc attttagcga tgcagatttg 600

attgatgtgc ttgcgattaa ggataataaa atcaatccac tgtctaaagc tgaccgttat 660

actcaagaaa agccggtgac cagtttagag aatttgcgtg ctaaatatct caatgcaggg 720

ttcgtgcgtt ttgagattaa agatgctaag cttaatatta atgaagataa aaaccgtatc 780

tttgttgaga tttcattgca tgaaggtgag caatatcgct ttggacagac acagcttttg 840

ggtaatttaa cctatactca agcagaactt gaggcactgc ttaaattcaa agcagaagaa 900

gggttttcac aagccatgct tgagcaaaca acaaacaata tcagtaccaa atttggtgac 960

gacggctatt attatgctca aatccgtcct gtaacacgca ttaatgatga aagtcgtacg 1020

gttgatgtgg aatattatat tgaccctgta caccctgtct atgtacgccg tattaatttt 1080

acaggtaac ttcaagaccca agatgaagta ctccgtcgtg agatgcgaca acttgaaggt 1140

gcgttggcat ctaatcaaaa aatccagctg tctcgtgcac gcttgatgcg gactgggttt 1200

tttaaacatg ttaccgttga tactcgtcca gtacccaact cacctgatca ggttgatgca 1260

aattttgtgg ttgaagaaca accttcagga tcatcaacca tcgcagcagg ctactctcaa 1320

agtggtggtg taacttttca atttgatgtt tttcaaaata actttatggg tacaggtaag 1380

cacgtcaatg cttcgttttc tcgctctgag acccgtgagg tgtatagttt gggtacgacc 1440

aacccatac tttaccgtaaa tggcgtctcg caaagcttga gtggctacta tcgtaaaacc 1500

aagtatgata acaagaacat tagtaattat gtacttgatt cttatggtgg ctcattaagc 1560

tatggatatc caattgatga aaatcaacgc ataagctttg gtctgaatgc tgacaatacc 1620

aagcttcatg gcggtcgttt catgggcatt agtaatgtca agcagctgat ggcagacggc 1680

ggcaaaattc aagtggataa taatggcatt cctgatttta agcatgatta cacaacctac 1740

aatgccattt tggggtggaa ttattcaagt ctagatcgcc ctgtatttcc aacccaaggc 1500

acgagtcatt ctgtagattt gacggttggt tttggtgata aaactcatca aaaagtggtt 1860

tatcaaggca atatctatcg cccatttatc aaaaaatcag tcttgcgcgg atacgccaag 1920

ttaggctatg gcaataattt accattttat gaaaatttct atgcaggcgg ctatggttcg 1980

gttcgtggct atgatcaatc ccctttgggt ccacgctcac aagcctattt gacagctcgt 2040

cgtggtcaac aaaccacact aggagaggtt gttggtggta atgctttggc aactttcgg 2100

agcgagctga ttttaccttt gccatttaaa ggtgattgga tagatcaggt gcgtccagtg 2160

atattcattg agggcggtca ggtctttgat acaacaggta tggataaaca aaccattgat 2220

ttaacccaat ttaaagaccc acaagcaaca gctgaacaaa atgcaaaagc agccaatcgc 2280

ccgccactaa cccaagataa acagccgcgt tatagtgctg gtgttggtgc aacttggtat 2340

acgcccattg gtcctttatc tattagctat gccaagccat tgaataaaaa acaaaatgat 2400

cagaccgata cggtacagtt ccagattggt agtgtctttt aa 2442

<210>2

<211>813

<212>PRT

＜213〉bacterium

<400>2

Met Arg Asn Ser Tyr Phe Lys Gly phe Gln Val Ser Ala Met Thr Met

1 5 10 15

Ala Val Met Met Val Met Ser Thr His Ala Gln Ala Ala Asp Phe Met

20 25 30

Ala Asn Asp Ile Thr Ile Thr Gly Leu Gln Arg Val Thr Ile Glu Ser

35 40 45

Leu Gln Ser Val Leu Pro Phe Arg Leu Gly Gln Val Val Ser Glu Asn

50 55 60

Glh Leu Ala Asp Gly Val Lys Ala Leu Tyr Ala Tnr Gly Asn Phe Ser

65 70 75 80

Asp Val Gln Val Tyr His Gln Glu Gly Arg Ile Ile Tyr Gln Val Thr

85 90 95

Glu Arg Pro Leu Ile Ala Glu Ile Asn Phe Glu Gly Asn Arg Leu Ilc

100 105 110

Pro Lys Glu Gly Leu Gln Glu Gly Leu Lys Asn Ala Gly Leu Ala Val

115 120 125

Gly Gln Pro Leu Lys Gln Ala Thr Val Gln Met Ile Glu Thr Glu Leu

130 135 140

Thr Asn Gln Tyr Ile Ser Gln Gly Tyr Tyr Asn Thr Glu Ile Thr Val

l45 150 155 160

Lys Gln Thr Met Leu Asp Gly Asn Arg Val Lys Leu Asp Met Thr Phe

165 170 175

Ala Glu Gly Lys Pro Ala Arg Val Val Asp Ile Asn Ile Ile Gly Asn

180 185 190

Gln His Phe Ser Asp Ala Asp Leu Ile Asp Val Leu Ala Ile Lys Asp

195 200 205

Asn Lys Ile Asn Pro Leu ser Lys Ala Asp Arg Tyr Thr Gln Glu Lys

210 215 220

Leu Val Thr ser Leu Glu Asn Leu Arg Ala Lys Tyr Leu Asn Ala Gly

225 230 235 240

Phe Val Arg Phe Glu Ile Lys Asp Ala Lys Leu Asn Ile Asn Glu Asp

245 250 255

Lys Asn Arg Ile Phe Val Glu Ile Ser Leu His Glu Gly Glu Gln Tyr

260 265 270

Arg Phe Gly Gln Thr Gln Phe Leu Gly Asn Leu Thr Tyr Thr Gln Ala

275 280 285

Glu Leu Glu Ala Leu Leu Lys Phe Lys Ala Glu Glu Gly Phe Ser Gln

290 295 300

Ala Met Leu Glu Gln Thr Thr Asn Asn Ile Ser Thr Lys Phe Gly Asp

305 310 315 320

Asp Gly Tyr Tyr Tyr Ala Gln Ile Arg Pro Val Thr Arg Ile Asn Asp

325 330 335

Glu Ser Arg Thr Val Asp Val Glu Tyr Tyr Ile Asp Pro Val His Pro

340 345 350

Val Tyr Val Arg Arg Ile Asn Phe Thr Gly Asn Phe Lys Thr Gln Asp

355 360 365

Glu Val Leu Arg Arg Glu Met Arg Gln Leu Glu Gly Ala Leu Ala Ser

370 375 380

Asn Gln Lys Ile Gln Leu Ser Arg Ala Arg Leu Met Arg Thr Gly Phe

385 390 395 400

Phe Lys His Val Tnr Val Asp Thr Arg Pro Val Pro Asn Ser Pro Asp

405 410 415

Gln Val Asp Val Asn Phe Val Val Glu Glu Gln Pro Ser Gly Ser Ser

420 425 430

Thr Ile Ala Ala Gly Tyr Ser Gln Ser Gly Gly Val Thr Phe Gln Pne

435 440 445

Asp Val Ser Gln Asn Asn Phe Met Gly Thr Gly Lys His Val Asn Ala

450 455 460

Ser Phe Sar Arg Ser Glu Thr Arg Glu Val Tyr Ser Leu Gly Met Thr

465 470 475 480

Asn Pro Tyr Phe Thr Val Asn Gly Val Ser Gln Ser Leu Ser Gly Tyr

485 490 495

Tyr Arg Lys Thr Lys Tyr Asp Asn Lys Asn Ile Ser Asn Tyr Val Leo

500 505 510

Asp Ser Tyr Gly Gly Ser Leu Ser Tyr Gly Tyr Pro Ile Asp Glu Asn

515 520 525

Gln Arg Ile Ser Phe Gly Leu Asn Ala Asp Asn Thr Lys Leu His Gly

530 535 540

Gly Arg Phe Met Gly Ile Ser Asn Val Lys Gln Leu Met Ala Asp Gly

545 550 555 560

Gly Lys Ile Gln Val Asp Ash Asn Gly Ile Pro Asp Phe Lys His Asp

565 570 575

Tyr Tbr Thr Tyr Asn Ala Ile Leu Gly Trp Asn Tyr Ser Ser Leu Asp

580 585 590

Arg Pro Val Phe Pro Thr Gln Gly Met Ser His Ser Val Asp Leu Thr

595 600 605

Val Gly Phe Gly Asp Lys Thr His Gln Lys Val Val Tyr Gln Gly Asn

610 615 620

Ile Tyr Arg Pro Phe Ile Lys Lys Ser Val Leu Arg Gly Tyr Ala Lys

625 630 635 640

Leu Gly Tyr Gly Asn Asn Leu Pro Phe Tyr Glu Asn Phe Tyr Ala Gly

645 650 655

Gly Tyr Gly Ser Val Arg Gly Tyr Asp Gln Ser Ser Leu Gly Pro Arg

660 665 670

Ser Gln Ala Tyr Leu Thr Ala Arg Arg Gly Gln Gln Thr Thr Leu Gly

675 680 685

Glu Val Val Gly Gly Asn Ala Leu Ala Thr Phe Gly Ser Glu Leu Ile

690 695 700

Leu Pro Leu Pro Phe Lys Gly Asp Trp Ile Asp Gln Val Arg Pro Val

705 710 715 720

Ile Phe Ile Glu Gly Gly Gln Val Phe Asp Thr Thr Gly Met AsP Lys

725 730 735

Gln Thr Ile Asp Leu Thr Gln Phe Lys Asp Pro Gln Ala Thr Ala Glu

740 745 750

Gln Asn Ala Lys Ala Ala Asn Arg Pro Leu Leu Thr Gln Asp Lys Gln

755 760 765

Leu Arg Tyr Ser Ala Gly Val Gly Ala Thr Trp Tyr Thr Pro Ile Gly

770 775 780

Pro Leu Ser Ile Ser Tyr Ala Lys Pro Leu Asn Lys Lys Gln Asn Asp

785 790 795 800

Gln Thr Asp Thr Val Gln Phe Gln Ile Gly Ser Val Phe

805 810

<210>3

<211>2442

<212>DNA

＜213〉bacterium

<400>3

atgcgtaatt catattttaa aggttttcag gtcagcgcaa tgacaacggc tgtcatgatg 60

gtaatgtcaa ctcatgcaca agcggcggat tttatggcaa atgacattgc catcacagga 120

ctacagcgag tgaccattga aagcttacaa agcgtgctgc cgtttcgctt gggtcaagtg 180

gtgagcgaag cacagttggc tgatggtgtc aaagcacttt atgcaacagg caatttttca 240

gacgtgcaag tctatcatca agaagggcgt atcatctatc aggcaaccga aaggccgtta 300

atcgctgaga ttaattttga gggcaatcgc ttaattccaa aagaaggtct acaagaaggg 360

ccaaaaaatg ctggcttagc tgtgggtcaa ccactaaaac aagccacagc acagatgatc 400

gaaaccgagc ccaccaatca atatatatca caaggctatt ataataccga aattactgtc 480

aaacagacga tgtttgatgg taatcgtgtt aagcttgatc cgacctttgc tgaaggcaaa 540

cctgcacggg tggttgatat taacatcatt ggcaatcagc tttttagcga tgcagattcg 600

attgatgtgc ttgcgattaa ggataataaa atcaatccac tgtctaaagc tgaccgttat 660

actcaagaaa agctggtgac cagtttagag aatttgcgtg ccaaatattc caacgcaggg 720

tctgtgcgtgtt gagattaa agatgctaag cttaatacca atgaagataa aaaccgtatc 790

tttgttgaga tctcactgca tgaaggtgag caatatcgcc ttggacagac acagtctttg 840

ggtaatttaa cttatactca agcagaactt gaggcactgc ttaaattcaa agcagaagaa 900

gggtttttac aagccatgct tgagcaaaca acaaacaata tcagtaccaa atttggtgac 960

gatggctatt attatgctca aatccgtcct gtaacacgca ttaatgatga aagtcgtacg 1020

gtcgatgtgg aatattatat tgaccctgta caccctgtcc atgtacgccg tattaatttt 1080

acaggtaact ttaagaccca agatgaagta ctccgtcgtg agatgcgaca acttgaaggt 1140

gcgttggcat ctaatcaaaa aatccagctg tctcgtgcac gcttgatgcg gactgggttt 1200

tttaaacatg ttaccgttga tactcgtcca gcacccaact cacctgatca ggttgatgta 1260

aattttgtgg ttgaagaaca accttcagga ccatcaacca tcgcagcagg ccactctcaa 1320

agtggtggtg taacttttca atttgatgtt tctcaaaata actttatggg tacaggtaag 1360

cacgtcaatg cttcgttttc tcgctctgag acccgtgagg tgtatagttt gggtacgacc 1440

aacccatact ttaccgtaaa tggcgtctcg caaagcttga gtggctacta tcgtaaaacc 1500

aagtatgata acaagaacat tagtaattat gtacttgatt cttatggtgg ctcattaagc 1560

tatggatatc caattgatga aaatcaacgc ataagctttg gtctgaatgc tgacaatacc 1620

aagcttcatg gcggtcgttt tatgggcatt agtaatgtca agcagctgat ggcagatggt 1680

ggcaaaattc aagtggataa taatggcatt cctgatttta agcatgatta cacaacctac 1740

aatgccattt tggggtggaa ttattcaagt ccagatcgcc ctgtatttcc aacccaaggc 1800

atgagtcatc ctgtagattt gacggttggt tttggtgata aaactcatca aaaagtggtt 1860

tatcaaggca atatctatcg cccatttatc aaaaaatcag tcttgcgtgg atacgccaag 1920

ttaggctatg gcaataattt accattttat gaaaatttcta tgcaggcgg ctatggttcg 1980

gttcgtggct atgatcaacc ctctttgggt ccacgctcac aagcctattt gacagctcgt 2040

cgcggtcaac aaaccacact aggagaggtt gttggtggta atgctttggc aactttcggc 2100

agtgagctga ttttaccttt gccatttaaa ggtgattgga tagatcaggt gcgtccagtg 2160

atattcattg agggcggtca ggtttttgat acaacaggta tggataaaca aaccattgat 2220

ttaacccaat ttaaagaccc acaagcaaca gctgaacaaa atgcaaaagc agccaaccgc 2280

ccgctactaa cccaagataa acagttgcgt tatagtgctg gtgttggcgc aacttggtat 2340

acgcccattg gccctttatc tattagctat gccaagccat tgaataaaaa acaaaatgat 2400

cagaccgata cggtacagtt ccagattggt agtgtctttt aa 2442

<210>4

<211>813

<212>PRT

＜213〉bacterium

<400>4

Mec Arg Asn Ser Tyr Phe Lys Gly Phe Gln Val Ser Ala Met Thr Met

1 5 10 15

Ala Val Met Met Val Met Ser Thr His Ala Gln Ala Ala Asp Phe Met

20 25 30

Ala Asn Asp Ile Ala Ile Thr Gly Leu Gln Arg Val Thr Ile Glu Ser

35 40 45

Leu Gln Ser Val Leu Pro Phe Arg Leu Gly Gln Val Val Ser Glu Ala

50 55 60

Gln Leu Ala Asp Gly Val Lys Ala Lau Tyr Ala Thr Gly Asn Pne Ser

65 70 75 80

Asp Val Gln Val Tyr His Gln Glu Gly Arg Ile Ile Tyr Gln Val Thr

85 90 95

Glu Arg Pro Leu Ile Ala Glu Ile Asn Phe Glu Gly Asn Arg Leu Ile

100 105 110

Pro Lys Glu Gly Leu Gln Glu Gly Leu Lys Asn Ala Gly Leu Ala Val

115 120 125

Gly Gln Pro Leu Lys Gln Ala Thr Val Gln Met Ile Glu Thr Glu Leu

130 135 140

Thr Asn Gln Tyr Ile Ser Gln Gly Tyr Tyr Asn Thr Glu Ile Tnr Val

145 150 155 160

Lys Gln Thr Met Leu Asp Gly Asn Arg Val Lys Leu Asp Met Tnr Pne

165 170 177

Ala Glu Gly Lys Pro Ala Arg Val Val Asp Ile Asn Ile Ile Gly Asn

180 185 190

Gln His Phe Ser Asp Ala Asp Leu Ile Asp Val Leu Ala Ile Lys Asp

195 200 205

Asn Lys Ile Asn Pro Leu Ser Lys Ala Asp Arg Tyr Thr Gln Glu Lys

210 215 220

Leu Val Thr Ser Leu Glu Asn Leu Arg Ala Lys Tyr Leu Asn Ala Gly

225 230 235 240

Phe Val Arg Phe Glu Ile Lys Asp Ala Lys Leu Asn Ile Asn Glu Asp

245 250 255

Lys Asn Arg Ile Phe Val Glu Ile Ser Leu His Glu Gly Glu Gln Tyr

260 265 270

Arg Phe Gly Gln Thr Gln Phe Leu Gly Asn Leu Thr Tyr Thr Gln Ala

275 280 285

Glu Leu Glu Ala Leu Leu Lys Phe Lys Ala Glu Glu Gly Phe Ser Gln

290 295 300

Ala Met Leu Glu Gln Thr Thr Asn Asn Ile Ser Thr Lys Phe Gly Asp

305 310 315 320

Asp Gly Tyr Tyr Tyr Ala Gln Ile Arg Pro Val Thr Arg Ile Asn Asp

325 330 335

Glu Ser Arg Thr Val Asp Val Glu Tyr Tyr Ile Asp Pro Val His Pro

340 345 350

Val Tyr Val Arg Arg Ile Asn Phe Thr Gly Asn Phe Lys Thr Gln Asp

355 360 365

Glu Val Leu Arg Arg Glu Met Arg Gln Leu Glu Gly Ala Leu Ala Ser

370 375 380

Asn Gln Lys Ile Gln Leu Ser Arg Ala Arg Leu Met Arg Thr Gly Phe

385 390 395 400

Phe Lys His Val Thr Val Asp Thr Arg Pro Val Pro Asn Ser Pro Asp

405 410 415

Gln Val Asp Val Asn Phe Val Val Glu Glu Gln Pro Ser Gly Ser Ser

420 425 430

Thr Ile Ala Ala Gly Tyr Ser Gln Ser Gly Gly Val Thr Phe Gln Phe

435 440 445

Asp Val Ser Gln Asn Asn Phe Met Gly Thr Gly Lys His Val Asn Ala

450 455 460

Ser Phe Ser Arg Ser Glu Thr Arg Glu Val Tyr Ser Leu Gly Met Thr

465 470 475 480

Asn Pro Tyr Phe Thr Val Asn Gly Val Ser Gln Ser Leu Ser Gly Tyr

485 490 495

Tyr Arg Lys Thr Lys Tyr Asp Asn Lys Asn Ile Ser Asn Tyr Val Leu

500 505 510

Asp Ser Tyr Gly Gly Ser Leu Ser Tyr Gly Tyr Pro Ile Asp Glu Asn

515 520 525

Gln Arg Ile Ser Phe Gly Leu Asn Ala Asp Asn Thr Lys Leu His Gly

530 535 540

Gly Arg Phe Met Gly Ile Ser Asn Val Lys Gln Leu Met Ala Asp Gly

545 550 555 560

Gly Lys Ile Gln Val Asp Asn Asn Gly Ile Pro Asp Phe Lys His Asp

565 570 575

Tyr Thr Thr Tyr Asn Ala Ile Leu Gly Trp Asn Tyr Ser Ser Leu Asp

580 585 590

Arg Pro Val Phe Pro Thr Gln Gly Met Ser His Ser Val Asp Leu Thr

595 600 605

Val Gly Phe Gly Asp Lys Thr His Gln Lys Val Val Tyr Gln Gly Asn

610 615 620

Ile Tyr Arg Pro Phe Ile Lys LyS Ser Val Leu Arg Gly Tyr Ala Lys

625 630 635 640

Leu Gly Tyr Gly Asn Asn Leu Pro Phe Tyr Glu Asn Phe Tyr Ala Gly

645 650 655

Gly Tyr Gly Ser Val Arg Gly Tyr Asp Gln Ser Ser Leu Gly Pro Arg

660 665 670

Ser Gln Ala Tyr Leu Thr Ala Arg Arg Gly Gln Gln Thr Thr Leu Gly

675 680 685

Glu Val Val Gly Gly Asn Ala Leu Ala Thr Phe Gly Ser Glu Leu Ile

690 695 700

Leu Pro Leu Pro Phe Lys Gly Asp Trp Ile Asp Gln Val Arg Pro Val

705 710 715 720

Ile Phe Ile Glu Gly Gly Gln Val Phe Asp Thr Thr Gly Met Asp Lys

725 730 735

Gln Thr Ile Asp Leu Thr Gln Phe Lys Asp Pro Gln Ala Thr Ala Glu

740 745 750

Gln Asn Ala Lys Ala Ala Asn Arg Pro Leu Leu Thr Gln Asp Lys Gln

755 760 765

Leu Arg Tyr Ser Ala Gly Val Gly Ala Thr Trp Tyr Thr Pro Ile Gly

770 775 780

Pro Leu Ser Ile Ser Tyr Ala Lys Pro Leu Asn Lys Lys Gln Asn Asp

785 790 795 800

Gln Thr Asp Thr Val Gln Phe Gln Ile Gly Ser ValPhe

805 810

<210>5

<211>17

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>5

accatagggc acgcgtg 17

<210>6

<211>20

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>6

cctgcgtttg tttgattgag 26

<210>7

<211>61

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>7

aagggcccaa ctacgcagag gggacccaca ggactacagc gagtgaccat tgaaagctta 60

c 61

<210>8

<211>67

<212>DNA

＜213〉artificial sequence

<220>

＜223〉oligonucleotide

<400>8

aagggcccaa tcacgcagag ggtcgactta ttaaaagaca ctaccaatct ggaactgtac 60

cgtatcg 67

<210>9

<211>16

<212>PRT

＜213〉artificial sequence

<220>

＜223〉oligopeptides

<400>9

Cys Tyr Ala Lys Pro Leu Asn Lys Lys Gln Asn Asp Gln Thr Asp Thr

1 5 10 15

<210>10

<211>17

<212>PRT

＜213〉artificial sequence

<220>

＜223〉oligopeptides

<400>10

Tyr Leu Thr Ala Arg Arg Gly Gln Gln Thr Thr Leu Gly Glu Val Val

1 5 10 15

Cys

Sequence information

BASB027 polynucleotide sequence and peptide sequence

SEQ ID NO：1

Morazella catarrhalis BASB027 polynucleotide sequence from strains A TCC43617

ATGCGTAATTCATATTTTAAAGGTTTTCAGGTCAGTGCAATGACAATGGCTGTCATGATG

GTAATGTCAACTCATGCACAAGCGGCGGATTTTATGGCAAATGACATTACCATCACAGGA

CTACAGCGAGTGACCATTGAAAGCTTACAAAGCGTGCTGCCGTTTCGCTTGGGTCAACTG

GTGAGCGAAAACCAGTTGGCTGATGGTGTCAAAGCACTTTATGCAACAGGCAATTTTTCA

GATGTGCAAGTCTATCATCAAGAAGGGCGTATCATCTATCAGGTAACCGAAAGGCCGTTA

ATCGCTGAGATTAATTTTGAGGGCAATCGCTTAATTCCAAAAGAAGGTCTACAAGAAGGG

CTAAAAAATGCTGGCTTAGCTGTGGGTCAACCACTAAAACAAGCCACAGTACAGATGATC

GAAACCGAGCTTACCAATCAATATATATCACAAGGCTATTATAATACCGAAATTACTGTC

AAACAGACGATGCTTGATGGTAATCGTGTTAAGCTTGATATGACCTTTGCTGAAGGTAAA

CCTGCACGGGTGGTTGATATTAATATCATTGGCAATCAGCATTTTAGCGATGCAGATTTG

ATTGATGTGCTTGCGATTAAGGATAATAAAATCAATCCACTGTCTAAAGCTGACCGTTAT

ACTCAAGAAAAGCTGGTGACCAGTTTAGAGAATTTGCGTGCTAAATATCTCAATGCAGGG

TTTGTGCGTTTTGAGATTAAAGATGCTAAGCTTAATATTAATGAAGATAAAAACCGTATC

TTTGTTGAGATTTCATTGCATGAAGGTGAGCAATATCGCTTTGGACAGACACAGTTTTTG

GGTAATTTAACTTATACTCAAGCAGAACTTGAGGCACTGCTTAAATTCAAAGCAGAAGAA

GGGTTTTCACAAGCCATGCTTGAGCAAACAACAAACAATATCAGTACCAAATTTGGTGAC

GATGGCTATTATTATGCTCAAATCCGTCCTGTAACACGCATTAATGATGAAAGTCGTACG

GTTGATGTGGAATATTATATTGACCCTGTACACCCTGTCTATGTACGCCGTATTAATTTT

ACAGGTAACTTTAAGACCCAAGATGAAGTACTCCGTCGTGAGATGCGACAACTTGAAGGT

GCGTTGGCATCTAATCAAAAAATCCAGCTGTCTCGTGCACGCTTGATGCGGACTGGGTTT

TTTAAACATGTTACCGTTGATACTCGTCCAGTACCCAACTCACCTGATCAGGTTGATGTA

AATTTTGTGGTTGAAGAACAACCTTCAGGATCATCAACCATCGCAGCAGGCTACTCTCAA

AGTGGTGGTGTAACTTTTCAATTTGATGTTTCTCAAAATAACTTTATGGGTACAGGTAAG

CACGTCAATGCTTCGTTTTCTCGCTCTGAGACCCGTGAGGTGTATAGTTTGGGTATGACC

AACCCATACTTTACCGTAAATGGCGTCTCGCAAAGCTTGAGTGGCTACTATCGTAAAACC

AAGTATGATAACAAGAACATTAGTAATTATGTACTTGATTCTTATGGTGGCTCATTAAGC

TATGGATATCCAATTGATGAAAATCAACGCATAAGCTTTGGTCTGAATGCTGACAATACC

AAGCTTCATGGCGGTCGTTTTATGGGCATTAGTAATGTCAAGCAGCTGATGGCAGATGGT

GGCAAAATTCAAGTGGATAATAATGGCATTCCTGATTTTAAGCATGATTACACAACCTAC

AATGCCATTTTGGGGTGGAATTATTCAAGTCTAGATCGCCCTGTATTTCCAACCCAAGGC

ATGAGTCATTCTGTAGATTTGACGGTTGGTTTTGGTGATAAAACTCATCAAAAAGTGGTT

TATCAAGGCAATATCTATCGCCCATTTATCAAAAAATCAGTCTTGCGTGGATACGCCAAG

TTAGGCTATGGCAATAATTTACCATTTTATGAAAATTTCTATGCAGGCGGCTATGGTTCG

GTTCGTGGCTATGATCAATCCTCTTTGGGTCCACGCTCACAAGCCTATTTGACAGCTCGT

CGTGGTCAACAAACCACACTAGGAGAGGTTGTTGGTGGTAATGCTTTGGCAACTTTCGGC

AGTGAGCTGATTTTACCTTTGCCATTTAAAGGTGATTGGATAGATCAGGTGCGTCCAGTG

ATATTCATTGAGGGCGGTCAGGTTTTTGATACAACAGGTATGGATAAACAAACCATTGAT

TTAACCCAATTTAAAGACCCACAAGCAACAGCTGAACAAAATGCAAAAGCAGCCAATCGC

CCGCTACTAACCCAAGATAAACAGTTGCGTTATAGTGCTGGTGTTGGTGCAACTTGGTAT

ACGCCCATTGGTCCTTTATCTATTAGCTATGCCAAGCCATTGAATAAAAAACAAAATGAT

CAGACCGATACGGTACAGTTCCAGATTGGTAGTGTCTTTTAA

SEQ ID NO：2

The morazella catarrhalis BASB027 that derives by the polynucleotide sequence of SEQ ID NO:1

Peptide sequence

MRNSYFKGFQVSAMTMAVMMVMSTHAQAADFMANDITITGLQRVTIESLQSVLRFRLGQV

VSENQLADGVKALYATGNFSDVQVYHQEGRIIYQVTERPLIAEINFEGNRLIPKEGLQEG

LKNAGLAVGQPLKQATVQMIETELTNQYISQGYYNTEITVKQTMLDGNRVKLDMTFAEGK

PARVVDINIIGNQHFSDADLIDVLAIKDNKINPLSKADRYTQEKLVTSLENLRAXYLNAG

FVRFEIKDAKLNINEDKNRIFVEISLHEGEQYRFGQTQFLGNLTYTQAELEALLKFKASE

GFSQANLEQTTNNISTKFGDDGYYYAQIRPVTRINDESRTVDVEYYIDPVHPVYVRRINF

TGNFKTQDEVLRREMRQLEGALASNQKIQLSRARLMRTGFFKHVTVDTRPVPNSPDQVDV

NFVVEEQPSGSSTIAAGYSQSGGVTFQFDVSQNNFMGTGKHVNASFSRSETREVYSLGMT

NPYFTVNGVSQSLSGYYRKTKYDNKNISNYVLDSYGGSLSYGYPIDENQRISFGLNADNT

KLHGGRFMGISNVKQLMADGGKIQVDNNGIPDFKHDYTTYNAILGWNYSSLDRFVFPTQG

MSHSVDLTVGFGDKTHQKVVYQGNIYRPFIKKSVLRGYAKLGYGNNLPFYENFYAGGYGS

VRGYDQSSLGPRSQAYLTARRGQQTTLGEVVGGNALATFGSELILPLPFKGDwIDQVRPV

IFIEGGQVFDTTGMDKQTIDLTQFKDPQATAEQNAKAANRPLLTQDKQLRYSAGVGATWY

TPIGPLSISYAKPLNKKQNDQTDTVQFQIGSVF

SEQ ID NO：3

Morazella catarrhalis BASB027 polynucleotide sequence from strains A TCC43617

ATGCGTAATTCATATTTTAAAGGTTTTCAGGTCAGTGCAATGACAATGGCTGTCATGATG

GTAATGTCAACTCATGCACAAGCGGCGGATTTTATGGCAAATGACATTACCATCACAGGA

CTACAGCGAGTGACCATTGAAAGCTTACAAAGCGTGCTGCCGTTTCGCTTGGGTCAAGTG

GTGAGCGAAAACCAGTTGGCTGATGGTGTCAAAGCACTTTATGCAACAGGCAATTTTTCA

GATGTGCAAGTCTATCATCAAGAAGGGCGTATCATCTATCAGGTAACCGAAAGGCCGTTA

ATCGCTGAGATTAATTTTGAGGGCAATCGCTTAATTCCAAAAGAAGGTCTACAAGAAGGG

CTAAAAAATGCTGGCTTAGCTGTGGGTCAACCACTAAAACAAGCCACAGTACAGATGATC

GAAACCGAGCTTACCAATCAATATATATCACAAGGCTATTATAATACCGAAATTACTGTC

AAACAGACGATGCTTGATGGTAATCGTGTTAAGCTTGATATGACCTTTGCTGAAGGTAAA

CCTGCACGGGTGGTTGATATTAATATCATTGGCAATCAGCATTTTAGCGATGCAGATTTG

ATTGATGTGCTTGCGATTAAGGATAATAAAATCAATCCACTGTCTAAAGCTGACCGTTAT

ACTCAAGAAAAGCTGGTGACCAGTTTAGAGAATTTGCGTGCTAAATATCTCAATGCAGGG

TTTGTGCGTTTTGAGATTAAAGATGCTAAGCTTAATATTAATGAAGATAAAAACCGTATC

TTTGTTGAGATTTCATTGCATGAAGGTGAGCAATATCGCTTTGGACAGACACAGTTTTTG

GGTAATTTAACTTATACTCAAGCAGAACTTGAGGCACTGCTTAAATTCAAAGCAGAAGAA

GGGTTTTCACAAGCCATGCTTGAGCAAACAACAAACAATATCAGTACCAAATTTGGTGAC

GATGGCTATTATTATGCTCAAATCCGTCCTGTAACACGCATTAATGATGAAAGTCGTACG

GTTGATGTGGAATATTATATTGACCCTGTACACCCTGTCTATGTACGCCGTATTAATTTT

ACAGGTAACTTTAAGACCCAAGATGAAGTACTCCGTCGTGAGATGCGACAACTTGAAGGT

GCGTTGGCATCTAATCAAAAAATCCAGCTGTCTCGTGCACGCTTGATGCGGACTGGGTTT

TTTAAACATGTTACCGTTGATACTCGTCCAGTACCCAACTCACCTGATCAGGTTGATGTA

AATTTTGTGGTTGAAGAACAACCTTCAGGATCATCAACCATCGCAGCAGGCTACTCTCAA

AGTGGTGGTGTAACTTTTCAATTTGATGTTTCTCAAAATAACTTTATGGGTACAGGTAAG

CACGTCAATGCTTCGTTTTCTCGCTCTGAGACCCGTGAGGTGTATAGTTTGGGTATGACC

AACCCATACTTTACCGTAAATGGCGTCTCGCAAAGCTTGAGTGGCTACTATCGTAAAACC

AAGTATGATAACAAGAACATTAGTAATTATGTACTTGATTCTTATGGTGGCTCATTAAGC

TATGGATATCCAATTGATGAAAATCAACGCATAAGCTTTGGTCTGAATGCTGACAATACC

AAGCTTCATGGCGGTCGTTTTATGGGCATTAGTAATGTCAAGCAGCTGATGGCAGATGGT

GGCAAAATTCAAGTGGATAATAATGGCATTCCTGATTTTAAGCATGATTACACAACCTAC

AATGCCATTTTGGGGTGGAATTATTCAAGTCTAGATCGCCCTGTATTTCCAACCCAAGGC

ATGAGTCATTCTGTAGATTTGACGGTTGGTTTTGGTGATAAAACTCATCAAAAAGTGGTT

TATCAAGGCAATATCTATCGCCCATTTATCAAAAAATCAGTCTTGCGTGGATACGCCAAG

TTAGGCTATGGCAATAATTTACCATTTTATGAAAATTTCTATGCAGGCGGCTATGGTTCG

GTTCGTGGCTATGATCAATCCTCTTTGGGTCCACGCTCACAAGCCTATTTGACAGCTCGT

CGTGGTCAACAAACCACACTAGGAGAGGTTGTTGGTGGTAATGCTTTGGCAACTTTCGGC

AGTGAGCTGATTTTACCTTTGCCATTTAAAGGTGATTGGATAGATCAGGTGCGTCCAGTG

ATATTCATTGAGGGCGGTCAGGTTTTTGATACAACAGGTATGGATAAACAAACCATTGAT

TTAACCCAATTTAAAGACCCACAAGCAACAGCTGAACAAAATGCAAAAGCAGCCAATCGC

CCGCTACTAACCCAAGATAAACAGTTGCGTTATAGTGCTGGTGTTGGTGCAACTTGGTAT

ACGCCCATTGGTCCTTTATCTATTAGCTATGCCAAGCCATTGAATAAAAAACAAAATGAT

CAGACCGATACGGTACAGTTCCAGATTGGTAGTGTCTTTTAA

SEQ ID NO：4

The morazella catarrhalis BASB027 that derives by the polynucleotide sequence of SEQID NO:3

Peptide sequence

MRNSYFKGFQVSAMTMAVMMVMSTHAQAADFMANDITITGLQRVTIESLQSVLPFRLGQV

VSENQLADGVKALYATGNFSDVQVYHQEGRIIYQVTERPLIAEINFEGNRLIPKEGLQEG

LKNAGLAVGQPLKQATVQMIETELTNQYISQGYYNTEITVKQTMLDGNRVKLDMTFAEGK

PARVVDINIIGNQHFSDADLIDVLAIKDNKINPLSKADRYTQEKLVTSLENLRAKYLNAG

FVRFEIKDAKLNINEDKNRIFVEISLHEGEQYRFGQTQFLGNLTYTQAELEALLKFKAEE

GFSQAMLEQTTNNISTKFGDDGYYYAQIRPVTRINDESRTVDVEYYIDPVHPVYVRRINF

TGNFKTQDEVLRREMRQLEGALASNQKIQLSRARLMRTGFFKHVTVDTRPVPNSPDQVDV

NFVVEEQPSGSSTIAAGYSQSGGVTFQFDVSQNNFMGTGKHVNASFSRSETREVYSLGMT

NPYFTVNGVSQSLSGYYRKTKYDNKNISNYVLDSYGGSLSYGYPIDENQRISFGLNADNT

KLHGGRFMGISNVKQLMADGGKIQVDNNGIPDFKHDYTTYNAILGWNYSSLDRPVFPTQG

MSHSVDLTVGFGDKTHQKVVYQGNIYRPFIKKSVLRGYAKLGYGNNLPFYENFYAGGYGS

VRGYDQSSLGPRSQAYLTARRGQQTTLGEVVGGNALATFGSELILPLPFKGDwIDQVRPV

IFIEGGQVFDTTGMDKQTIDLTQFKDPQATAEQNAKAANRPLLTQDKQLRYSAGVGATWY

TPIGPLSISYAKPLNKKQNDQTDTVQFQIGSVF

SEQ ID NO：5

ACT ATA GGG CAC GCG TG

SEQ ID NO：6

CCT GCG TTT GTT TGA TTG AG

SEQ ID NO：7

AAG GGC CCA ATT ACG CAG AGG GGA TCC ACA GGA CTA CAG CGA GTG

ACC ATT GAA AGC TTA C

SEQ ID NO：8

AAG GGC CCA ATT ACGCAG AGG GTC GAC TTA TTA AAA GAC ACT ACC

AAT CTG GAA CTG TAC CGT ATC G

SEQ ID NO：9

CYAKPLNKKQNDQTDT

SEQ ID NO：10

YLTARRGQQTTLGEVVC

Claims (24)

1. one section aminoacid sequence that isolated polypeptide, described polypeptide comprise has at least 85% identity with the aminoacid sequence of being selected by SEQ ID NO:2, SEQ ID NO:4.

2. as claim 1 isolated polypeptide required for protection, wherein said aminoacid sequence has at least 95% identity with the aminoacid sequence of being selected by SEQ ID NO:2, SEQ ID NO:4.

3. as claim 1 polypeptide required for protection, described polypeptide comprises the aminoacid sequence of being selected by following: SEQ ID NO:2, SEQ ID NO:4.

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

5. as the immunogenic fragments of each polypeptide required for protection in the claim 1 to 4, the polypeptide of the immunogenicity of wherein said immunogenic fragments and SEQ ID NO:2, SEQ ID NO:4 is basic identical.

6. isolating polynucleotide or with described isolating polynucleotide complementary nucleotide sequence, the nucleotide sequence coded polypeptide that at least 85% identity is arranged with SEQ ID NO:2,4 aminoacid sequence on SEQ ID NO:2,4 total length respectively of a section of comprising of described isolating polynucleotide.

7. isolating polynucleotide or with described isolating polynucleotide complementary nucleotide sequence, one section nucleotide sequence that described isolating polynucleotide comprise and coding SEQ ID NO:2,4 nucleotides sequence are listed at least 85% identity on the whole coding region.

8. isolating polynucleotide or with described isolating polynucleotide complementary nucleotide sequence, one section nucleotide sequence that described isolating polynucleotide comprise is shown at least 85% identity with SEQ ID NO:1,3 nucleotides sequence respectively on SEQ ID NO:1,3 total length.

9. as each isolating polynucleotide required for protection in the claim 6 to 8, wherein said and SEQ ID NO:1,3 identity are at least 95%.

10. isolating polynucleotide, described isolating polynucleotide comprise the nucleotide sequence of the polypeptide of coding SEQ ID NO:2, SEQ ID NO:4.

11. comprise the isolating polynucleotide of the polynucleotide of SEQ ID NO:1, SEQ ID NO:3.

12. isolating polynucleotide, one section nucleotide sequence coded passing through under stringent hybridization condition that described polynucleotide comprise uses to have the sequence of SEQ ID NO:1, SEQ ID NO:3 or the polypeptide that their segmental label probe screens suitable library obtainable SEQ ID NO:2, SEQ ID NO:4.

13. the live microorganism of expression vector or reorganization, the live microorganism of described expression vector or reorganization comprise according to each isolating polynucleotide among the claim 6-12.

14. the subcellular fraction of host cell or described host cell expression isolated polypeptide or film, described host cell comprises the expression vector of claim 13, and described isolated polypeptide comprises the aminoacid sequence that at least 85% identity is arranged with the aminoacid sequence of being selected by SEQ ID NO:2, SEQ ID NO:4.

15. produce the method for polypeptide, described polypeptide comprises the aminoacid sequence that at least 85% identity is arranged with the aminoacid sequence of being selected by SEQ ID NO:2, SEQ IDNO:4, described method is included in the host cell of cultivating claim 14 under the condition that is enough to produce described polypeptide, reclaims described polypeptide then from substratum.

16. express the method for each polynucleotide among the claim 6-12, comprise with comprising expression vector transformed host cell at least a in the described polynucleotide, be enough to express the described host cell of cultivation under the condition any in the described polynucleotide then.

17. vaccine composition comprises in the claim 1 to 5 of significant quantity each polypeptide and a kind of pharmaceutically acceptable carrier.

18. vaccine composition comprises in the claim 6 to 12 of significant quantity each polynucleotide and a kind of pharmaceutically effective carrier.

19. according to each vaccine composition in claim 17 or 18, wherein said composition comprises at least a other morazella catarrhalis antigen.

20. to the antibody that has immunologic opsonin as each claimed polypeptide or immune fragment in the claim 1 to 5.

21. the method that the diagnosis morazella catarrhalis infects; comprise that evaluation is subjected in a kind of like this biological sample of infected animals the existence as each polypeptide required for protection among the claim 1-5 from suspection, or described polypeptide is had the existence of the antibody of immunologic opsonin.

22. composition is in the purposes of preparation in the medicine, described composition comprise immune significant quantity as each polypeptide required for protection among the claim 1-5, described medicine is used for producing immunne response animal.

23. composition is in the purposes of preparation in the medicine, described composition comprise immune significant quantity as each polynucleotide required for protection among the claim 6-12, described medicine is used for producing immunne response animal.

24. can be used for treating the mankind's that suffer from the morazella catarrhalis disease therapeutic composition, described composition comprises antibody and a kind of suitable pharmaceutical carriers of at least a polypeptide at claim 1-5.

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