CN106749556A - A kind of bovine respiratory born of the same parents zoarium viral antigen proteins - Google Patents

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

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CN106749556A
CN106749556A CN201611065884.4A CN201611065884A CN106749556A CN 106749556 A CN106749556 A CN 106749556A CN 201611065884 A CN201611065884 A CN 201611065884A CN 106749556 A CN106749556 A CN 106749556A
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ser
leu
val
asn
ile
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汤斌
邱啟欲
朱云
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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

Abstract

The invention discloses a kind of bovine respiratory born of the same parents zoarium viral antigen proteins, the amino acid sequence of the bovine respiratory born of the same parents zoarium viral antigen proteins is substituted compared at least two sites of SEQ AAA42804.1 by cysteine, and by disulfide bond between the cysteine after substituting, carry out interior disulfide bond, hole filling and single-stranded connection etc. again on this basis to improve, while there is provided the preparation method of such antigen protein.The technology and theory of the antigen protein application protein crystallography, determine structure change of the albumen in virus infection and pathogenic course, result further according to structure change carries out genetic engineering transformation to this crucial virus protein, become and only infect proteantigen that is not pathogenic and causing animal body antibody highly effective reaction, the fit virus infection of bovine respiratory born of the same parents can effectively be prevented and treated, preferable viral vaccine can be developed based on this, the loss that effectively control and strick precaution virus infection bring, with great economic implications and social effect.

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 draw Cattle respiratory disease is played, is listed in EU member country and is only second to bovine mucosal disease（BVD）And infectious bovine rhinotrachetis（IBR）'s One of important cattle disease.This disease is popular in world wide, and its harmfulness is that BRSV the incidence of infection is up to 60%-80%, extremely Rate of dying reaches more than 20% in some outbursts, and very big economic loss is caused 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 various inactivations of BRSV, weak poison, the report of restructuring and DNA vaccination, but also have certain distance, current state apart from Clinical practice Still without preferable viral vaccine on border.
The development of traditional vaccine and production are mainly by changing condition of culture, or passage makes cause on different host animals 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 is needed to be cultivated in zooblast, and this is caused 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 can cause to contain strong toxicity morbid substance in vaccine, and then cause that 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 and Tripolymer furcella is formed, it occurs conformation change under activation.The fusion of the protein mediated viruses of F- and cell membrane, allows virus nucleocapsid Body enters cytoplasm, suppresses the protein mediated steps of F- and prevents the initial period of infectious cycle and neutralize 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 From BRSV infection.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 the shortcoming for overcoming prior art to exist, there is provided a kind of bovine respiratory born of the same parents zoarium viral 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, the result further according to structure change carries out genetic engineering and changes to this crucial virus protein Make, become and only infect proteantigen that is not pathogenic and causing animal body antibody highly effective reaction.
To achieve these goals, the technical scheme adopted by the invention to solve the technical problem is 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 Amino acid sequence abbreviation SEQ AAA42804.1, particular sequence is MATTTMRMIISIILISTYVPHITLCQNITEEFYQSTCSAVSRGYLSALRTGWYTSVVTIELSKIQKNVCNGTDSKVK LIKQELERYNNAVAELQSLMQNEPTSSSRAKRGIPESIHYTRNSTKKFYGLMGKKRKRRFLGFLLGIGSAIASGVAV SKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKELLPKVNNHDCRISNIATVIEFQQKNNRLL EIAREFSVNAGITTPLSTYMLTNSELLSIINDMPITNDQKKLMSVCQIVRQQSYSIMSVLREVIAYVVQLPLYGVID TPCWKLHTSPLCTTDNKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPTDVNLCNTDIF NSKYDCKIMTSKTDISSSVITSIGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEG KALYIKGEPIINYYNPLVFPSDEFDASIAQVNAKINQSLAFIRRSDELLHSVDVGKSTTNVVITTIIIVIVVVILML ITVGLLFYCKTRSTPIMLGKDQLSSINNLSFSK, it is characterised in that：The bovine respiratory born of the same parents zoarium viral antigen proteins Amino acid sequence is substituted compared at least two sites of SEQ AAA42804.1 by cysteine, and substitute after cysteine it Between by disulfide bond.
Used as optimization, the site is the 148th valine of amino acid sequence and 288 isoleucines.
Used as optimization, the site is the 158th leucine of amino acid sequence and 290 serines.
Used as optimization, the amino acid sequence of the bovine respiratory born of the same parents zoarium viral antigen proteins compares SEQ AAA42804.1, the leucine in the 260th site is substituted by cysteine, and realizes that hole is filled in the site.
Used as optimization, the amino acid sequence for bovine respiratory born of the same parents zoarium viral antigen proteins rejects SEQ The amino acid sequence in 103 sites to 138 sites in AAA42804.1 sequences, 102 sites and 139 sites are connected using sgsgs.
Used as optimization, the amino acid sequence for bovine respiratory born of the same parents zoarium viral antigen proteins rejects SEQ The amino acid sequence in 103 sites to 142 sites in AAA42804.1 sequences, 102 sites and 143 sites are connected using sgsgs.
Used as optimization, the amino acid sequence of the coding bovine respiratory born of the same parents zoarium viral antigen proteins compares SEQ AAA42804.1 sequences, the 99th site aspartic acid is substituted by cysteine, and the 362nd site serine is substituted by cysteine, And disulfide bond in being connected between the cysteine after replacement.
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 infects front and rear 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 through various antigen tests and stability characteristic detection；
（4）Corresponding albumen synthesis, expression, purification will be carried out by the antigen protein sequence of checking and detected, obtain for making It is animal experiment and clinical test 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 ox born of the same parents and cell membrane, allows viral nucleocapsid to enter cell.Pin It is that the structure change before and after being infected according to F- albumen sets to the antigen protein of bovine respiratory born of the same parents zoarium viral design, State before making F- protein structures remain at fusion on the basis of genetic engineering, makes it have and only infect 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 that tripolymer furcella state is kept before and after infecting（Closure state）, only infected Proteantigen that is not pathogenic and causing animal's antibody highly effective reaction；In order to the F- albumen ensured to come from pathogenic virus is in The preinfective state of stabilization, to the connects chain that series is added on F- albumen, 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 effectively control and strick precaution virus infection bring, with great economic implications and society Meaning.
Brief 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 ox syncytial virus F- albumen Figure, left figure is tripolymer 3-D solid structure simulation drawing before contaminating, and right figure is the partial enlarged drawing of left figure lower right corner square frame position.
Fig. 2 ox syncytial virus Respiroviruses of the present invention infect the crucial F- protein structures of front and rear process Change schematic diagram.Before display is infected in the figure（It is left）, F- albumen tripolymer is presented tripolymer 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 ox body simultaneously It is set cause a disease.
Fig. 3 Electronic Speculum that Niu Tihou is infected by the successful bovine respiratory born of the same parents zoarium proteantigen of transformation of the present invention Photo state.The bovine respiratory born of the same parents zoarium proteantigen of left figure display RD-BRSV-DB1-CF1 transformations infects the state of Niu Tihou, It can be seen that tripolymer furcella state before stable fusion is presented, right figure is without the bovine respiratory born of the same parents zoarium virus infection ox of transformation Electromicroscopic photograph after body.
Connects chain schematic diagram, including interior disulfide bond, disulfide bond, hole filling are added on Fig. 4 F- albumen of the present invention With single-stranded connection, these links guarantee F- albumen holding tripolymer furcella states.
Specific embodiment
Below by embodiment and with reference to accompanying drawing, the present invention is further described.
Embodiment 1：SEQ AAA42804.1 are carried out into following transformation and obtains a kind of fit disease of bovine respiratory born of the same parents of the invention Malicious antigen protein：
Bovine respiratory born of the same parents zoarium virus F- protein amino acid sequence SEQ the 148th valine of AAA42804.1 is replaced by cysteine Generation, the 288th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond, is 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 amino acid sequence is referred to as It is SEQ DB1.
Bovine respiratory born of the same parents zoarium virus F- protein amino acid sequence SEQ the 158th leucine of AAA42804.1 is by half Guang ammonia Acid is substituted, and the 290th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond, is carried out corresponding egg White synthesis, expression, purification, the bovine respiratory born of the same parents zoarium viral antigen proteins abbreviation RD-BRSV-DB2 for obtaining, its amino acid sequence Referred to as SEQ DB2.
Bovine respiratory born of the same parents zoarium virus F- protein amino acid sequence SEQ the 148th valine of AAA42804.1 is by half Guang ammonia Acid is substituted, and the 288th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond, while by the 260th site Leucine is substituted by cysteine, and realizes that hole is filled in the site, is carried out corresponding albumen synthesis, expression, purification, The bovine respiratory born of the same parents zoarium viral antigen proteins abbreviation RD-BRSV-DB1-CF1 for obtaining, its amino acid sequence is referred to as SEQ DB1-CF1。
Bovine respiratory born of the same parents zoarium virus F- protein amino acid sequence SEQ the 158th leucine of AAA42804.1 is by half Guang ammonia Acid is substituted, and the 290th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond, while by the 260th site Leucine is substituted by cysteine, and realizes that hole is filled in the site, is carried out corresponding albumen synthesis, expression, purification, The bovine respiratory born of the same parents zoarium viral antigen proteins abbreviation RD-BRSV-DB2-CF1 for obtaining, its amino acid sequence is referred to as SEQ DB2-CF1。
Bovine respiratory born of the same parents zoarium virus F- protein amino acid sequence SEQ the 148th valine of AAA42804.1 is by half Guang ammonia Acid is substituted, and the 288th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond；Simultaneously by the 260th site Leucine is substituted by cysteine, and realizes that hole is filled in the site；Reject 103 to site in SEQ AAA42804.1 sequences The amino acid sequence in 138 sites, 102 and 139 sites are connected using sgsgs, are carried out corresponding albumen synthesis, express, carry Bovine respiratory born of the same parents zoarium viral antigen proteins abbreviation RD-BRSV-DB1-CF1-SC1 that is pure, obtaining, its amino acid sequence is referred to as SEQ DB1-CF1-SC1。
Bovine respiratory born of the same parents zoarium virus F- protein amino acid sequence SEQ the 158th leucine of AAA42804.1 is by half Guang ammonia Acid is substituted, and the 290th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond, while by the 260th site Leucine is substituted by cysteine, and realizes that hole is filled in the site；Reject 103 to site in SEQ AAA42804.1 sequences The amino acid sequence in 138 sites, 102 and 139 sites are connected using sgsgs, are carried out corresponding albumen synthesis, express, carry Bovine respiratory born of the same parents zoarium viral antigen proteins abbreviation RD-BRSV-DB2-CF1-SC1 that is pure, obtaining, its amino acid sequence is referred to as SEQ DB2-CF1-SC1。
Bovine respiratory born of the same parents zoarium virus F- protein amino acid sequence SEQ the 148th valine of AAA42804.1 is by half Guang ammonia Acid is substituted, and the 288th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond；Simultaneously by the 260th site Leucine is substituted by cysteine, and realizes that hole is filled in the site；Reject 103 to site in SEQ AAA42804.1 sequences The amino acid sequence in 138 sites, 102 and 139 sites are connected using sgsgs, and the 99th site aspartic acid is substituted by cysteine, 362nd site serine is substituted by cysteine, and disulfide bond in connection between cysteine after replacement, 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 ID1, its amino acid sequence is referred to as SEQ DB1-CF1-SC1-ID1.
Bovine respiratory born of the same parents zoarium virus F- protein amino acid sequence SEQ the 148th valine of AAA42804.1 is by half Guang ammonia Acid is substituted, and the 288th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond；Simultaneously by the 260th site Leucine is substituted by cysteine, and realizes that hole is filled in the site；Reject 103 to site in SEQ AAA42804.1 sequences The amino acid sequence in 142 sites, 102 and 143 sites are connected using sgsgs, and the 99th site aspartic acid is substituted by cysteine, 362nd site serine is substituted by cysteine, and disulfide bond in connection between cysteine after replacement, 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-SC2- for obtaining ID1, its amino acid sequence is referred to as SEQ DB1-CF1-SC2-ID1.
Bovine respiratory born of the same parents zoarium virus F- protein amino acid sequence SEQ the 158th leucine of AAA42804.1 is by half Guang ammonia Acid is substituted, and the 290th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond, while by the 260th site Leucine is substituted by cysteine, and realizes that hole is filled in the site；Reject 103 to site in SEQ AAA42804.1 sequences The amino acid sequence in 138 sites, 102 and 139 sites are connected using sgsgs；99th site aspartic acid is substituted by cysteine, 362nd site serine is substituted by cysteine, and disulfide bond in connection between cysteine after replacement.It 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 amino acid sequence is referred to as SEQ DB2-CF1-SC1-ID1.
Bovine respiratory born of the same parents zoarium virus F- protein amino acid sequence SEQ the 158th leucine of AAA42804.1 is by half Guang ammonia Acid is substituted, and the 290th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond, while by the 260th site Leucine is substituted by cysteine, and realizes that hole is filled in the site；Reject 103 to site in SEQ AAA42804.1 sequences The amino acid sequence in 142 sites, 102 and 143 sites are connected using sgsgs；99th site aspartic acid is substituted by cysteine, 362nd site serine is substituted by cysteine, and disulfide bond in connection between cysteine after replacement.It 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 amino acid sequence is referred to as SEQ DB2-CF1-SC2-ID1.
Immunogenicity refers to that antigenic stimulus body produces the characteristic of immune response ability also known as antigenicity.Immunoreactivity refers to Antigen molecule can be with the product of corresponding immune response（Antibody or sensitized lymphocyte）, specifically bind in vivo or in vitro Performance, the affinity of antigen and antibody-immune is tested using ELISA method.ELISA method has sensitive, special, letter Single, quick, stabilization and the features such as be easily operated automatically, as a new technology in immunodiagnosis, has been applied successfully to The quantitative determination of macromolecular 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.Compatibility represents that K values are bigger, affine with equilibrium constant K Property is higher, also more firm with antigen binding.Table 1 give the present invention preparation antigen protein respectively Site, SiteQP, The compatibility data that Site V, the sites of Site II are combined with antibody.
The Characteristics Detection of the embodiment bovine respiratory born of the same parents of table 1 zoarium viral antigen proteins
The present invention has antibody library compatibility higher by the F- albumen of design improvement.All protected by improved antigen protein Hold tripolymer furcella state before fusion（Trimer）, and purpose antigen albumen can be obtained.
The antigen protein that the present invention obtains purifying is specifically bound with D25 antibody, through protein immunization electrophoresis and ELISA Method detection, as a result shows not only there is preferable immunogenicity, and being capable of inducing mouse generation antibody level higher, table The 2 immunogenicity weighted averages for giving D25 antibody and antigen protein of the present invention specific binding, control group is unmanifest open country Non-hibernating eggs.
The Characteristics Detection that the embodiment bovine respiratory born of the same parents of table 2 zoarium viral antigen proteins specifically bind 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 pressure, Detailed data is shown in Table 3, and data are the active ratio with optimal antigen protein activity of antigen protein under corresponding conditionses, knot in table Fruit shows that the antigen protein by design improvement is presented preferable stability to temperature, pH value, osmotic pressure etc., with higher anti- Should activity.
The reactivity stability of the embodiment bovine respiratory born of the same parents of table 3 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）, Electronic Speculum, The present invention is observed protein structure using x-ray method.It is serial by target egg using the plux of Solution maker 600 Crystallized in vain, the target protein after crystallization is used into X-ray system（SER-CAT22）Collect crystallization volume data, and make its into Picture, obtains the electronic cloud of protein structure, obtains key protein F- albumen and infects front and rear data variation, 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）Carried 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 AAA42804.1 is designed.
The protein sequence that will be designed is translated into DNA sequence dna, the Expi293F conducts provided using invitrogen companies Cell carrier carries out culture 5 days in 293Fectin culture mediums, collects cell culture fluid, and target protein is carried out into purification The BRSV F- albumen of the purifying for being designed, i.e. RD-BRSV-DB1-CF1-SC1-ID1, improved bovine respiratory born of the same parents close Body protein antigen infects tripolymer furcella state before Niu Tihou is presented stable fusion and sees Fig. 3（It is left）.
Above-mentioned specific embodiment is only specific case of the invention, and scope of patent protection of the invention is included 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 it, should all fall into Scope of patent protection of the 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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275 280 285
Leu His Thr Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly Ser Asn
290 295 300
Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly
305 310 315 320
Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn
325 330 335
Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp Val
340 345 350
Asn Leu Cys Asn Thr Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile
355 360 365
Met Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr Ser Ile Gly
370 375 380
Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys
385 390 395 400
Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser
405 410 415
Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val
420 425 430
Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile Ile
435 440 445
Asn Tyr Tyr Asn Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser
450 455 460
Ile Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg
465 470 475 480
Arg Ser Asp Glu Leu Leu His
485
<210> 3
<211> 487
<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 Asn Gly 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 Ala Glu Leu Gln Ser Leu Met Gln Asn Glu Pro Thr Ser Ser Ser
65 70 75 80
Arg Ala Lys Arg Gly Ile Pro Glu Ser 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 Ile Ala Ser Gly Val Ala
115 120 125
Val Ser Lys Val Cys His Leu Glu Gly Glu Val Asn Lys Ile Lys Asn
130 135 140
Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val
145 150 155 160
Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys
165 170 175
Glu Leu Leu Pro Lys Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile
180 185 190
Ala 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 Ile Ile Asn Asp Met Pro
225 230 235 240
Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Val Cys Gln Ile Val Arg
245 250 255
Gln Gln Ser Tyr Ser Ile Met Cys Val Leu Arg Glu Val Ile Ala Tyr
260 265 270
Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys
275 280 285
Leu His Thr Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly Ser Asn
290 295 300
Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly
305 310 315 320
Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn
325 330 335
Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp Val
340 345 350
Asn Leu Cys Asn Thr Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile
355 360 365
Met Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr Ser Ile Gly
370 375 380
Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys
385 390 395 400
Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser
405 410 415
Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val
420 425 430
Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile Ile
435 440 445
Asn Tyr Tyr Asn Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser
450 455 460
Ile Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg
465 470 475 480
Arg Ser Asp Glu Leu Leu His
485
<210> 4
<211> 487
<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 Asn Gly 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 Ala Glu Leu Gln Ser Leu Met Gln Asn Glu Pro Thr Ser Ser Ser
65 70 75 80
Arg Ala Lys Arg Gly Ile Pro Glu Ser 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 Lys Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile
180 185 190
Ala 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 Val Cys Gln Ile Val Arg
245 250 255
Gln Gln Ser Tyr Ser Cys Met Ser Val Leu Arg Glu Val Ile Ala Tyr
260 265 270
Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys
275 280 285
Leu His Thr Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly Ser Asn
290 295 300
Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly
305 310 315 320
Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn
325 330 335
Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp Val
340 345 350
Asn Leu Cys Asn Thr Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile
355 360 365
Met Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr Ser Ile Gly
370 375 380
Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys
385 390 395 400
Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser
405 410 415
Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val
420 425 430
Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile Ile
435 440 445
Asn Tyr Tyr Asn Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser
450 455 460
Ile Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg
465 470 475 480
Arg Ser Asp Glu Leu Leu His
485
<210> 5
<211> 487
<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 Asn Gly 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 Ala Glu Leu Gln Ser Leu Met Gln Asn Glu Pro Thr Ser Ser Ser
65 70 75 80
Arg Ala Lys Arg Gly Ile Pro Glu Ser 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 Ile Ala Ser Gly Val Ala
115 120 125
Val Ser Lys Val Cys His Leu Glu Gly Glu Val Asn Lys Ile Lys Asn
130 135 140
Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val
145 150 155 160
Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys
165 170 175
Glu Leu Leu Pro Lys Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile
180 185 190
Ala 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 Val Cys Gln Ile Val Arg
245 250 255
Gln Gln Ser Tyr Ser Ile Met Cys Val Leu Arg Glu Val Ile Ala Tyr
260 265 270
Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys
275 280 285
Leu His Thr Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly Ser Asn
290 295 300
Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly
305 310 315 320
Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn
325 330 335
Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp Val
340 345 350
Asn Leu Cys Asn Thr Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile
355 360 365
Met Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr Ser Ile Gly
370 375 380
Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys
385 390 395 400
Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser
405 410 415
Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val
420 425 430
Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile Ile
435 440 445
Asn Tyr Tyr Asn Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser
450 455 460
Ile Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg
465 470 475 480
Arg Ser Asp Glu Leu Leu His
485
<210> 6
<211> 456
<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 Asn Gly 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 Ala Glu Leu Gln Ser Leu Met Gln Asn Glu Pro Thr Ser Gly Ser
65 70 75 80
Gly Ser Gly Phe Leu Leu Gly Ile Gly Ser Ala Cys Ala Ser Gly Val
85 90 95
Ala Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys
100 105 110
Asn Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly
115 120 125
Val Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp
130 135 140
Lys Glu Leu Leu Pro Lys Val Asn Asn His Asp Cys Arg Ile Ser Asn
145 150 155 160
Ile Ala 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 Val Cys Gln Ile Val
210 215 220
Arg Gln Gln Ser Tyr Ser Cys Met Ser Val Leu Arg Glu Val Ile Ala
225 230 235 240
Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp
245 250 255
Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly Ser
260 265 270
Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala
275 280 285
Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Ser
290 295 300
Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp
305 310 315 320
Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys
325 330 335
Ile Met Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr Ser Ile
340 345 350
Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn
355 360 365
Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val
370 375 380
Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr
385 390 395 400
Val Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile
405 410 415
Ile Asn Tyr Tyr Asn Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala
420 425 430
Ser Ile Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile
435 440 445
Arg Arg Ser Asp Glu Leu Leu His
450 455
<210> 7
<211> 452
<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 Asn Gly 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 Ala Glu Leu Gln Ser Leu Met Gln Asn Glu Pro Thr Ser Gly Ser
65 70 75 80
Gly Ser Gly Ile Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys
85 90 95
Val Cys His Leu Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu
100 105 110
Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu
115 120 125
Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys Glu Leu Leu
130 135 140
Pro Lys Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile Ala 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 Val Cys Gln Ile Val Arg Gln Gln Ser
210 215 220
Tyr Ser Ile Met Cys Val Leu Arg Glu Val Ile Ala Tyr Val Val Gln
225 230 235 240
Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr
245 250 255
Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly Ser Asn Ile Cys Leu
260 265 270
Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser
275 280 285
Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe
290 295 300
Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp Val Asn Leu Cys
305 310 315 320
Asn Thr Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser
325 330 335
Lys Thr Asp Ile Ser Ser Ser Val Ile Thr Ser Ile Gly Ala Ile Val
340 345 350
Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly
355 360 365
Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly
370 375 380
Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu
385 390 395 400
Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile Ile Asn Tyr Tyr
405 410 415
Asn Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ala Gln
420 425 430
Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp
435 440 445
Glu Leu Leu His
450
<210> 8
<211> 456
<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 Asn Gly 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 Ala Glu Leu Gln Ser Leu Met Gln Cys Glu Pro Thr Ser Gly Ser
65 70 75 80
Gly Ser Gly Phe Leu Leu Gly Ile Gly Ser Ala Cys Ala Ser Gly Val
85 90 95
Ala Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys
100 105 110
Asn Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly
115 120 125
Val Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp
130 135 140
Lys Glu Leu Leu Pro Lys Val Asn Asn His Asp Cys Arg Ile Ser Asn
145 150 155 160
Ile Ala 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 Val Cys Gln Ile Val
210 215 220
Arg Gln Gln Ser Tyr Ser Cys Met Ser Val Leu Arg Glu Val Ile Ala
225 230 235 240
Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp
245 250 255
Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly Ser
260 265 270
Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala
275 280 285
Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Cys
290 295 300
Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp
305 310 315 320
Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys
325 330 335
Ile Met Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr Ser Ile
340 345 350
Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn
355 360 365
Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val
370 375 380
Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr
385 390 395 400
Val Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile
405 410 415
Ile Asn Tyr Tyr Asn Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala
420 425 430
Ser Ile Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile
435 440 445
Arg Arg Ser Asp Glu Leu Leu His
450 455
<210> 9
<211> 452
<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 Asn Gly 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 Ala Glu Leu Gln Ser Leu Met Gln Cys Glu Pro Thr Ser Gly Ser
65 70 75 80
Gly Ser Gly Ile Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys
85 90 95
Val Cys His Leu Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu
100 105 110
Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu
115 120 125
Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys Glu Leu Leu
130 135 140
Pro Lys Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile Ala 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 Val Cys Gln Ile Val Arg Gln Gln Ser
210 215 220
Tyr Ser Ile Met Cys Val Leu Arg Glu Val Ile Ala Tyr Val Val Gln
225 230 235 240
Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr
245 250 255
Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly Ser Asn Ile Cys Leu
260 265 270
Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser
275 280 285
Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Cys Asn Arg Val Phe
290 295 300
Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp Val Asn Leu Cys
305 310 315 320
Asn Thr Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser
325 330 335
Lys Thr Asp Ile Ser Ser Ser Val Ile Thr Ser Ile Gly Ala Ile Val
340 345 350
Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly
355 360 365
Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly
370 375 380
Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu
385 390 395 400
Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile Ile Asn Tyr Tyr
405 410 415
Asn Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ala Gln
420 425 430
Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp
435 440 445
Glu Leu Leu His
450
<210> 10
<211> 456
<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 Asn Gly 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 Ala Glu Leu Gln Ser Leu Met Gln Cys Glu Pro Thr Ser Gly Ser
65 70 75 80
Gly Ser Gly Phe Leu Leu Gly Ile Gly Ser Ala Cys Ala Ser Gly Val
85 90 95
Ala Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys
100 105 110
Asn Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly
115 120 125
Val Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp
130 135 140
Lys Glu Leu Leu Pro Lys Val Asn Asn His Asp Cys Arg Ile Ser Asn
145 150 155 160
Ile Ala 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 Val Cys Gln Ile Val
210 215 220
Arg Gln Gln Ser Tyr Ser Cys Met Ser Val Leu Arg Glu Val Ile Ala
225 230 235 240
Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp
245 250 255
Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly Ser
260 265 270
Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala
275 280 285
Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Cys
290 295 300
Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp
305 310 315 320
Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys
325 330 335
Ile Met Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr Ser Ile
340 345 350
Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn
355 360 365
Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val
370 375 380
Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr
385 390 395 400
Val Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile
405 410 415
Ile Asn Tyr Tyr Asn Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala
420 425 430
Ser Ile Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile
435 440 445
Arg Arg Ser Asp Glu Leu Leu His
450 455
<210> 11
<211> 452
<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 Asn Gly 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 Ala Glu Leu Gln Ser Leu Met Gln Cys Glu Pro Thr Ser Gly Ser
65 70 75 80
Gly Ser Gly Ile Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys
85 90 95
Val Cys His Leu Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu
100 105 110
Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu
115 120 125
Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys Glu Leu Leu
130 135 140
Pro Lys Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile Ala 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 Val Cys Gln Ile Val Arg Gln Gln Ser
210 215 220
Tyr Ser Ile Met Cys Val Leu Arg Glu Val Ile Ala Tyr Val Val Gln
225 230 235 240
Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr
245 250 255
Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly Ser Asn Ile Cys Leu
260 265 270
Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser
275 280 285
Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Cys Asn Arg Val Phe
290 295 300
Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp Val Asn Leu Cys
305 310 315 320
Asn Thr Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser
325 330 335
Lys Thr Asp Ile Ser Ser Ser Val Ile Thr Ser Ile Gly Ala Ile Val
340 345 350
Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly
355 360 365
Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly
370 375 380
Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu
385 390 395 400
Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile Ile Asn Tyr Tyr
405 410 415
Asn Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ala Gln
420 425 430
Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp
435 440 445
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 The amino acid sequence planted is abbreviation SEQ AAA42804.1, and particular sequence is MATTTMRMIISIILISTYVPHITLCQNITEEFYQSTCSAVSRGYLSALRTGWYTSVVTIELSKIQKNVCNGTDSKVK LIKQELERYNNAVAELQSLMQNEPTSSSRAKRGIPESIHYTRNSTKKFYGLMGKKRKRRFLGFLLGIGSAIASGVAV SKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKELLPKVNNHDCRISNIATVIEFQQKNNRLL EIAREFSVNAGITTPLSTYMLTNSELLSIINDMPITNDQKKLMSVCQIVRQQSYSIMSVLREVIAYVVQLPLYGVID TPCWKLHTSPLCTTDNKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPTDVNLCNTDIF NSKYDCKIMTSKTDISSSVITSIGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEG KALYIKGEPIINYYNPLVFPSDEFDASIAQVNAKINQSLAFIRRSDELLHSVDVGKSTTNVVITTIIIVIVVVILML ITVGLLFYCKTRSTPIMLGKDQLSSINNLSFSK, it is characterised in that：The bovine respiratory born of the same parents zoarium viral antigen proteins Amino acid sequence is substituted compared at least two sites of SEQ AAA42804.1 by cysteine, 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 valine of amino acid sequence and 288 isoleucines.
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 amino acid sequence and 290 serines.
4. a kind of fit viral antigen proteins of bovine respiratory born of the same parents according to claim 1, it is characterised in that：The ox exhales The amino acid sequence for inhaling road born of the same parents zoarium viral antigen proteins compares SEQ AAA42804.1, the 260th site leucine by half Cystine is substituted, and realizes that hole is filled in the site.
5. the fit viral antigen proteins of any one bovine respiratory born of the same parents according to claim 1 ~ 3, it is characterised in that：It is described For 103 sites in the amino acid sequence rejecting SEQ AAA42804.1 sequences of bovine respiratory born of the same parents zoarium viral antigen proteins extremely The amino acid sequence in 138 sites, 102 sites and 139 sites are connected using sgsgs.
6. the fit viral antigen proteins of any one bovine respiratory born of the same parents according to claim 1 ~ 3, it is characterised in that：It is described For 103 sites in the amino acid sequence rejecting SEQ AAA42804.1 sequences of bovine respiratory born of the same parents zoarium viral antigen proteins extremely The amino acid sequence in 142 sites, 102 sites and 143 sites are connected using sgsgs.
7. the fit viral antigen proteins of any one bovine respiratory born of the same parents according to claim 1 ~ 3, it is characterised in that：It is described The amino acid sequence of coding bovine respiratory born of the same parents' zoarium viral antigen proteins compares SEQ AAA42804.1 sequences, the 99th site asparagus fern Propylhomoserin is substituted by cysteine, and the 362nd site serine is substituted by cysteine, and is connected between cysteine after replacement Interior 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 infects front and rear 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 through various antigen tests and stability characteristic detection；
（4）Corresponding albumen synthesis, expression, purification will be carried out by the antigen protein sequence of checking and detected, obtain for making It is animal experiment and clinical test for bovine respiratory born of the same parents zoarium viral antigen proteins.
CN201611065884.4A 2016-11-28 2016-11-28 A kind of bovine respiratory born of the same parents zoarium viral antigen proteins Pending CN106749556A (en)

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CN1264425A (en) * 1997-07-17 2000-08-23 皮埃尔法博赫药品公司 Syncytial respiratory virus epitopes and antibodies comprising them, useful in diagnosis and therapy
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Application publication date: 20170531