CN106432439A - Bovine respiratory syncytial virus antigen protein - Google Patents

Abstract

The invention discloses bovine respiratory syncytial virus antigen protein. Compared with SEQ AAB28458.1, an amino acid sequence of the bovine respiratory syncytial virus antigen protein is characterized in that at least two sites are replaced by cysteines, the replaced cysteines are in disulfide linkage, and improvements such as disulfide linkage, hole filling and single-strand ligation on this basis. The invention further provides a method for preparing the bovine respiratory syncytial virus antigen protein. The bovine respiratory syncytial virus antigen protein and the method for preparing the same have the advantages that technologies and theories of protein crystallography are applied, structural changes of protein in a virus infection and pathopoiesis process are determined, and the key viral protein is subjected to genetic engineering modifications according to structural change results so as to become the antigen protein which is infectious but not pathogenic and capable of causing high-efficiency reaction of animal antigens; the bovine respiratory syncytial virus antigen protein is capable of effectively preventing and treating bovine respiratory syncytial virus infection and is developed into satisfactory viral vaccines on the basis to control and prevent loss brought by virus infection, thereby being of great economic and social significance.

Description

A kind of bovine respiratory born of the same parents zoarium viral antigen proteins

Technical field

The invention belongs to biological product preparing technical field, be related to a kind of bovine respiratory born of the same parents zoarium viral antigen proteins and itsPreparation method.

Background technology

The fit virus of bovine respiratory born of the same parents（Bovine Respiration Syncytial Virus – BRSV）Infection can be drawnCattle respiratory disease is played, is listed in EU member country and is only second to bovine mucosal disease（BVD）And infectious bovine rhinotrachetiss（IBR）'sOne of important cattle disease.Primary disease is popular in world wide, and its hazardness is that BRSV the incidence of infection is up to 60%-80%, extremelyRate of dying reaches more than 20% in some outbursts, causes very big economic loss to cattle-raising.Although this BRSV is from eighties of last centuryBegin to the seventies list research and development category in, do not develop preferable medicine and vaccine for various reasons.Although relevantIn multiple inactivations of BRSV, weak poison, the report of restructuring and DNA vaccination, but there is certain distance apart from Clinical practice, current stateStill without preferable viral vaccine on border.

The development of traditional vaccine and production are mainly by changing condition of culture, or pass on different host animals and make causeSick Ecotoxicology weakens, or inactivated to complete by physics, chemical method.With the continuous progress of human knowledge,The limitation of traditional vaccine is also increasingly revealed：(1) virus of animals and humans needs to cultivate in zooblast, and this causesThe cost of production of vaccine is very high；(2) morbid substance in vaccine is possible in vaccine production process not kill completely or fillDivide attenuation, this can cause containing strong toxicity morbid substance in vaccine, and then disease is propagated in the larger context；(3) subtractToxic bacterial strain is possible to undergo mutation, and causes disease.

Bovine respiratory born of the same parents zoarium virus is the member of family of paramyxovirus section, F- albumen thereon be highly conserved simultaneouslyTrimer furcella is formed, which occurs conformation change under activation.The protein mediated virus of F- and the fusion of cell membrane, allow virus nucleocapsidBody enters Cytoplasm, and the step for suppressing F- protein mediated is prevented the initial period of infectious cycle and neutralizes viral infection and can haveImitated prevents virus infraction, i.e., for the antibody of F- albumen, suppress its activity, can neutralize viral infection and can protectInfect from BRSV.The open beginning for having created Chinese structure Basic of Biology theoretical research is applied, the present invention is by structure biologyIt is applied in practice, synthesis bovine respiratory born of the same parents' zoarium viral antigen proteins.

Content of the invention

It is an object of the invention to the shortcoming for overcoming prior art to exist, provides a kind of bovine respiratory born of the same parents zoarium virus antigenAlbumen and preparation method thereof, the technology of the antigen protein application protein crystallography and theory, determine albumen in virus infection andStructure change in pathogenic course, the result further according to structure change carries out genetic engineering to this crucial virus protein and changesMake so as to become and only infect proteantigen that is not pathogenic and causing animal body antibody highly effective reaction.

To achieve these goals, the present invention solves its technical problem and is adopted the technical scheme that：

A kind of bovine respiratory born of the same parents zoarium viral antigen proteins, the fit virus key protein F- albumen wild species of coding bovine respiratory born of the same parentsAminoacid sequence abbreviation SEQ AAB28458.1, particular sequence isMGTTAMRMVISIIFISTYVTHITLCQNITEEFYQSTCSAVSRGYLSALRTGWYTSVVTIELSKIQKNVCKSTDSKVKLIKQELERYNNAVIELQSLMQNEPASFSRAKRGIPELIHYPRNSTKRFYGLMGKKRKRRFLGFLLGIGSAIASGVAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKELLPKVNNHDCRISNIGTVIEFQQKNNRLLEIAREFSVNAGITTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSVVKEEVIAYEVQLPIYGVIDTPCWKIHTSPLCTTDNKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPTDVNLCNTDIFNTKYDCKIMTSKTDISSSVITSIGAIVSCYGKTKCTASNKNRGIIKTFPIGCDYVSNKGVDTVSVGNTLYYVNKLEGKALYIKGEPIINYYDPLVFPSDEFDASIAQVNAKINQSLAFIRRSDELLHSVDVGKSTTNVVITTIIIVIVVVILMLIAVGLLFYCKTRSTPIMLGKDQLSGINNLSFSK, it is characterised in that：The bovine respiratory born of the same parents zoarium viral antigen proteinsAminoacid sequence compare at least two sites of SEQ AAB28458.1 and substituted by cysteine, and the cysteine after substitutingBetween by disulfide bond.

Used as optimized, the site is the 148th L-Valine of aminoacid sequence and 288 isoleucine.

Used as optimized, the site is the 158th Leucine of aminoacid sequence and 290 serines.

Used as optimized, the aminoacid sequence of the bovine respiratory born of the same parents zoarium viral antigen proteins compares SEQAAB28458.1, is substituted by cysteine in the Leucine in the 260th site, and realizes hole filling in the site.

Used as optimized, the aminoacid sequence for bovine respiratory born of the same parents zoarium viral antigen proteins rejects SEQThe aminoacid sequence in 103 sites in AAB28458.1 sequence to 138 sites, connects 102 sites and 139 sites using sgsgs.

Used as optimized, the aminoacid sequence for bovine respiratory born of the same parents zoarium viral antigen proteins rejects SEQThe aminoacid sequence in 103 sites in AAB28458.1 sequence to 142 sites, connects 102 sites and 143 sites using sgsgs.

Used as optimized, the aminoacid sequence of the coding bovine respiratory born of the same parents zoarium viral antigen proteins compares SEQAAB28458.1 sequence, the 99th site aspartic acid is substituted by cysteine, and the 362nd site serine is substituted by cysteine,And between the cysteine after replacement connect in disulfide bond.

A kind of preparation method of bovine respiratory born of the same parents zoarium viral antigen proteins, it is characterised in that：Concrete technology is as follows：

（1）According to the principle of structure biology, protein crystal is carried out, observed with X-ray after crystallization, determine and infect virus keyCause of disease F- albumen infect before and after structure change；

（2）According to step（1）Structure change designs the sequence of antigen protein and structure；

（3）Determine and need the qualification of antigen protein through multiple antigen tests and stability characteristic detection；

（4）Antigen protein sequence through checking is carried out corresponding albumen synthesis, expression, purification and is detected, obtain for makingFor animal experiment and clinical trial for bovine respiratory born of the same parents zoarium viral antigen proteins.

The protein mediated virus of cattle born of the same parents zoarium virus key F- and the fusion of cell membrane, allow viral nucleocapsid enter cell.PinAntigen protein to bovine respiratory born of the same parents zoarium viral design, is that the structure change before and after being infected according to F- albumen sets,State before making F- protein structure remain at fusion on the basis of genetic engineering so as to only infecting not pathogenic and drawRise animal body antibody highly effective reaction proteantigen the features such as.

The present invention can make F- albumen keep trimer furcella state before and after infecting（Closure state）, only infectedProteantigen that is not pathogenic and causing animal's antibody highly effective reaction；In order to ensure that coming from the F- albumen on pathogenic virus is inStable preinfective state, to the connects chain for adding series on F- albumen, including interior disulfide bond, disulfide bond, hole fillingWith single-stranded connection.

Compared with prior art, its advantage is the present invention：The bovine respiratory for being prepared using protein crystallography methodBorn of the same parents' zoarium viral antigen proteins can effectively prevent and treat the fit virus infection of bovine respiratory born of the same parents, can open based on thisSend preferable viral vaccine；The loss that effective control and strick precaution virus infection bring, with great economic implications and societyMeaning.

Description of the drawings

Fig. 1 the present invention relates to the use of Phenix software and carry out the 3-D solid structure simulation of cattle syncytial viruses F- albumenFigure, left figure is trimer 3-D solid structure simulation drawing before contaminating, and right figure is the partial enlarged drawing of left figure lower right corner square frame position.

Fig. 2 cattle syncytial viruses Respiroviruses according to the present invention infect the F- protein structure of the key of before and after's processChange schematic diagram.Before in figure shows and infects（Left）, F- albumen trimer assumes trimer furcella state, after infecting（Right）F- eggIt is activated in vain, occurred conformation changes, the fusion of virus and cell membrane, viral nucleocapsid enters Cytoplasm, and then infects cattle body simultaneouslySo which is caused a disease.

Fig. 3 Electronic Speculum for infecting Niu Tihou through the successful bovine respiratory born of the same parents zoarium proteantigen of transformation according to the present inventionPhoto state.Left figure shows that the bovine respiratory born of the same parents zoarium proteantigen of RD-BRSV-DB1-CF1 transformation infects the state of Niu Tihou,It can be seen that assuming trimer furcella state before stable fusion, right figure is the bovine respiratory born of the same parents zoarium virus infection cattle without transformationElectromicroscopic photograph after body.

Add connects chain schematic diagram on Fig. 4 F- albumen according to the present invention, including interior disulfide bond, disulfide bond, hole fillingWith single-stranded connection, these links guarantee F- albumen holding trimer furcella state.

Specific embodiment

Below by embodiment and combine accompanying drawing the present invention is further described.

Embodiment 1：SEQ AAB28458.1 is carried out a kind of bovine respiratory born of the same parents zoarium disease that following transformation obtains the present inventionMalicious antigen protein：

Bovine respiratory born of the same parents zoarium virus F- protein amino acid sequence SEQ the 148th L-Valine of AAB28458.1 is replaced by cysteineIn generation, the 288th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond, is carried out corresponding albumen conjunctionBecome, express, purify, the bovine respiratory born of the same parents zoarium viral antigen proteins abbreviation RD-BRSV-DB1 for obtaining, its aminoacid sequence is referred to asFor SEQ DB1.

Bovine respiratory born of the same parents zoarium virus F- protein amino acid sequence SEQ the 158th Leucine of AAB28458.1 is by half Guang ammoniaAcid is substituted, and the 290th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond, is carried out corresponding eggWhite synthesis, expression, purification, the bovine respiratory born of the same parents zoarium viral antigen proteins abbreviation RD-BRSV-DB2 for obtaining, its aminoacid sequenceReferred to as SEQ DB2.

Bovine respiratory born of the same parents zoarium virus F- protein amino acid sequence SEQ the 148th L-Valine of AAB28458.1 is by half Guang ammoniaAcid is substituted, and the 288th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond, while by the 260th siteLeucine is substituted by cysteine, and realizes hole filling in the site, is carried out corresponding albumen synthesis, expression, purification,The bovine respiratory born of the same parents zoarium viral antigen proteins abbreviation RD-BRSV-DB1-CF1 for obtaining, its aminoacid sequence is referred to as SEQDB1-CF1.

Bovine respiratory born of the same parents zoarium virus F- protein amino acid sequence SEQ the 158th Leucine of AAB28458.1 is by half Guang ammoniaAcid is substituted, and the 290th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond, while by the 260th siteLeucine is substituted by cysteine, and realizes hole filling in the site, is carried out corresponding albumen synthesis, expression, purification,The bovine respiratory born of the same parents zoarium viral antigen proteins abbreviation RD-BRSV-DB2-CF1 for obtaining, its aminoacid sequence is referred to as SEQDB2-CF1.

Bovine respiratory born of the same parents zoarium virus F- protein amino acid sequence SEQ the 148th L-Valine of AAB28458.1 is by half Guang ammoniaAcid is substituted, and the 288th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond；Simultaneously by the 260th siteLeucine is substituted by cysteine, and realizes hole filling in the site；Reject 103 to site in SEQ AAB28458.1 sequenceThe aminoacid sequence in 138 sites, connects 102 and 139 sites using sgsgs, is carried out corresponding albumen synthesis, express, carryBovine respiratory born of the same parents zoarium viral antigen proteins abbreviation RD-BRSV-DB1-CF1-SC1 that is pure, obtaining, its aminoacid sequence is referred to asSEQ DB1-CF1-SC1.

Bovine respiratory born of the same parents zoarium virus F- protein amino acid sequence SEQ the 158th Leucine of AAB28458.1 is by half Guang ammoniaAcid is substituted, and the 290th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond, while by the 260th siteLeucine is substituted by cysteine, and realizes hole filling in the site；Reject 103 to site in SEQ AAB28458.1 sequenceThe aminoacid sequence in 138 sites, connects 102 and 139 sites using sgsgs, is carried out corresponding albumen synthesis, express, carryBovine respiratory born of the same parents zoarium viral antigen proteins abbreviation RD-BRSV-DB2-CF1-SC1 that is pure, obtaining, its aminoacid sequence is referred to asSEQ DB2-CF1-SC1.

Bovine respiratory born of the same parents zoarium virus F- protein amino acid sequence SEQ the 148th L-Valine of AAB28458.1 is by half Guang ammoniaAcid is substituted, and the 288th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond；Simultaneously by the 260th siteLeucine is substituted by cysteine, and realizes hole filling in the site；Reject 103 to site in SEQ AAB28458.1 sequenceThe aminoacid sequence in 138 sites, connects 102 and 139 sites using sgsgs, and the 99th site aspartic acid is substituted by cysteine,362nd site serine is substituted by cysteine, and disulfide bond in connecting between the cysteine after replacement, is carried out correspondingAlbumen synthesis, expression, purification, the bovine respiratory born of the same parents zoarium viral antigen proteins abbreviation RD-BRSV-DB1-CF1-SC1- for obtainingID1, its aminoacid sequence is referred to as SEQ DB1-CF1-SC1-ID1.

Bovine respiratory born of the same parents zoarium virus F- protein amino acid sequence SEQ the 148th L-Valine of AAB28458.1 is by half Guang ammoniaAcid is substituted, and the 288th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond；Simultaneously by the 260th siteLeucine is substituted by cysteine, and realizes hole filling in the site；Reject 103 to site in SEQ AAB28458.1 sequenceThe aminoacid sequence in 142 sites, connects 102 and 143 sites using sgsgs, and the 99th site aspartic acid is substituted by cysteine,362nd site serine is substituted by cysteine, and disulfide bond in connecting between the cysteine after replacement, is carried out correspondingAlbumen synthesis, expression, purification, the bovine respiratory born of the same parents zoarium viral antigen proteins abbreviation RD-BRSV-DB1-CF1-SC2- for obtainingID1, its aminoacid sequence is referred to as SEQ DB1-CF1-SC2-ID1.

Bovine respiratory born of the same parents zoarium virus F- protein amino acid sequence SEQ the 158th Leucine of AAB28458.1 is by half Guang ammoniaAcid is substituted, and the 290th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond, while by the 260th siteLeucine is substituted by cysteine, and realizes hole filling in the site；Reject 103 to site in SEQ AAB28458.1 sequenceThe aminoacid sequence in 138 sites, connects 102 and 139 sites using sgsgs；99th site aspartic acid is substituted by cysteine,362nd site serine is substituted by cysteine, and disulfide bond in connecting between the cysteine after replacement.Which carries out correspondingAlbumen synthesis, expression, purification, the bovine respiratory born of the same parents zoarium viral antigen proteins abbreviation RD-BRSV-DB2-CF1-SC1- for obtainingID1, its aminoacid sequence is referred to as SEQ DB2-CF1-SC1-ID1.

Bovine respiratory born of the same parents zoarium virus F- protein amino acid sequence SEQ the 158th Leucine of AAB28458.1 is by half Guang ammoniaAcid is substituted, and the 290th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond, while by the 260th siteLeucine is substituted by cysteine, and realizes hole filling in the site；Reject 103 to site in SEQ AAB28458.1 sequenceThe aminoacid sequence in 142 sites, connects 102 and 143 sites using sgsgs；99th site aspartic acid is substituted by cysteine,362nd site serine is substituted by cysteine, and disulfide bond in connecting between the cysteine after replacement.Which carries out correspondingAlbumen synthesis, expression, purification, the bovine respiratory born of the same parents zoarium viral antigen proteins abbreviation RD-BRSV-DB2-CF1-SC2- for obtainingID1, its aminoacid sequence is referred to as SEQ DB2-CF1-SC2-ID1.

Also known as antigenicity, immunogenicity refers to that antigenic stimulus body produces the characteristic of immunne response ability.Immunoreactivity refers toAntigen molecule can be with the product of corresponding immunne response（Antibody or primed lymphocyte）, specifically bind in vivo or in vitroPerformance, the affinity-immunity using ELISA method to antigen with antibody tests.ELISA method has sensitive, special, simpleSingly, quickly the features such as, stablizing and be easily operated automatically, as a new technique in immunologic diagnosises, is applied successfully toMacromole antigen and the quantitative determination of small molecule antigens.The antibody of high affinity and the adhesion of antigen are strong, i.e., antigen concentration is veryAlso more antibodies bind antigen forms immune complex when low.Affinity is represented with equilibrium constant K, and K value is bigger, affineProperty is higher, also more firm with antigen binding.Table 1 give the present invention preparation antigen protein respectively Site, SiteQP,The affinity data of Site V, II site of Site and antibodies.

The Characteristics Detection of 1 embodiment bovine respiratory born of the same parents of table zoarium viral antigen proteins

The present invention has higher antibody library affinity through the F- albumen of design improvement.All protect through improved antigen proteinHold trimer furcella state before fusion（Trimer）, and purpose antigen albumen can be obtained.

The present invention is obtained the antigen protein of purification and is specifically bound with D25 antibody, through protein immunization electrophoresis and ELISAMethod is detected, as a result shows not only there is preferable immunogenicity, and can inducing mouse produce higher antibody horizontal, table2 give the immunogenicity weighted mean that D25 antibody is specifically bound with antigen protein of the present invention, and matched group is unmanifest open countryNon-hibernating eggs.

The Characteristics Detection that 2 embodiment bovine respiratory born of the same parents of table zoarium viral antigen proteins are specifically bound with D25

Physical and chemical stability is carried out to the antigen protein in the present invention to be measured, including aspects such as temperature, pH, osmotic pressuries,Detailed data is shown in Table 3, and in table, data are the active ratio with optimal antigen protein activity of antigen protein under corresponding conditionses, knotFruit shows to assume preferable stability through the antigen protein of design improvement to temperature, pH value, osmotic pressure etc., with higher anti-Should activity.

The reactivity stability of 3 embodiment bovine respiratory born of the same parents of table zoarium viral antigen proteins

Embodiment 2：Prepare the concrete technology of RD-BRSV-DB1-CF1-SC1-ID1 in embodiment 1.See albumen mechanism and shapeCan be by following three kinds of approach, respectively x- ray（x-ray Crystallography）, nuclear magnetic resonance, NMR（NMR）, Electronic Speculum,The present invention is observed to protein structure using x-ray method.Serial by target egg using 600 plux of Solution makerCrystallized in vain, the target protein after crystallization is adopted X-ray system（SER-CAT22）Crystallization volume data is collected, and becomes whichPicture, obtains the electronic cloud of protein structure, obtains the data variation before and after key protein F- albumen infects, and infects front-end geometry changeChange as shown in Figure 2.

Data processing of racking is entered to above-mentioned electronic cloud, using corresponding software（Phenix）Carry out with wild protein sequenceDigital simulation, forms the 3-D solid structure simulation figure of protein structure.According to the three-dimensional protein structure that protein sequence is simulatedFigure obtains the Trimeric structures of F- albumen, refers to Fig. 1.

According to the principle of crystal chemistry, antigen protein structure design is carried out using simulation softward, its design is included as Fig. 4 instituteThe four kinds of modes that shows：Interior disulfide bond（intra disulphide bond）, disulfide bond（disulphide bond）, hole filling（cavity filling）With single-stranded connection（single chain）, wild species AAB28458.1 is designed.

The protein sequence for designing is translated into DNA sequence, the Expi293F conduct for providing using invitrogen companyCell carrier carries out in 293Fectin culture medium cultivating 5 days, collects cell culture fluid, target protein is carried out purificationObtain the BRSV F- albumen of purification for designing, i.e. RD-BRSV-DB1-CF1-SC1-ID1, improved bovine respiratory born of the same parents closeBody protein antigen infects trimer furcella state before Niu Tihou assumes stable fusion and sees Fig. 3（Left）.

Above-mentioned specific embodiment is only the concrete case of the present invention, and the scope of patent protection of the present invention is included but is not limited toThe product form of above-mentioned specific embodiment and style, a kind of any bovine respiratory born of the same parents zoarium for meeting claims of the present inventionAppropriate change or modification that viral antigen proteins and any person of an ordinary skill in the technical field are done to which, should all fall intoThe scope of patent protection of the present invention.

SEQUENCE LISTING

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<120>A kind of bovine respiratory born of the same parents zoarium viral antigen proteins

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Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

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Arg Gln Gln Ser Tyr Ser Ile Met Cys Val Val Lys Glu Glu Val Ile

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Ala Tyr Glu Val Gln Leu Pro Ile Tyr Gly Val Ile Asp Thr Pro Cys

275 280 285

Trp Lys Ile His Thr Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly

290 295 300

Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn

305 310 315 320

Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln

325 330 335

Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr

340 345 350

Asp Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Thr Lys Tyr Asp Cys

355 360 365

Lys Ile Met Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr Ser

370 375 380

Ile Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser

385 390 395 400

Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Pro Ile Gly Cys Asp Tyr

405 410 415

Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr

420 425 430

Tyr Val Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro

435 440 445

Ile Ile Asn Tyr Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp

450 455 460

Ala Ser Ile Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe

465 470 475 480

Ile Arg Arg Ser Asp Glu Leu Leu His

485

<210> 4

<211> 489

<212> PRT

<213>Artificial sequence

<400> 4

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

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

20 25 30

Thr Ile Glu Leu Ser Lys Ile Gln Lys Asn Val Cys Lys Ser Thr Asp

35 40 45

Ser Lys Val Lys Leu Ile Lys Gln Glu Leu Glu Arg Tyr Asn Asn Ala

50 55 60

Val Ile Glu Leu Gln Ser Leu Met Gln Asn Glu Pro Ala Ser Phe Ser

65 70 75 80

Arg Ala Lys Arg Gly Ile Pro Glu Leu Ile His Tyr Pro Arg Asn Ser

85 90 95

Thr Lys Arg Phe Tyr Gly Leu Met Gly Lys Lys Arg Lys Arg Arg Phe

100 105 110

Leu Gly Phe Leu Leu Gly Ile Gly Ser Ala Cys Ala Ser Gly Val Ala

115 120 125

Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys Asn

130 135 140

Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val

145 150 155 160

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

165 170 175

Glu Leu Leu Pro Lys Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile

180 185 190

Gly Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile

195 200 205

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

210 215 220

Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Phe Ile Asn Asp Met Pro

225 230 235 240

Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Ser Asn Val Gln Ile Val

245 250 255

Arg Gln Gln Ser Tyr Ser Cys Met Ser Val Val Lys Glu Glu Val Ile

260 265 270

Ala Tyr Glu Val Gln Leu Pro Ile Tyr Gly Val Ile Asp Thr Pro Cys

275 280 285

Trp Lys Ile His Thr Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly

290 295 300

Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn

305 310 315 320

Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln

325 330 335

Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr

340 345 350

Asp Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Thr Lys Tyr Asp Cys

355 360 365

Lys Ile Met Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr Ser

370 375 380

Ile Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser

385 390 395 400

Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Pro Ile Gly Cys Asp Tyr

405 410 415

Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr

420 425 430

Tyr Val Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro

435 440 445

Ile Ile Asn Tyr Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp

450 455 460

Ala Ser Ile Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe

465 470 475 480

Ile Arg Arg Ser Asp Glu Leu Leu His

485

<210> 5

<211> 489

<212> PRT

<213>Artificial sequence

<400> 5

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

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

20 25 30

Thr Ile Glu Leu Ser Lys Ile Gln Lys Asn Val Cys Lys Ser Thr Asp

35 40 45

Ser Lys Val Lys Leu Ile Lys Gln Glu Leu Glu Arg Tyr Asn Asn Ala

50 55 60

Val Ile Glu Leu Gln Ser Leu Met Gln Asn Glu Pro Ala Ser Phe Ser

65 70 75 80

Arg Ala Lys Arg Gly Ile Pro Glu Leu Ile His Tyr Pro Arg Asn Ser

85 90 95

Thr Lys Arg Phe Tyr Gly Leu Met Gly Lys Lys Arg Lys Arg Arg Phe

100 105 110

Leu Gly Phe Leu Leu Gly Ile Gly Ser Ala Ile Ala Ser Gly Val Ala

115 120 125

Val Ser Lys Val Cys His Leu Glu Gly Glu Val Asn Lys Ile Lys Asn

130 135 140

Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val

145 150 155 160

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

165 170 175

Glu Leu Leu Pro Lys Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile

180 185 190

Gly Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile

195 200 205

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

210 215 220

Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Phe Ile Asn Asp Met Pro

225 230 235 240

Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Ser Asn Val Gln Ile Val

245 250 255

Arg Gln Gln Ser Tyr Ser Ile Met Cys Val Val Lys Glu Glu Val Ile

260 265 270

Ala Tyr Glu Val Gln Leu Pro Ile Tyr Gly Val Ile Asp Thr Pro Cys

275 280 285

Trp Lys Ile His Thr Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly

290 295 300

Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn

305 310 315 320

Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln

325 330 335

Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr

340 345 350

Asp Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Thr Lys Tyr Asp Cys

355 360 365

Lys Ile Met Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr Ser

370 375 380

Ile Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser

385 390 3 95 400

Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Pro Ile Gly Cys Asp Tyr

405 410 415

Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr

420 425 430

Tyr Val Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro

435 440 445

Ile Ile Asn Tyr Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp

450 455 460

Ala Ser Ile Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe

465 470 475 480

Ile Arg Arg Ser Asp Glu Leu Leu His

485

<210> 6

<211> 458

<212> PRT

<213>Artificial sequence

<400> 6

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

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

20 25 30

Thr Ile Glu Leu Ser Lys Ile Gln Lys Asn Val Cys Lys Ser Thr Asp

35 40 45

Ser Lys Val Lys Leu Ile Lys Gln Glu Leu Glu Arg Tyr Asn Asn Ala

50 55 60

Val Ile Glu Leu Gln Ser Leu Met Gln Asn Glu Pro Ala Ser Gly Ser

65 70 75 80

Gly Ser Gly Phe Leu Leu Gly Ile Gly Ser Ala Cys Ala Ser Gly Val

85 90 95

Ala Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys

100 105 110

Asn Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

115 120 125

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

130 135 140

Lys Glu Leu Leu Pro Lys Val Asn Asn His Asp Cys Arg Ile Ser Asn

145 150 155 160

Ile Gly Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu

165 170 175

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

180 185 190

Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Phe Ile Asn Asp Met

195 200 205

Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Ser Asn Val Gln Ile

210 215 220

Val Arg Gln Gln Ser Tyr Ser Cys Met Ser Val Val Lys Glu Glu Val

225 230 235 240

Ile Ala Tyr Glu Val Gln Leu Pro Ile Tyr Gly Val Ile Asp Thr Pro

245 250 255

Cys Trp Lys Ile His Thr Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu

260 265 270

Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp

275 280 285

Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val

290 295 300

Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro

305 310 315 320

Thr Asp Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Thr Lys Tyr Asp

325 330 335

Cys Lys Ile Met Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr

340 345 350

Ser Ile Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala

355 360 365

Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Pro Ile Gly Cys Asp

370 375 380

Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu

385 390 395 400

Tyr Tyr Val Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu

405 410 415

Pro Ile Ile Asn Tyr Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe

420 425 430

Asp Ala Ser Ile Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala

435 440 445

Phe Ile Arg Arg Ser Asp Glu Leu Leu His

450 455

<210> 7

<211> 454

<212> PRT

<213>Artificial sequence

<400> 7

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

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

20 25 30

Thr Ile Glu Leu Ser Lys Ile Gln Lys Asn Val Cys Lys Ser Thr Asp

35 40 45

Ser Lys Val Lys Leu Ile Lys Gln Glu Leu Glu Arg Tyr Asn Asn Ala

50 55 60

Val Ile Glu Leu Gln Ser Leu Met Gln Asn Glu Pro Ala Ser Gly Ser

65 70 75 80

Gly Ser Gly Ile Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys

85 90 95

Val Cys His Leu Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu

100 105 110

Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu

115 120 125

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

130 135 140

Pro Lys Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile Gly Thr Val

145 150 155 160

Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile Ala Arg Glu

165 170 175

Phe Ser Val Asn Ala Gly Ile Thr Thr Pro Leu Ser Thr Tyr Met Leu

180 185 190

Thr Asn Ser Glu Leu Leu Ser Phe Ile Asn Asp Met Pro Ile Thr Asn

195 200 205

Asp Gln Lys Lys Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln

210 215 220

Ser Tyr Ser Ile Met Cys Val Val Lys Glu Glu Val Ile Ala Tyr Glu

225 230 235 240

Val Gln Leu Pro Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Ile

245 250 255

His Thr Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly Ser Asn Ile

260 265 270

Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser

275 280 285

Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg

290 295 300

Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp Val Asn

305 310 315 320

Leu Cys Asn Thr Asp Ile Phe Asn Thr Lys Tyr Asp Cys Lys Ile Met

325 330 335

Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr Ser Ile Gly Ala

340 345 350

Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn

355 360 365

Arg Gly Ile Ile Lys Thr Phe Pro Ile Gly Cys Asp Tyr Val Ser Asn

370 375 380

Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn

385 390 395 400

Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile Ile Asn

405 410 415

Tyr Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile

420 425 430

Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg

435 440 445

Ser Asp Glu Leu Leu His

450

<210> 8

<211> 458

<212> PRT

<213>Artificial sequence

<400> 8

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

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

20 25 30

Thr Ile Glu Leu Ser Lys Ile Gln Lys Asn Val Cys Lys Ser Thr Asp

35 40 45

Ser Lys Val Lys Leu Ile Lys Gln Glu Leu Glu Arg Tyr Asn Asn Ala

50 55 60

Val Ile Glu Leu Gln Ser Leu Met Gln Cys Glu Pro Ala Ser Gly Ser

65 70 75 80

Gly Ser Gly Phe Leu Leu Gly Ile Gly Ser Ala Cys Ala Ser Gly Val

85 90 95

Ala Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys

100 105 110

Asn Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

115 120 125

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

130 135 140

Lys Glu Leu Leu Pro Lys Val Asn Asn His Asp Cys Arg Ile Ser Asn

145 150 155 160

Ile Gly Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu

165 170 175

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

180 185 190

Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Phe Ile Asn Asp Met

195 200 205

Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Ser Asn Val Gln Ile

210 215 220

Val Arg Gln Gln Ser Tyr Ser Cys Met Ser Val Val Lys Glu Glu Val

225 230 235 240

Ile Ala Tyr Glu Val Gln Leu Pro Ile Tyr Gly Val Ile Asp Thr Pro

245 250 255

Cys Trp Lys Ile His Thr Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu

260 265 270

Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp

275 280 285

Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val

290 295 300

Gln Cys Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro

305 310 315 320

Thr Asp Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Thr Lys Tyr Asp

325 330 335

Cys Lys Ile Met Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr

340 345 350

Ser Ile Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala

355 360 365

Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Pro Ile Gly Cys Asp

370 375 380

Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu

385 390 395 400

Tyr Tyr Val Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu

405 410 415

Pro Ile Ile Asn Tyr Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe

420 425 430

Asp Ala Ser Ile Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala

435 440 445

Phe Ile Arg Arg Ser Asp Glu Leu Leu His

450 455

<210> 9

<211> 454

<212> PRT

<213>Artificial sequence

<400> 9

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

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

20 25 30

Thr Ile Glu Leu Ser Lys Ile Gln Lys Asn Val Cys Lys Ser Thr Asp

35 40 45

Ser Lys Val Lys Leu Ile Lys Gln Glu Leu Glu Arg Tyr Asn Asn Ala

50 55 60

Val Ile Glu Leu Gln Ser Leu Met Gln Cys Glu Pro Ala Ser Gly Ser

65 70 75 80

Gly Ser Gly Ile Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys

85 90 95

Val Cys His Leu Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu

100 105 110

Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu

115 120 125

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

130 135 140

Pro Lys Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile Gly Thr Val

145 150 155 160

Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile Ala Arg Glu

165 170 175

Phe Ser Val Asn Ala Gly Ile Thr Thr Pro Leu Ser Thr Tyr Met Leu

180 185 190

Thr Asn Ser Glu Leu Leu Ser Phe Ile Asn Asp Met Pro Ile Thr Asn

195 200 205

Asp Gln Lys Lys Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln

210 215 220

Ser Tyr Ser Ile Met Cys Val Val Lys Glu Glu Val Ile Ala Tyr Glu

225 230 235 240

Val Gln Leu Pro Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Ile

245 250 255

His Thr Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly Ser Asn Ile

260 265 270

Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser

275 280 285

Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Cys Asn Arg

290 295 300

Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp Val Asn

305 310 315 320

Leu Cys Asn Thr Asp Ile Phe Asn Thr Lys Tyr Asp Cys Lys Ile Met

325 330 335

Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr Ser Ile Gly Ala

340 345 350

Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn

355 360 365

Arg Gly Ile Ile Lys Thr Phe Pro Ile Gly Cys Asp Tyr Val Ser Asn

370 375 380

Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn

385 390 395 400

Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile Ile Asn

405 410 415

Tyr Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile

420 425 430

Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg

435 440 445

Ser Asp Glu Leu Leu His

450

<210> 10

<211> 458

<212> PRT

<213>Artificial sequence

<400> 10

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

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

20 25 30

Thr Ile Glu Leu Ser Lys Ile Gln Lys Asn Val Cys Lys Ser Thr Asp

35 40 45

Ser Lys Val Lys Leu Ile Lys Gln Glu Leu Glu Arg Tyr Asn Asn Ala

50 55 60

Val Ile Glu Leu Gln Ser Leu Met Gln Cys Glu Pro Ala Ser Gly Ser

65 70 75 80

Gly Ser Gly Phe Leu Leu Gly Ile Gly Ser Ala Cys Ala Ser Gly Val

85 90 95

Ala Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys

100 105 110

Asn Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

115 120 125

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

130 135 140

Lys Glu Leu Leu Pro Lys Val Asn Asn His Asp Cys Arg Ile Ser Asn

145 150 155 160

Ile Gly Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu

165 170 175

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

180 185 190

Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Phe Ile Asn Asp Met

195 200 205

Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Ser Asn Val Gln Ile

210 215 220

Val Arg Gln Gln Ser Tyr Ser Cys Met Ser Val Val Lys Glu Glu Val

225 230 235 240

Ile Ala Tyr Glu Val Gln Leu Pro Ile Tyr Gly Val Ile Asp Thr Pro

245 250 255

Cys Trp Lys Ile His Thr Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu

260 265 270

Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp

275 280 285

Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val

290 295 300

Gln Cys Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro

305 310 315 320

Thr Asp Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Thr Lys Tyr Asp

325 330 335

Cys Lys Ile Met Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr

340 345 350

Ser Ile Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala

355 360 365

Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Pro Ile Gly Cys Asp

370 375 380

Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu

385 390 395 400

Tyr Tyr Val Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu

405 410 415

Pro Ile Ile Asn Tyr Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe

420 425 430

Asp Ala Ser Ile Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala

435 440 445

Phe Ile Arg Arg Ser Asp Glu Leu Leu His

450 455

<210> 11

<211> 454

<212> PRT

<213>Artificial sequence

<400> 11

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

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

20 25 30

Thr Ile Glu Leu Ser Lys Ile Gln Lys Asn Val Cys Lys Ser Thr Asp

35 40 45

Ser Lys Val Lys Leu Ile Lys Gln Glu Leu Glu Arg Tyr Asn Asn Ala

50 55 60

Val Ile Glu Leu Gln Ser Leu Met Gln Cys Glu Pro Ala Ser Gly Ser

65 70 75 80

Gly Ser Gly Ile Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys

85 90 95

Val Cys His Leu Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu

100 105 110

Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu

115 120 125

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

130 135 140

Pro Lys Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile Gly Thr Val

145 150 155 160

Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile Ala Arg Glu

165 170 175

Phe Ser Val Asn Ala Gly Ile Thr Thr Pro Leu Ser Thr Tyr Met Leu

180 185 190

Thr Asn Ser Glu Leu Leu Ser Phe Ile Asn Asp Met Pro Ile Thr Asn

195 200 205

Asp Gln Lys Lys Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln

210 215 220

Ser Tyr Ser Ile Met Cys Val Val Lys Glu Glu Val Ile Ala Tyr Glu

225 230 235 240

Val Gln Leu Pro Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Ile

245 250 255

His Thr Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly Ser Asn Ile

260 265 270

Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser

275 280 285

Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Cys Asn Arg

290 295 300

Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp Val Asn

305 310 315 320

Leu Cys Asn Thr Asp Ile Phe Asn Thr Lys Tyr Asp Cys Lys Ile Met

325 330 335

Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr Ser Ile Gly Ala

340 345 350

Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn

355 360 365

Arg Gly Ile Ile Lys Thr Phe Pro Ile Gly Cys Asp Tyr Val Ser Asn

370 375 380

Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn

385 390 395 400

Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile Ile Asn

405 410 415

Tyr Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile

420 425 430

Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg

435 440 445

Ser Asp Glu Leu Leu His

450

Claims (8)

1. a kind of bovine respiratory born of the same parents zoarium viral antigen proteins, encode the fit virus key protein F- albumen of bovine respiratory born of the same parents wildThe aminoacid sequence that plants is abbreviation SEQ AAB28458.1, and particular sequence isMGTTAMRMVISIIFISTYVTHITLCQNITEEFYQSTCSAVSRGYLSALRTGWYTSVVTIELSKIQKNVCKSTDSKVKLIKQELERYNNAVIELQSLMQNEPASFSRAKRGIPELIHYPRNSTKRFYGLMGKKRKRRFLGFLLGIGSAIASGVAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKELLPKVNNHDCRISNIGTVIEFQQKNNRLLEIAREFSVNAGITTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSVVKEEVIAYEVQLPIYGVIDTPCWKIHTSPLCTTDNKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPTDVNLCNTDIFNTKYDCKIMTSKTDISSSVITSIGAIVSCYGKTKCTASNKNRGIIKTFPIGCDYVSNKGVDTVSVGNTLYYVNKLEGKALYIKGEPIINYYDPLVFPSDEFDASIAQVNAKINQSLAFIRRSDELLHSVDVGKSTTNVVITTIIIVIVVVILMLIAVGLLFYCKTRSTPIMLGKDQLSGINNLSFSK, it is characterised in that：The bovine respiratory born of the same parents zoarium viral antigen proteinsAminoacid sequence compare at least two sites of SEQ AAB28458.1 and substituted by cysteine, and the cysteine after substitutingBetween by disulfide bond.

2. a kind of bovine respiratory born of the same parents zoarium viral antigen proteins according to claim 1, it is characterised in that：The site isThe 148th L-Valine of aminoacid sequence and 288 isoleucine.

3. a kind of bovine respiratory born of the same parents zoarium viral antigen proteins according to claim 1, it is characterised in that：The site isThe 158th Leucine of aminoacid sequence and 290 serines.

4. a kind of fit viral antigen proteins of bovine respiratory born of the same parents according to claim 1, it is characterised in that：The cattle exhalesThe aminoacid sequence for inhaling road born of the same parents zoarium viral antigen proteins compares SEQ AAB28458.1, the 260th site Leucine by halfCystine is substituted, and realizes hole filling in the site.

5. the fit viral antigen proteins of any one bovine respiratory born of the same parents according to claim 1 ~ 3, it is characterised in that：DescribedFor 103 sites in the aminoacid sequence rejecting SEQ AAB28458.1 sequence of bovine respiratory born of the same parents zoarium viral antigen proteins extremelyThe aminoacid sequence in 138 sites, connects 102 sites and 139 sites using sgsgs.

6. the fit viral antigen proteins of any one bovine respiratory born of the same parents according to claim 1 ~ 3, it is characterised in that：DescribedFor 103 sites in the aminoacid sequence rejecting SEQ AAB28458.1 sequence of bovine respiratory born of the same parents zoarium viral antigen proteins extremelyThe aminoacid sequence in 142 sites, connects 102 sites and 143 sites using sgsgs.

7. the fit viral antigen proteins of any one bovine respiratory born of the same parents according to claim 1 ~ 3, it is characterised in that：DescribedThe aminoacid sequence of coding bovine respiratory born of the same parents' zoarium viral antigen proteins compares SEQ AAB28458.1 sequence, the 99th site Radix AsparagiPropylhomoserin is substituted by cysteine, and the 362nd site serine is substituted by cysteine, and is connected between the cysteine after replacementInterior disulfide bond.

8. the preparation method of a kind of bovine respiratory born of the same parents zoarium viral antigen proteins, it is characterised in that：Concrete technology is as follows：

（1）According to the principle of structure biology, protein crystal is carried out, observed with X-ray after crystallization, determine and infect virus keyCause of disease F- albumen infect before and after structure change；

（2）According to step（1）Structure change designs the sequence of antigen protein and structure；

（3）Determine and need the qualification of antigen protein through multiple antigen tests and stability characteristic detection；

（4）Antigen protein sequence through checking is carried out corresponding albumen synthesis, expression, purification and is detected, obtain for makingFor animal experiment and clinical trial for bovine respiratory born of the same parents zoarium viral antigen proteins.