CN106518988A

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Info

Publication number: CN106518988A
Application number: CN201611065117.3A
Authority: CN
Inventors: 汤斌; 邱啟欲; 朱宁新; 赵斌
Original assignee: Yantai Royal Emperor Biological Engineering Co Ltd
Current assignee: Yantai Royal Emperor Biological Engineering Co Ltd
Priority date: 2016-11-28
Filing date: 2016-11-28
Publication date: 2017-03-22

Abstract

The invention discloses a bovine respiratory syncytial cell virus antigen protein. Compared with SEQ 048055.1, an amino acid sequence of the bovine respiratory syncytial cell virus antigen protein is provided with at least two loci substituted by cysteines, the substitutive cysteines are connected through a disulfide bond, improvements such as internal disulfide bond, hole filling and single stranded connection are also performed on the basis, and meanwhile a preparation method of the antigen protein is provided. The antigen protein applies the technique and theory of protein crystallography, the structural change of the protein in virus infection and pathopoiesis process is determined, and then genetic engineering modification is conducted on the key virus protein according to a structural change result, so that the protein becomes a protein antigen only infecting without pathopoiesis and causing efficient reaction of animal body antibodies. The antigen protein can effectively prevent and treat bovine respiratory syncytial cell virus infection, an ideal viral vaccine can be developed on the basis, loss brought by virus inflection can be effectively controlled and prevented, and the antigen protein has great economic significance 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 up to arrives 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 the report of various inactivations of BRSV, weak poison, restructuring and DNA vaccination, but there are certain distance, current state apart from Clinical practiceStill no 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 is 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 to not kill completely or fill in vaccine production processDivide attenuation, this contains strong toxicity morbid substance in causing 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 it is highly conserved simultaneouslyTrimer furcella is formed, which occurs conformation change under activation.The fusion of the protein mediated viruses of F- and cell membrane, allows 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 haveWhat is imitated 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, it is of the invention by structure biologyIt is applied in practice, synthesis bovine respiratory born of the same parents' zoarium viral antigen proteins.

The content of the invention

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

To achieve these goals, its technical problem of present invention solution 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 048055.1, particular sequence isMATTAMRMIISIIFISTYVTHITLCQNITEEFYQSTCSAVSRGYLSALRTGWYTSVVTIELSKIQKNVCKSTDSKVKLIKQELERYNNAVVELQSLMQNEPASFSRAKRGIPELIHYTRNSTKKFYGLMGKKRKRRFLGFLLGIGSAVASGVAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKELLPQVNNHDCRISNIETVIEFQQKNNRLLEIAREFSVNAGITTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSVVKEEVIAYVVQLPIYGVIDTPCWKLHTSPLCTTDNKEGSNICLTRTDRGWYCDNAGSVSFFPQTETCKVQSNRVFCDTMNSLTLPTDVNLCNTDIFNTKYDCKIMTSKTDISSSVITSIGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKALYIKGEPIINYYDPLVFPSDEFDASIAQVNAKINQSLAFIRRSDELLHSVDVGKSTTNVVITTIIIVIVVVILMLIAVGLLFYCKTKSTPIMLGKDQLSGINNLSFSK, 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 048055.1 by cysteine substitute, and substitute after cysteine itBetween by disulfide bond.

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

Used as optimization, the site is the 158th leucine of aminoacid sequence and 290 serines.

Used as optimization, the aminoacid sequence of the bovine respiratory born of the same parents zoarium viral antigen proteins compares SEQ048055.1, the leucine in the 260th site is substituted by cysteine, and realizes that hole is filled in the site.

Used as optimization, the aminoacid sequence for bovine respiratory born of the same parents zoarium viral antigen proteins rejects SEQThe aminoacid sequence in 103 sites in 048055.1 sequence to 138 sites, connects 102 sites and 139 sites using sgsgs.

Used as optimization, the aminoacid sequence for bovine respiratory born of the same parents zoarium viral antigen proteins rejects SEQThe aminoacid sequence in 103 sites in 048055.1 sequence to 142 sites, connects 102 sites and 143 sites using sgsgs.

Used as optimization, the aminoacid sequence of the coding bovine respiratory born of the same parents zoarium viral antigen proteins compares SEQ 048055.1Sequence, the 99th site aspartic acid are substituted by cysteine, and the 362nd site serine is substituted by cysteine, and after replacementCysteine between connection 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, it is determined that infecting viral keyCause of disease F- albumen infect before and after structure change；

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

（3）The qualification for needing antigen protein is determined in many kinds of antigen tests of Jing and stability characteristic detection；

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

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

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

Compared with prior art, its advantage is the present invention：The bovine respiratory 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 softwares and carry out the 3-D solid structure simulation of cattle syncytial viruses F- albumenFigure, left figure are trimer 3-D solid structure simulation drawing before contaminating, partial enlarged drawing of the right figure for left figure lower right corner square frame position.

Fig. 2 cattle syncytial viruses Respiroviruses according to the present invention infect the F- protein structures of the key of before and after's processChange schematic diagram.Show in figure before infecting（It is left）, F- albumen trimer is presented trimer furcella state, after infecting（It is right）F- eggsIt is activated in vain, occurred conformation change, the fusion of virus and cell membrane, viral nucleocapsid enters Cytoplasm, and then infects cattle body simultaneouslyMake which cause a disease.

Fig. 3 Electronic Speculum that Niu Tihou is infected 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 transformations infects the state of Niu Tihou,It can be seen that trimer furcella state before stable fusion is presented, 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, including interior disulfide bond, disulfide bond, hole filling on Fig. 4 F- albumen according to the present inventionWith single-stranded connection, these links guarantee F- albumen holding trimer furcella states.

Specific embodiment

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

Embodiment 1：SEQ 048055.1 is carried out into the fit virus of a kind of bovine respiratory born of the same parents that following transformation obtains the present inventionAntigen protein：

Bovine respiratory born of the same parents zoarium virus F- protein amino acid sequences the 148th L-Valine of SEQ 048055.1 is replaced by cysteineIn generation, the 288th isoleucine, are substituted by cysteine, and are connected two sites by disulfide bond, are carried out corresponding albumen conjunctionInto, expression, purification, 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 sequences the 158th leucine of SEQ 048055.1 is by cysteineSubstitute, the 290th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond, is carried out corresponding albumenSynthesis, expression, purification, the bovine respiratory born of the same parents zoarium viral antigen proteins abbreviation RD-BRSV-DB2 for obtaining, its aminoacid sequence letterReferred to as SEQ DB2.

Bovine respiratory born of the same parents zoarium virus F- protein amino acid sequences the 148th L-Valine of SEQ 048055.1 is by cysteineSubstitute, the 288th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond, while by the bright of the 260th sitePropylhomoserin is substituted by cysteine, and realizes that hole is filled in the site, is carried out corresponding albumen synthesis, expression, purification, is obtainedThe bovine respiratory born of the same parents zoarium viral antigen proteins abbreviation RD-BRSV-DB1-CF1 for arriving, its aminoacid sequence are referred to as SEQ DB1-CF1。

Bovine respiratory born of the same parents zoarium virus F- protein amino acid sequences the 158th leucine of SEQ 048055.1 is by cysteineSubstitute, the 290th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond, while by the bright of the 260th sitePropylhomoserin is substituted by cysteine, and realizes that hole is filled in the site, is carried out corresponding albumen synthesis, expression, purification, is obtainedThe bovine respiratory born of the same parents zoarium viral antigen proteins abbreviation RD-BRSV-DB2-CF1 for arriving, its aminoacid sequence are referred to as SEQ DB2-CF1。

Bovine respiratory born of the same parents zoarium virus F- protein amino acid sequences the 148th L-Valine of SEQ 048055.1 is by cysteineSubstitute, the 288th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond；Simultaneously by the bright of the 260th sitePropylhomoserin is substituted by cysteine, and realizes that hole is filled in the site；Reject 103 to site 138 in 048055.1 sequences of SEQThe aminoacid sequence in site, connects 102 and 139 sites using sgsgs, 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-SC1 for obtaining, its aminoacid sequence are referred to as SEQDB1-CF1-SC1。

Bovine respiratory born of the same parents zoarium virus F- protein amino acid sequences the 158th leucine of SEQ 048055.1 is by cysteineSubstitute, the 290th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond, while by the bright of the 260th sitePropylhomoserin is substituted by cysteine, and realizes that hole is filled in the site；Reject 103 to site 138 in 048055.1 sequences of SEQThe aminoacid sequence in site, connects 102 and 139 sites using sgsgs, 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-SC1 for obtaining, its aminoacid sequence are referred to as SEQDB2-CF1-SC1。

Bovine respiratory born of the same parents zoarium virus F- protein amino acid sequences the 148th L-Valine of SEQ 048055.1 is by cysteineSubstitute, the 288th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond；Simultaneously by the bright of the 260th sitePropylhomoserin is substituted by cysteine, and realizes that hole is filled in the site；Reject 103 to site 138 in 048055.1 sequences of SEQThe aminoacid sequence in site, connects 102 and 139 sites using sgsgs, and the 99th site aspartic acid is substituted by cysteine, the362 site serines are substituted by cysteine, and disulfide bond in connecting between the cysteine after replacement, carry out corresponding eggWhite synthesis, expression, purification, the bovine respiratory born of the same parents zoarium viral antigen proteins abbreviation RD-BRSV-DB1-CF1-SC1-ID1 for obtaining,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 sequences the 148th L-Valine of SEQ 048055.1 is by cysteineSubstitute, the 288th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond；Simultaneously by the bright of the 260th sitePropylhomoserin is substituted by cysteine, and realizes that hole is filled in the site；Reject 103 to site 142 in 048055.1 sequences of SEQThe aminoacid sequence in site, connects 102 and 143 sites using sgsgs, and the 99th site aspartic acid is substituted by cysteine, the362 site serines are substituted by cysteine, and disulfide bond in connecting between the cysteine after replacement, carry out corresponding eggWhite synthesis, expression, purification, the bovine respiratory born of the same parents zoarium viral antigen proteins abbreviation RD-BRSV-DB1-CF1-SC2-ID1 for obtaining,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 sequences the 158th leucine of SEQ 048055.1 is by cysteineSubstitute, the 290th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond, while by the bright of the 260th sitePropylhomoserin is substituted by cysteine, and realizes that hole is filled in the site；Reject 103 to site 138 in 048055.1 sequences of SEQThe aminoacid sequence in site, connects 102 and 139 sites using sgsgs；99th site aspartic acid is substituted by cysteine, the362 site serines are 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 are referred to as SEQ DB2-CF1-SC1-ID1.

Bovine respiratory born of the same parents zoarium virus F- protein amino acid sequences the 158th leucine of SEQ 048055.1 is by cysteineSubstitute, the 290th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond, while by the bright of the 260th sitePropylhomoserin is substituted by cysteine, and realizes that hole is filled in the site；Reject 103 to site 142 in 048055.1 sequences of SEQThe aminoacid sequence in site, connects 102 and 143 sites using sgsgs；99th site aspartic acid is substituted by cysteine, the362 site serines are 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 are 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）, there is specific binding in vivo or in vitroPerformance, the affinity-immunity using ELISA method to antigen with antibody tests.ELISA method has sensitive, special, simpleIt is single, quick, stable and the features such as be easily operated automatically, as immunologic diagnosises in a new technique, be applied successfully toThe quantitative determination of macromole antigen and 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 represents that with equilibrium constant K K values are 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 sites 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, Jing protein immunizations electrophoresis and ELISAMethod detects, as a result shows not only there is preferable immunogenicity, and can inducing mouse produce higher antibody horizontal, tableThe 2 immunogenicity weighted means for giving D25 antibody and antigen protein of the present invention specific binding, matched group are 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 antigen protein activity and the antigen protein activity in optimum state under corresponding conditionsesRatio, as a result show that the antigen protein through design improvement is presented preferable stability, tool to temperature, pH value, osmotic pressure etc.There is higher reactivity.

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- rays（x-ray Crystallography）, nuclear magnetic resonance, NMR（NMR）, Electronic Speculum,The present invention is observed to protein structure using x-ray method.It is serial by target egg using 600 plux of Solution makerCrystallized in vain, the target protein after crystallization is adopted into X-ray system（SER-CAT22）Collect crystallization volume data, and make its intoPicture, 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 such as Fig. 4 institutesThe four kinds of modes shown：Interior disulfide bond（intra disulphide bond）, disulfide bond（disulphide bond）, hole filling（cavity filling）With single-stranded connection（single chain）, wild species 048055.1 are designed.

The protein sequence for designing is translated into into DNA sequence, the Expi293F conducts provided using invitrogen companiesCell carrier carries out culture 5 days in 293Fectin culture medium, collects cell culture fluid, target protein is carried out purificationThe BRSV F- albumen of purification for designing is obtained, i.e. RD-BRSV-DB1-CF1-SC1-ID1, improved bovine respiratory born of the same parents are closedBody protein antigen infects trimer furcella state before Niu Tihou is presented stable fusion and sees Fig. 3（It is left）.

Above-mentioned specific embodiment be only the present invention concrete case, the present invention scope of patent protection include but is not limited toThe product form and style of above-mentioned specific embodiment, a kind of any bovine respiratory born of the same parents for meeting claims of the present invention are fitAppropriate 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

<110>Yantai Ruo Di biological engineering company limited

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

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<170> PatentIn version 3.3

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<212> PRT

<213>Bovine respiratory born of the same parents zoarium virus F- albumen wild species

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Met Ala Thr Thr Ala Met Arg Met Ile Ile Ser Ile Ile Phe Ile Ser

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Thr Tyr Val Thr His Ile Thr Leu Cys Gln Asn Ile Thr Glu Glu Phe

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

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Arg Thr Gly Trp Tyr Thr Ser Val Val Thr Ile Glu Leu Ser Lys Ile

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

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

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Met Gln Asn Glu Pro Ala Ser Phe Ser Arg Ala Lys Arg Gly Ile Pro

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Glu Leu Ile His Tyr Thr Arg Asn Ser Thr Lys Lys Phe Tyr Gly Leu

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Ala Gly Ile Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Ile Arg Arg Ser Asp Glu Leu Leu His

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

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

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

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

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

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

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

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

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

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

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

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

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

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Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu 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 Val Val Gln Leu Pro Ile Tyr Gly Val Ile Asp Thr Pro Cys

275 280 285

Trp Lys Leu 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 Thr 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 Ser Asn 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

<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 Val 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 Thr Arg Asn Ser

85 90 95

Thr Lys Lys 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 Gln Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile

180 185 190

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

275 280 285

Trp Lys Leu 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 Thr 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 Ser Asn 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

<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 Val 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 Thr Arg Asn Ser

85 90 95

Thr Lys Lys 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 Val 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 Gln Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile

180 185 190

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

275 280 285

Trp Lys Leu 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 Thr 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 Ser Asn 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

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

65 70 75 80

Gly Ser Ser Phe Ser Arg Ala Lys Arg Gly Ile Pro Glu Leu Ile His

85 90 95

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

100 105 110

Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Ile Gly Ser Ala Cys

115 120 125

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

130 135 140

Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser

145 150 155 160

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

165 170 175

Asn Tyr Ile Asp Lys Glu Leu Leu Pro Gln Val Asn Asn His Asp Cys

180 185 190

Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Cys Met Ser Val Val

260 265 270

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

275 280 285

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

290 295 300

Asp Asn Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly

305 310 315 320

Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Thr Glu

325 330 335

Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser

340 345 350

Leu Thr Leu Pro Thr Asp Val Asn Leu Cys Asn Thr Asp Ile Phe Asn

355 360 365

Thr Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Ile Ser Ser

370 375 380

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

385 390 395 400

Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser

405 410 415

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

420 425 430

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

435 440 445

Ile Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro Leu Val Phe Pro

450 455 460

Ser Asp Glu Phe Asp Ala Ser Ile Ala Gln Val Asn Ala Lys Ile Asn

465 470 475 480

Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu Leu His

<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 Val 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 Val 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 Gln Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile Glu 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 Val

225 230 235 240

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

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 Thr 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 Ser Asn 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

<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 Val 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 Gln Val Asn Asn His Asp Cys Arg Ile Ser Asn

145 150 155 160

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

245 250 255

Cys Trp Lys Leu 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 Thr 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 Ser Asn 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

<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 Val 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 Val 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 Gln Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile Glu 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 Val

225 230 235 240

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

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 Thr 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 Ser Asn 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

<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 Val 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 Gln Val Asn Asn His Asp Cys Arg Ile Ser Asn

145 150 155 160

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

245 250 255

Cys Trp Lys Leu 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 Thr 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 Ser Asn 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

<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 Val 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 Val 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 Gln Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile Glu 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 Val

225 230 235 240

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

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 Thr 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 Ser Asn 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

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 wildKind aminoacid sequence be abbreviation SEQ 048055.1, particular sequence isMATTAMRMIISIIFISTYVTHITLCQNITEEFYQSTCSAVSRGYLSALRTGWYTSVVTIELSKIQKNVCKSTDSKVKLIKQELERYNNAVVELQSLMQNEPASFSRAKRGIPELIHYTRNSTKKFYGLMGKKRKRRFLGFLLGIGSAVASGVAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKELLPQVNNHDCRISNIETVIEFQQKNNRLLEIAREFSVNAGITTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSVVKEEVIAYVVQLPIYGVIDTPCWKLHTSPLCTTDNKEGSNICLTRTDRGWYCDNAGSVSFFPQTETCKVQSNRVFCDTMNSLTLPTDVNLCNTDIFNTKYDCKIMTSKTDISSSVITSIGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKALYIKGEPIINYYDPLVFPSDEFDASIAQVNAKINQSLAFIRRSDELLHSVDVGKSTTNVVITTIIIVIVVVILMLIAVGLLFYCKTKSTPIMLGKDQLSGINNLSFSK, 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 048055.1 by cysteine substitute, and substitute after cysteine itBetween 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. according to a kind of bovine respiratory born of the same parents zoarium viral antigen proteins described in claim 1, it is characterised in that：The cattle exhalesInhale road born of the same parents zoarium viral antigen proteins aminoacid sequence compare SEQ 048055.1, the 260th site leucine by half GuangPropylhomoserin is substituted, and realizes that hole is filled in the site.

5. according to any one bovine respiratory born of the same parents zoarium viral antigen proteins described in claim 1 ~ 3, it is characterised in that：It is describedFor 103 sites in aminoacid sequence rejecting 048055.1 sequences of SEQ 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. according to any one bovine respiratory born of the same parents zoarium viral antigen proteins described in claim 1 ~ 3, it is characterised in that：It is describedFor 103 sites in aminoacid sequence rejecting 048055.1 sequences of SEQ 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. according to any one bovine respiratory born of the same parents zoarium viral antigen proteins described in claim 1 ~ 3, it is characterised in that：It is describedThe aminoacid sequence of coding bovine respiratory born of the same parents' zoarium viral antigen proteins compares 048055.1 sequences of SEQ, the 99th site Radix Asparagi ammoniaAcid is substituted by cysteine, and the 362nd site serine is substituted by cysteine, and in connecting between the cysteine after replacementDisulfide 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：