CN102580076A - Synthetic peptide vaccine for O-type foot and mouth disease of swine and preparation method thereof - Google Patents
Synthetic peptide vaccine for O-type foot and mouth disease of swine and preparation method thereof Download PDFInfo
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- CN102580076A CN102580076A CN2011104373040A CN201110437304A CN102580076A CN 102580076 A CN102580076 A CN 102580076A CN 2011104373040 A CN2011104373040 A CN 2011104373040A CN 201110437304 A CN201110437304 A CN 201110437304A CN 102580076 A CN102580076 A CN 102580076A
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Abstract
The invention discloses synthetic peptide vaccine for O-type foot and mouth disease of swine and the preparation method thereof. In the invention, synthetic peptide genes are expressed by selecting an eukaryotic expression system, then an expression product is emulsified into vaccine, since the application of genetic engineering technology, the synthetic peptide for O-type foot and mouth disease of swine can be efficiently and stably expressed, the production cost is low, the amplification is easy, and the toxic side effect and worries about biosafety can be avoided. Therefore, the synthetic peptide vaccine for the O-type foot and mouth disease of swine is suitable for large scale industrial production, and is classified as novel synthetic peptide vaccine for O-type foot and mouth disease of swine, is used for clinically preventing, controlling and curing foot and mouth disease.
Description
Technical field
The present invention relates to the genetic engineering field, be specifically related to boar O type aftosa synthetic peptide vaccine and preparation method thereof.
Background technology
Foot and mouth disease (Foot-and-Mouth Disease; FMD) be by foot and mouth disease virus (Foot and mouth virus; FMDV) cause a kind of acute hot height contagious disease that artiodactyls such as pig, cattle, sheep are beastly; The productivity of serious harm domestic animal and livestock products quality safety are classified as the important infectious disease of category-A by OIE.At present the anti-system developed country for this disease mainly takes slaughter policy, and developing country takes to isolate and the immunity inoculation policy, and wherein the preceding immunity inoculation of disease popularity is that specificity protects domestic animal to exempt from the effective means of infection.China controls the popular of this disease through some new generation vaccines such as inoculation inactivated vaccine and synthetic peptide vaccines at present.But all there are some defectives in existing these vaccines.Like the inactivated vaccine major defect is biological safety problem and stability problem, and virus must be produced in the equipment of requirement for height, with anti-scattered poison; Though existing synthetic peptide Seedling has the excellent protection effect, complex manufacturing, the cost height is unfavorable for reducing cost.Thereby a kind of cheapness, safety, the exploitation of production of vaccine system efficiently have great application prospect.
Summary of the invention
The objective of the invention is to the complex manufacturing that exists according in the existing synthetic peptide vaccine, the problem that cost is high provides a kind of cheapness, safety, pig O type aftosa synthetic peptide vaccine efficiently.
Another purpose of the present invention is to provide the method for preparing of above-mentioned vaccine.
Above-mentioned purpose of the present invention is achieved through following technical scheme:
One boar O type aftosa synthetic peptide vaccine, its aminoacid sequence is shown in SEQ ID NO:1, and gene order is shown in SEQ ID NO:2; What said aminoacid sequence adopted is to have antigenic 141-160 position and 200-213 amino acids peptide section in the pig O type foot and mouth disease VP1 albumen, and it is formed the cascaded structure of multicopy, adds amino acid polypeptide in peptide section junction as joint; Antigen polypeptide adopts following cascaded structure: Y-M-X-N-Y, and wherein " X " and " Y " represents pig O type foot and mouth disease VP1 protein 14 1-160,200-213 amino acids peptide section respectively; " M " and " N " represents " Pro-Gly " two peptide linkers and 11 peptide linkers respectively, and the aminoacid sequence of said 11 peptide linkers is shown in SEQ ID NO:3.
The method for preparing of the above-mentioned pig O of the present invention type aftosa synthetic peptide vaccine comprises the steps:
(1) use recombinant DNA technology, the DNA sequence of composite coding pig O type aftosa synthetic peptide makes up pGAPZ α A-SP expression vector.
(2) in the recombinant expression carrier pGAPZ α A-SP transformed host cell Pichia sp., construction expression engineering bacteria: with competence
P.pastorisX33 (80 μ L) mixes with the linearizing pGAPZ α of Bln A-SP (10 μ g) mutually, 1.5 kV, 200 electric shocks, 5 ms.Yeast cells after transforming is laid on the YPDS dull and stereotyped (containing 100 μ g/ml Zeocin) of prepared fresh, flat board is inverted, in 30 ℃ of incubators, be cultured to single bacterium colony and (needing 3-5 d) occur.Employing boils-freezes-and cooking method prepares PCR template analysis P.pastoris transformant, and clone's that can amplify about 200bp with primer is decided to be positive transformant.Again through the high copy clone of the YPD of variable concentrations Zeocin plate screening, to be used for high efficiency expressing destination protein.
(3) the fermentation culture recombinant yeast is expressed, and expression product is made SDS-PAGE and Western-blot Analysis and Identification;
(4) expression product purification, and select for use adjuvant emulsion to process novel pig O type aftosa synthetic peptide vaccine.
As a kind of preferred version, in the said method, said expression vector is a yeast expression vector.
As a kind of preferred version; In the said method, said step (3) is specially with the tall and slender single bacterium colony with Zeocin resistance that screens of sterilization toothpick, chooses and in the YPD of 5 mL fluid medium, carries out the one-level cultivation; 28 ℃-30 ℃; 200 r/min shaken overnight, to OD600=2-6, this moment, cell was in exponential phase; Get 1 mL one-level culture fluid, be resuspended among the YPD of 30 mL, continue shaken cultivation, add two-layer newspaper wrapping with four layers of clean gauze.Cultivated 72 hours, centrifugal 5 min of 3000 r/min collect the culture fluid supernatant, promptly get purifying protein.
As a kind of preferred version, in the said method, adjuvant is the import oil emulsion adjuvant described in the said step (4).
Compared with prior art, the present invention has following beneficial effect:
Pichia yeast has higher expression as the exogenous gene eukaryotic expression system.Produce protein with the Pichia sp. gene expression system and have very big development prospect, existing abroad multiple protein enzyme and antibody gene have obtained efficiently expressing some commercialization productions.Yeast has than the more complete gene expression regulation of escherichia coli mechanism with to the processing modification and the secretion capacity of expression product, and can not produce endotoxin, is eukaryotic gene recipient bacterium good in the genetic engineering.The present invention selects wild type Pichia yeast X33 for use, fast growth, and also self secretion endogenous protein is seldom, and this has just significantly reduced our purification to exogenous gene expression product in the culture medium, helps to improve expression.PGAPZ α A is a kind of non-secretion inducing type expression vector, does not need methanol induction, and expressed heterologous protein direct secretion is convenient to the purification of destination gene expression product in culture medium, and culture medium is cheap, is more conducive to realize suitability for industrialized production.
The appearance of outstanding recombinant vaccine often depends on the abundant research to the antigen site of this sick pathogen.So since the eighties in last century, people pay attention to the analysis to each antigen site of FMDV more.Wherein the research of structural protein VP1 is the most general, and obtains it for this important conclusion of main immunogenic antigen, and VP1 G-H ring and C-terminal contain important antigen site, and have carried out a large amount of research.The foot-and-mouth disease gene engineering vaccine of existing research all is based on VP1 gene and epitope thereof.As: the 141-160AA of chemosynthesis VP1 and 200-213AA; In live vector, express the VP1 fusion rotein; Be developed into nucleic acid vaccine with the 141-160AA of coding VP1 and the DNA of 200-213A; With expression of plants VP1 albumen, be developed into plant and can raise vaccine etc.141-160AA and these two amino acid peptide sections of 200-213AA are selected in this research equally, and it is formed the cascaded structure of 141-160AA ~ 200-213AA ~ 141-160AA, add suitable amino acid polypeptide in peptide section junction as joint.
Summarize said; Vaccine described in the present invention has B cell antigen epi-position and T cell antigen epitope simultaneously; Has better immunogenicity; The present invention uses eukaryotic expression system (pichia yeast expression system) to express synthetic peptide gene in addition, obtains behind the destination gene expression modifying, and has strengthened the immunogenicity of peptide vaccine.Using gene engineering technique of the present invention has efficiently expressed pig O type aftosa synthetic peptide in eukaryotic cell, show that through experiment this expression product has better immunogenicity.And expression efficiency of the present invention is high, and separation and purification is simple, and production cost is low, is prone to amplify, and good stability is suitable for large-scale industrial production.Therefore, the present invention has broad application prospects for pig O type foot and mouth disease diseases prevention and treatment clinically provide novel vaccine production method with treatment.
Description of drawings
Fig. 1 is a recombiant plasmid pGAPZ α A-SP electrophoretogram, M:DNA molecular mass standard; 1,2: recombiant plasmid pGAPZ α A-SP.
Fig. 2 is the sub-PCR electrophoretogram of recombination yeast positive colony, M:DNA molecular mass standard; 1,2: recombinant yeast pGAPZ α A-SP/X33; 3: recombinant yeast pGAPZ α A/X33.
Fig. 3 is that expression product SDS-PAGE analyzes M: non-preparatory dsred protein molecular weight standard; 1: the expression supernatant of recombinant yeast pGAPZ α A/X33; 2,3,4: the expression supernatant of recombinant yeast pGAPZ α A-SP/X33 bacterium.
Fig. 4 is expression product Western-blot result, M: non-preparatory dsred protein molecular weight standard; 1,2: the expression supernatant of recombinant yeast pGAPZ α A-SP/X33 bacterium; 3: the expression supernatant of recombinant yeast pGAPZ α A/X33.
The specific embodiment
Come further to explain the present invention below in conjunction with embodiment, but embodiment does not do any type of qualification to the present invention.
(1) makes up recombinant expression carrier: according to the MCS of the inclined to one side preferendum of Pichia sp. gene, synthetic peptide SP aminoacid sequence and expression vector pGAPZ α A; Design and the synthetic SP gene of full gene; Be connected on the plasmid Puc57 carrier; Double digestion Puc57 recombinant vector and expression vector pGAPZ α A connect conversion then and make up recombinant expression carrier (Fig. 1).Other designs two primers and is used for detection, and primer is following: p1:SEQ ID NO:4, p2:SEQ ID NO:5, restriction enzyme site are EcoR I and Not I.
(2) make up genetic engineering bacterium: transform the host bacterium with the above-mentioned recombinant expression carrier that builds, the host bacterium is Pichia sp. X33.
(3) expression of the synthetic peptide of recombinant yeast: with the tall and slender single bacterium colony that screens of sterilization toothpick with Zeocin resistance; Choose and in the YPD of 5 mL fluid medium, carry out one-level cultivation, 30 ℃, 200 r/min shaken overnight; To OD600=2-6, this moment, cell was in exponential phase.Get 1 mL one-level culture fluid, be resuspended among the YPD of 30 mL, continue shaken cultivation, add two-layer newspaper wrapping, cultivated about 72 hours, after other gets a part of bacterium liquid employing " boiling-freeze-boil " processing, make PCR and detect (Fig. 2) with four layers of clean gauze.
(4) purification: express centrifugal 5 min of supernatant 3 000 r/min, purifying protein.
The preparation of pig O type aftosa synthetic peptide vaccine: the expression supernatant carries out SDS-PAGE and Western-Blot analyzes (Fig. 3 ~ 4) to appealing; Then penicillin, streptomycin are mixed with the albumen of separation and purification; The mixture pH value is transferred to biological value; Be PH7.0-7.5, promptly prepared pig O type aftosa synthetic peptide vaccine, adjuvant is selected the import oil emulsion adjuvant for use.
Pig O type aftosa synthetic peptide expression is measured: SP expresses the supernatant determination of protein concentration and adopts Bradford determination of protein concentration test kit, has a spot of tropina owing to express in the supernatant, so the concentration of measuring is total protein concentration, concrete steps are following:
1. complete soluble protein standard substance BSA (5mg/mL) gets 10 μ L and is diluted to 100 μ L, and making final concentration is 0.5mg/mL.With PBS dilution standard article.
2. the standard substance after will diluting are added in the standard substance hole of 96 orifice plates by 0,1,2,4,8,12,16,20 μ L, add the PBS diluent and supply 20 μ L.
3. will be respectively that the synthetic peptide supernatant 10 μ L of 48h, 72h, 96h are added in the sample well of 96 orifice plates expression time, add PBS diluent to 20 μ L.
4. each hole adds 200 μ L G250 dyeing liquors, and room temperature was placed 3-5 minute.
5. use ELIASA to measure the absorbance of wavelength as 595nm.
6. calculate the protein concentration in the synthetic peptide supernatant of different expression time according to standard curve.
The concentration that said method is measured is the total protein concentration in the supernatant, accounts for the percentage ratio of total protein again according to purpose band albumen in the scanning densitometer scanning SDS-PAGE glue, can calculate the expression of synthetic peptide at different time.
1. experimental program result: according to the OD595 value of standard protein BSA under variable concentrations; The total protein expression of calculating at 48h, 72h, 96h different time is respectively 75mg/L, 90mg/L, 120mg/L; Account for the total protein different weight percentage through the synthetic peptide content of scanning densitometer scanning again; Can calculate the synthetic peptide expression of 48h, 72h, 96h and be respectively 40mg/L, 57mg/L, 80mg/L, confirm that thus optimum expression time is 96h, promptly confirm the incubation time of synthetic peptide large scale fermentation.The soluble protein standard substance are got 10 μ l and are diluted to 100 μ l fully, and making final concentration is 0.5mg/ml.Protein sample is in what solution, and what solution dilution standard substance also should use.But for for simplicity, also can be with 0.9%NaCl or PBS dilution standard article.The soluble protein standard substance are got 10 μ l and are diluted to 100 μ l fully, and making final concentration is 0.5mg/ml.Protein sample is in what solution, and what solution dilution standard substance also should use.But for for simplicity, also can be with 0.9%NaCl or PBS dilution standard article.
Embodiment 4
Zoopery (serum ELISA detection):
1.1 grouping of mice and immunity
30 of mices are divided into 5 groups at random, 5 every group.Head exempts from, two exempt from and three exempt from each and be 14d at interval, before each immunity mouse tail vein blood sampling separation of serum put-20 ℃ subsequent use.The immunity group has A:pGAPZ α A-SP import oil emulsion group; B:pGAPZ α A-SP does not have the adjuvant group; C:pGAPZ α A group; D: positive controls; The E:PBS matched group.Immunization protocol is following:
(1) each group is all immune 3 times, immunity 14 days at interval.
(2) pGAPZ α A-SP import oil emulsion group is processed Emulsion with the synthetic peptide of import oil emulsion adjuvant and purification, and making synthetic peptide protein concentration is 50 μ g/mL, the intramuscular injection immunity, and 200 μ l/only/inferior.
(3) pGAPZ α A-SP does not have the adjuvant group and processes Emulsion with the synthetic peptide of aseptic PBS and purification, and making synthetic peptide protein concentration is 50 μ g/mL, the intramuscular injection immunity, and 200 μ l/only/inferior.
(4) pGAPZ α A group is mixed and made into Emulsion with aseptic PBS and empty carrier albumen, and making final concentration is 50 μ g/mL, intramuscular injection, and 200 μ l/only/inferior.
(5) as positive controls, calculate ID according to the description of commodity animal weight is 20 μ l/only/inferior to commodity with synthetic peptide vaccine.
(6) the PBS matched group uses aseptic PBS, intramuscular injection, and 200 μ l/only/inferior.
1.2 vaccine is to the laboratory animal safety testing
Observe the mice of immunity inoculation continuously, part and general reaction situation after the investigation injected in mice.
1.3 the mice antigen-specific antibodies detects
Dock respectively and take a blood sample in after the immunity 14 days at every turn, detects the variation of antigen-specific antibody horizontal with O type foot and mouth disease ELISA detection kit.ELIAS secondary antibody is with the anti-mouse antibodies of HRP labelled goat of 1:2500 dilution.Make negative control with normal mouse serum (PBS immune group).OD450nm value when serum 1:100 is doubly diluted is carried out statistical analysis, the poor opposite sex.
2. result
2.1 laboratory animal safety experiment
After 25 mices are inoculated, observed continuously 4 days, spirit, appetite is normal, all do not occur explaining that because of death, obviously redness, local ulcer and general reaction that vaccinate causes vaccine is safe to mice.
2.2 the testing result of antigen-specific antibodies level
14d after each immunity, docking blood sampling respectively, separation of serum detects the variation of antigen-specific antibody horizontal with ELISA.The result sees table
The ELISA testing result of the anti-VP1 protein antibodies of table 1 immune serum (OD450nm value)
| The immunity group | Head exempts from back 14d | Two exempt from back 14d | Three exempt from back 14d |
| PGAPZ α A-SP import oil emulsion group | 0.433 ± 0.115 | 0.760 ± 0.076 | 1.610 ± 0.115 |
| PGAPZ α A-SP does not have the adjuvant group | 0.510 ± 0.083 | 0.633 ± 0.230 | 0.649 ± 0.083 |
| PGAPZ α A group | 0.323 ± 0.050 | 0.327 ± 0.067 | 0.334 ± 0.050 |
| Positive controls | 0.635 ± 0.054 | 1.671 ± 0.042 | 2.764 ± 0.054 |
| The PBS matched group | 0.297 ± 0.040 | 0.305 ± 0.043 | 0.313 ± 0.040 |
Visible by table 1, three exempt from back 14d, and positive controls and other are respectively organized antibody horizontal there were significant differences (P＜0.05); PGAPZ α A-SP import oil emulsion group is exempted from the back antibody horizontal from two and is risen comparatively fast, and three exempt from the back does not have adjuvant group, empty carrier group and PBS matched group with pGAPZ α A-SP all there were significant differences; PGAPZ α A-SP does not have the antibody that the adjuvant group has produced certain level, but three immune change in values are little; PGAPZ α A group and PBS matched group do not have pig O type foot and mouth disease specific antibody and produce.Above presentation of results, the pig O type aftosa synthetic peptide vaccine production process that is provided among the present invention need not to contact any foot and mouth disease and live malicious, safe and reliable.Simultaneously, the adjuvant side effect of being adopted is little, can increase substantially the specific immune response of body to synthetic peptide.In a word, this vaccine immunogenicity is good, and is safe, and low production cost has good using value and promotion prospect.
SEQUENCE LISTING
< 110>Guangzhou is from bio tech ltd far away
< 120>one boar O type aftosa synthetic peptide vaccines and preparation method thereof
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<170> PatentIn version 3.2
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< 213>pig O type aftosa synthetic peptide aminoacid sequence
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Val Ala Asn Val Arg Gly Asp Leu Gln Val Leu Thr Pro Lys Ala Ala
1 5 10 15
Arg Thr Leu Pro Pro Gly Arg His Lys Gln Lys Ile Val Ala Pro Val
20 25 30
Lys Gln Ser Leu Gln Phe Glu Leu Glu Phe Met Val Pro Ser Arg Val
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Ala Asn Val Arg Gly Asp Leu Gln Val Leu Thr Pro Lys Ala Ala Arg
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Thr Leu Pro
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< 213>nucleotide sequence of coding pig O type aftosa synthetic peptide
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ccaggtagac acaagcaaaa gatcgttgct ccagttaagc aatccttgca attcgagttg 120
gagttcatgg ttccatccag agttgctaac gttagaggtg acttgcaagt tttgactcca 180
aaggctgcta gaactttgcc a 201
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< 213>ten one peptide linkers
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Gln Phe Glu Leu Glu Phe Met Val Pro Ser Arg
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gctgaagctg aattcgttg 19
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Claims (6)
1. a boar O type aftosa synthetic peptide vaccine is characterized in that its aminoacid sequence shown in SEQ ID NO:1, and gene order is shown in SEQ ID NO:2; What said aminoacid sequence adopted is to have antigenic 141-160 position and 200-213 amino acids peptide section in the pig O type foot and mouth disease VP1 albumen, and it is formed the cascaded structure of multicopy, adds amino acid polypeptide in peptide section junction as joint; Antigen polypeptide adopts following cascaded structure: Y-M-X-N-Y, and wherein " X " and " Y " represents pig O type foot and mouth disease VP1 protein 14 1-160,200-213 amino acids peptide section respectively; " M " and " N " represents " Pro-Gly " two peptide linkers and 11 peptide linkers respectively, and the aminoacid sequence of said 11 peptide linkers is shown in SEQ ID NO:3.
2. the method for preparing of the said pig O of claim 1 type aftosa synthetic peptide vaccine is characterized in that comprising the steps:
(1) use recombinant DNA technology, the DNA sequence of composite coding pig O type aftosa synthetic peptide makes up pGAPZ α A-SP expression vector;
(2) in the recombinant expression carrier pGAPZ α A-SP transformed host cell Pichia sp., the construction expression engineering bacteria;
(3) the fermentation culture recombinant yeast is expressed, and expression product is made SDS-PAGE and Western-blot Analysis and Identification;
(4) expression product purification, and select for use adjuvant emulsion to process novel pig O type aftosa synthetic peptide vaccine.
3. according to the method for preparing of the said pig O of claim 2 type aftosa synthetic peptide vaccine, it is characterized in that said expression vector is a yeast expression vector.
4. according to the method for preparing of the said pig O of claim 2 type aftosa synthetic peptide vaccine; It is characterized in that said step (3) is specially with the tall and slender single bacterium colony with Zeocin resistance that screens of sterilization toothpick; Choose and in the YPD of 5 mL fluid medium, carry out one-level cultivation, 28 ℃-30 ℃, 200 r/min shaken overnight; To OD600=2-6, this moment, cell was in exponential phase; Get 1 mL one-level culture fluid, be resuspended among the YPD of 30 mL, continue shaken cultivation, add two-layer newspaper wrapping with four layers of clean gauze.
5. cultivated 72 hours, centrifugal 5 min of 3000 r/min collect the culture fluid supernatant, promptly get purifying protein.
6. according to the method for preparing of the said pig O of claim 2 type aftosa synthetic peptide vaccine, it is characterized in that adjuvant is the import oil emulsion adjuvant described in the said step (4).
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103214560A (en) * | 2013-03-25 | 2013-07-24 | 中国牧工商(集团)总公司 | Polypeptide used for preparing bovine foot and mouth disease type O peptide vaccine, and preparation method and application thereof |
| CN103773803A (en) * | 2014-01-23 | 2014-05-07 | 中国农业科学院特产研究所 | Recombined cattle parainfluenza carrier for expressing protein VP1 of porcine O type foot-and-mouth disease virus |
| CN103848902A (en) * | 2014-03-07 | 2014-06-11 | 中牧实业股份有限公司 | Synthetic peptide vaccine and preparation method thereof |
| CN105820217A (en) * | 2014-03-07 | 2016-08-03 | 中牧实业股份有限公司 | Synthetic peptide vaccine and preparation method thereof |
| CN110885362A (en) * | 2019-11-22 | 2020-03-17 | 中牧实业股份有限公司 | O-type synthetic peptide vaccine for foot-and-mouth disease and preparation method and application thereof |
| CN110894214A (en) * | 2019-11-22 | 2020-03-20 | 中牧实业股份有限公司 | Foot-and-mouth disease O-type epitope polypeptide and preparation method and application thereof |
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| CN101082051A (en) * | 2006-06-02 | 2007-12-05 | 广东出入境检验检疫局检验检疫技术中心 | Recombined foot-and-mouth disease virus polypeptide fragment and and uses thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103214560A (en) * | 2013-03-25 | 2013-07-24 | 中国牧工商(集团)总公司 | Polypeptide used for preparing bovine foot and mouth disease type O peptide vaccine, and preparation method and application thereof |
| CN103773803A (en) * | 2014-01-23 | 2014-05-07 | 中国农业科学院特产研究所 | Recombined cattle parainfluenza carrier for expressing protein VP1 of porcine O type foot-and-mouth disease virus |
| CN103848902A (en) * | 2014-03-07 | 2014-06-11 | 中牧实业股份有限公司 | Synthetic peptide vaccine and preparation method thereof |
| CN105820217A (en) * | 2014-03-07 | 2016-08-03 | 中牧实业股份有限公司 | Synthetic peptide vaccine and preparation method thereof |
| CN105906693A (en) * | 2014-03-07 | 2016-08-31 | 中牧实业股份有限公司 | Synthetic peptide vaccine as well as preparation method and application thereof |
| CN105820217B (en) * | 2014-03-07 | 2019-06-07 | 中牧实业股份有限公司 | Synthetic peptide vaccine and preparation method thereof |
| CN110885362A (en) * | 2019-11-22 | 2020-03-17 | 中牧实业股份有限公司 | O-type synthetic peptide vaccine for foot-and-mouth disease and preparation method and application thereof |
| CN110894214A (en) * | 2019-11-22 | 2020-03-20 | 中牧实业股份有限公司 | Foot-and-mouth disease O-type epitope polypeptide and preparation method and application thereof |
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Application publication date: 20120718 |