CN106518988A - Bovine respiratory syncytial cell virus antigen protein - Google Patents

Bovine respiratory syncytial cell virus antigen protein Download PDF

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CN106518988A
CN106518988A CN201611065117.3A CN201611065117A CN106518988A CN 106518988 A CN106518988 A CN 106518988A CN 201611065117 A CN201611065117 A CN 201611065117A CN 106518988 A CN106518988 A CN 106518988A
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val
leu
asn
thr
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汤斌
邱啟欲
朱宁新
赵斌
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Yantai Royal Emperor Biological Engineering Co Ltd
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Yantai Royal Emperor Biological Engineering Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18522—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

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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 its Preparation method.
Background technology
The fit virus of bovine respiratory born of the same parents（Bovine Respiration Syncytial Virus – BRSV）Infection can be drawn Cattle respiratory disease is played, is listed in EU member country and is only second to bovine mucosal disease（BVD）And infectious bovine rhinotrachetiss（IBR）'s One 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%, extremely Rate 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 century Begin to the seventies list research and development category in, do not develop preferable medicine and vaccine for various reasons.Although relevant In the report of various inactivations of BRSV, weak poison, restructuring and DNA vaccination, but there are certain distance, current state apart from Clinical practice Still 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 cause Sick 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 causes The cost of production of vaccine is very high；(2) morbid substance in vaccine is possible to not kill completely or fill in vaccine production process Divide attenuation, this contains strong toxicity morbid substance in causing vaccine, and then disease is propagated in the larger context；(3) subtract Toxic 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 simultaneously Trimer furcella is formed, which occurs conformation change under activation.The fusion of the protein mediated viruses of F- and cell membrane, allows virus nucleocapsid Body enters Cytoplasm, and the step for suppressing F- protein mediated is prevented the initial period of infectious cycle and neutralizes viral infection and can have What is imitated prevents virus infraction, i.e., for the antibody of F- albumen, suppress its activity, can neutralize viral infection and can protect Infect from BRSV.The open beginning for having created Chinese structure Basic of Biology theoretical research is applied, it is of the invention by structure biology It 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 antigen Albumen and preparation method thereof, the technology and theory of the antigen protein application protein crystallography, determine albumen in virus infection and Structure change in pathogenic course, carries out genetic engineering to this crucial virus protein and changes further according to the result of structure change Make 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 parents Aminoacid sequence abbreviation SEQ 048055.1, particular sequence is MATTAMRMIISIIFISTYVTHITLCQNITEEFYQSTCSAVSRGYLSALRTGWYTSVVTIELSKIQKNVCKSTDSKVK LIKQELERYNNAVVELQSLMQNEPASFSRAKRGIPELIHYTRNSTKKFYGLMGKKRKRRFLGFLLGIGSAVASGVAV SKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKELLPQVNNHDCRISNIETVIEFQQKNNRLL EIAREFSVNAGITTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSVVKEEVIAYVVQLPIYGV IDTPCWKLHTSPLCTTDNKEGSNICLTRTDRGWYCDNAGSVSFFPQTETCKVQSNRVFCDTMNSLTLPTDVNLCNTD IFNTKYDCKIMTSKTDISSSVITSIGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKL EGKALYIKGEPIINYYDPLVFPSDEFDASIAQVNAKINQSLAFIRRSDELLHSVDVGKSTTNVVITTIIIVIVVVIL MLIAVGLLFYCKTKSTPIMLGKDQLSGINNLSFSK, it is characterised in that：The bovine respiratory born of the same parents zoarium viral antigen proteins Aminoacid sequence compare at least two sites of SEQ 048055.1 by cysteine substitute, and substitute after cysteine it Between 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 SEQ 048055.1, 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 SEQ The 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 SEQ The 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 048055.1 Sequence, the 99th site aspartic acid are substituted by cysteine, and the 362nd site serine is substituted by cysteine, and after replacement Cysteine 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 key Cause 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 making It 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.Pin Antigen 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 draw The 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 infected It 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 in Stable preinfective state, to add on F- albumen series connects chain, including interior disulfide bond, disulfide bond, hole filling With single-stranded connection.
Compared with prior art, its advantage is the present invention：The bovine respiratory prepared using protein crystallography method Born 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 this Send preferable viral vaccine；The loss that effective control and strick precaution virus infection bring, with great economic implications and society Meaning.
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- albumen Figure, 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 process Change schematic diagram.Show in figure before infecting（It is left）, F- albumen trimer is presented trimer furcella state, after infecting（It is right）F- eggs It is activated in vain, occurred conformation change, the fusion of virus and cell membrane, viral nucleocapsid enters Cytoplasm, and then infects cattle body simultaneously Make 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 invention Photo 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 transformation Electromicroscopic 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 invention With 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 invention Antigen 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 cysteine In generation, the 288th isoleucine, are substituted by cysteine, and are connected two sites by disulfide bond, are carried out corresponding albumen conjunction Into, 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 as For 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 cysteine Substitute, the 290th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond, is carried out corresponding albumen Synthesis, expression, purification, the bovine respiratory born of the same parents zoarium viral antigen proteins abbreviation RD-BRSV-DB2 for obtaining, its aminoacid sequence letter Referred 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 cysteine Substitute, the 288th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond, while by the bright of the 260th site Propylhomoserin is substituted by cysteine, and realizes that hole is filled in the site, is carried out corresponding albumen synthesis, expression, purification, is obtained The 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 cysteine Substitute, the 290th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond, while by the bright of the 260th site Propylhomoserin is substituted by cysteine, and realizes that hole is filled in the site, is carried out corresponding albumen synthesis, expression, purification, is obtained The 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 cysteine Substitute, the 288th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond；Simultaneously by the bright of the 260th site Propylhomoserin is substituted by cysteine, and realizes that hole is filled in the site；Reject 103 to site 138 in 048055.1 sequences of SEQ The 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 SEQ DB1-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 cysteine Substitute, the 290th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond, while by the bright of the 260th site Propylhomoserin is substituted by cysteine, and realizes that hole is filled in the site；Reject 103 to site 138 in 048055.1 sequences of SEQ The 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 SEQ DB2-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 cysteine Substitute, the 288th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond；Simultaneously by the bright of the 260th site Propylhomoserin is substituted by cysteine, and realizes that hole is filled in the site；Reject 103 to site 138 in 048055.1 sequences of SEQ The aminoacid sequence in site, connects 102 and 139 sites using sgsgs, and the 99th site aspartic acid is substituted by cysteine, the 362 site serines are substituted by cysteine, and disulfide bond in connecting between the cysteine after replacement, carry out corresponding egg White 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 cysteine Substitute, the 288th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond；Simultaneously by the bright of the 260th site Propylhomoserin is substituted by cysteine, and realizes that hole is filled in the site；Reject 103 to site 142 in 048055.1 sequences of SEQ The aminoacid sequence in site, connects 102 and 143 sites using sgsgs, and the 99th site aspartic acid is substituted by cysteine, the 362 site serines are substituted by cysteine, and disulfide bond in connecting between the cysteine after replacement, carry out corresponding egg White 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 cysteine Substitute, the 290th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond, while by the bright of the 260th site Propylhomoserin is substituted by cysteine, and realizes that hole is filled in the site；Reject 103 to site 138 in 048055.1 sequences of SEQ The aminoacid sequence in site, connects 102 and 139 sites using sgsgs；99th site aspartic acid is substituted by cysteine, the 362 site serines are substituted by cysteine, and disulfide bond in connecting between the cysteine after replacement.Which carries 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 ID1, 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 cysteine Substitute, the 290th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond, while by the bright of the 260th site Propylhomoserin is substituted by cysteine, and realizes that hole is filled in the site；Reject 103 to site 142 in 048055.1 sequences of SEQ The aminoacid sequence in site, connects 102 and 143 sites using sgsgs；99th site aspartic acid is substituted by cysteine, the 362 site serines are substituted by cysteine, and disulfide bond in connecting between the cysteine after replacement.Which carries out corresponding Albumen synthesis, expression, purification, the bovine respiratory born of the same parents zoarium viral antigen proteins abbreviation RD-BRSV-DB2-CF1-SC2- for obtaining ID1, 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 to Antigen molecule can be with the product of corresponding immunne response（Antibody or primed lymphocyte）, there is specific binding in vivo or in vitro Performance, the affinity-immunity using ELISA method to antigen with antibody tests.ELISA method has sensitive, special, simple It is single, quick, stable and the features such as be easily operated automatically, as immunologic diagnosises in a new technique, be applied successfully to The 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 very Also more antibodies bind antigen forms immune complex when low.Affinity represents that with equilibrium constant K K values are bigger, affine Property 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 protein Hold 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 ELISA Method detects, as a result shows not only there is preferable immunogenicity, and can inducing mouse produce higher antibody horizontal, table The 2 immunogenicity weighted means for giving D25 antibody and antigen protein of the present invention specific binding, matched group are unmanifest open country Non-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 conditionses Ratio, 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 shape Can 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 maker Crystallized in vain, the target protein after crystallization is adopted into X-ray system（SER-CAT22）Collect crystallization volume data, and make its into Picture, obtains the electronic cloud of protein structure, obtains the data variation before and after key protein F- albumen infects, and infects front-end geometry change Change 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 sequence Digital simulation, forms the 3-D solid structure simulation figure of protein structure.According to the three-dimensional protein structure that protein sequence is simulated Figure 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 institutes The 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 companies Cell carrier carries out culture 5 days in 293Fectin culture medium, collects cell culture fluid, target protein is carried out purification The 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 closed Body 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 to The 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 fit Appropriate 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 into The 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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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 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 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
485
<210> 3
<211> 489
<212> PRT
<213>Artificial sequence
<400> 3
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 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
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 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
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 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
485
<210> 6
<211> 494
<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 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
485 490
<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 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
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 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
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 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
<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 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
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 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 (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 wild Kind aminoacid sequence be abbreviation SEQ 048055.1, particular sequence is MATTAMRMIISIIFISTYVTHITLCQNITEEFYQSTCSAVSRGYLSALRTGWYTSVVTIELSKIQKNVCKSTDSKVK LIKQELERYNNAVVELQSLMQNEPASFSRAKRGIPELIHYTRNSTKKFYGLMGKKRKRRFLGFLLGIGSAVASGVAV SKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKELLPQVNNHDCRISNIETVIEFQQKNNRLL EIAREFSVNAGITTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSVVKEEVIAYVVQLPIYGV IDTPCWKLHTSPLCTTDNKEGSNICLTRTDRGWYCDNAGSVSFFPQTETCKVQSNRVFCDTMNSLTLPTDVNLCNTD IFNTKYDCKIMTSKTDISSSVITSIGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKL EGKALYIKGEPIINYYDPLVFPSDEFDASIAQVNAKINQSLAFIRRSDELLHSVDVGKSTTNVVITTIIIVIVVVIL MLIAVGLLFYCKTKSTPIMLGKDQLSGINNLSFSK, it is characterised in that：The bovine respiratory born of the same parents zoarium viral antigen proteins Aminoacid sequence compare at least two sites of SEQ 048055.1 by cysteine substitute, and substitute after cysteine it Between 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 is The 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 is The 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 exhales Inhale road born of the same parents zoarium viral antigen proteins aminoacid sequence compare SEQ 048055.1, the 260th site leucine by half Guang Propylhomoserin 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 described For 103 sites in aminoacid sequence rejecting 048055.1 sequences of SEQ of bovine respiratory born of the same parents zoarium viral antigen proteins extremely The 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 described For 103 sites in aminoacid sequence rejecting 048055.1 sequences of SEQ of bovine respiratory born of the same parents zoarium viral antigen proteins extremely The 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 described The 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 ammonia Acid is substituted by cysteine, and the 362nd site serine is substituted by cysteine, and in connecting between the cysteine after replacement 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, it is determined that infecting viral key Cause 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 making It is animal experiment and clinical trial for bovine respiratory born of the same parents zoarium viral antigen proteins.
CN201611065117.3A 2016-11-28 2016-11-28 Bovine respiratory syncytial cell virus antigen protein Pending CN106518988A (en)

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