WO2005034993A1 - Vp1 fusion protein vaccin of recombinant foot-and-mouth disease virus - Google Patents

Vp1 fusion protein vaccin of recombinant foot-and-mouth disease virus Download PDF

Info

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
Authority
WO
WIPO (PCT)
Prior art keywords
foot
mouth disease
disease virus
fusion protein
recombinant
Prior art date
Application number
PCT/CN2004/001168
Other languages
French (fr)
Chinese (zh)
Inventor
Yongli Yu
Original Assignee
Beijing Hydvax Biotechnology Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beijing Hydvax Biotechnology Co., Ltd. filed Critical Beijing Hydvax Biotechnology Co., Ltd.
Publication of WO2005034993A1 publication Critical patent/WO2005034993A1/en

Links

Classifications

    • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Molecular Biology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention provides and recombinant VP1 fusion protein vaccin which is an recombinant fusion protein that fused the coding genes of vp1 peptides, two poly-Glycines, six poly-Histidines of foot-and-mouth disease virus and produced in Ecoli. The invention provides the anino acid sequence and nucleiotide sequence of the recombinant VP1 fusion protein of foor-and-mouth disease virus. The invention also provides the using of the recombinant VP1 fusion protein of foot-and-mouth disease virus to prevent foot-and-mouth disease virus infection.

Description

重组口蹄疫病毒 VPl融合蛋白疫苗 发明领域  Recombinant foot and mouth disease virus VPl fusion protein vaccine Field of the invention
本发明涉及基因工程领域， 具体涉及基因工程重组蛋白疫苗， 特别是涉 及一种预防动物口蹄疫病毒感染的重组口蹄疫病毒 VP1融合蛋白疫苗 （下文 有时也称为重组口蹄疫病毒 VP1融合蛋白）。 它是将口蹄疫病毒 VP1肽段、 2 聚甘氨酸、 6聚组氨酸编码基因融合， 在大肠杆菌生产的重组融合蛋白， 在应 用于动物机体后可以诱发机体产生针对口蹄疫病毒的中和性抗体， 具有预防 动物口蹄疫病毒感染的生物学活性。  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. 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.
发明背景 Background of the invention
口蹄疫 (foot-and-mouthdisease， FMD)是由口蹄疫病毒引起的烈性传染病。 猪、 牛、 羊都可感染口蹄疫病毒而发生口蹄疫， 轻者在口、 舌、 唇、 蹄、 乳 房等部位出现水泡、 破溃及烂斑等病变， 重者发生死亡。 口蹄疫的发生和流 行会造成巨大的直接和间接的经济损失 (江鹏斐， 1999)。 国际兽疫局 (OIE)将 口蹄疫列为 A类家畜传染病。世界各国对预防和控制口蹄疫都十分重视。 除 屠杀感染口蹄疫的家畜外， 接种疫苗是预防和控制口蹄疫的主要措施 (M. Wool ouse, 2001)。 传统的口蹄疫疫苗由灭活的初步纯化的口蹄疫病毒加油 佐剂乳化制成的灭活苗， 对易感家畜有相当的保护作用， 但存在灭活不充分 而引发口蹄疫的可能。 此外， 在生产这种疫苗过程中动用的有活力的口蹄疫 病毒是一种不可忽视的潜在传染源。  Foot-and-mouth disease (FMD) 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. In addition to the slaughter of livestock infected with FMD, vaccination is the main measure for the prevention and control of FMD (M. Wool ouse, 2001). Traditional foot-and-mouth disease vaccines are inactivated vaccines prepared by inactivating the preliminary purified foot-and-mouth disease virus with an adjuvant emulsification, which has considerable protective effects on susceptible livestock, but there is a possibility that foot-and-mouth disease may be caused by insufficient inactivation. In addition, the vibrant foot-and-mouth disease virus used in the production of this vaccine is a potential source of infection that cannot be ignored.
二十世纪 80年代初以来，许多国家投入了大量人力、物力和财力研制新 型口蹄疫疫苗， 如重组蛋白疫苗， 合成肽疫苗， 活载体疫苗和 D N A疫苗等 (Broel huigsen M P, 1987; Morgan D O, 1990； Ward, G， 1997； Berinstein A， 2000)。 一些疫苗显示了较好的效果， 但大部分仍在实验室的探索之中。  Since the early 1980s, many countries have invested a lot of human, material and financial resources to develop new 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.
口蹄疫病毒 (foot-and-mouth disease virus, FMDV)是口蹄疫的病原体， 是 小 R A病毒科 (Picomaviridae)口蹄疫病毒属 (Ap ovirus)的成员， 现已发现 了 7个血清型， 即 0、 A、 C型 (称欧洲型)， SAT1、 SAT2、 SAT3型 (南非 1、 2、 3型， 也称非洲型)和 Asial型 (亚洲 I型， 称亚洲型)。 0型口蹄疫病毒是 近年来世界范围内流行较广的血清型。 研究表明， 针对的体液免疫在机体抗 口蹄疫病毒感染中起主要作用。 在家畜体内， 口蹄疫病毒中和抗体的水平与 其抵抗口蹄疫病毒攻击的能力呈明显的正相关关系 (Pay TW, 1987)。基于这种 观察， 许多实验室做了大量实验， 在口蹄疫病毒上寻找能剌激机体产生病毒 中和抗体的靶点， 发现在口蹄疫病毒的外壳蛋白 (VP)存在五个侯选的结构 (Crowther JR, 1993； Kitson JD, 1990)， 其中的三个位于外壳蛋白 l(Vpl)中。 实验证明，从口蹄疫病毒中分离出的 Vpl蛋白具有良好的免疫原性 (Bachrach HL, 1975； Kleid DG, 1981； Stro maier K, 1982； Rodriguez A, 1994)。 Foot-and-mouth disease virus (FMDV) 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). Based on this observation, many laboratories have done a lot of experiments to find targets for FMD virus that can stimulate the body to produce virus neutralizing antibodies, and found that there are five candidate structures in the coat protein (VP) of FMD virus (Crowther JR, 1993; Kitson JD, 1990), three of which are located in coat protein l (Vpl). Experiments have shown that the Vpl protein isolated from foot-and-mouth disease virus has good immunogenicity (Bachrach HL, 1975; Kleid DG, 1981; Stro maier K, 1982; Rodriguez A, 1994).
用 Vpl蛋白的肽段 (141-160和 200-213)免疫豚鼠和牛产生的中和性抗体 能保护动物免于感染口蹄疫病毒 (Bittle JL, 1982； Pfaff E5 1982; DiMarchi R， 1986)cVPl 140-160氨基酸构成的 "环"状结构暴露在病毒表面，其中的 R-G-D 序列在口蹄疫病毒高度保守， 是口蹄疫病毒结合细胞表面受体的关键结构， 可能介导口蹄疫病毒进入被感染的细胞 (Jackson T, 2002; Almeida MR, 1998)。 依据 VP1 环状结构合成的肽段能诱导产生高水平的中和抗体。 也有研究表 明， 用从口蹄疫病毒分离出来的或基因工程方法得到的 Vpl蛋白免疫动物， 可以产生部分保护作用。通过基因工程手段把 Vpl蛋白与某些来源于其他微 生物的蛋白融合在一起可以提高 Vpl 蛋白的免疫原性 (Clarke BE, 1987； Corchero JL, 1996)。 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. Studies have also shown that 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).
参考文献： references:
江鹏斐，赵启祖，谢庆阁， 口蹄疫研離展。中国农业科学。 1999年； 32(6):93〜100。 艾米达 （Almeida) 等， 构建及评价一种减毒的口蹄疫病毒疫苗： 在对 01血 清型病毒采用主要蛋白酶删除策略中存在的困难。 病毒研究 （Virus. Res.)。 1998年 5月; 55(1):49-60。 Jiang Pengfei, Zhao Qizu, Xie Qingge, FMD Research and Exhibition. Chinese Agricultural Sciences. 1999; 32 (6): 93 ~ 100. Almeida et al., Constructed and evaluated an attenuated foot-and-mouth disease virus vaccine: difficulties in adopting a major protease deletion strategy for 01 serotype viruses. Virus Research (Virus. Res.). May 1998; 55 (1): 49-60.
贝克瑞史 (Bachrach)等，对一种分离的口蹄疫病毒包膜蛋白所激发的免疫和抗 体反应。 免疫学杂志 (Virus. J. Immunol.)。 1975年 12月； 115(6):1636-41。 Bachrach et al., Immune and antibody responses to an isolated FMD virus envelope protein. Journal of Immunology (Virus. J. Immunol.). Dec 1975; 115 (6): 1636-41.
柏林斯坦 (Berinstein)等，重组牛痘病毒所诱导的抗口蹄疫的保护性免疫反应。 疫苗 (Vaccinia Virus Vaccine)。 2000年 4月； 18(21):2231-8。 Berinstein et al., Protective immune response against foot-and-mouth disease induced by recombinant vaccinia virus. Vaccinia Virus Vaccine. April 2000; 18 (21): 2231-8.
贝托 (Bittle)等， 由病毒核苷酸序列预测后经化学合成的肽免疫所激发的抗口 蹄疫保护反应。 自然 ( ature)o 1982年 7月; 298(5869):30-3。 Bittle et al., Anti-foot-and-mouth disease protection response triggered by chemically synthesized peptide immunization predicted by the viral nucleotide sequence. Atureo July 1982; 298 (5869): 30-3.
伯克维森 (Broekhuigsen)等，含有多拷贝口蹄疫病毒表位的融合蛋白能够保护 自然宿主和实验动物。基因病毒学杂志 (J. Gen. Virol.) o 1987年；（68): 3137-430 克拉克 (Clarke)等， 肽表位与乙型肝炎病毒核心蛋白融合后免疫原性大大改 善。 自然 (Nature)。 1987年 11月— 12月; 330(6146):381-4。 Broekhuigsen et al., Fusion proteins containing multiple copies of FMD epitope can protect Natural hosts and experimental animals. Journal of Virology o 1987 Nian gene; (68) (J. Gen. Virol .): 3137-43 0 Clarke (Clarke), etc., after peptide epitopes of hepatitis B virus core protein fusion greatly improved immunogenicity. Nature. November-December 1987; 330 (6146): 381-4.
克史诺 (Corchero)等， /3-半乳糖苷酶融合蛋白中的病毒肽的抗原性受到同型伴 侣蛋白存在的影响。 FEMS微生物通报 (FEMS Microbiol. Lett.)。 1996年 11 月； 145(1):77-82。 Corchero et al., The antigenicity of viral peptides in the / 3-galactosidase fusion protein is affected by the presence of homopartite proteins. FEMS Microbiol. Lett. November 1996; 145 (1): 77-82.
克朗茨 (Crowther)等，由单克隆抗体和五倍单克隆抗体逃逸变异所确定的第五 个 0型口蹄疫病毒中和位点。 基因病毒学杂志 (J. Gen. Virol.)。 1993年 8月; 74(Pt8) : 1 547-53 o Crowther et al., The fifth type 0 foot-and-mouth disease virus neutralization site identified by monoclonal antibodies and five-fold monoclonal antibody escape mutations. Journal of Gene Virology (J. Gen. Virol.). August 1993; 74 (Pt8): 1 547-53 o
德马其 (DiMarchi)等， 合成肽对牲畜口蹄疫感染的保护作用。 科学 (Science)。 1986年 5月; 232(4750):639-41。 DiMarchi et al., Protective effect of synthetic peptides on foot-and-mouth disease infection in livestock. Science. May 1986; 232 (4750): 639-41.
杰克逊 (Jackson)等， 整合素 0 ^是口蹄疫病毒的受体。 病毒学杂志 (J. Virol.)。 2002年 2月; 76(3):935-41。 Jackson et al., Integrin ^ is a receptor for foot-and-mouth disease virus. Journal of Virology (J. Virol.). 2002 Feb; 76 (3): 935-41.
卡特孙 (Kitson)等， O 型口蹄疫病毒抵抗变异的单克隆抗体序列分析:在四个 抗原位点中涉及三个表面暴露的包膜蛋白的证据。 病毒学 (Virology)。 1990 年 11月； 179(1):26-34。 Kitson et al. Sequence analysis of monoclonal antibodies against foot-and-mouth disease virus variant O: evidence of involvement of three surface-exposed envelope proteins in four antigenic sites. Virology. November 1990; 179 (1): 26-34.
克雷德 (Kleid)等， 克隆的口蹄疫病毒蛋白疫苗 ： 在牲畜和猪的反应。 科学 (Science)。 1981年 12月; 214(4525): 1125-9。 Kleid et al., Cloning of Foot-and-Mouth Disease Virus Protein Vaccine: Responses in Livestock and Pigs. Science. December 1981; 214 (4525): 1125-9.
勒布莱斯 (Lubroth)等， 分类的口蹄疫病毒疫苗中蛋白 2C 的缺乏提供了区分 免疫动物和恢复期的依据。 疫苗 (Vaccine)。 1996年 4月；14(5):419-27。 The lack of protein 2C in classified foot-and-mouth disease virus vaccines provided a basis for distinguishing between immunized animals and recovery periods, according to Lubroth et al. Vaccine. April 1996; 14 (5): 419-27.
乌赫斯 (Woolhouse)等， 管理口蹄疫： 控制疾病爆发的科学。 自然 (Nature)。 410(2001)； 第 515-516页。 Woolhouse et al. Managing Foot-and-Mouth Disease: The Science of Controlling Outbreaks. Nature (Natur e ). 410 (2001); 515-516.
摩根 (Morgan)等， 应用合成肽疫苗保护牲畜和猪免遭口蹄疫袭击。 美国兽医 研究杂志 (Am. J. Vet. Res.)。 1990年； 51(l):40-5。 Morgan et al. Applied synthetic peptide vaccines to protect livestock and pigs from foot-and-mouth disease. American Journal of Veterinary Research (Am. J. Vet. Res.). 1990; 51 (l): 40-5.
裴 (Pay)等， 140S抗原计量和血清中中和抗体反应之间的相关性及在牲畜中由 口蹄疫疫苗所诱导的保护程度。 疫苗 (Vaccine)。 1987年 3月; 5(1)·.60-4。 Pay et al., Correlation between 140S antigen metering and serum neutralizing antibody response and the degree of protection induced by foot-and-mouth disease vaccine in livestock. Vaccine. March 1987; 5 (1) ·. 60-4.
普法普 (Pfaff)等， 针对选择性肽的抗体识别并中和口蹄疫病毒。 EMBO杂志 (EMBO. J.) o 1982年; 1(7):869-74。 Pfaff et al., Antibodies against selective peptides recognize and neutralize FMD virus. EMBO Magazine (EMBO. J.) o 1982; 1 (7): 869-74.
霍兹 (Ward)等，编码复制口蹄疫病毒基因组的质粒 DNA能够在猪诱导抗病毒 的免疫反应。 病毒学杂志 (Journal of Virology) 1997; 71:7442-7。 Ward et al., Plasmid DNA encoding the genome of the replicated foot-and-mouth disease virus can induce anti-virus in pigs Immune response. Journal of Virology 1997; 71: 7442-7.
罗德洛戈斯 (Rodriguez)等， 针对口蹄疫病毒结构蛋白的猪 T淋巴细胞反应的 抗原特异性： VP1中辅助 T细胞表位的确定。 病毒学 (Virology)。 1994年 11 月; 205(1):24-33。 Rodriguez et al. Antigen specificity of porcine T-lymphocyte response to foot-and-mouth disease virus structural protein: identification of helper T-cell epitopes in VP1. Virology. November 1994; 205 (1): 24-33.
史多梅尔 (Strohmaier)等， 口蹄疫病毒免疫蛋白中抗原表位的存在区域和特 性。 基因病毒学杂志 (J. Gen. Virol.)。 1982年 4月; 59(Pt 2):295-306。 Strohmaier et al. Existing regions and characteristics of antigenic epitopes in foot-and-mouth disease virus immune proteins. Journal of Gene Virology (J. Gen. Virol.). April 1982; 59 (Pt 2): 295-306.
发明内容 Summary of the invention
本发明的一方面是提供一种重组口蹄疫病毒 VP1融合蛋白疫苗， 它是将 口蹄疫病毒 VP1肽段、 2聚甘氨酸和 6聚组氨酸编码基因融合， 在大肠杆菌 生产的重组融合蛋白。  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.
本发明的另一方面提供了重组口蹄疫病毒 VP 1融合蛋白的氨基酸序列及 其核苷酸序列。  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.
本发明的另一方面提供了口蹄疫病毒 VP1 BT1融合肽的核苷酸序列及其 编码的氨基酸序列。  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.
本发明的另一方面提供了口蹄疫病毒 VP1 BT2融合肽的核苷酸序列及其 编码的氨基酸序列。  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.
本发明的另一方面提供了口蹄疫病毒 VP1 BT1融合肽的连接方式，包括 2 聚甘氨酸的存在、 连接方式及位置。  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.
本发明的另一方面提供了口蹄疫病毒 VP1 BT2融合肽的连接方式，包括 2 聚甘氨酸的存在、 连接方式及位置。  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.
本发明的另一方面提供了两侧有 6聚组氨酸的口蹄疫病毒 VP1肽段融合 蛋白。  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.
本发明的另一方面涉及重组口蹄疫病毒 VP1融合蛋白按照权利要求 1所 述的重组口蹄疫病毒 VP 1融合蛋白， 它在应用于动物机体后可以诱发机体产 生针对口蹄疫病毒的中和性抗体， 具有预防动物口蹄疫病毒的感染的生物学 活性。  Another aspect of the present invention relates to a recombinant foot-and-mouth disease virus VP1 fusion protein. The recombinant foot-and-mouth disease virus VP 1 fusion protein according to claim 1, which can induce the body to produce a neutralizing antibody against the foot-and-mouth disease virus after being applied to an animal body, and has prevention Biological Activity of Animal Foot-and-Mouth Disease Virus Infection.
另外， 需要指出的是， 在本申请的上下文的公开内容的基础上， 本发明 的其它具有实质性特点的方面和其它具有创造性的有益效果对本领域的普通 技术人员来说是显而易见的。 附图简要说明 In addition, it should be pointed out that, on the basis of the disclosure in the context of the present application, other aspects of the present invention that have substantial features and other creative beneficial effects will be apparent to those of ordinary skill in the art. Brief description of the drawings
附图 1： 表达的重组口蹄疫病毒 VP 1融合蛋白 SDS-PAGE电泳图 Figure 1: SDS-PAGE electrophoresis of the expressed recombinant foot-and-mouth disease virus VP 1 fusion protein
附图 2: 纯化的重组口蹄疫病毒 VP1融合蛋白 SDS-PAGE电泳图 Figure 2: SDS-PAGE electrophoresis of purified recombinant foot-and-mouth disease virus VP1 fusion protein
附图 3: 重组口蹄疫病毒 VP1融合蛋白免疫豚鼠 (1)血清对乳鼠的保护作用 附图 4: 重组口蹄疫病毒 VP1融合蛋白免疫豚鼠 (2)血清对乳鼠的保护作用 附图 5: 重组口蹄疫病毒 VP1融合蛋白免疫豚鼠 (3)血清对乳鼠的保护作用 具体实施方式 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 Protective effect of virus VP1 fusion protein on guinea pig (3) serum on suckling rats
在本发明的上下文中， 所使用的术语除非另外说明， 一般具有本领域的 普通技术人员通常理解的含义。 特别地， 下列术语具有如下的含义. - 重组蛋白疫苗. · 是利用分子克隆技术， 将编码具有免疫原性的蛋白或多 肽的基因克隆到原核或真核细胞的表达载体上， 用此载体在细菌或真核细胞 (酵母细胞或哺乳动物细胞)生产的具有免疫原性的蛋白质或多肽， 这种蛋白 质或多肽在应用于机体后可剌激机体 (人或动物)发生特异性的体液免疫应答 和 /或细胞免疫应答。若将病毒如口蹄疫病毒基因编码的具有免疫原性的蛋白 或多肽的基因克隆到原核或真核细胞的表达载体上， 用其在细菌或真核细胞 (酵母细胞或哺乳动物细胞)生产的由病毒基因编码的蛋白质或多肽是具有抗 病毒如口蹄疫病毒作用的重组蛋白疫苗。 这种疫苗在应用于动物后可诱导机 体发生特异性的体液免疫应答和 /或细胞免疫应答，防止病毒如口蹄疫病毒引 起的感染。  In the context of the present invention, the terms used have the meanings commonly understood by a person of ordinary skill in the art unless otherwise stated. In particular, the following terms have the following meanings.-Recombinant protein vaccines. · The use of molecular cloning technology to clone a gene encoding a protein or polypeptide having immunogenicity into an expression vector of a prokaryotic or eukaryotic cell. Immunogenic protein or polypeptide produced by bacteria or eukaryotic cells (yeast cells or mammalian cells). This protein or polypeptide can stimulate the body (human or animal) to generate a specific humoral immune response after being applied to the body And / or cellular immune response. If 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 (yeast cell or mammalian 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.
动物的口蹄疫病毒（FMDV)感染： 是由 FMDV引起的偶蹄类动物共患 的接触性传染病。 FMDV是一种 RNA病毒， 其基因组约包含 7500-8000个 碱基。 FMDV主要以唾液的方式传播， 传播的速度很快， 易引发大面积的流 行。 牛、 猪、 羊、 鹿等均为口蹄疫病毒的感染动物。  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.
口蹄疫病毒 VP1抗原： 是 FMD病毒壳蛋白的一部分， 它可诱导机体产 生具有保护作用的口蹄疫病毒中和抗体。  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.
口蹄疫病毒 VP1 肽段包括口蹄疫病毒 VP1 BT1 融合肽和口蹄疫病毒 VP1 BT2融合肽。  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.
口蹄疫病毒 VP1 BT1融合肽是指具有如 SEQ ID NO : 2所示氨基酸序列 的多肽 （SEQ ID N0 2)。 口蹄疫病毒 VPl BT2融合肽是指具有如 SEQ ID NO 4所示氨基酸序列 的多肽 （SEQ ID N04)。 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聚甘氨酸是由 2个相邻的甘氨酸组成的 2肽。  2-Polyglycine is a 2 peptide composed of two adjacent glycines.
口蹄疫病毒 VP1肽段融合蛋白编码基因是指具有如 SEQ ID NO : 5 所示 的核苷酸序列的 DNA， 其编码的蛋白质为口蹄疫病毒 VP1肽段融合蛋白。  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.
重组口蹄疫病毒 VP1融合蛋白疫苗编码基因具有如 SEQ ID NO： 8 所示 的核苷酸序列，其编码的蛋白质为重组口蹄疫病毒 VP1融合蛋白疫苗或称重 组口蹄疫病毒 VP1融合蛋白， 它具有如 SEQ ID NO： 9 所示的氨基酸序列。  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.
本发明提供了一种重组口蹄疫病毒 VP1融合蛋白疫苗，它是将口蹄疫病 毒 VP1肽段、 2聚甘氨酸、 6聚组氨酸编码基因融合， 在大肠杆菌生产的重 组融合蛋白。本发明的重组口蹄疫病毒 VP1融合蛋白疫苗具有 SEQ ID NO : 8 所示的核苷酸序列和 SEQ ID NO : 9所示的氨基酸序列。  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.
本发明提供的重组口蹄疫病毒 VP1融合蛋白中的口蹄疫病毒 VPl BT1融 合肽具有 SEQ ID ΝΟ:1所示的核苷酸序列和 SEQ ID NO： 2所示的氨基酸序 列。  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.
本发明提供的重组口蹄疫病毒 VP1 融合蛋白中的口蹄疫病毒 VPl BT2 融合肽具有 SEQ ID NO:3所示的核苷酸序列和 SEQ ID NO： 4所示的氨基酸 序列。  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.
本发明提供的重组口蹄疫病毒 VP1融合蛋白中的口蹄疫病毒 VP1肽段融 合蛋白编码基因具有如 SEQ ID NO： 5所示的核苷酸序列， 其编码的蛋白质 为口蹄疫病毒 VP1肽段融合蛋白。  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.
本发明提供的重组口蹄疫病毒 VP1融合蛋白中的口蹄疫病毒 VPl BT1融 合肽具有 SEQ ID NO： 2所示的连接方式， 包括 2聚甘氨酸的存在、 连接方式 及位置。  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.
本发明提供的重组口蹄疫病毒 VP1融合蛋白中的口蹄疫病毒 VPl BT2融 合肽具有 SEQ ID NO： 4所示的连接方式， 包括 2聚甘氨酸的存在、 连接方式 及位置。  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.
本发明提供的重组口蹄疫病毒 VP1融合蛋白中的口蹄疫病毒 VP1肽段融 合蛋白具有如 SEQ ID NO : 5 所示的连接方式。 本发明提供的重组口蹄疫病毒 VP1融合蛋白中的口蹄疫病毒 VP1肽段融 合蛋白的两侧有 6聚组氨酸的序列。 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.
本发明提供的重组口蹄疫病毒 VP1融合蛋白采用大肠杆菌生产。  The recombinant foot-and-mouth disease virus VP1 fusion protein provided by the present invention is produced by E. coli.
本发明提供的重组口蹄疫病毒 VP1融合蛋白在应用于动物后可以诱发机 体产生针对口蹄疫病毒的中和性抗体， 具有预防动物口蹄疫病毒的感染的生 物学活性。  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.
本发明还涉及该基因工程重组蛋白在制备用于预防动物口蹄疫病毒的 感染的疫苗制品中的用途以及含有该基因工程重组蛋白的疫苗制品。 本领域 技术人员可以理解的是，这些疫苗制品可用本领域周知的各种常规方法制备。  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. Those skilled in the art will understand that these vaccine preparations can be prepared by various conventional methods known in the art.
本发明的重组蛋白疫苗可通过皮下注射的方式给动物接种， 接种的剂量 为 100-5000μ§。为了加强效果，可在第一次免疫后 14或 21天进行 1次加强免疫。 The recombinant protein vaccine of the present invention can be vaccinated to animals by subcutaneous injection, and the vaccinated dose is 100-5000 μ § . In order to enhance the effect, the booster can be performed once every 14 or 21 days after the first immunization.
下面结合具体的制备实施例和生物学效果实施例， 并参照附图进一步详 细地描述本发明。 应理解， 这些实施例只是为了举例说明本发明， 而非以任 何方式限制本发明的范围。  The present invention will be described in further detail below with reference to specific preparation examples and biological effect examples, and with reference to the drawings. It should be understood that these examples are only intended to illustrate the present invention, and not to limit the scope of the present invention in any way.
实施例 Examples
在如下实施例中， 未详细描述的各种过程和方法是本领域中公知的常规 方法，例如《分子克隆》一书（J. Sambrook, Cold Spring Harbor Laboratory Press, Molecular cloning, 1989) 所述的方法。  In the following examples, various processes and methods that are not described in detail are conventional methods well known in the art, such as described in the book "Molecular Cloning" (J. Sambrook, Cold Spring Harbor Laboratory Press, Molecular cloning, 1989) method.
实施例 1、 合成 VP1 BT1融合肽编码基因 Example 1.Synthesis of VP1 BT1 fusion peptide encoding gene
釆用 PCR方法合成 VP1 BT1融合肽编码基因。 设计并合成了具有下 述序列的 PCR引物：  PCRThe VP1 BT1 fusion peptide coding gene was synthesized by PCR method. A PCR primer with the following sequence was designed and synthesized:
引物 1 : Primer 1:
5'CTGC 5'CTGC
GAAGAAAACTACGGTGGT3  GAAGAAAACTACGGTGGT3
引物 2: Primer 2:
CCGTAGTTTTCTTCGCC3 CCGTAGTTTTCTTCGCC3
引物 3: CTGGCTCAA3' Primer 3: CTGGCTCAA3 '
引物 4:Primer 4:
Figure imgf000009_0001
Figure imgf000009_0001
AGATGTCAGTGTG3' AGATGTCAGTGTG3 '
采用引物 1， 2合成具有下述序列的 DNA片段 （片段 1， 2)：  A DNA fragment (fragments 1, 2) with the following sequence was synthesized using primers 1, 2:
CTGCAAGTAC TGGCTCAAAA AGCAGAACGT GCTCTGCCGG GTGGCGAAGA AAACTACGGT GGTGAAACTC AGGTTCAGCG TCGTCAGCAC ACTGACATCT CCTTCA CTGCAAGTAC TGGCTCAAAA AGCAGAACGT GCTCTGCCGG GTGGCGAAGA AAACTACGGT GGTGAAACTC AGGTTCAGCG TCGTCAGCAC ACTGACATCT CCTTCA
以片段 1， 2为摸板，采用引物 3, 4合成两侧带限制性内切酶切点的 VP1 BT1融合肽编码基因， 其序列如 SEQ ID NO: l所示。  Using fragments 1 and 2 as a template, 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.
采用的 PCR操作程序是：  The PCR procedures used are:
在一个 500 μΐ微量离心管中加入下列试剂：  Add the following reagents to a 500 μΐ microcentrifuge tube:
lOxPCR缓冲液 5 μ 1  lOxPCR buffer 5 μ 1
(500匪 ol/L KCl,100mmol/L Tris-Cl ,  (500 bands ol / L KCl, 100 mmol / L Tris-Cl,
15匪 ol/L MgCl2) 15 Bandol / L MgCl 2 )
dNTPs(10謹 ol/L) 1 μ 1  dNTPs (10 ol / L) 1 μ 1
5'端和 3'端引物 (0.01 mmol/L)各 0.5 μ 1  5 'and 3' primers (0.01 mmol / L) each 0.5 μ 1
Taq DNA聚合酶 (5u/ μ 1) 0.25 μ 1  Taq DNA polymerase (5u / μ 1) 0.25 μ 1
加去离子水至终体积 50 μ 1  Add deionized water to a final volume of 50 μ 1
混合后加入矿物油 3滴  Add 3 drops of mineral oil after mixing
反应条件： 94°C,30";55°C，1';72°C,2'， 30个循环周期后， 72°C延伸 10分钟。 将 PCR产物做 2%琼脂糖凝胶电泳 (lxTAE, 150-200mA,0.5小时：)。采用北京 鼎国公司的 DNA回收试剂盒回收用 PCR合成的 VPl BT1融合肽编码基因 DNA片段。 从琼脂糖凝胶上切下含 DNA片段的凝胶， 放入一个离心管中。加 入 3倍体积的溶胶液 (北京鼎国公司)， 45-55°C水浴 5-10ηώι使胶完全融化。 加 入 lOul玻璃奶 (北京鼎国公司)，轻弹管底混匀,然后在 45-55°C水浴 5-10min,期间 每 2-3min混匀一次。 5000g离心 60秒,弃上清。 加 400ul漂洗液，轻弹管底混匀玻 璃奶,然后同上离心， 弃上清。 再加入 400ul漂洗液，轻弹管底混匀玻璃奶,然后 同上离心弃上清,并用加样器尽量除净漂洗液。 然后室温晾干玻璃奶。 加 10-30μ1无菌双蒸水将玻璃奶悬浮起来, 45-55 °C水浴 5-10分钟。 10000g离心 min,回收上清 (含 VP1 BT1融合肽编码基因 DNA片段)备用。 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 5-10ηι to completely melt the glue. Add lOul glass milk (Beijing Dingguo Company), and flick the bottom mix Mix well, then mix in a water bath at 45-55 ° C for 5-10min, and mix once every 2-3min. Centrifuge at 5000g for 60 seconds, discard the supernatant. Add 400ul of rinsing solution, flick the bottom of the tube to mix the glass milk, and then centrifuge as above Discard the supernatant. Add 400ul of rinsing solution, flick the bottom of the tube to mix the glass milk, and then discard the supernatant by centrifugation, and remove the rinsing solution as much as possible with a sampler. Then dry the glass milk at room temperature. Add 10-30μ1 sterile double distilled water to suspend the glass milk, 45-55 ° C water bath for 5-10 minutes. After centrifugation at 10,000 g for min, the supernatant (containing the DNA fragment encoding the VP1 BT1 fusion peptide encoding gene) was recovered for use.
将 VP1 BT1融合肽编码基因 DNA片段克隆入 pMD18-T Vect质粒 (TakaRa公司） 。  The DNA fragment of the VP1 BT1 fusion peptide encoding gene was cloned into the pMD18-T Vect plasmid (TakaRa).
连接反应体系：  Connection reaction system:
VP1 BT1融合肽编码基因 DNA片段 (序列参见 SEQ ID NO: 1)0.05-0.3 pmol pMD18-T Vect (TakaRa公司） 0.5-1μ1  VP1 BT1 fusion peptide encoding gene DNA fragment (see SEQ ID NO: 1 for sequence) 0.05-0.3 pmol pMD18-T Vect (TakaRa) 0.5-1 μ1
Solution I ( TakaRa公司） 5μ1  Solution I (TakaRa) 5μ1
双蒸水 加齐到 10 μΐ  Double distilled water, add to 10 μΐ
16°C反应 1小时  16 ° C reaction for 1 hour
将克隆有 VP1 BT1融合肽编码基因 DNA片段的 pMD18-T Vect质粒转化 入大肠杆菌 JM109 (Novagen公司） 。  The pMD18-T Vect plasmid cloned with the VP1 BT1 fusion peptide-encoding DNA fragment was transformed into E. coli JM109 (Novagen).
感受态大肠杆菌 JM109细胞的制备方法是： 取一大肠杆菌 JM109单菌落 于 2ml LB培养基中, 37°C， 225rpm速度震荡培养 12-16小时;取 lml上述培养物 接种于 100ml LB培养基中, 37°C， 225rpm的速度震荡培养直至 OD值为 0.5左右 (大约 3小时)；将菌液冰浴 2小时,然后 2,500 X g,4°C离心 20分钟收集菌体； 加入 100ml冰冷的 Trituration缓冲液 (100mmol/L CaCl2,70mmol/L MgCl2， 40mmol/L 醋酸钠 ,pH5.5),混勾,置冰上 45分钟； 1,800 Xg,4°C离心 10分钟,弃上清，加入 10ml冰冷的 Trituration缓冲液悬浮细胞； 按每份 200μ1分装, 4°C可保存 1-2周。 若需长期保存，可加甘油至终浓度为 15%，置 -70°C备用。 . 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-cold Trituration buffer to suspend cells; aliquot 200μ1 each, and store at 4 ° C for 1-2 weeks. For long-term storage, add glycerin to a final concentration of 15% and set at -70 ° C until use. .
' 转化的方法是： 将 200μ1感受态细胞置冰上融化，然后加入 3^ DMSO或 β -巯基乙醇， 混合后，加入 2 μ ΐ连接反应液 (含重组质粒)， 混匀，置冰上 30分 钟; 42°C 45秒,然后迅速放回冰中 1_2分钟;加入 21111 1^培养液,37°( ， 225rpm的 速度摇荡培养 1小时; 4,000 X g离心 10秒钟,弃上清，用 200μ1 LB培养液重悬菌 体;将菌液铺于含有抗生素的 LB琼脂培养板上,涂匀,室温放置 20-30 分钟,倒 置于 37°C孵箱中培养 12-16小时。  'The transformation method is: 200μ1 competent cells are thawed on ice, and then 3 ^ DMSO or β-mercaptoethanol is added. After mixing, 2 μΐ 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 200μ1 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.
挑选阳性克隆， 提取质粒 （J. Sambrook, Cold Spring Harbor Laboratory Press, Molecular cloning, 1989) 。  Select positive clones and extract plasmids (J. Sambrook, Cold Spring Harbor Laboratory Press, Molecular cloning, 1989).
实施例 2、 合成 VP1 BT2融合肽编码基因 采用 PCR方法合成 VP1 BT1融合肽编码基因。 设计并合成了具有下述 序列的 PCR引物： Example 2. Synthesis of VP1 BT2 fusion peptide coding gene VP1 BT1 fusion peptide coding gene was synthesized by PCR method. PCR primers with the following sequences were designed and synthesized:
引物 7: Primer 7:
5'GTa 5'GTa
GGTCCGTCT3'  GGTCCGTCT3 '
引物 8: Primer 8:
5'CGQ 5'CGQ
GCA3' GCA3 '
引物 9: Primer 9:
5'CCA] 5'CCA]
AAGTACTGGC3'  AAGTACTGGC3 '
引物 10: Primer 10:
5'AAGC 5'AAGC
CGATTTTCTG3' CGATTTTCTG3 '
采用引物 7, 8 合成具有下述序列的 DNA片段 （片段 7， 8)：  DNA fragments with the following sequences were synthesized using primers 7, 8 (fragments 7, 8):
GTGACCTGCA AGTACTGGCT CAAAAAGCTA AACGTGCTCT GCCGGGTGGT CCGTCTGACG CTCGTCACAA ACAGAAAATC GTTGCTCCG GTGACCTGCA AGTACTGGCT CAAAAAGCTA AACGTGCTCT GCCGGGTGGT CCGTCTGACG CTCGTCACAA ACAGAAAATC GTTGCTCCG
以片段 7， 8为摸板， 采用引物 9， 10合成两侧带限制性内切酶切点的 VP1 BT2融合肽编码基因， 具有如 SEQ ID NO:3所示序列。  Using fragments 7, 8 as a template, 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.
采用的 PCR操作程序和实施例 1类同。回收、克隆 VP1 BT2融合肽编码基因 的方法和实施例 1类同。 将含 VP1 BT2融合肽编码基因的 pMD18-T Vect质粒转 化入感受态大肠杆菌 JM109细胞 (方法类同于实施例 1)。 挑选阳性克隆， 提取质 粒 (J. Sambrook, Cold Spring Harbor Laboratory Press, Molecular cloning, 1989)。 实施例 3、 构建 VP1肽段融合蛋白编码基因  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). Example 3.Construction of VP1 peptide fusion protein encoding gene
用 BamHI消化 pMD18-T Vect质粒上的 VP1 BT1融合肽编码基因， 用 Bglll消化 pMD18-T Vect质粒上的 BT2融合肽编码基因， 用 T4连接酶将两 个消化片段连接得到 BT1 融合肽编码基因 -BT2 融合肽编码基因融合基因 SEQ ID NO:5 c 用 BamHI和 Bglll分别消化 VPl BTl融合肽编码基因 -VPl BT2融合肽 编码基因融合基因， 得到 BamHI消化的 VPl BT1融合肽编码基因 -VPl BT2 融合肽编码基因融合基因片段和 Bglll消化的 VPl BT1融合肽编码基因 -VP1 BT2融合肽编码基因融合基因片段， 用 T4连接酶将两个消化片段连接得到 VPl BT1融合肽编码基因 -BT2融合肽编码基因融合基因的 2聚体基因 SEQ ID NO:6。 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, and 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 The coding gene-VP1 BT2 fusion peptide encodes a gene fusion gene fragment, and two digested fragments are ligated with T4 ligase to obtain a VP1 BT1 fusion peptide-encoding gene-BT2 fusion peptide-encoding gene 2-mer gene SEQ ID NO: 6.
用 BamHI消化 VPl BT1融合肽编码基因 -VPl BT2融合肽编码基因融合 基因的 2聚体基因，用 Bglll消化 VPl BT1融合肽编码基因 -VPl BT2融合肽 编码基因融合基因， 用 T4连接酶将两个消化片段连接得到 VPl BT1融合肽 编码基因 -VPl BT2融合肽编码基因融合基因的 3聚体基因 SEQ ID NO:7。  Digest the VP1 BT1 fusion peptide coding gene-VPl BT2 fusion peptide coding gene dimer gene with BamHI and digest the VPl BT1 fusion peptide coding gene-VPl BT2 fusion peptide coding gene fusion gene with Bglll. The digested fragments were ligated to obtain the VP1 BT1 fusion peptide-encoding gene-VPl BT2 fusion peptide-encoding 3-mer gene SEQ ID NO: 7.
VPl BTl融合肽编码基因 -VPl BT2融合肽编码基因融合基因的 3聚体基 因既为 VP1肽段融合蛋白编码基因。  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.
将 VP1肽段融合蛋白编码基因克隆入 PMD18-T Vect质粒，采用的方法和 实施例 1所用的类同。 将含 W1肽段融合蛋白编码基因的 pMD18-T Vect质粒 转化入感受态大肠杆菌 JM109细胞 (方法类同于实施例 1)。挑选阳性克隆，提取 质粒 (J. Sambrook, Cold Spring Harbor Laboratory Press, Molecular cloning, 1989)。 实施例 4、 构建重组口蹄疫病毒 VPl融合蛋白编码基因  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). Example 4.Construction of recombinant foot-and-mouth disease virus VPl fusion protein encoding gene
用 EcoRI和 Hindlll 消化含 VPl肽段融合蛋白编码基因（SEQ ID NO:5) 的 pMD18-T Vect质粒和 pET28质粒。  The pMD18-T Vect plasmid and pET28 plasmid containing the VPl peptide fusion protein encoding gene (SEQ ID NO: 5) were digested with EcoRI and Hindlll.
含 VPl肽段融合蛋白编码基因 （SEQ ID NO:5 ) 的 pMD18-T Vect质粒 PMD18-T Vect plasmid containing VPl peptide fusion protein encoding gene (SEQ ID NO: 5)
DNA的消化反应： DNA digestion response:
质粒 DNA 1 μ g  Plasmid DNA 1 μ g
10x 缓冲液 （ΙΟχΜ buffer Lot: A1032, TaKaRa) Ι μ ΐ  10x buffer solution (ΙΟχΜ buffer Lot: A1032, TaKaRa) Ι μΐ
限制性内切酶 Hindlll (10单位 /μ 1) 1 μ 1  Restriction enzyme Hindlll (10 units / μ 1) 1 μ 1
限制性内切酶 EcoRI (10单位 /μ 1) 1 μ 1  Restriction enzyme EcoRI (10 units / μ 1) 1 μ 1
用双蒸水补齐至 10 μ ΐ  Make up to 10 μ with double distilled water ΐ
混合后于 37°C温育 30-120分钟。  After mixing, incubate at 37 ° C for 30-120 minutes.
pET-28a(+)质粒的消化反应：  Digestion reaction of pET-28a (+) plasmid:
质粒 DNA 1 μ g 10x 缓冲液 （ΙΟχΚ buffer Lot: A1018, TaKa a) Ι μ ΐ 限制性内切酶 EcoRI(10单位 /μ 1) 1 μ 1 Plasmid DNA 1 μ g 10x Buffer (ΙΟχΚ buffer Lot: A1018, TaKa a) Ι μ ΐ Restriction enzyme EcoRI (10 units / μ 1) 1 μ 1
限制性内切酶 Hindlll (10单位 / μ 1) 1 μ 1  Restriction enzyme Hindlll (10 units / μ 1) 1 μ 1
用双蒸水补齐至 10 μ ΐ  Make up to 10 μ with double distilled water ΐ
混合后， 于 37°C温育 30-120分钟。  After mixing, incubate at 37 ° C for 30-120 minutes.
将用 EcoRI和 Hindlll消化的 VP1肽段融合蛋白编码基因 DNA片段和 经限制性内切酶 EcoRI和 Hindlll消化的原核细胞表达载体 pET-28a(+)质 粒 （美国 Novagen公司） 相连接。  A DNA fragment encoding the VP1 peptide fusion protein digested with EcoRI and Hindlll and a prokaryotic cell expression vector pET-28a (+) digested with the restriction enzymes EcoRI and Hindlll (Novagen, USA) were connected.
连接反应：  Connection reaction:
pET-28a(+)质粒 (0.5 u g/ l) 2 μ 1  pET-28a (+) plasmid (0.5 u g / l) 2 μ 1
VP1肽段融合蛋白编码基因 DNA片段 DNA(300ng/ 1) 5 μ 1  VP1 peptide fusion protein encoding gene DNA fragment DNA (300ng / 1) 5 μ 1
10χ 连接缓冲液  10χ ligation buffer
(T4 Ligation Solution Lot: CA2901 , TaKaRa) l l  (T4 Ligation Solution Lot: CA2901, TaKaRa) l l
T4 DNA连接酶 （T4 Ligation Code No: D2011A5TaKaRa) 1 μ 1 用双蒸水补齐至 10 μ ΐ 混合后置 14-16°C水浴 6-12小时。 T4 DNA ligase (T4 Ligation Code No: D2011A 5 TaKaRa) 1 μ 1 Make up to 10 μ with double distilled water. Ϊ́ Mix and place in a water bath at 14-16 ° C for 6-12 hours.
将含口蹄疫病毒 VP1肽段融合蛋白编码基因的重组 pET-28a(+)质粒转化 入大肠杆菌表达菌 BL21DE3。  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.
对 pET-28a(+)质粒中的插入片段进行 DNA测序。 利用 pET-28a(+)质粒 EcoRI和 Hindlll切点两侧的序列融合出重组口蹄疫病毒 VP1融合蛋白编石 a 基因 SEQ ID NO:8。 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.
此基因 (1092个碱基)编码由 363个氨基酸残基组成的重组口蹄疫病毒 VP 融合蛋白， 此重组蛋白的氨基酸序列如 SEQ ID NO:9所示。  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.
实施例 5、 重组口蹄疫病毒 VP1融合蛋白疫苗的表达及裂菌 Example 5.Expression of recombinant foot-and-mouth disease virus VP1 fusion protein vaccine and Schizophyllum
将单菌落的细菌接种于 100 ml LB培养基，于 500ml三角烧瓶中， 37°C 水浴震荡培养至 OD600为 0.6。加入 IPTG使其终浓度为 lmM， 37°C水浴 震荡培养 3小时。 将三角烧瓶于冰上 5分钟， 4°C离心 5分钟（5000 x g)。 弃上清， 收集细菌， 立即使用或冻存。 取样做 SDS-PAGE分析重组口蹄疫 病毒 VP1融合蛋白的表达 （附图 1 ) 。 将细菌重悬于细胞裂解液 (20mM Tris， 0.5M NaCl, 5mM咪唑， 调 pH至 7.9)中， 1克湿菌 /3ml细胞裂解液， 加 DNA酶致终浓度 2毫克 /毫升， 力口 ImM Mg2SO4(10 μΐ/ml), 加 50Mm PMSF(0.5 μ1/η 1)， 冰上孵育 30分， 5,000rpm， 离 心 15min， 弃上清。 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). Resuspend the bacteria in cell lysate (20mM Tris, 0.5M NaCl, 5mM imidazole, adjust the pH to 7.9), 1 gram of wet bacteria / 3ml of cell lysate, add DNase to a final concentration of 2mg / ml, LiMu ImM Mg 2 SO 4 (10 μΐ / ml), add 50Mm PMSF (0.5 μ1 / η 1), incubate on ice for 30 minutes, 5,000 rpm, centrifuge for 15 min, and discard the supernatant.
对沉淀加结合液， 1克湿菌产生的沉淀加 6 ml结合液 (20mM Tris， 0.5 M NaCl, 5mM咪唑， 6M尿素， 调 pH至 7.9)。 冰浴震摇 2小时。 5,000rpm, 离心 30min, 收上清， 用上清上柱。  Add the 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.
施例 6、 重组口蹄疫病毒 VP1融合蛋白疫苗的纯化 Example 6.Purification of recombinant foot-and-mouth disease virus VP1 fusion protein vaccine
( 1 ) 、 亲合层析  (1), affinity chromatography
装柱：  Packing:
用磷酸盐缓冲液装柱 （20 mM phosphate, pH 7.2 IM NaCl) ， 层析介质 为 Sepharose4B-Ni2+ (Pharmacia) 并平衡。 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.
用 5个柱床体积的双蒸水洗柱。  Wash the column with 5 bed volumes of double distilled water.
将 3-5个柱床体积的 20mM金属离子溶液 （ lOOmM镍离子溶液的配制： NiS04-6H20 13. lg, 双馏水加到 1000ml) 加到柱中。 Add 3-5 column bed volumes of 20 mM metal ion solution (preparation of 100 mM nickel ion solution: NiS0 4 -6H 2 0 13.1 g, double distilled water to 1000 ml) to the column.
用 5个柱床体积的洗脱缓冲液（20mM Tris, pH7.9， 5M NaCl， 0.5M咪唑， Use 5 column volumes of elution buffer (20mM Tris, pH7.9, 5M NaCl, 0.5M imidazole,
4M尿素） 洗柱。 4M urea) Wash the column.
用 5个柱床体积的吸附缓冲液 (20mM Tris，pH7.9， 0.5M NaCl, 5mM咪唑) 洗柱。 将样品加到金属螯合的柱中。  Wash the column with 5 bed volumes of adsorption buffer (20 mM Tris, pH 7.9, 0.5 M NaCl, 5 mM imidazole). The sample was applied to a metal-chelated column.
用洗涤 1缓冲液 (20mM Tris， pH7.9, 0.5M NaCl， 20mM咪唑， 4M尿素） 洗柱。  Wash the column with Wash 1 buffer (20 mM Tris, pH 7.9, 0.5 M NaCl, 20 mM imidazole, 4 M urea).
洗脱：  Elution:
用洗脱 2缓冲液 （20mM Tris, pH7.9， 0.5M NaCl,l M咪唑， 4 M尿素） 洗 脱， 收集洗脱的蛋白。直到吸收值不再下降为止,用容器接取不同时段的流出 液,并进行 SDS电泳,判定纯化效果， 纯度可达 50%以上。  Eluted with 2 buffer (20 mM Tris, pH 7.9, 0.5 M NaCl, 1 M imidazole, 4 M urea) to collect the eluted protein. Until the absorption value no longer drops, use the container to collect the effluent from different periods and perform SDS electrophoresis to determine the purification effect. The purity can reach more than 50%.
(2)、 除盐  (2) Desalting
j¾ Sephadex G25 (Pharmacia) 装柱。 用 2个柱床体积的双蒸水洗柱。 j¾ Sephadex G25 (Pharmacia) packing. Wash the column with 2 bed volumes of double distilled water.
用 2个柱床体积的 10 mM PBS, pH 7.2 洗柱。  Wash the column with 2 bed volumes of 10 mM PBS, pH 7.2.
上样。  Load.
用 10 mM PBS, pH 7.2洗脱，  Eluted with 10 mM PBS, pH 7.2,
收集洗脱的蛋白， 此蛋白为重组口蹄疫病毒 VP1融合蛋白。  The eluted protein was collected, and this protein was a recombinant foot-and-mouth disease virus VP1 fusion protein.
冻干保存重组口蹄疫病毒 VP1融合蛋白。  The recombinant VP1 fusion protein of FMDV was lyophilized.
用 SDS-PAGE对纯化的重组口蹄疫病毒 VP1融合蛋白进行鉴定，其纯度 达 95% (见附图 2) 。  The purified recombinant FMD virus VP1 fusion protein was identified by SDS-PAGE, and its purity was 95% (see Figure 2).
实施例 7、 重组口蹄疫病毒 VP1融合蛋白的免疫 Example 7.Immunization of recombinant foot-and-mouth disease virus VP1 fusion protein
免疫程序：  Immunization program:
三次免疫， 间隔 14天， 初次免疫加铝佐剂， 两次加强免疫不加铝佐剂。 每次免疫的剂量均为 50微克 /只 /次。设重组口蹄疫病毒 VP1融合蛋白免疫组、 口蹄疫病毒灭活苗 （内蒙生物制药厂组） 组和 PBS对照组， 每组 3只豚鼠。  Three immunizations, with an interval of 14 days, the first immunization with aluminum adjuvant, and two booster immunization without aluminum adjuvant. The dose of each immunization was 50 micrograms / piece / time. The recombinant foot-and-mouth disease virus VP1 fusion protein immunization group, foot-and-mouth disease virus inactivated vaccine (Inner Mongolia Biopharmaceutical Factory group) group and PBS control group were set up, each group had 3 guinea pigs.
配制铝佐剂：  Formulated aluminum adjuvant:
10%12水硫酸铝钾溶液 10毫升移入 50毫升锥形瓶中。 逐滴加入 0.25N 氢氧化钠 22.8毫  10% of 12% aluminum potassium sulfate solution in water was transferred into a 50 ml Erlenmeyer flask. Add 0.25N sodium hydroxide 22.8 milligrams dropwise
升， 边加入边震荡混匀。 Liters, shake and mix while adding.
室温孵育 10分钟。 lOOOg离心 10分钟。 弃上清。 加入 50毫升蒸馏水重悬沉 淀的氢氧化铝。 Incubate at room temperature for 10 minutes. Centrifuge at 1,000 g for 10 minutes. Discard the supernatant. Add 50 ml of distilled water to resuspend the precipitated aluminum hydroxide.
分装上述氢氧化铝悬液至 20个离心管中， 每管 1毫升。 lOOOg离心 10 分钟,弃上清。  Aliquot the above-mentioned aluminum hydroxide suspension into 20 centrifuge tubes, each with 1 ml. Centrifuge at 1000g for 10 minutes and discard the supernatant.
将 200微克重组口蹄疫病毒 VP1融合蛋白溶于 400微升制备的铝佐剂， 吹打混匀， 室温孵育 20分钟。 腹腔注射免疫， 100微升 /每只豚鼠。  200 μg of the recombinant FMDV VP1 fusion protein was dissolved in 400 μl of the prepared aluminum adjuvant, mixed by pipetting, and incubated at room temperature for 20 minutes. Intraperitoneal immunization, 100 μl / guinea pig.
在腹腔注射免疫后第 21天， 做加强免疫。 用 PBS溶解重组口蹄疫病毒 VP1融合蛋白， 浓度为 50微克 /0.2毫升。 静脉（阴茎静脉）注射， 每只豚鼠 0.2毫升。 对照组豚鼠注射 0.2毫升 PBS。  On the 21st day after intraperitoneal immunization, booster was performed. The VP1 fusion protein of recombinant foot-and-mouth disease virus was dissolved in PBS at a concentration of 50 μg / 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.
在腹腔注射免疫后第 42天， 做第二次加强免疫。 用 PBS溶解重组口蹄 疫病毒 VP1融合蛋白， 浓度为 50微克 /0.2毫升。 静脉 （阴茎静脉） 注射， 每只豚鼠 0.2毫升。 对照组豚鼠注射 0.2毫升 PBS。 在腹腔注射免疫后第 46天。 无菌取豚鼠心脏血。 放置于试管中。 37°C 1 小时。 4°C倾斜放置过夜， 待血清充分析出后， 收集血清， 无菌分装， -20°C 保存。 On the 42nd day after intraperitoneal injection, a second booster was performed. Recombinant foot-and-mouth disease virus VP1 fusion protein was dissolved in PBS at a concentration of 50 μg / 0.2 ml. Intravenous (penile vein) injection, 0.2 ml per guinea pig. Control group guinea pigs were injected with 0.2 ml of PBS. Day 46 after intraperitoneal injection. Take guinea pig heart blood aseptically. Place in a test tube. 37 ° C for 1 hour. It was placed at 4 ° C over night for oblique analysis. After the serum was fully analyzed, the serum was collected, aliquoted, and stored at -20 ° C.
实施例 8、 口蹄疫病毒特异性抗体的检测 （ELISA法） Example 8 Detection of Foot-and-Mouth Disease Virus Specific Antibodies (ELISA)
口蹄疫病毒的纯化在内蒙生物制药厂进行。 取经 BHK单层细胞培养扩 增的新鲜 01型口蹄疫病毒液 (内蒙生物制药厂）， 4000转 /分离心 20分钟， 去除细胞碎片。 在上清中逐滴加入饱和的聚乙二醇 6000至终浓度为 7%， 边 滴加边搅拌， 4°C静止过夜。 5000转 /分离心 30分钟， 收集沉淀。 用 pH7.6 的 PB液将沉淀重悬， 在 15%-45%的蔗糖密度梯度上进一步纯化 （离心力为 35000克力， 时间为 3小时， 温度为 4°C )。 分段取样， 用紫外监测仪测各段 样品 260nm吸收值， 146s的病毒颗粒出现在 30%-35%的蔗糖中。 将其用 PB 液稀释 5倍， 在含 20%蔗糖的 PB液上离心 2小时， 离心力为 35000克力， 温度为 4°C， 收集沉淀。 将沉淀用 PB液重悬， 用 BCA法测定纯化的口蹄疫 病毒浓度。  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. At 5,000 rpm for 30 minutes, 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). Sampling was performed in sections, and the absorption value at 260 nm of each sample was measured with a UV monitor. 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.
用纯化的口蹄疫病毒抗原包板。 包被液为 0.05M, PH值 9.6的碳酸盐缓 冲液， 每孔 100μ1， 含口蹄疫病毒抗原 1μβ， 4°C包被过夜。 用含 0.05%吐温 20的 PBS (PBS-T) 洗液洗板 3次后， 加入封闭液 （含 2%BSA的 PBS-T) 200μ1于 37Ό孵箱放置 1小时。 洗板 3次后将稀释（1 : 2048)好的待检血清 ΙΟΟμΙ加入各孔， 于 37°C孵箱放置 1小时。 设 3个复孔。 洗板 3次， 加入酶 标抗体 （辣根过氧化物酶标记的羊抗豚鼠 IgG) 工作液， 37°C孵育 1小时， 加入 TMB底物溶液及 ¾02 37°C放置 10-30分钟，用酶标仪测 OD值 ( A492), 用各组 OD值的均值和标准差表示结果。 Plate with purified FMD virus antigen. The coating solution was 0.05M, carbonate buffer with a pH value of 9.6, 100 μl per well, containing 1 μ β 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 μ1 of blocking solution (PBS-T containing 2% BSA) and place in a 37Ό incubator for 1 hour. After washing the plate 3 times, 100 μl 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. Wash the plate 3 times, add enzyme-labeled antibody (horseradish peroxidase-labeled goat anti-guinea pig IgG) working solution, incubate at 37 ° C for 1 hour, add TMB substrate solution and ¾0 2 at 37 ° C, and let stand for 10-30 minutes. The OD value was measured with a microplate reader (A492), and the results were expressed by the mean and standard deviation of the OD values of each group.
结果：  Results:
空白对照： OD值: 0.005±0.0005  Blank control: OD value: 0.005 ± 0.0005
重组口蹄疫病毒 VP1融合蛋白免疫豚鼠 1 : 1.55±0.013  Recombinant foot-and-mouth disease virus VP1 fusion protein immunizes guinea pig 1: 1.55 ± 0.013
重组口蹄疫病毒 VP1融合蛋白免疫豚鼠 2: 1.231±0.021  Recombinant foot and mouth disease virus VP1 fusion protein immunizes guinea pig 2: 1.231 ± 0.021
重组口蹄疫病毒 VP1融合蛋白免疫豚鼠 3 : 1.001±0.013  Recombinant foot and mouth disease virus VP1 fusion protein immunizes guinea pigs 3: 1.001 ± 0.013
3只 PBS对照组小鼠： 0.403±0.014  3 PBS control mice: 0.403 ± 0.014
实验结果表明， 重组口蹄疫病毒 VP1融合蛋白免疫豚鼠血清中存在高水 平的抗口蹄疫病毒的抗体。 The experimental results show that high water levels are present in the serum of guinea pigs immunized with the recombinant foot-and-mouth disease virus VP1 fusion protein. Flat antibodies against foot and mouth disease virus.
实施例 9、 重组口蹄疫病毒 VP1融合蛋白免疫血清的乳鼠中和实验 Example 9: Neutralization experiment on sera from recombinant foot-and-mouth disease virus VP1 fusion protein
将 2倍倍比稀释待检测血清和口蹄疫病毒液 （100 TCID50/0.1ml) 等体 积混合， 37Ό孵育 1小时。 经乳鼠项背部皮下注射 0.2ml上述混合液， 连续 观察 3天， 记录各组发病、 死亡情况。  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.
乳鼠中和实验在内蒙生物制药厂进行， 采用 "O"型口蹄疫病毒毒株。 病毒的浓度为 1毫升 1000LD50。 倍比稀释豚鼠血清。 将 ΙΟΟ μ Ι不同稀释度 豚鼠血清和 ΙΟΟ μ Ι病毒稀释液混合， 37°C孵育 1小时。在乳鼠背部注射上述 混合物。  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 μl guinea pig serum and 100 μl 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.
观察 48小时。 记录存活的乳鼠数目。  Observe for 48 hours. The number of surviving suckling rats was recorded.
结果 （如附图 3， 4， 5所示）： 重组口蹄疫病毒 VP1融合蛋白免疫豚鼠 的血清在稀释至 1 : 2048时仍能完全保护乳鼠。  Results (as shown in Figures 3, 4, and 5): The sera of guinea pigs immunized with the recombinant VP1 fusion protein of foot-and-mouth disease virus can completely protect the suckling rats when diluted to 1: 2048.
结论： 重组口蹄疫病毒 VP1融合蛋白剌激动物产生中和抗体， 此中和抗 体可预防动物感染口蹄疫病毒。  Conclusion: Recombinant foot-and-mouth disease virus VP1 fusion peptone agonist produces neutralizing antibodies. This neutralizing antibody can prevent animals from being infected with foot-and-mouth disease virus.

Claims

权利要求书 Claim
1. 一种重组口蹄疫病毒 VPl融合蛋白疫苗， 它是将口蹄疫病毒 VP1肽 段、 2聚甘氨酸和 6聚组氨酸编码基因融合， 在大肠杆菌生产的重组融合蛋 白。 1. A recombinant protein vaccine of foot-and-mouth disease virus VPl fusion protein, which is a recombinant fusion protein produced by fusing VP1 peptide, 2-glycine and 6-histidine-encoding genes of foot-and-mouth disease virus.
2. 按照权利要求 1所述的重组口蹄疫病毒 VP1融合蛋白， 其具有选自 如下的任一核苷酸序列和氨基酸序列：  2. The recombinant foot-and-mouth disease virus VP1 fusion protein according to claim 1, which has a nucleotide sequence and an amino acid sequence selected from the group consisting of:
1 ) SEQ ID NO： 8所示的核苷酸序列和 SEQ ID NO： 9所示的氨基酸序 列和  1) the nucleotide sequence shown in SEQ ID NO: 8 and the amino acid sequence shown in SEQ ID NO: 9 and
2) 由在严谨的杂交条件下与编码如 1 )的氨基酸序列和核苷酸序列杂交 的核苷酸序列及其所编码的氨基酸序列。  2) The nucleotide sequence and its encoded amino acid sequence are hybridized with the amino acid sequence and nucleotide sequence encoding 1) under stringent hybridization conditions.
3. 按照权利要求 1所述的重组口蹄疫病毒 VT1融合蛋白， 其中所述的 口蹄疫病毒 VPl BT1融合肽具有 SEQ ID NO： 1所示的核苷酸序列， 在重组 口蹄疫病毒 VP1融合蛋白疫苗中编码具有 SEQ ID NO : 2所示氨基酸序列的 口蹄疫病毒 VP1 BT1融合肽。  3. The recombinant foot-and-mouth disease virus VT1 fusion protein according to claim 1, wherein the foot-and-mouth disease virus VP1 BT1 fusion peptide has a nucleotide sequence shown in SEQ ID NO: 1 and is encoded in a recombinant foot-and-mouth disease virus VP1 fusion protein vaccine. Foot-and-mouth disease virus VP1 BT1 fusion peptide having the amino acid sequence shown in SEQ ID NO: 2.
4. 按照权利要求 1所述的重组口蹄疫病毒 VP1融合蛋白， 其中所述的 口蹄疫病毒 VPl BT2融合肽具有 SEQ ID NO : 3所示的核苷酸序列， 在重组 口蹄疫病毒 VP1融合蛋白疫苗中编码具有 SEQ ID NO : 4所示氨基酸序列的 口蹄疫病毒 VPl BT2融合肽。  4. The recombinant foot-and-mouth disease virus VP1 fusion protein according to claim 1, wherein the foot-and-mouth disease virus VP1 BT2 fusion peptide has a nucleotide sequence shown in SEQ ID NO: 3 and is encoded in a recombinant foot-and-mouth disease virus VP1 fusion protein vaccine. Foot-and-mouth disease virus VP1 BT2 fusion peptide having the amino acid sequence shown in SEQ ID NO: 4.
5. 按照权利要求 1所述的重组口蹄疫病毒 VP1融合蛋白， 其中所述的 口蹄疫病毒 VPl BT1融合肽具有 SEQ ID NO : 2所示的连接方式， 包括 2聚 甘氨酸的存在、 连接方式及位置。  5. The recombinant foot-and-mouth disease virus VP1 fusion protein according to claim 1, wherein the foot-and-mouth disease virus VPl BT1 fusion peptide has a connection mode shown in SEQ ID NO: 2, including the presence, connection mode, and position of 2-polyglycine.
6. 按照权利要求 1所述的重组口蹄疫病毒 VP1融合蛋白， 其中所述口 蹄疫病毒 VPl BT2融合肽具有 SEQ ID NO： 4所示的连接方式， 包括 2聚甘 氨酸的存在、 连接方式及位置。  6. The recombinant foot-and-mouth disease virus VP1 fusion protein according to claim 1, wherein the foot-and-mouth disease virus VPl BT2 fusion peptide has a connection mode shown in SEQ ID NO: 4, including the presence, connection mode, and position of 2 polyglycine.
7. 按照权利要求 1所述的重组口蹄疫病毒 VP1融合蛋白， 其中的口蹄 疫病毒 VP1肽段融合蛋白编码基因具有如 SEQ ID NO : 5所示的序列和连接 方式。  7. The recombinant foot-and-mouth disease virus VP1 fusion protein according to claim 1, wherein the foot-and-mouth disease virus VP1 peptide fusion protein-encoding gene has a sequence and a linkage manner as shown in SEQ ID NO: 5.
8. 按照权利要求 1所述的重组口蹄疫病毒 VP1融合蛋白， 其中的口蹄 疫病毒 VP1肽段融合蛋白的两侧有 6聚组氨酸的序列。 8. The recombinant foot-and-mouth disease virus VP1 fusion protein according to claim 1, wherein the foot-and-mouth disease virus VP1 peptide fusion protein is flanked by 6-polyhistidine sequences on both sides.
9.按照权利要求 1所述的重组口蹄疫病毒 VP1融合蛋白， 它在应用于 动物机体后可以诱发机体产生针对口蹄疫病毒的中和性抗体， 具有预防动物 口蹄疫病毒感染的生物学活性。 The recombinant foot-and-mouth disease virus VP1 fusion protein according to claim 1, which, after being applied to an animal body, can induce the body to produce neutralizing antibodies against the foot-and-mouth disease virus, and has biological activity for preventing the animal's foot-and-mouth disease virus infection.
PCT/CN2004/001168 2003-10-15 2004-10-14 Vp1 fusion protein vaccin of recombinant foot-and-mouth disease virus WO2005034993A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CNB2003101001751A CN1290579C (en) 2003-10-15 2003-10-15 Recombinant foot-and-mouth disease virus VP1 confluent protein vaccine
CN200310100175.1 2003-10-15

Publications (1)

Publication Number Publication Date
WO2005034993A1 true WO2005034993A1 (en) 2005-04-21

Family

ID=34427783

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2004/001168 WO2005034993A1 (en) 2003-10-15 2004-10-14 Vp1 fusion protein vaccin of recombinant foot-and-mouth disease virus

Country Status (2)

Country Link
CN (1) CN1290579C (en)
WO (1) WO2005034993A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013001285A3 (en) * 2011-06-30 2013-03-14 The Pirbright Institute Fmdv vp1 peptides

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102138780B1 (en) * 2009-11-02 2020-07-29 더 트러스티스 오브 더 유니버시티 오브 펜실바니아 Foot and mouth disease virus (fmdv) consensus proteins, coding sequences therefor and vaccines made therefrom
CN102212137A (en) * 2010-04-07 2011-10-12 高地 Genetically engineered recombinant polypeptide for cloven-hoofed animals
CN102372766B (en) * 2011-07-13 2013-12-25 青岛红桥明勤生物科技有限公司 O-type foot-and-mouth disease multi-epitope vaccine
CN104474542A (en) * 2014-11-18 2015-04-01 天津瑞普生物技术股份有限公司 Preparation method of bi-combined inactivated vaccine

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1354674A (en) * 1998-06-20 2002-06-19 美国联合生物医学公司 Synthetic peptide vaccines for foot-and-mouth disease
CN1440296A (en) * 2000-06-29 2003-09-03 梅瑞尔公司 Vaccine against foot-and-mouth disease

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1354674A (en) * 1998-06-20 2002-06-19 美国联合生物医学公司 Synthetic peptide vaccines for foot-and-mouth disease
CN1440296A (en) * 2000-06-29 2003-09-03 梅瑞尔公司 Vaccine against foot-and-mouth disease

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013001285A3 (en) * 2011-06-30 2013-03-14 The Pirbright Institute Fmdv vp1 peptides
US9457075B2 (en) 2011-06-30 2016-10-04 The Pirbright Institute Modified foot and mouth disease virus (FMDV) VP1 capsid protein

Also Published As

Publication number Publication date
CN1607004A (en) 2005-04-20
CN1290579C (en) 2006-12-20

Similar Documents

Publication Publication Date Title
AU2006203358B2 (en) Vaccines containing ribavirin and methods of use thereof
JP2006020637A (en) Immunological adjuvant protein originated from klebsiella pneumoniae
KR102673090B1 (en) Vaccine composition based on synthetic polypeptide epitope
WO2010045881A1 (en) A vaccine composition against foot-and-mouth disease and the preparation method thereof
KR20080091759A (en) Novel plant virus particles and methods of inactivation thereof
CN109232720B (en) Foot-and-mouth disease O type virus sIgA antibody ELISA detection kit and application thereof
WO2021254287A1 (en) Novel coronavirus tandem epitope polypeptide vaccine and use thereof
TW200948959A (en) Preparation of soluble capsid proteins of picornavirus using SUMO fusion technology
WO2020238458A1 (en) Cell strain for expressing e2 protein and application thereof, and e2 protein and application thereof
CA2892221A1 (en) Canine parvovirus type 2c isolates and methods of use
JPH0768267B2 (en) Flavivirus antigen
JP5132780B2 (en) Fishery subunit vaccine
JP5568017B2 (en) Recombinant chicken infectious coryza vaccine and method for producing the same
WO2005034993A1 (en) Vp1 fusion protein vaccin of recombinant foot-and-mouth disease virus
NZ198388A (en) Dna molecule comprising sequence substantially corresponding to at least part of foot and mouth disease virus rna;transformed host cell;antigen and vaccine
KR102332725B1 (en) Pentamer-based recombinant protein vaccine platform and expressing system there of
JP3245169B2 (en) Canine coronavirus S gene and uses thereof
Liu et al. Quadruple antigenic epitope peptide producing immune protection against classical swine fever virus
Lorenzo et al. Expression and immunological evaluation of the Escherichia coli‐derived hepatitis C virus envelope E1 protein
US7217784B2 (en) VP1 of foot-and-mouth disease virus
US4743554A (en) Recombinant DNA expression vector encoding for foot and mouth disease virus proteins
Shao et al. Immune potential of a novel multiple-epitope vaccine to FMDV type Asia 1 in guinea pigs and sheep
JPH04504422A (en) Rickettsial antigens for vaccines and diagnostics
KR102451412B1 (en) Novel immunopotent recombinant protein with simultaneous induction of cellular and humoral immune responses and broad spectrum of protective efficacy, and foot-and-mouth disease (FMD) vaccine composition comprising the same
CN112724205B (en) Method for preparing virus-like particles from C hepatitis E virus 239 protein and application thereof

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase