WO2005021035A1 - Vaccin peptidique bivalent dirige contre fmd, ses procedes de preparation et ses applications - Google Patents

Vaccin peptidique bivalent dirige contre fmd, ses procedes de preparation et ses applications Download PDF

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WO2005021035A1
WO2005021035A1 PCT/CN2004/001016 CN2004001016W WO2005021035A1 WO 2005021035 A1 WO2005021035 A1 WO 2005021035A1 CN 2004001016 W CN2004001016 W CN 2004001016W WO 2005021035 A1 WO2005021035 A1 WO 2005021035A1
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seq
vaccine
nucleic acid
type
foot
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PCT/CN2004/001016
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Chinese (zh)
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Yukun Sun
Dengxi Wu
Jin Xia
Weiling Gu
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Shanghai Huayi Bio-Tech Lab
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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • 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
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • 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
    • A61K2039/70—Multivalent vaccine
    • 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
    • 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
    • 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
    • C12N2770/32134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/582—Recycling of unreacted starting or intermediate materials

Definitions

  • the invention relates to a novel foot-and-mouth disease vaccine, in particular to a bivalent polypeptide vaccine produced by a genetic engineering method, a preparation method thereof and use thereof. Background technique
  • Foot-and-mouth disease is a highly contagious disease in the livestock industry.
  • Artiodactyls such as cattle, pigs, sheep, and camels are susceptible to infection. So far, the world's countries have had a large-scale epidemic of foot-and-mouth disease except North America and Australia. The epidemic has been popular in Germany (1937-38), Europe (1951-52), Turkey (1964-65), countries such as the United Kingdom (1967-68), Austria (1973), France (1974), and South Korea (2002) caused huge economic losses.
  • the pathogen of foot-and-mouth disease is a pico-RNA virus, the foot-and-mouth disease virus (FMDV).
  • the virus particle contains a single-stranded single-stranded RNA consisting of 8500 nucleotides.
  • the ruthenium membrane of the viral particle includes four structural proteins VP1 (molecular weight 34,000), VP 2 (30, 000), VP 3 (26, 000), and VP 4 (13500) surrounding the single-stranded RNA.
  • Each of the four structural proteins of 60 molecules constitutes an icosahedral virion.
  • the apex of the icosahedron consists of the structural protein VPt.
  • VP an antibody to the protein neutralizes the virus and prevents the infection of the foot-and-mouth disease virus, but the ability to neutralize the virus is lower than that produced by the intact virion.
  • the immunoglobulin region of the structural protein VP! is the 141-160 region and the 200-21 3 region.
  • the antibodies produced by chemically linking the polypeptide fragments of these two regions to the carrier protein neutralize the virions (Na ture, 1982, 298 30-33).
  • FMDV FMDV-induced pulmonary disease
  • pharynx After infection through the respiratory tract, primary replication of FMDV is in the pharynx, then infects adjacent lymph nodes and enters the bloodstream, which in turn spreads to various organs and tissues. In most cases, clinical symptoms appear 2-14 days after infection. Older animals rarely die after infection with FMDV, but their reproductive capacity, growth and health are greatly affected. Moreover, although some animals develop high antibody titers after infection with FMDV, these healthy animals may secrete FMDV and infect other animals.
  • FMDV has significant antigenic diversity. FMDV has been found to have seven serotypes, each of which can continue to be divided into multiple subtypes, as shown in the table below:
  • foot-and-mouth disease virus is easy to spread, has the ability to infect a variety of animals and has multiple antigenic forms, making the foot-and-mouth disease outbreak difficult to control.
  • the development of vaccines to immunize animals is one of the effective methods to control FMDV.
  • the traditional inactivated FMDV vaccine is produced by culturing animal cells in large quantities, then infecting foot-and-mouth disease virus, culturing, isolating the virus, and inactivating the virus with chemical reagents.
  • France Mer i eux is the largest inactivated FMDV vaccine manufacturer. Brazil, Argentina, Russia and other countries also use the same technology to produce this vaccine, which has a good protective effect.
  • the disadvantage of preparing an inactivated FMDV vaccine is the need to establish a tightly closed environment to prevent leakage and contaminate the surrounding environment. This potential hazard limits the use of this vaccine.
  • inactivated FMDV vaccines although not often anaphylactic shock or coma, can be severe enough if it occurs.
  • the foot-and-mouth disease genetic engineering peptide vaccine has achieved good application results after more than ten years of research. It takes only 20 hours to use Escherichia coli as the host cell for fermentation, and the cycle is much shorter than the production of inactivated FMDV vaccine by animal cells. Genetically engineered vaccines are environmentally friendly, have no risk of leakage, are very safe, and produce vaccines that do not require refrigeration during storage, transportation, and use, and have stable titers. However, the polypeptide vaccine has a lower protective effect than the whole virus vaccine. For example, the amino acid of the VP1 protein 141-160 is synthesized, and the prepared polypeptide vaccine is less than 100-1 000 times less than the corresponding amount of the inactivated FMDV vaccine.
  • the current FMDV vaccine whether inactivated FMDV vaccine or peptide vaccine, cannot distinguish between immunized animals and infected animals by serological experiments, and the confusion between immunized animals and infected animals will greatly affect the market import and export. influences.
  • the present inventors based on the VP of the two types of FMDV of 0, A, the amino acid sequence of the immunological determinant and T- The cell-assisted sequence, a deoxyoligonucleotide fragment is designed, and the gene is expressed in tandem and expressed in Bacillus megaterium to obtain an immunogenic polypeptide. Moreover, after the amino acid sequence of the F? 1 immunological determinant polypeptide is reversed (from the N-terminus to the C-terminus), an immunoassay is performed, and a good effect is obtained, and the time for producing the neutralizing antibody is greatly prolonged, and the polypeptide is The half-life in the body is also prolonged.
  • the preparation of the vaccine by the polypeptide of the present invention can prevent both the 0 and A types of foot-and-mouth disease, and can conveniently distinguish between the immunized animal and the infected animal.
  • Figure 1 Single copy forward gene sequence F1 (N), capable of encoding immunological determinants that produce positively linked type 0 and type A foot-and-mouth disease viruses.
  • Figure 2 Single copy reverse gene sequence F1 (R), capable of encoding immunological determinants of type 0 and type A foot-and-mouth disease viruses that produce reverse junctions.
  • FIG. 3 T-cell helper sequences and the amino acids encoded thereby.
  • Figure 4 Schematic diagram of the construction process of plasmid pUC8 /Fl.
  • Fig. 5 is a schematic diagram showing the construction of the plasmid pUC8/F1, which was ligated into the expression vector PGEX-6P-3 after the T-he l per sequence was ligated.
  • Figure 6 SDS-PAGE of the fusion protein after pGEX-6P-3/F4 (N/R) was transferred to E. coli.
  • 1 is pGEX-6p-3 carrier protein (control)
  • 2-3 is fusion expression product of pGEX-6p-3/F4 (R)
  • 4-6 is P GEX-6p-3/F4 (N) Fusion of expression products.
  • Figure 7 is a growth map of engineering bacteria containing pGEX-6P-3/F4 (N/R) during fermentation.
  • Figure 8 shows the results of a bivalent peptide vaccine (type 0).
  • NO. 9 is a control well, only sensitized red blood cells are added, and no antigen preparation is added.
  • Figure 9 shows the results of measurement of a two-way immunodiffusion reaction (antigen-antibody reaction).
  • the antigens are 1 and 2, wherein 1 is a polypeptide obtained by purifying the expression product of pGEX-6P-3/F4 Q),
  • 2 is a polypeptide obtained by purifying pGEX-6P-3/F4 (N) expression product
  • the antibody is A, which is a type A FMD agar expansion positive serum supplied by Lanzhou Veterinary Medicine, X 1 is a stock solution, and X 5 is a 5-fold dilution;
  • ® is a blank control.
  • the antigen is a type A FMD agar expansion antigen of Lanzhou Veterinary Institute, X 1 is a stock solution, X 5 is a 5-fold dilution; the antibodies are A and F, wherein A is a purified product of PGEX-6P-3/F4(R) expression product.
  • the polypeptide is immunized with sera obtained from guinea pigs, and F is a purified protein of pGEX-6P-3/F4(N) expression product, and the sera obtained from guinea pigs are immunized.
  • the antigens are 1 and 2, wherein 1 is a polypeptide obtained by purifying the expression product of PGEX-6P-3/F4(R), and 2 is a polypeptide obtained by purifying pGEX-6P-3/F4(N);
  • the antibodies are A and D, wherein A is a PGEX-6P-3/F4(R) expression product purified polypeptide immunized guinea pig serum, and D is pGEX-6P-3/F4 (N) expression product purified polypeptide Immune serum obtained from guinea pigs;
  • ⁇ is type 0 vesicular disease antigen "+" (provided by Lanzhou Veterinary Institute), ® is a blank control.
  • FIG. 10 shows the results of detection by immunoelectrophoresis.
  • Antibody B is an anti-serum prepared by immunizing guinea pigs with purified protein of PGEX-6P-3/F4 (R) expression product, and the antigen is a type 0 "+" reagent provided by Lanzhou Veterinary.
  • Antibody A is a FMDV agar-positive serum (provided by Lanzhou Veterinary Medicine), and the antigen is a polypeptide obtained by purifying the expression product of pGEX-6P-3/F4 (N).
  • Antibody A is a type A FMD agar-positive serum (Lanzhou Veterinary Institute); the antigen is pGEX-6P-3/F4 (R) The polypeptide obtained after purification of the expressed product.
  • Antibody E is a purified guinea pig serum obtained by purifying the pGEX-6P-3/F4 (N) expression product.
  • the antigen is a type 0 "+" reagent provided by Lanzhou Veterinary. Detailed description of the invention
  • the present invention provides a novel foot-and-mouth disease genetically engineered polypeptide vaccine comprising 2 n - 1 linked polypeptide encoded by the nucleic acid sequence of SEQ ID NO. 1 or SEQ ID NO. 2, wherein Is a 1 - 5 0 integer.
  • SEQ ID NO. 1 contains type 0 and type A foot-and-mouth disease virus? a coding sequence of a protein immunological determinant and a T-cell helper sequence, which encodes a forward-expressed antigenic determinant of type A and type 0 foot-and-mouth disease virus, the expression product of which can stimulate the production of antibodies against type A and type 0 foot-and-mouth disease;
  • SEQ ID NO. 2 encodes an antigenic determinant of inverted-expressed type A and type 0 foot-and-mouth disease viruses, the expression product of which also stimulates the production of antibodies against type A and type 0 foot-and-mouth disease.
  • the vaccine of the present invention comprises two polypeptides encoded by SEQ ID NO: 1 or SEQ ID NO: 2 in which n is an integer of from 2 to 4.
  • the vaccine of the present invention comprises 2 n - 1 of the polypeptide encoded by SEQ ID NO: 1 or SEQ ID NO: 2, wherein n is 3.
  • the vaccine of the invention comprises 4 polypeptides encoded by SEQ ID NO: 2 in tandem.
  • the vaccine of the present invention contains the protein expressed by the plasmid carried in strain CGMCC No. 0893.
  • the plasmid contains four nucleic acid sequences of SEQ ID NO. 1 in series.
  • the vaccine of the present invention contains the protein expressed by the plasmid carried in the strain CGMCC No. 0894.
  • the plasmid contains four nucleic acid sequences of SEQ ID NO. 2 in tandem.
  • the vaccine of the present invention may further contain a pharmaceutically acceptable carrier, excipient, and/or immunoadjuvant for the purpose of improving bioavailability, enhancing immunity, and the like.
  • An immunological adjuvant is a substance that enhances an immune response, and can be used in combination with an antigen, which not only contributes to deposition or aggregation of an injectable substance, but also enhances an antibody reaction.
  • the substances that can be used as immunoadjuvants are: 1 microorganisms and their products, commonly used microorganisms such as mycobacteria, Corynebacterium parvum, B. pertussis, and extracts of the gram-negative bacillus, lipopolysaccharide, and mycobacterial extract material wall Acyl dipeptide and the like.
  • 3 Freund's adjuvant Including Freund's incomplete adjuvant (mixing the aqueous solution of the antigen with an oil (paraffin or vegetable oil) in an equal amount, adding an emulsifier (lanolin or Tween 80) to the water-in-oil antigen emulsion) and Fuchs Agent (adding mycobacteria, such as BCG) to incomplete adjuvants.
  • immunological adjuvants including: 1 bacterial toxins such as cholera toxin (CT) and Escherichia coli heat labile enterotoxin (LT); 2 attenuated derivatives or variants of CT and LT 3 human endogenous immunoregulatory factors, such as IL-2, IL-12, GM-GSF; 4 hormones; 5 lipopeptides; 6 saponins, saponin derivatives QS-21; 7 synthesis containing CpG mot if Oligonucleotide fragment (CpG ODN); a derivative of 8 lipid A, such as a lipopolysaccharide derivative monophosphoryl lipid A (MPL); a derivative of 9 muramyl dipeptide (MDP).
  • 1 bacterial toxins such as cholera toxin (CT) and Escherichia coli heat labile enterotoxin (LT)
  • certain delivery systems with intrinsic immunostimulatory activity can also be used as immunoadjuvants for vaccine construction, including but not limited to: liposomes, emulsions, cochlea te, viral particles, microparticles. And immunostimulating complexes (ISCOMs).
  • immunoadjuvants including but not limited to: liposomes, emulsions, cochlea te, viral particles, microparticles.
  • ISCOMs immunostimulating complexes
  • immunoadjuvants described above can be used in the present invention.
  • the adjuvant may be used in combination with the immunogenic polypeptide of the invention in a suitable dosage to form a vaccine of the invention.
  • the vaccine of the present invention contains Freund's complete adjuvant or A1(OH) 3 , and more preferably, the vaccine of the present invention contains Freund's complete adjuvant as an immunopotentiator.
  • the ratio of lanolin to paraffin oil in Freund's complete adjuvant varies slightly with the seasons. For example, the ratio of lanolin to paraffin oil in spring, summer and autumn is about 3:7; the ratio between the two is about 1 in winter. 5 : 8. 5.
  • a further aspect of the invention provides a method of preparing the genetically engineered polypeptide vaccine comprising cloning 2 n - 1 of the nucleic acid sequence of SEQ ID NO. 1 or SEQ ID NO. 2 into a vector The step of expressing in a suitable expression system, wherein n is an integer from 1 to 5.
  • nucleic acid sequences represented by SEQ ID NO. 1 or SEQ ID NO. 2 are expressed in series, wherein n is an integer of 2 - 4.
  • nucleic acid sequences represented by SEQ ID NO. 1 or SEQ ID NO. 2 are expressed in series to prepare a vaccine of the present invention.
  • nucleic acid sequences represented by SEQ ID NO. 2 are expressed in series to prepare a vaccine of the present invention.
  • the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 of the present invention may be directly synthesized, or several oligonucleotide fragments may be synthesized first, and ligated to produce SEQ ID NO: 1 or SEQ ID NO: 2
  • the nucleic acid sequence shown For the convenience of subsequent operations, when designing oligonucleotide fragments, Appropriate cleavage sites are ligated into both ends of the sequence to clone the target sequence into a suitable vector.
  • F4 (N), F8 (N), and F16 (N) can be cloned into vectors in turn, which in turn contain 4, 8, and 16 consecutive F1 (N) sequences, in appropriate expression.
  • the resulting polypeptides contained 4, 8, and 16 positively linked immunomorphic determinants of type 0 and type A FMDV, respectively.
  • the inventors designed to synthesize four oligonucleotide fragments and the sequence F1 (R) generated after ligation comprises the nucleic acid sequence shown in SEQ ID NO: 2 and a suitable restriction. The cleavage point is cut.
  • F1 (R) was cloned into the vector by genetic engineering, and then F2 (R) (containing 2 tandem F1 (R) sequences) by restriction endonuclease with complementary matching ends ) cloning into a vector, the expressed polypeptide contains two immunological determinants of inverted type 0 and type A FMDV.
  • F4 (R), F8 (R), and F16 (R) can be cloned into vectors in turn, which in turn contain four, eight, and sixteen consecutive F1 (R) sequences, in appropriate expression.
  • the resulting polypeptides contained 4, 8, and 16 inverted immunological determinants of type 0 and type A FMDV, respectively.
  • the expression system for polypeptide expression may be a prokaryotic expression system or a eukaryotic expression system.
  • Expression systems include suitable host cells, as well as plasmids or vectors that are capable of replicating and stably present in the host cell.
  • Examples which may be used as host cells include, but are not limited to, bacterial cells such as Escherichia coli, Streptomyces, Salmonella typhimurium, and the like; Fungal cells: such as yeast.
  • Vectors that can be used can include chromosome-derived, non-chromosomal-derived, and synthetic DNA sequences.
  • chromosome-derived non-chromosomal-derived, and synthetic DNA sequences.
  • phage DNA baculovirus
  • bacterial plasmid bacterial plasmid
  • yeast sac granule yeast sac granule
  • vector derived from a combination of plasmid, phage and viral DNA derived from a combination of plasmid, phage and viral DNA.
  • the polypeptide of the invention is expressed in a prokaryotic expression system. More preferably, the polypeptide of the present invention can be cloned into a highly efficient expression vector (e.g., a commercially available expression vector) for fusion expression with a carrier protein.
  • a highly efficient expression vector e.g., a commercially available expression vector
  • the present invention also relates to a genetically engineered strain which carries a plasmid comprising two nucleic acid sequences of SEQ ID NO. 1 or SEQ ID NO. 2 in series, wherein n is an integer from 1 to 5.
  • the plasmid carried by the engineered bacteria contains two nucleic acid sequences of SEQ ID NO. 1 or SEQ ID NO. 2 in series, wherein n is an integer of 2 - 4.
  • the plasmid carried by the engineered bacteria contains SEQ ID NO. 1 or SEQ ID in tandem
  • the genetically engineered strain comprising four nucleic acid sequences of SEQ ID NO. 1 and SEQ ID NO. 2 in series is in accordance with the Budapest Treaty on the Internationally Recognized Collection of Microorganisms for Patent Procedures.
  • the regulations are deposited with the General Microbiology Center of the China Microbial Culture Collection Management Committee (CGMCC is deposited on February 25, 2003, and the deposit numbers are CGMCC No. 0893 and CGMCC No. 0894, respectively).
  • the deposit CGMCC No. 0893 was prepared by transferring the plasmid pGEX/F4 (N-T) containing the T-hel per sequence into Escherichia coli BL21.
  • pGEX/F4 (NT) was digested with EcoRI and Sa l I to pUC18/F4 (N-T), and the F4 (NT) fragment was recovered and cloned into the same pGEX vector (GenBank Acces s ion No.). U78874) Made.
  • pGEX/F4 (NT) contains four forward tandem nucleic acid sequences encoding type A and type 0 FMDV epitopes and contains a T-he l per sequence, ie four tandem SEQ ID NO.
  • the expression product contains four forward-connected type A and type 0 FMDV epitopes.
  • the deposit CGMCC No. 0894 was prepared by transferring the shield granule pGEX/F4 (R-T) into E. coli BL21.
  • pGEX/F4 (R-T) was obtained by digesting pUC18/F4 (R_T) with EcoRI and Sa i l, and then recovering the F4 (R-T) fragment and cloning into the same pGEX vector.
  • pGEX/F4 (RT) contains four reversely linked nucleic acid sequences encoding type A and type 0 FMDV epitopes and contains the T-hel per sequence, ie four tandem SEQ ID NO. II, expression The product contains 4 reverse-linked type A and type 0 FMDV epitopes.
  • the deposit is for the purpose of fully disclosing the invention and is convenient for those skilled in the art. Any act of manufacturing, using or selling the deposit requires permission from the inventor and no such license is granted here.
  • the invention also provides the use of the genetically engineered polypeptide vaccine for preventing and treating foot and mouth disease.
  • the vaccine of the present invention can be injected into an animal to stimulate the production of specific antibodies against type 0 and type A foot-and-mouth disease in the animal, thereby achieving the purpose of prevention and treatment.
  • the invention provides the genetically engineered polypeptide vaccine in distinguishing between infected animals and immunized animals Use. Since animals naturally infected with foot-and-mouth disease usually produce only a single antibody (a serotype), and the vaccine of the present invention is injected into an animal to stimulate the production of specific antibodies against type 0 and type A foot-and-mouth disease in the animal, the vaccine of the present invention can Used to distinguish between immunized animals and infected animals.
  • the use of the vaccine of the present invention has the following advantages: 1.
  • the safety of the vaccine of the present invention is a genetically engineered product, not an inactivated vaccine, and there is no risk of a disease outbreak due to leakage of a trace amount of live virus.
  • mice and guinea pigs were injected subcutaneously with higher doses of vaccine. During the longer observation period, the experimental animals survived healthily and specific antibodies were produced in vivo.
  • the vaccine of the present invention is suitable for mass production by genetic engineering methods, which reduces the cost.
  • the vaccine of the present invention makes it easy to distinguish between infected animals and immunized animals.
  • the vaccine of the invention has both an A-type antigen and an immuno-determinant of type 0 antigen. After the immunization, the antibody produced in the animal has both type A and type 0, thereby distinguishing between infected animals and immunized animals, thereby avoiding giving animals Exports cause confusion. 4
  • the vaccine of the present invention contains type A and type 0 FMDV? !
  • the polypeptide expressed by the immune determinant after inversion (from the N-terminus to the C-terminus) has a good effect in the immunoassay, and the half-life of the polypeptide in the body is prolonged, and the time for producing the neutralizing antibody is also prolonged.
  • Example 1 Construction of plasmid pUC8/Fl (N) encoding positively aligned type 0 and type A epitopes
  • the 141-160 amino acid sequence of the protein was synthesized into four oligonucleotide fragments, which were amplified by PCR and cloned into a vector to form a recombinant plasmid pUC8/Fl (N) containing positively aligned type 0 and type A epitopes. .
  • the chemical synthesis consists of a four-segment DNA fragment of the forward FMDV gene, the sequence of which is as follows:
  • Taq enzyme (5 U/ ⁇ 1) 1 ⁇ ⁇
  • the reaction product was loaded onto a 1.2% agarose gel, electrophoresed at 80 V for 30 min, and purified using a QIAquick Gel Purification Kit. Cut the agarose block containing the DNA of interest, put it into a 1.5 ml centrifuge tube, add QG buffer (according to 100 mg agarose plus 300 ⁇ 1 QG), set a water bath at 50 ° C for 10 min, and mix it every 2 min. Times ⁇ Add 1/3 QG buffer volume of isopropanol, mix and hook after 55 °C warm bath for 1 min—move the melted agarose solution into the adsorption column, centrifuge at 12000 g for 1 min at room temperature ⁇ discard the liquid in the collection tube.
  • QG buffer accordinging to 100 mg agarose plus 300 ⁇ 1 QG
  • adsorption column into the collection tube ⁇ add 500 yl QG buffer to the adsorption column, centrifuge at 12000 g for 1 min at room temperature ⁇ discard the liquid in the collection tube, place the adsorption column into the collection tube ⁇ add in the adsorption column 750 ⁇ ⁇ ⁇ buffer, centrifuge at 12000 g for 1 min at room temperature ⁇ place the adsorption column in another 1.5 ml clean centrifuge tube ⁇ add 30 ⁇ l Tl (10 mM Tris H 8.0) buffer to the center of the adsorption membrane and let stand. Centrifuge at 12000 g for 1 min after 1 min. The purified DNA was collected and stored frozen at -20 °C.
  • Both the purified PCR product and the PUC18 vector were digested with EcoR I and Sal I.
  • Each reaction system was added with ddH20 to a total volume of 40 ⁇ l, and digested at 37 °C for 2 h. 1.2% agarose After separation by gel electrophoresis, the purified DNA fragment was recovered by QIAquick kit and finally eluted with 30 ⁇ l of T1.
  • the F1(N) gene fragment was reacted with T4 DNA ligase at 16 ° C for 5 h and inserted into the PUC18 vector.
  • the frozen E. coli JM109 was scooped with sterile platinum wire and colonized on LB agar plates, and cultured overnight with YTC.
  • ⁇ Pick a monoclonal strain into 3.5 ml LB medium, shake to 37°C to log phase ⁇ take 100 ⁇ 1000 ⁇ ⁇ and transfer to 100 ml LB, shake at 37 °C to A600 « 0.3 ⁇
  • the culture was ice-bathed for 10 minutes, divided into two portions, and centrifuged at 4 ° C, 3000 g for 5 min, and the supernatant was removed.
  • the centrifuge tube was inverted for 1 minute ⁇ 10 ml of pre-cooled 0. ltnol/L calcium chloride sensitizing solution was used.
  • Suspension cells ice bath for 10 minutes, centrifuge at 4 ° C, 4000 g for 10 min, collect cells ⁇ add 2 ml each in two centrifuge tubes 0. lmol / L calcium chloride combined with two centrifuge tubes and add glycerol 420 ⁇ ⁇ —Package 100 ⁇ of competent cells per Eppendorf tube, stored at 80 °C.
  • the single-copy cloned plasmids identified by sequencing were digested with BamH I, Hind III and Bgl II, Hind III, respectively.
  • Buffer E 2 ⁇ 1 Buffer B 2 ⁇ ⁇ Add ddH20 to a total volume of 20 ⁇ l and digest at 37 °C for 2 h. After purification by 1.2% agarose gel electrophoresis, the purified DNA fragment was recovered by QIAquick kit and finally eluted with 20 ⁇ Tl (10 mM Tris pH 8.0).
  • the small fragment NF1 (single-copy forward FMDV gene) was reacted at 16 °C for 5 h under the action of T4 DNA ligase, and inserted into the NF1-PUC18 (PUC18 containing a single copy of the forward FMDV gene) vector, which will result in a double copy.
  • the PUC18 vector of the forward FMDV gene was reacted at 16 °C for 5 h under the action of T4 DNA ligase, and inserted into the NF1-PUC18 (PUC18 containing a single copy of the forward FMDV gene) vector, which will result in a double copy.
  • the PUC18 vector of the forward FMDV gene was reacted at 16 °C for 5 h under the action of T4 DNA ligase, and inserted into the NF1-PUC18 (PUC18 containing a single copy of the forward FMDV gene) vector, which will result in a double copy.
  • the PUC18 vector of the forward FMDV gene was re
  • the total volume is 20 ⁇ 1 .
  • Transformation, plasmid extraction method identification method is the same as single copy gene cloning.
  • the plasmid containing the correct copy of the double-copy clone was subjected to the method described in 2 to obtain a clone containing the four-copy gene.
  • a clone of the eight-copy gene can be obtained, and thus, 16, 32, and 64 copies of the clone can be obtained by the cycle.
  • Example 3 Construction of plasmid pUC8/Fl (R) encoding reverse-aligned type 0 and scorpion antigenic determinants
  • oligonucleotide fragments were synthesized according to the reverse alignment sequence of amino acids 141 - 160 of type 1 and type A foot-and-mouth disease virus VP1 protein, which were amplified by PCR and cloned into vector to form type 0 and A with reverse alignment.
  • Recombinant plasmid P UC8/F1 (R) of the type antigenic determinant Recombinant plasmid P UC8/F1 (R) of the type antigenic determinant.
  • Each 0D DNA fragment was dissolved in 20 ⁇ l of ddH 2 0 and phosphorylated according to the following reaction system.
  • the reaction was carried out at 37 ° C for 1 h, placed in a 92 ° C water bath, allowed to cool naturally, to complete the enzyme inactivation and the annealing of the DNA strand.
  • the correct cloned clone was a clone of the four-copy forward/reverse FMDV gene with the T-helper sequence (N/R F4-T-PUC18).
  • pGEX-6P-3 Pharmacia biotech pGEX-6P-3 was used as an expression vector.
  • pGEX-6P-3 contains an inducible tac promoter, a lacl q gene for expression in E. coli, and a recognition site for PreScission enzyme for cleavage of a fusion protein (a fusion expression product of GST and a protein of interest).
  • N/R F4-T-PUC18 and PGEX-6P-3 carriers were double digested with EcoR I and Sal I, and the recovered N/R F4-T (EcoR I/Sal I ) and PGEX-6P-3 (EcoR) were recovered.
  • I/Sal I) DNA fragment which was ligated under the action of ligase, was transformed into E. coli JM109, the same procedure as before.
  • Each reaction system was digested with ddH 2 0 to a total volume of 40 ⁇ l and digested at 37 ° C for 2 h. After purification by 1.2% agarose gel electrophoresis, the purified DNA fragment was recovered by QIAquick kit, and finally 30 ⁇ 1 T1 was used. Elution.
  • the resulting clone was transformed, and the plasmid was extracted and identified by double digestion with EcoR I and Sal I.
  • the recombinant plasmid (N/R F4-T-PGEX-6P-3) was transformed into E. coli c600, and cultured at 37 ° C, and induced with IPTG at a final concentration of 0.2 mmol/L.
  • the loading amount is about 50 ⁇ g, the current is 10-15 mA, the voltage is 100 V, electrophoresis is 1-1.5 hours, staining, and the results are observed. The results are shown in Fig. 6.
  • 250 ml seed culture solution (1QG0 ml seed culture solution containing peptone 10 g, yeast extract powder 5 g, 0.02 mol/L phosphate buffer 20 ml, pH 7.0) in a 1000 ml flask, sterilized at 120 °C 2 After 0 minutes, 5 ml of a 20% glucose solution was added after cooling. Add 1 ml of the strain stored in glycerol at low temperature to the above solution, add ampicillin to a final concentration of 50 g/ml, incubate at 37 ° C, 200 rpm. 12-14 hours as a seed culture for expanded culture.
  • the 1000 ml seed culture solution contains 20 g of peptone, 10 g of yeast extract powder, 20 mol / L of citric acid buffer 20 ml, pH 7.0, and trace elements CaC12, NiN03, CoC13, MgS04, FeC13 each 1 mg, 12 (TC sterilization for 20 minutes, after cooling to 37 ° C, add ampicillin to a final concentration of 50 mg.
  • the cells were collected by centrifugation at 4000 rpm (Beckman J6-HC centrifuge) for 30 minutes.
  • the cells were suspended in a solution containing 1% sodium chloride, 1 crypt ol / L EDTA, 20 ⁇ / L potassium phosphate pH 7.0 in a ratio of 1000 g / 3 liter. 1 g of lysozyme was added to the suspension, and the mixture was stirred at room temperature for 1 hour to break the cells. The bacterial suspension was centrifuged at 10,000 rpm for 30 minutes, and the supernatant was discarded.
  • the above precipitate was added to a 6 mol/L guanidine hydrochloride solution at a rate of 250 g/liter. Stir and extract overnight. Centrifuge at 20, OOO rpm for 30 minutes, take the supernatant, and rinse the dialysis bag with water.
  • Precipitation occurs on water dialysis. Centrifuge at 10,000 rpm for 30 minutes, take a pellet, and homogenize with sterile water for use.
  • the sensitizing red blood cell type 0 foot-and-mouth disease titer assay kit is a product of Lanzhou Animal Husbandry and Veterinary Institute.
  • Type 0 foot-and-mouth disease vaccine polypeptide protein (forward amino acid sequence), a solution dissolved with 0.1% sodium dodecyl sulfate.
  • Type 0 foot-and-mouth disease vaccine polypeptide protein (reverse amino acid sequence), a solution dissolved with 0.1% sodium dodecyl sulfate.
  • the positive serum in the test kit and the negative serum were diluted by the same two-fold dilution method, and the same amount of 20 ⁇ l of the 5-fold diluted sensitized red blood cell solution was added, and the detection was compared at the same time, and the results are shown in Fig. 8.
  • the antigen and the antibody can be used to produce a precipitation band or a precipitation ring at a concentration ratio, and the immunogenicity of the vaccine of the present invention can be measured by a two-way immunodiffusion method.
  • a semi-solid substrate e.g., a gel
  • Test article The pGEX-6P_3/F4 (R) and pGEX-6P-3/F4 (N) of the present invention express the purified protein.
  • Test article 1 mg of the protein purified by pGEX-6P-3/F4 0 and pGEX-6P-3/F4 (N) of the present invention, and 1 ml of Freund's complete adjuvant to form a water-in-oil emulsion, subcutaneously injected into the guinea pig , serum collected after 3 weeks of blood collection.
  • the purified protein of the pGEX-6P-3/F4 (R) and P GEX_6P-3/F4 (N) expression products of the present invention is an antigen
  • the A-type FMDV antiserum (standard) is an antibody. It was observed that both the forward and reverse expressed F4 proteins and the type A FMDV antibodies were immunoprecipitated.
  • Fig. 9B the type A FMD agar expansion antigen (standard) is used as an antigen, and the pGEX-6P-3/F4 (R) and pGEX-6P-3/F4 (N) expression products of the present invention are purified, and the guinea pig is immunized.
  • the serum prepared after the preparation was an antibody, and an immunoprecipitation reaction between the antigen and the antibody was observed.
  • the positive control of type 0 porcine blister lyophilized positive hemagglutination diagnostic fluid antigen is type 0 antigen
  • the well without added reagent is a blank control
  • the purified protein of PGEX-6P-3/F4 (N) expression product is the antigen to be detected, and expressed by pGEX-6P-3/F4 (R) and pGEX-6P-3/F4 (N) of the present invention.
  • the serum prepared by immunizing the guinea pig is an antibody, and an immunoprecipitation reaction occurs between the antigen to be tested and the antibody.
  • the genetically engineered polypeptide vaccine prepared by the invention has both antigenic determinants of type A FMDV and type 0 FMDV, and the antibodies produced after the immunoinjection are resistant to both type A and type 0 foot and mouth disease. Therefore, it can be used to distinguish between infected animals and immunized animals.
  • agarose solution 0.5% agarose solution was prepared with 0. 05 mo l /L (pH 8.6) barbital sodium-hydrochloric acid buffer solution, heated and thawed and spread on a glass slide, after cooling Punch holes and fill holes with hot agarose solution.
  • the antibody was added to the middle well and the antigen was added to both wells.
  • the slides were placed in an electrophoresis tank, and the + and - poles were electrophoresed for 1 hour at a current of 10 mA.
  • the precipitated strip was directly observed after soaking for a few hours with 0.9% sodium chloride solution, or the precipitated strip was observed after staining with amino black.
  • FIG. 10 shows that the FMDV vaccine of the present invention can induce both type 0 and type A antibodies, whereas animals naturally infected with foot-and-mouth disease usually produce only a single antibody (a serotype), so the present invention Vaccines can be used to distinguish between vaccinated animals and infected animals.
  • Three male Kunming mice each weighing about 20 g, were purchased from the Shanghai Animal Center of the Chinese Academy of Sciences. 1 ⁇ / ⁇ The lmg of the purified antigen protein was suspended in 1 ml of sterile water, and added 0. 1 ° /. The SDS promotes protein dissolution. A 100 ⁇ l solution was taken and the mice were injected subcutaneously. Mice were observed within 1 week and serum was collected for testing.

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Abstract

L'invention concerne un vaccin peptidique bivalent, ses procédés de préparation et ses applications. Ce vaccin, qui est obtenu par génie biologique, comprend un peptide codé par 2n-1 séquences nucléotidiques tandem de SEQ ID NO: 1 ou SEQ ID NO: 2, n étant un entier de 1 à 5. Le vaccin selon l'invention est sûr et présente une immunoréactivité élevée. Ce vaccin convient à une fabrication en masse et permet de prévenir ou de traiter efficacement la fièvre aphteuse. Ce vaccin peut également permettre d'établir une distinction entre des animaux infectés et vaccinés.
PCT/CN2004/001016 2003-09-03 2004-09-03 Vaccin peptidique bivalent dirige contre fmd, ses procedes de preparation et ses applications WO2005021035A1 (fr)

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CN100425291C (zh) * 2005-04-06 2008-10-15 中国农业科学院兰州兽医研究所 O型口蹄疫病毒多基因复制缺陷型腺病毒活载体疫苗及制备方法
CN101659695B (zh) * 2008-08-27 2012-08-29 中牧实业股份有限公司 O型口蹄疫合成肽疫苗
CN101643500B (zh) * 2009-05-19 2012-06-06 中牧实业股份有限公司 一种亚洲一型口蹄疫合成肽疫苗
CN102274496B (zh) * 2010-06-12 2015-05-06 吴晓琰 一种O/Asia I型口蹄疫病毒两价基因工程多肽疫苗及制备方法和用途
CN102380095A (zh) * 2010-09-03 2012-03-21 吴晓琰 一种口蹄疫三价多肽疫苗及制备方法和用途
CN102772797B (zh) * 2011-05-13 2013-11-06 吴晓琰 O型口蹄疫病毒中4个亚型基因工程多肽疫苗及其用途

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WO2002000251A1 (fr) * 2000-06-29 2002-01-03 Merial Vaccin contre la fievre aphteuse
CN1408349A (zh) * 2002-09-16 2003-04-09 复旦大学 一种口蹄疫基因工程多肽疫苗及其制备方法

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CN1090203A (zh) * 1993-10-21 1994-08-03 复旦大学 家畜口蹄疫病素多肽疫苗及其制备方法
WO2002000251A1 (fr) * 2000-06-29 2002-01-03 Merial Vaccin contre la fievre aphteuse
CN1408349A (zh) * 2002-09-16 2003-04-09 复旦大学 一种口蹄疫基因工程多肽疫苗及其制备方法

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