WO2005034993A1 - Vaccin de proteine hybride vp1 de virus recombine de la fievre aphteuse - Google Patents

Vaccin de proteine hybride vp1 de virus recombine de la fievre aphteuse Download PDF

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Publication number
WO2005034993A1
WO2005034993A1 PCT/CN2004/001168 CN2004001168W WO2005034993A1 WO 2005034993 A1 WO2005034993 A1 WO 2005034993A1 CN 2004001168 W CN2004001168 W CN 2004001168W WO 2005034993 A1 WO2005034993 A1 WO 2005034993A1
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foot
mouth disease
disease virus
fusion protein
recombinant
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PCT/CN2004/001168
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Chinese (zh)
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Yongli Yu
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Beijing Hydvax Biotechnology 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to the field of genetic engineering, and particularly to a genetically engineered recombinant protein vaccine, and in particular, to a recombinant foot-and-mouth disease virus VP1 fusion protein vaccine (hereinafter sometimes referred to as a recombinant foot-and-mouth disease virus VP1 fusion protein) for preventing animal foot-and-mouth disease virus infection.
  • a recombinant foot-and-mouth disease virus VP1 fusion protein vaccine hereinafter sometimes referred to as a recombinant foot-and-mouth disease virus VP1 fusion protein
  • It is a recombinant fusion protein produced by fusing the VP1 peptide, 2 polyglycine, and 6 polyhistidine-encoding genes of foot-and-mouth disease virus, produced in E. coli, which can induce the body to produce neutralizing antibodies against foot-and-mouth disease virus. It has biological activity to prevent animal foot-and-mouth disease virus infection.
  • Foot-and-mouth disease is a severe infectious disease caused by foot-and-mouth disease. Foot-and-mouth disease can occur in pigs, cattle, and sheep due to the infection of the foot-and-mouth disease virus. In mild cases, blisters, ulcers, and plaques appear in the mouth, tongue, lips, hoofs, and breasts. In severe cases, death occurs. The occurrence and spread of foot-and-mouth disease can cause huge direct and indirect economic losses (Jiang Pengfei, 1999). Foot-and-mouth disease is classified as a Class A livestock infectious disease by the International Organisation for Animal Diseases (OIE). countries around the world attach great importance to the prevention and control of foot-and-mouth disease.
  • OFE International Organisation for Animal Diseases
  • FMD vaccines such as recombinant protein vaccines, synthetic peptide vaccines, live vector vaccines and DNA vaccines (Broel huigsen MP, 1987; Morgan DO, 1990 Ward, G, 1997; Berinstein A, 2000). Some vaccines have shown good results, but most are still being explored in the laboratory.
  • Foot-and-mouth disease virus is a pathogen of foot-and-mouth disease virus, and is a member of the genus Apovirus of the small RA virus family (Picomaviridae). Seven serotypes have been found, namely 0, A, Type C (called European type), SAT1, SAT2, SAT3 (South Africa 1, 2, 3, also called African type) and Asial (Asian I type, called Asian type). Foot-and-mouth disease virus type 0 is a serotype that has spread widely worldwide in recent years. Studies show that targeted humoral immunity Foot-and-mouth disease virus plays a major role. In domestic animals, the level of FMD virus neutralizing antibodies is significantly positively related to their ability to resist FMD virus attack (Pay TW, 1987).
  • Vpl coat protein l
  • Neutralizing antibodies produced by immunizing guinea pigs and cattle with peptides (141-160 and 200-213) of Vpl protein can protect animals from foot-and-mouth disease virus (Bittle JL, 1982; Pfaff E 5 1982; DiMarchi R, 1986) cVPl 140
  • the "loop" -like structure composed of -160 amino acids is exposed on the surface of the virus.
  • the RGD sequence is highly conserved among the foot-and-mouth disease virus. , 2002; Almeida MR, 1998).
  • the peptides synthesized based on the VP1 loop structure can induce high levels of neutralizing antibodies.
  • immunizing animals with Vpl protein isolated from foot-and-mouth disease virus or genetically engineered methods can provide partial protection. Fusion of Vpl protein with proteins derived from other microorganisms through genetic engineering can improve the immunogenicity of Vpl protein (Clarke BE, 1987; Corchero JL, 1996).
  • Integrin ⁇ is a receptor for foot-and-mouth disease virus. Journal of Virology (J. Virol.). 2002 Feb; 76 (3): 935-41.
  • One aspect of the present invention is to provide a recombinant foot-and-mouth disease virus VP1 fusion protein vaccine, which is a recombinant fusion protein produced by fusing the VP1 peptide of foot-and-mouth disease virus, 2 polyglycine, and 6 polyhistidine-encoding genes.
  • Another aspect of the present invention provides an amino acid sequence of a recombinant foot-and-mouth disease virus VP 1 fusion protein and a nucleotide sequence thereof.
  • Another aspect of the present invention provides the nucleotide sequence of the foot-and-mouth disease virus VP1 BT1 fusion peptide and the amino acid sequence encoded thereby.
  • Another aspect of the present invention provides the nucleotide sequence of the foot-and-mouth disease virus VP1 BT2 fusion peptide and its encoded amino acid sequence.
  • Another aspect of the present invention provides a linking mode of the foot-and-mouth disease virus VP1 BT1 fusion peptide, including the presence, linking mode and position of 2 polyglycine.
  • Another aspect of the present invention provides a linking mode of the foot-and-mouth disease virus VP1 BT2 fusion peptide, including the presence, linking mode and position of 2 polyglycine.
  • Another aspect of the present invention provides a VP1 peptide fusion protein of foot-and-mouth disease virus VP1 with 6 polyhistidines on both sides.
  • Another aspect of the present invention relates to a recombinant foot-and-mouth disease virus VP1 fusion protein.
  • Figure 1 SDS-PAGE electrophoresis of the expressed recombinant foot-and-mouth disease virus VP 1 fusion protein
  • FIG. 1 SDS-PAGE electrophoresis of purified recombinant foot-and-mouth disease virus VP1 fusion protein
  • Figure 3 Protective effect of recombinant foot-and-mouth disease virus VP1 fusion protein on guinea pigs (1) serum on suckling mice
  • Figure 4 Protective effect of recombinant foot-and-mouth disease virus VP1 fusion protein on guinea pigs (2) serum on suckling mice
  • a gene such as a foot-and-mouth disease virus gene with an immunogenic protein or polypeptide is cloned into an expression vector of a prokaryotic or eukaryotic cell
  • the gene produced by the bacterium or eukaryotic cell is used.
  • the protein or polypeptide encoded by the viral gene is a recombinant protein vaccine with antiviral effects such as foot-and-mouth disease virus.
  • the vaccine can induce specific humoral and / or cellular immune responses in animals after being applied to animals, and prevent infections caused by viruses such as foot-and-mouth disease virus.
  • FMDV infection in animals A contact infectious disease common to artiodactyls caused by FMDV.
  • FMDV is an RNA virus whose genome contains approximately 7500-8000 bases.
  • FMDV is mainly transmitted in the form of saliva, and the speed of transmission is very fast, which is likely to cause widespread circulation.
  • Cows, pigs, sheep, deer, etc. are all infected animals of foot-and-mouth disease virus.
  • Foot-and-mouth disease virus VP1 antigen It is a part of FMD virus shell protein, which can induce the body to produce protective antibodies against foot-and-mouth disease virus.
  • Foot-and-mouth disease virus VP1 peptides include foot-and-mouth disease virus VP1 BT1 fusion peptide and foot-and-mouth disease virus VP1 BT2 fusion peptide.
  • Foot-and-mouth disease virus VP1 BT1 fusion peptide refers to a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 (SEQ ID NO 2).
  • Foot-and-mouth disease virus VP1 BT2 fusion peptide refers to a polypeptide having the amino acid sequence shown in SEQ ID NO 4 (SEQ ID N04).
  • 2-Polyglycine is a 2 peptide composed of two adjacent glycines.
  • Foot-and-mouth disease virus VP1 peptide fusion protein-encoding gene refers to DNA having a nucleotide sequence shown in SEQ ID NO: 5, and the encoded protein is a foot-and-mouth disease virus VP1 peptide fusion protein.
  • the gene encoding the recombinant foot-and-mouth disease virus VP1 fusion protein vaccine has a nucleotide sequence as shown in SEQ ID NO: 8, and the encoded protein is a recombinant foot-and-mouth disease virus VP1 fusion protein vaccine or a recombinant foot-and-mouth disease virus VP1 fusion protein. NO: 9 amino acid sequence.
  • the present invention provides a recombinant foot-and-mouth disease virus VP1 fusion protein vaccine, which is a recombinant fusion protein produced by fusing VP1 peptides, 2-polyglycine, and 6-histidine-encoding genes of foot-and-mouth disease virus.
  • the recombinant foot-and-mouth disease virus VP1 fusion protein vaccine of the present invention has a nucleotide sequence shown in SEQ ID NO: 8 and an amino acid sequence shown in SEQ ID NO: 9.
  • the foot-and-mouth disease virus VPl BT1 fusion peptide in the recombinant foot-and-mouth disease virus VP1 fusion protein provided by the present invention has the nucleotide sequence shown in SEQ ID NO: 1 and the amino acid sequence shown in SEQ ID NO: 2.
  • the foot-and-mouth disease virus VPl BT2 fusion peptide in the recombinant foot-and-mouth disease virus VP1 fusion protein provided by the present invention has the nucleotide sequence shown in SEQ ID NO: 3 and the amino acid sequence shown in SEQ ID NO: 4.
  • the foot-and-mouth disease virus VP1 peptide fusion protein encoding gene of the recombinant foot-and-mouth disease virus VP1 fusion protein provided by the present invention has a nucleotide sequence as shown in SEQ ID NO: 5, and the encoded protein is a foot-and-mouth disease virus VP1 peptide fusion protein.
  • the foot-and-mouth disease virus VPl BT1 fusion peptide in the recombinant foot-and-mouth disease virus VP1 fusion protein provided by the present invention has a connection manner shown in SEQ ID NO: 2, including the presence, connection manner and position of 2 polyglycine.
  • the foot-and-mouth disease virus VPl BT2 fusion peptide in the recombinant foot-and-mouth disease virus VP1 fusion protein provided by the present invention has a connection manner shown in SEQ ID NO: 4, including the presence, connection manner and position of 2 polyglycine.
  • the foot-and-mouth disease virus VP1 peptide fusion protein in the recombinant foot-and-mouth disease virus VP1 fusion protein provided by the present invention has a connection manner as shown in SEQ ID NO: 5.
  • the FM1 VP1 peptide fusion protein of the recombinant FM1 virus VP1 fusion protein provided by the present invention has a 6-polyhistidine sequence on both sides of the fusion protein.
  • the recombinant foot-and-mouth disease virus VP1 fusion protein provided by the present invention is produced by E. coli.
  • the recombinant VP1 fusion protein provided by the present invention can induce the body to produce a neutralizing antibody against the foot-and-mouth disease virus after being applied to an animal, and has the biological activity of preventing the animal's foot-and-mouth disease virus infection.
  • the invention also relates to the use of the genetically engineered recombinant protein in the preparation of a vaccine product for preventing infection of animal foot-and-mouth disease virus, and a vaccine product containing the genetically engineered recombinant protein.
  • these vaccine preparations can be prepared by various conventional methods known in the art.
  • the recombinant protein vaccine of the present invention can be vaccinated to animals by subcutaneous injection, and the vaccinated dose is 100-5000 ⁇ ⁇ .
  • the booster can be performed once every 14 or 21 days after the first immunization.
  • PCRThe VP1 BT1 fusion peptide coding gene was synthesized by PCR method.
  • a PCR primer with the following sequence was designed and synthesized:
  • a DNA fragment (fragments 1, 2) with the following sequence was synthesized using primers 1, 2:
  • primers 3 and 4 were used to synthesize a VP1 BT1 fusion peptide-encoding gene with restriction sites on both sides. The sequence is shown in SEQ ID NO: 1.
  • Reaction conditions 94 ° C, 30 "; 55 ° C, 1 '; 72 ° C, 2', after 30 cycles, extended at 72 ° C for 10 minutes.
  • the PCR products were subjected to 2% agarose gel electrophoresis (lxTAE , 150-200mA, 0.5 hours :).
  • the DNA fragment of the VPl BT1 fusion peptide encoding gene synthesized by PCR was recovered using the DNA recovery kit of Beijing Dingguo Company.
  • the gel containing the DNA fragment was excised from the agarose gel and placed Into a centrifuge tube. Add 3 times the volume of sol solution (Beijing Dingguo Company), 45-55 ° C water bath for 5-10 ⁇ to completely melt the glue.
  • the DNA fragment of the VP1 BT1 fusion peptide encoding gene was cloned into the pMD18-T Vect plasmid (TakaRa).
  • the pMD18-T Vect plasmid cloned with the VP1 BT1 fusion peptide-encoding DNA fragment was transformed into E. coli JM109 (Novagen).
  • the method for preparing competent E. coli JM109 cells is: take a single colony of E. coli JM109 in 2ml LB medium, culture at 37 ° C, 225 rpm, and shake for 12-16 hours; take 1ml of the above culture and inoculate it into 100ml LB medium , 37 ° C, shaking culture at 225 rpm until the OD value is about 0.5 (about 3 hours); the bacterial solution is ice-bathed for 2 hours, and then centrifuged at 2,500 X g, 4 ° C for 20 minutes to collect the bacterial cells; add 100ml ice-cold Trituration Buffer (100mmol / L CaCl 2 , 70mmol / L MgCl 2 , 40mmol / L sodium acetate, pH 5.5), mixed with hook, and placed on ice for 45 minutes; 1,800 Xg, centrifuged at 4 ° C for 10 minutes, discarded the supernatant, added 10ml of ice-
  • the transformation method is: 200 ⁇ 1 competent cells are thawed on ice, and then 3 ⁇ DMSO or ⁇ -mercaptoethanol is added. After mixing, 2 ⁇ ligation reaction solution (containing recombinant plasmid) is added, mixed, and placed on ice 30 Minutes; 42 ° C for 45 seconds, then quickly put back on ice for 1_2 minutes; add 21111 1 ⁇ culture medium, shake and culture for 1 hour at 37 ° (, 225rpm); centrifuge at 4,000 X g for 10 seconds, discard the supernatant, and use 200 ⁇ 1
  • the LB medium was resuspended; the bacteria liquid was spread on the LB agar culture plate containing antibiotics, spread evenly, and left at room temperature for 20-30 minutes, and then placed in a 37 ° C incubator for 12-16 hours.
  • primers 9, 10 were used to synthesize a VP1 BT2 fusion peptide coding gene with restriction enzyme cleavage points on both sides, which has the sequence shown in SEQ ID NO: 3.
  • Example 3 The PCR operation procedure used was the same as in Example 1.
  • the method for recovering and cloning the VP1 BT2 fusion peptide-encoding gene is similar to that in Example 1.
  • the pMD18-T Vect plasmid containing the gene encoding the VP1 BT2 fusion peptide was transformed into competent E. coli JM109 cells (the method was similar to that in Example 1). Select positive clones and extract plasmids (J. Sambrook, Cold Spring Harbor Laboratory Press, Molecular cloning, 1989).
  • the VP1 BT1 fusion peptide encoding gene on the pMD18-T Vect plasmid was digested with BamHI
  • the BT2 fusion peptide encoding gene on the pMD18-T Vect plasmid was digested with BglHI
  • the two digested fragments were ligated with T4 ligase to obtain the BT1 fusion peptide encoding gene- BT2 fusion peptide encoding gene fusion gene
  • SEQ ID NO: 5 c BamHI and Bglll were used to digest the VPl BTl fusion peptide-encoding gene-VPl BT2 fusion peptide-encoding gene fusion gene to obtain BamHI-digested VPl BT1 fusion peptide-encoding gene-VPl BT2 fusion peptide-encoding gene fusion gene fragment and Bglll-digested VPl BT1 fusion peptide
  • VPl BT1 fusion peptide-encoding gene The VP1 BT2 fusion peptide-encoding gene is a 3-mer gene that is both a VP1 peptide fusion protein-encoding gene.
  • the gene encoding the VP1 peptide fusion protein was cloned into the PMD18-T Vect plasmid using the same method as used in Example 1.
  • the pMD18-T Vect plasmid containing the gene encoding the W1 peptide fusion protein was transformed into competent E. coli JM109 cells (the method was similar to that in Example 1). Select positive clones and extract plasmids (J. Sambrook, Cold Spring Harbor Laboratory Press, Molecular cloning, 1989).
  • the pMD18-T Vect plasmid and pET28 plasmid containing the VPl peptide fusion protein encoding gene were digested with EcoRI and Hindlll.
  • Plasmid DNA 1 ⁇ g 10x Buffer ( ⁇ buffer Lot: A1018, TaKa a) ⁇ ⁇ ⁇ Restriction enzyme EcoRI (10 units / ⁇ 1) 1 ⁇ 1
  • T4 DNA ligase (T4 Ligation Code No: D2011A 5 TaKaRa) 1 ⁇ 1 Make up to 10 ⁇ with double distilled water. ⁇ Mix and place in a water bath at 14-16 ° C for 6-12 hours.
  • the recombinant pET-28a (+) plasmid containing the gene encoding the VP1 peptide fusion protein of foot-and-mouth disease virus was transformed into the E. coli expression strain BL21DE3.
  • DNA sequencing was performed on the insert in the pET-28a (+) plasmid.
  • the pET-28a (+) plasmid EcoRI and Hindlll cleavage sequences were used to fuse the recombinant foot-and-mouth disease virus VP1 fusion protein arranging a gene SEQ ID NO: 8.
  • This gene (1092 bases) encodes a recombinant foot-and-mouth disease virus VP fusion protein consisting of 363 amino acid residues.
  • the amino acid sequence of this recombinant protein is shown in SEQ ID NO: 9.
  • a single colony of bacteria was inoculated into 100 ml of LB medium, and cultured in a 500 ml Erlenmeyer flask with 37 ° C water bath shaking to an OD600 of 0.6. IPTG was added to a final concentration of lmM, and cultured in a 37 ° C water bath with shaking for 3 hours. Place the Erlenmeyer flask on ice for 5 minutes and centrifuge at 4 ° C for 5 minutes (5000 xg). Discard the supernatant and collect bacteria for immediate use or frozen storage. Samples were taken for SDS-PAGE analysis of the expression of the recombinant foot-and-mouth disease virus VP1 fusion protein ( Figure 1).
  • binding solution to the precipitate, and add 1 ml of the binding solution (6 mM Tris, 0.5 M NaCl, 5 mM imidazole, 6 M urea, adjust the pH to 7.9). Shake in an ice bath for 2 hours. Centrifuge at 5,000 rpm for 30 min. Collect the supernatant and use the supernatant to the column.
  • 1 ml of the binding solution (6 mM Tris, 0.5 M NaCl, 5 mM imidazole, 6 M urea, adjust the pH to 7.9). Shake in an ice bath for 2 hours. Centrifuge at 5,000 rpm for 30 min. Collect the supernatant and use the supernatant to the column.
  • the column was packed with phosphate buffer (20 mM phosphate, pH 7.2 IM NaCl), and the chromatography medium was Sepharose4B-Ni 2+ (Pharmacia) and equilibrated.
  • Wash the column with Wash 1 buffer (20 mM Tris, pH 7.9, 0.5 M NaCl, 20 mM imidazole, 4 M urea).
  • the eluted protein was collected, and this protein was a recombinant foot-and-mouth disease virus VP1 fusion protein.
  • the recombinant VP1 fusion protein of FMDV was lyophilized.
  • the purified recombinant FMD virus VP1 fusion protein was identified by SDS-PAGE, and its purity was 95% (see Figure 2).
  • VP1 fusion protein of recombinant foot-and-mouth disease virus was dissolved in PBS at a concentration of 50 ⁇ g / 0.2 ml.
  • Intravenous (penile vein) injection 0.2 ml per guinea pig.
  • a control group of guinea pigs was injected with 0.2 ml of PBS.
  • Foot-and-mouth disease virus was purified at the Inner Mongolia Biopharmaceutical Plant.
  • Fresh BMD virus type 01 fluid (Inner Mongolia Biopharmaceutical Factory), which was expanded by BHK monolayer cell culture, was taken at 4000 rpm for 20 minutes to remove cell debris.
  • Saturated polyethylene glycol 6000 was added dropwise to the supernatant to a final concentration of 7%, while stirring, the solution was left to stand at 4 ° C overnight.
  • the pellet was collected.
  • the pellet was resuspended in PB solution at pH 7.6, and further purified on a sucrose density gradient of 15% -45% (centrifugal force was 35,000 grams-force, time was 3 hours, and temperature was 4 ° C).
  • Virus particles of 146s appeared in 30% -35% sucrose. It was diluted 5 times with PB solution, centrifuged on 20% sucrose-containing PB solution for 2 hours, the centrifugal force was 35,000 grams force, and the temperature was 4 ° C. The precipitate was collected. The pellet was resuspended in PB solution, and the concentration of purified FMD virus was determined by BCA method.
  • the coating solution was 0.05M, carbonate buffer with a pH value of 9.6, 100 ⁇ l per well, containing 1 ⁇ ⁇ of foot-and-mouth disease virus antigen, and coated overnight at 4 ° C. After washing the plate three times with a PBS (PBS-T) washing solution containing 0.05% Tween 20, add 200 ⁇ 1 of blocking solution (PBS-T containing 2% BSA) and place in a 37 ⁇ incubator for 1 hour. After washing the plate 3 times, 100 ⁇ l of the diluted (1: 2048) serum to be tested was added to each well and placed in a 37 ° C incubator for 1 hour. Set 3 compound holes.
  • Blank control OD value: 0.005 ⁇ 0.0005
  • Example 9 Neutralization experiment on sera from recombinant foot-and-mouth disease virus VP1 fusion protein
  • the volume of the serum to be tested and the foot-and-mouth disease virus solution (100 TCID50 / 0.1ml) were diluted by a factor of two, and incubated at 37 ° C for 1 hour. 0.2ml of the above-mentioned mixed solution was injected subcutaneously through the back of a suckling rat's neck, and observation was continued for 3 days, and the incidence and death of each group were recorded.
  • Neutralization experiments in suckling mice were performed at the Inner Mongolia Biopharmaceutical Plant using "O" foot-and-mouth disease virus strains.
  • the concentration of the virus is 1 ml 1000LD50.
  • Dilute guinea pig serum Mix 100 ⁇ l guinea pig serum and 100 ⁇ l virus dilution at different dilutions and incubate at 37 ° C for 1 hour. The above mixture was injected into the back of a suckling rat.

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Abstract

L'invention concerne un vaccin de protéine hybride VP1 recombinée, qui est une protéine hybride recombinée ayant réuni par fusion, les gènes de codage de peptides vp1, deux poly-Glycines, six poly-Histidines du virus de la fière aphteuse et qui est produite dans E. coli. L'invention concerne également la séquence d'aminoacide et la séquence de nucléotide de la protéine hybride VP1 recombinée du virus de la fièvre aphteuse. L'invention concerne en outre l'utilisation de la protéine hybride VP1 recombinée du virus de la fière aphteuse pour prévenir l'infection par le virus de la fière aphteuse.
PCT/CN2004/001168 2003-10-15 2004-10-14 Vaccin de proteine hybride vp1 de virus recombine de la fievre aphteuse WO2005034993A1 (fr)

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CNB2003101001751A CN1290579C (zh) 2003-10-15 2003-10-15 重组口蹄疫病毒vp1融合蛋白疫苗
CN200310100175.1 2003-10-15

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Publication number Priority date Publication date Assignee Title
WO2013001285A3 (fr) * 2011-06-30 2013-03-14 The Pirbright Institute Peptide

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CN102212137A (zh) * 2010-04-07 2011-10-12 高地 一种用于偶蹄类动物的重组基因工程多肽
CN102372766B (zh) * 2011-07-13 2013-12-25 青岛红桥明勤生物科技有限公司 一种o型口蹄疫多表位疫苗
CN104474542A (zh) * 2014-11-18 2015-04-01 天津瑞普生物技术股份有限公司 一种二联灭活疫苗制备方法

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CN1354674A (zh) * 1998-06-20 2002-06-19 美国联合生物医学公司 用于口蹄疫的合成肽疫苗
CN1440296A (zh) * 2000-06-29 2003-09-03 梅瑞尔公司 抗口蹄疫疫苗

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Publication number Priority date Publication date Assignee Title
CN1354674A (zh) * 1998-06-20 2002-06-19 美国联合生物医学公司 用于口蹄疫的合成肽疫苗
CN1440296A (zh) * 2000-06-29 2003-09-03 梅瑞尔公司 抗口蹄疫疫苗

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013001285A3 (fr) * 2011-06-30 2013-03-14 The Pirbright Institute Peptide
US9457075B2 (en) 2011-06-30 2016-10-04 The Pirbright Institute Modified foot and mouth disease virus (FMDV) VP1 capsid protein

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