CN106432435A - Bovine respiration syncytial virus antigen protein - Google Patents
Bovine respiration syncytial virus antigen protein Download PDFInfo
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Abstract
The invention discloses bovine respiration syncytial virus antigen protein. Through comparison between the amino acid sequence of the bovine respiration syncytial virus antigen protein and SEQ AAL49399.1, at least two sites are replaced by cysteine and the two pieces of cysteine after replacement are connected by a disulfide bond; on the basis, improvement, such as the internal disulfide bond, cavity filling, single-chain connection and the like, is conducted; meanwhile, a preparation method of the antigen protein is provided. According to the antigen protein, the structure change of the protein in the virus infection and pathogenic processes is determined by applying the technology and the theory of protein crystallography, and the key virus protein is subjected to genetic engineering reformation according to the result of the structure change to become protein antigen which is only infected, but is not pathogenic and causes animal body antibody high-efficiency reaction, so bovine respiration syncytial virus infection can be effectively prevented and treated, an ideal virus vaccine can be developed on the basis, loss caused by virus infection is effectively controlled and prevented, and economic significance and social significance are achieved.
Description
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
Play cattle respiratory disease, be listed in EU member country and be only second to bovine mucosal disease（BVD）And infectious bovine rhinotrachetiss（IBR）'s
One of important cattle disease.Primary disease is in that world wide is popular, 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 multiple inactivations of BRSV, weak poison, the report of restructuring and DNA vaccination, but also has certain distance apart from Clinical practice, current state
Preferable viral vaccine is not still had on border.
The development of traditional vaccine and produce and mainly pass through to change condition of culture, or pass on different host animals and make cause
Sick Ecotoxicology weakens, or is inactivated by physics, chemical method and to complete.With the continuous progress of human knowledge,
The limitation of traditional vaccine also increasingly reveals：(1) virus of animals and humans needs to cultivate in zooblast, and this makes
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 lead to contain strong toxicity morbid substance in 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 highly conserved simultaneously
Form trimer furcella, it occurs conformation change under activation.The protein mediated virus of F- and the fusion of cell membrane, allow virus nucleocapsid
Body enters Cytoplasm, and the suppression protein mediated step of F- is prevented the initial period of infectious cycle and neutralized viral infection and can have
The antibody preventing virus infraction, that is, being directed to F- albumen of effect, suppresses its activity, can neutralize viral infection and can protect
From BRSV infection.Apply the open beginning having created Chinese structure Basic of Biology theoretical research, the present invention is by structure biology
It is applied in practice, synthesis bovine respiratory born of the same parents zoarium viral antigen proteins.
Content of the invention
It is an object of the invention to the shortcoming overcoming prior art to exist, provide a kind of bovine respiratory born of the same parents zoarium virus antigen
Albumen and preparation method thereof, the technology of this antigen protein application protein crystallography and theory, determine albumen in virus infection and
Structure change in pathogenic course, the result further according to structure change carries out genetic engineering to this crucial virus protein and changes
Make and only infect proteantigen that is not pathogenic and causing animal body antibody highly effective reaction so as to become.
To achieve these goals, the present invention solves its technical problem and is adopted the technical scheme that：
A kind of bovine respiratory born of the same parents zoarium viral antigen proteins, coding bovine respiratory born of the same parents fit virus key protein F- albumen wild species
Aminoacid sequence abbreviation SEQ AAL49399.1, particular sequence is
MATTAMRMIISIIFISTYVTHITLCQNITEEFYQSTCSAVSRGYLSALRTGWYTSVVTIELSKIQKNVCNSTDSKVK
LIKQELERYNNAVVELQSLMQNEPASFSRAKRGIPELIHYTRNSTKKFYGLMGKKRKRRFLGFLLGIGSAIASGVAV
SKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKELLPKVNNHDCRISKIETVIEFQQKNNRLL
EIAREFSVNAGITTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSVVKEEVIAYVVQLPIYGV
IDTPCWKLHTSPLCTTDNKEGSNICLTRTDRGWYCDNAGSVSFFPQTETCKVQSNRVFCDTMNSLTLPTDVNLCNTD
IFNTKYDCKIMTSKTDISSSVITSIGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKL
EGKALYIKGEPIINYYDPLVFPSDEFDASIAQVNAKINQSLAFIRRSDELLHSVDVGKSTTNVVITTIIIVIVVVIL
MLIAVGLLFYCKTKSTPIMLGKDQLSGINNLSFSK it is characterised in that：Described bovine respiratory born of the same parents zoarium viral antigen proteins
Aminoacid sequence compare at least two sites of SEQ AAL49399.1 and substituted by cysteine, and the cysteine after substituting
Between by disulfide bond.
As optimize, described site is the 148th L-Valine of aminoacid sequence and 288 isoleucine.
As optimize, described site is the 158th leucine of aminoacid sequence and 290 serines.
As optimize, the aminoacid sequence of described bovine respiratory born of the same parents zoarium viral antigen proteins compares SEQ
AAL49399.1, the leucine in the 260th site is substituted by cysteine, and realizes hole filling in this site.
As optimize, the described aminoacid sequence for bovine respiratory born of the same parents zoarium viral antigen proteins rejects SEQ
The aminoacid sequence in 103 sites in AAL49399.1 sequence to 138 sites, connects 102 sites and 139 sites using sgsgs.
As optimize, the described aminoacid sequence for bovine respiratory born of the same parents zoarium viral antigen proteins rejects SEQ
The aminoacid sequence in 103 sites in AAL49399.1 sequence to 142 sites, connects 102 sites and 143 sites using sgsgs.
As optimize, the aminoacid sequence of described coding bovine respiratory born of the same parents zoarium viral antigen proteins compares SEQ
AAL49399.1 sequence, the 99th site aspartic acid is substituted by cysteine, and the 362nd site serine is substituted by cysteine,
And disulfide bond in connection 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, carry out protein crystal, observed with X-ray after crystallization, determine and infect virus key
Cause of disease F- albumen infect before and after structure change；
（2）According to step（1）Structure change designs sequence and the structure of antigen protein；
（3）Determine and need the qualification of antigen protein through multiple antigen tests and stability characteristic detection；
（4）Antigen protein sequence through checking is carried out corresponding albumen synthesis, expression, purification and detects, obtain for making
For animal experiment and clinical trial for bovine respiratory born of the same parents zoarium viral antigen proteins.
The protein mediated virus of cattle born of the same parents fit virus key F- and the fusion of cell membrane, allow viral nucleocapsid enter cell.Pin
Antigen protein to bovine respiratory born of the same parents zoarium viral design, is that the structure change before and after being infected according to F- albumen sets,
Make F- protein structure remain at the state before fusion on the basis of genetic engineering only to infect not pathogenic and draw so as to have
Rise animal body antibody highly effective reaction proteantigen the features such as.
The present invention can make F- albumen keep trimer furcella state before and after infecting（Closure state）, only infected
Proteantigen that is not pathogenic and causing animal's antibody highly effective reaction；In order to ensure that coming from the F- albumen on pathogenic virus is in
Stable preinfective state, to add on F- albumen series connects chain, including in disulfide bond, disulfide bond, hole filling
With single-stranded connection.
Compared with prior art, its advantage is the present invention：Bovine respiratory using the preparation of protein crystallography method
Born of the same parents' zoarium viral antigen proteins can effectively prevent and treat bovine respiratory born of the same parents fit virus infection, can open based on this
Send preferable viral vaccine；Effective control and strick precaution virus infect the loss bringing, and have great economic implications and society
Meaning.
Brief description
Fig. 1 the present invention relates to the use of the 3-D solid structure simulation that Phenix software carries out cattle syncytial viruses F- albumen
Figure, left figure is trimer 3-D solid structure simulation drawing before dip-dye, and right figure is the partial enlarged drawing of left figure lower right corner square frame position.
Fig. 2 cattle according to the present invention syncytial viruses Respiroviruses infect the F- protein structure of the key of before and after's process
Change schematic diagram.Before in figure shows and infects（Left）, F- albumen trimer assumes trimer furcella state, after infecting（Right）F- egg
It is activated in vain, occurred conformation changes, the fusion of virus and cell membrane, viral nucleocapsid enters Cytoplasm, and then infects cattle body simultaneously
So that it is caused a disease.
Fig. 3 Electronic Speculum infecting Niu Tihou through transformation successful bovine respiratory born of the same parents zoarium proteantigen 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 transformation infects the state of Niu Tihou,
It can be seen that assuming trimer furcella state before stable fusion, right figure is the bovine respiratory born of the same parents zoarium virus infection cattle without transformation
Electromicroscopic photograph after body.
On Fig. 4 F- according to the present invention albumen add connects chain schematic diagram, including in disulfide bond, disulfide bond, hole filling
With single-stranded connection, these link 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 AAL49399.1 is transformed as follows the fit disease of a kind of bovine respiratory born of the same parents obtaining the present invention
Malicious antigen protein：
Bovine respiratory born of the same parents fit virus the 148th L-Valine of F- protein amino acid sequence SEQ AAL49399.1 is replaced by cysteine
In generation, the 288th isoleucine is substituted by cysteine, and is connected two sites by disulfide bond, is carried out corresponding albumen conjunction
Become, express, purify, the bovine respiratory born of the same parents zoarium viral antigen proteins abbreviation RD-BRSV-DB1 obtaining, its aminoacid sequence is referred to as
For SEQ DB1.
Bovine respiratory born of the same parents fit virus the 158th leucine of F- protein amino acid sequence SEQ AAL49399.1 is by half Guang ammonia
Acid substitutes, 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 obtaining, its aminoacid sequence
Referred to as SEQ DB2.
Bovine respiratory born of the same parents fit virus the 148th L-Valine of F- protein amino acid sequence SEQ AAL49399.1 is by half Guang ammonia
Acid substitutes, 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 hole filling in this 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 obtaining, its aminoacid sequence is referred to as SEQ
DB1-CF1.
Bovine respiratory born of the same parents fit virus the 158th leucine of F- protein amino acid sequence SEQ AAL49399.1 is by half Guang ammonia
Acid substitutes, and the 290th 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 hole filling in this 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 obtaining, its aminoacid sequence is referred to as SEQ
DB2-CF1.
Bovine respiratory born of the same parents fit virus the 148th L-Valine of F- protein amino acid sequence SEQ AAL49399.1 is by half Guang ammonia
Acid substitutes, 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 hole filling in this site；Reject 103 to site in SEQ AAL49399.1 sequence
The aminoacid sequence in 138 sites, connects 102 and 139 sites using sgsgs, is 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 aminoacid sequence is referred to as
SEQ DB1-CF1-SC1.
Bovine respiratory born of the same parents fit virus the 158th leucine of F- protein amino acid sequence SEQ AAL49399.1 is by half Guang ammonia
Acid substitutes, and the 290th 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 hole filling in this site；Reject 103 to site in SEQ AAL49399.1 sequence
The aminoacid sequence in 138 sites, connects 102 and 139 sites using sgsgs, is 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 aminoacid sequence is referred to as
SEQ DB2-CF1-SC1.
Bovine respiratory born of the same parents fit virus the 148th L-Valine of F- protein amino acid sequence SEQ AAL49399.1 is by half Guang ammonia
Acid substitutes, 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 hole filling in this site；Reject 103 to site in SEQ AAL49399.1 sequence
The aminoacid sequence in 138 sites, connects 102 and 139 sites using sgsgs, and the 99th site aspartic acid is substituted by cysteine,
362nd site serine is substituted by cysteine, and disulfide bond in connection between the cysteine after replacement, carries out corresponding
Albumen synthesis, expression, purification, the bovine respiratory born of the same parents zoarium viral antigen proteins abbreviation RD-BRSV-DB1-CF1-SC1- obtaining
ID1, its aminoacid sequence is referred to as SEQ DB1-CF1-SC1-ID1.
Bovine respiratory born of the same parents fit virus the 148th L-Valine of F- protein amino acid sequence SEQ AAL49399.1 is by half Guang ammonia
Acid substitutes, 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 hole filling in this site；Reject 103 to site in SEQ AAL49399.1 sequence
The aminoacid sequence in 143 sites, connects 102 and 143 sites using sgsgs, and the 99th site aspartic acid is substituted by cysteine,
362nd site serine is substituted by cysteine, and disulfide bond in connection between the cysteine after replacement, carries out corresponding
Albumen synthesis, expression, purification, the bovine respiratory born of the same parents zoarium viral antigen proteins abbreviation RD-BRSV-DB1-CF1-SC2- obtaining
ID1, its aminoacid sequence is referred to as SEQ DB1-CF1-SC2-ID1.
Bovine respiratory born of the same parents fit virus the 158th leucine of F- protein amino acid sequence SEQ AAL49399.1 is by half Guang ammonia
Acid substitutes, and the 290th 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 hole filling in this site；Reject 103 to site in SEQ AAL49399.1 sequence
The aminoacid sequence in 138 sites, connects 102 and 139 sites using sgsgs；99th site aspartic acid is substituted by cysteine,
362nd site serine is substituted by cysteine, and disulfide bond in connection between the cysteine after replacement.It carries out corresponding
Albumen synthesis, expression, purification, obtain bovine respiratory born of the same parents zoarium viral antigen proteins abbreviation RD-BRSV-DB2-CF1-SC1-
ID1, its aminoacid sequence is referred to as SEQ DB2-CF1-SC1-ID1.
Bovine respiratory born of the same parents fit virus the 158th leucine of F- protein amino acid sequence SEQ AAL49399.1 is by half Guang ammonia
Acid substitutes, and the 290th 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 hole filling in this site；Reject 103 to site in SEQ AAL49399.1 sequence
The aminoacid sequence in 142 sites, connects 102 and 143 sites using sgsgs；99th site aspartic acid is substituted by cysteine,
362nd site serine is substituted by cysteine, and disulfide bond in connection between the cysteine after replacement.It carries out corresponding
Albumen synthesis, expression, purification, obtain bovine respiratory born of the same parents zoarium viral antigen proteins abbreviation RD-BRSV-DB2-CF1-SC2-
ID1, its aminoacid sequence is referred to as SEQ DB2-CF1-SC2-ID1.
Immunogenicity, also known as antigenicity, refers to the characteristic that antigenic stimulus body produces immunne response ability.Immunoreactivity refers to
Antigen molecule can be with the product of corresponding immunne response（Antibody or primed lymphocyte）, specifically bind in vivo or in vitro
Performance, using ELISA method, the affinity-immunity of antigen and antibody is tested.ELISA method has sensitive, special, simple
Singly, quickly the features such as, stablize and be easily operated automatically, as a new technique in immunologic diagnosises, is applied successfully to
Macromole antigen and the quantitative determination of small molecule antigens.The antibody of high affinity and the adhesion of antigen are strong, and that is, antigen concentration is very
Also more antibodies bind antigen is had to form immune complex when low.Affinity is represented with equilibrium constant K, and K value is 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, Site II site and antibodies.
The Characteristics Detection of table 1 embodiment bovine respiratory born of the same parents 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 antigen protein that the present invention obtains purification is specifically bound with D25 antibody, through protein immunization electrophoresis and ELISA
Method detects, result shows not only there is preferable immunogenicity, and can produce higher antibody horizontal, table by inducing mouse
The 2 immunogenicity weighted means giving D25 antibody and antigen protein specific binding of the present invention, matched group is unmanifest open country
Non-hibernating eggs.
The Characteristics Detection that table 2 embodiment bovine respiratory born of the same parents zoarium viral antigen proteins are specifically bound with D25
Carry out physical and chemical stability 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 is the ratio of the antigen protein active and optimal antigen protein activity under corresponding conditionses, knot
Fruit shows that the antigen protein through design improvement assumes preferable stability to temperature, pH value, osmotic pressure etc., has higher anti-
Should activity.
The reactivity stability of table 3 embodiment bovine respiratory born of the same parents zoarium viral antigen proteins
Embodiment 2：The concrete technology of RD-BRSV-DB1-CF1-SC1-ID1 in preparation embodiment 1.See albumen mechanism and shape
Can be by following three kinds of approach, respectively x- ray（x-ray Crystallography）, nuclear magnetic resonance, NMR（NMR）, Electronic Speculum,
The present invention is observed to protein structure using x-ray method.Using Solution maker 600 plux series by target egg
Crystallized in vain, the target protein after crystallization is adopted X-ray system（SER-CAT22）Collect crystallization volume data, and so that it is become
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 and becomes
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.The three-dimensional protein structure being simulated according to protein sequence
Figure obtains the Trimeric structures of F- albumen, refers to Fig. 1.
According to the principle of crystal chemistry, carry out antigen protein structure design using simulation softward, its design is included as Fig. 4 institute
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 AAL49399.1 is designed.
The protein sequence designing is translated into DNA sequence, the Expi293F conduct providing using invitrogen company
Cell carrier carries out cultivating 5 days in 293Fectin culture medium, collects cell culture fluid, target protein is carried out purification
Obtain the BRSV F- albumen of purification designing, i.e. RD-BRSV-DB1-CF1-SC1-ID1, improved bovine respiratory born of the same parents close
Body protein antigen infects trimer furcella state before Niu Tihou assumes stable fusion and sees Fig. 3（Left）.
Above-mentioned specific embodiment is only the concrete case of the present invention, and the scope of patent protection of the present invention includes but is not limited to
The product form of above-mentioned specific embodiment and style, a kind of any bovine respiratory born of the same parents zoarium meeting claims of the present invention
Suitable change or modification that viral antigen proteins and any person of an ordinary skill in the technical field are done to it, all should fall into
The scope of patent protection of the present invention.
SEQUENCE LISTING
<110>Yantai Ruo Di biological engineering company limited
<120>A kind of bovine respiratory born of the same parents zoarium viral antigen proteins
<130>
<160> 11
<170> PatentIn version 3.3
<210> 1
<211> 574
<212> PRT
<213>Bovine respiratory born of the same parents fit virus F- albumen wild species
<400> 1
Met Ala Thr Thr Ala Met Arg Met Ile Ile Ser Ile Ile Phe Ile Ser
1 5 10 15
Thr Tyr Val Thr His Ile Thr Leu Cys Gln Asn Ile Thr Glu Glu Phe
20 25 30
Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu
35 40 45
Arg Thr Gly Trp Tyr Thr Ser Val Val Thr Ile Glu Leu Ser Lys Ile
50 55 60
Gln Lys Asn Val Cys Asn Ser Thr Asp Ser Lys Val Lys Leu Ile Lys
65 70 75 80
Gln Glu Leu Glu Arg Tyr Asn Asn Ala Val Val Glu Leu Gln Ser Leu
85 90 95
Met Gln Asn Glu Pro Ala Ser Phe Ser Arg Ala Lys Arg Gly Ile Pro
100 105 110
Glu Leu Ile His Tyr Thr Arg Asn Ser Thr Lys Lys Phe Tyr Gly Leu
115 120 125
Met Gly Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Gly Ile
130 135 140
Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Leu His Leu
145 150 155 160
Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys
165 170 175
Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val
180 185 190
Leu Asp Leu Lys Asn Tyr Ile Asp Lys Glu Leu Leu Pro Lys Val Asn
195 200 205
Asn His Asp Cys Arg Ile Ser Lys Ile Glu Thr Val Ile Glu Phe Gln
210 215 220
Gln Lys Asn Asn Arg Leu Leu Glu Ile Ala Arg Glu Phe Ser Val Asn
225 230 235 240
Ala Gly Ile Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu
245 250 255
Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys
260 265 270
Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile
275 280 285
Met Ser Val Val Lys Glu Glu Val Ile Ala Tyr Val Val Gln Leu Pro
290 295 300
Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro
305 310 315 320
Leu Cys Thr Thr Asp Asn Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg
325 330 335
Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe
340 345 350
Pro Gln Thr Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp
355 360 365
Thr Met Asn Ser Leu Thr Leu Pro Thr Asp Val Asn Leu Cys Asn Thr
370 375 380
Asp Ile Phe Asn Thr Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr
385 390 395 400
Asp Ile Ser Ser Ser Val Ile Thr Ser Ile Gly Ala Ile Val Ser Cys
405 410 415
Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile
420 425 430
Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp
435 440 445
Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly
450 455 460
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465 470 475 480
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Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu
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Thr Ile Ile Ile Val Ile Val Val Val Ile Leu Met Leu Ile Ala Val
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Gly Leu Leu Phe Tyr Cys Lys Thr Lys Ser Thr Pro Ile Met Leu Gly
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Lys Asp Gln Leu Ser Gly Ile Asn Asn Leu Ser Phe Ser Lys
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<212> PRT
<213>Artificial sequence
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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 Ser Thr Asp
35 40 45
Ser Lys Val Lys Leu Ile Lys Gln Glu Leu Glu Arg Tyr Asn Asn Ala
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Val Val Glu Leu Gln Ser Leu Met Gln Asn Glu Pro Ala Ser Phe Ser
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Arg Ala Lys Arg Gly Ile Pro Glu Leu Ile His Tyr Thr Arg Asn Ser
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Thr Lys Lys Phe Tyr Gly Leu Met Gly Lys Lys Arg Lys Arg Arg Phe
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 Lys 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 Asn 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 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 Lys 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 Asn 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 Lys Val Asn Asn His Asp Cys Arg Ile Ser Lys 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 Asn 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 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 Lys 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 Asn 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 Lys Val Asn Asn His Asp Cys
180 185 190
Arg Ile Ser Lys 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 Asn 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 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 Lys 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 Asn 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 Lys Val Asn Asn His Asp Cys Arg Ile Ser Lys
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 Asn 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 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 Lys 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 Asn 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 Lys Val Asn Asn His Asp Cys Arg Ile Ser Lys
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 Asn 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 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 Lys 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, coding bovine respiratory born of the same parents fit virus key protein F- albumen is wild
The aminoacid sequence planted is abbreviation SEQ AAL49399.1, and particular sequence is
MATTAMRMIISIIFISTYVTHITLCQNITEEFYQSTCSAVSRGYLSALRTGWYTSVVTIELSKIQKNVCNSTDSKVK
LIKQELERYNNAVVELQSLMQNEPASFSRAKRGIPELIHYTRNSTKKFYGLMGKKRKRRFLGFLLGIGSAIASGVAV
SKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKELLPKVNNHDCRISKIETVIEFQQKNNRLL
EIAREFSVNAGITTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSVVKEEVIAYVVQLPIYGV
IDTPCWKLHTSPLCTTDNKEGSNICLTRTDRGWYCDNAGSVSFFPQTETCKVQSNRVFCDTMNSLTLPTDVNLCNTD
IFNTKYDCKIMTSKTDISSSVITSIGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKL
EGKALYIKGEPIINYYDPLVFPSDEFDASIAQVNAKINQSLAFIRRSDELLHSVDVGKSTTNVVITTIIIVIVVVIL
MLIAVGLLFYCKTKSTPIMLGKDQLSGINNLSFSK it is characterised in that：Described bovine respiratory born of the same parents zoarium viral antigen proteins
Aminoacid sequence compare at least two sites of SEQ AAL49399.1 and substituted by cysteine, and the cysteine after substituting
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：Described 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：Described site is
The 158th leucine of aminoacid sequence and 290 serines.
4. a kind of bovine respiratory born of the same parents zoarium viral antigen proteins according to claim 1 it is characterised in that：Described cattle exhales
The aminoacid sequence inhaling road born of the same parents zoarium viral antigen proteins compares SEQ AAL49399.1, and the leucine in the 260th site is by partly
Cystine substitutes, and realizes hole filling in this site.
5. according to claim 1 ~ 3 any one bovine respiratory born of the same parents zoarium viral antigen proteins it is characterised in that：Described
For 103 sites in the aminoacid sequence rejecting SEQ AAL49399.1 sequence 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 claim 1 ~ 3 any one bovine respiratory born of the same parents zoarium viral antigen proteins it is characterised in that：Described
For 103 sites in the aminoacid sequence rejecting SEQ AAL49399.1 sequence 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 claim 1 ~ 3 any one bovine respiratory born of the same parents zoarium viral antigen proteins it is characterised in that：Described
The aminoacid sequence of coding bovine respiratory born of the same parents zoarium viral antigen proteins compares SEQ AAL49399.1 sequence, the 99th site Radix Asparagi
Propylhomoserin is substituted by cysteine, and the 362nd site serine is substituted by cysteine, and connects between the cysteine after replacement
Interior disulfide bond.
8. a kind of bovine respiratory born of the same parents zoarium viral antigen proteins preparation method it is characterised in that：Concrete technology is as follows：
（1）According to the principle of structure biology, carry out protein crystal, observed with X-ray after crystallization, determine and infect virus key
Cause of disease F- albumen infect before and after structure change；
（2）According to step（1）Structure change designs sequence and the structure of antigen protein；
（3）Determine and need the qualification of antigen protein through multiple antigen tests and stability characteristic detection；
（4）Antigen protein sequence through checking is carried out corresponding albumen synthesis, expression, purification and detects, obtain for making
For animal experiment and clinical trial for bovine respiratory born of the same parents zoarium viral antigen proteins.
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| CN201611064357.1A CN106432435A (en) | 2016-11-28 | 2016-11-28 | Bovine respiration syncytial virus antigen protein |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN103842374A (en) * | 2011-05-13 | 2014-06-04 | 诺华股份有限公司 | Pre-fusion rsv f antigens |
-
2016
- 2016-11-28 CN CN201611064357.1A patent/CN106432435A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN103842374A (en) * | 2011-05-13 | 2014-06-04 | 诺华股份有限公司 | Pre-fusion rsv f antigens |
Non-Patent Citations (1)
| Title |
|---|
| YUNUS,A.S.等: "fusion protein [Bovine respiratory syncytial virus ATCC51908]", 《GENBANK DATABSE》 * |
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