CN117462665A - Preparation method and application of porcine parvovirus and epidemic encephalitis B bigeminal subunit vaccine - Google Patents

Preparation method and application of porcine parvovirus and epidemic encephalitis B bigeminal subunit vaccine Download PDF

Info

Publication number
CN117462665A
CN117462665A CN202210896463.5A CN202210896463A CN117462665A CN 117462665 A CN117462665 A CN 117462665A CN 202210896463 A CN202210896463 A CN 202210896463A CN 117462665 A CN117462665 A CN 117462665A
Authority
CN
China
Prior art keywords
protein
porcine
japanese encephalitis
encephalitis virus
vaccine
Prior art date
Legal status (The legal status 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 status listed.)
Pending
Application number
CN202210896463.5A
Other languages
Chinese (zh)
Inventor
张冬冬
林艳
郑琴勤
岳丰雄
刘汉平
陈莉群
夏嘉鑫
项聪英
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Chengdu Shiji Biopharmaceutical Co ltd
Original Assignee
Chengdu Shiji Biopharmaceutical 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 Chengdu Shiji Biopharmaceutical Co ltd filed Critical Chengdu Shiji Biopharmaceutical Co ltd
Priority to CN202210896463.5A priority Critical patent/CN117462665A/en
Publication of CN117462665A publication Critical patent/CN117462665A/en
Pending legal-status Critical Current

Links

Classifications

    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • 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
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vectore
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14311—Parvovirus, e.g. minute virus of mice
    • C12N2750/14322—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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14311—Parvovirus, e.g. minute virus of mice
    • C12N2750/14334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24122—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
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Mycology (AREA)
  • Biotechnology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Plant Pathology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention relates to an immunogen composition, which consists of porcine parvovirus VP2 protein and porcine Japanese encephalitis virus prME protein; the porcine parvovirus VP2 protein is a protein coded by a nucleotide sequence shown in SEQ ID NO. 1; the porcine Japanese encephalitis virus premE protein is a protein coded by a nucleotide sequence shown in SEQ ID NO. 2. According to the invention, codon sequence optimization is carried out on parvovirus VP2 and Japanese encephalitis virus prME genes, and the porcine parvovirus VP2 protein and the porcine Japanese encephalitis virus prME protein expressed by specific gene sequences are used as antigens, so that the prepared bivalent subunit vaccine has good immune effect, higher immune efficacy than that of commercially available vaccines, high safety and practical popularization and application values.

Description

Preparation method and application of porcine parvovirus and epidemic encephalitis B bigeminal subunit vaccine
Technical Field
The invention belongs to the technical field of gene recombination vaccines, and particularly relates to a preparation method and application of a porcine parvovirus and epidemic encephalitis B bigeminal subunit vaccine.
Background
Porcine parvovirus disease is a viral infectious disease caused by infection with porcine parvovirus (Porcine parvovirus, PPV), which is mainly responsible for abortion, malformation and stillbirth in sows, and can be transmitted to piglets through placenta barriers; skin inflammation, toxemia and even death occur after infection of piglets. Epidemic encephalitis B virus is also called Japanese encephalitis B (Japanese encephalitis virus, JEV), which is a mosquito-borne zoonotic virus and causes meningitis and brain tissue inflammation, and can cause dead fetus, abortion, mummy fetus, orchitis of boar and the like of any sow. The disease is transmitted mainly between mosquitoes and pigs, which are the main host for storage and proliferation. In recent years, the porcine parvovirus disease is mostly mixed with the porcine Japanese encephalitis to cause great economic loss to pig farms. At present, the whole-virus bivalent inactivated vaccine is used for preventing porcine parvovirus disease and porcine Japanese encephalitis disease in the market, but the risk caused by incomplete virus inactivation exists, and the porcine parvovirus is easy to scatter in the production process to cause workshop pollution and other product pollution, and the porcine Japanese encephalitis also has the risk of operator infection. There is a trend, therefore, to develop effective subunit genetically engineered vaccines. The expressed cultured virus-like particles (VLPs) are similar in morphology and structure to the natural virus, and are free of nucleic acid, and have good safety and immunogenicity.
Patent CN112168959a discloses a bivalent subunit vaccine for porcine parvovirus and encephalitis b, which can prevent porcine parvovirus and encephalitis b simultaneously, but the effect of preventing encephalitis b is not improved compared with that of single vaccine, and the quality of the bivalent subunit vaccine needs to be further improved.
Disclosure of Invention
The invention aims at providing an immunogen composition which is prepared by the method that the hemagglutination efficiency is 2: 10～18 ：2 10～18 is composed of porcine parvovirus VP2 protein and porcine Japanese encephalitis virus prME protein;
the porcine parvovirus VP2 protein is a protein coded by a nucleotide sequence shown in SEQ ID NO. 1;
the porcine Japanese encephalitis virus premE protein is a protein coded by a nucleotide sequence shown in SEQ ID NO. 2.
Further, the hemagglutination efficiency ratio of the porcine parvovirus VP2 protein and the porcine Japanese encephalitis virus prME protein is 2 10 ～14 ：2 10～14 Preferably 2 10 ：2 10 。
Further, the amino acid sequence of the porcine parvovirus VP2 protein is shown as SEQ ID NO. 3; the amino acid sequence of the porcine Japanese encephalitis virus preME protein is shown as SEQ ID NO. 4.
The invention also provides a preparation method of the immunogen composition, which comprises the following steps:
(1) Respectively taking SEQ ID NO:1 and SEQ ID NO:2, connecting the gene segment of the nucleotide sequence shown in the formula 2 with an expression vector, then introducing into escherichia coli, extracting recombinant bacmid, transfecting into insect cells sf9, culturing, and collecting the supernatant to obtain recombinant baculovirus;
(2) Respectively taking the recombinant baculovirus obtained in the step 2), inoculating the recombinant baculovirus into insect cells Highfive, culturing, and collecting porcine parvovirus VP2 protein and porcine Japanese encephalitis virus prME protein;
(3) Mixing the porcine parvovirus VP2 protein obtained in the step 2) and the porcine Japanese encephalitis virus prME protein according to a proportion.
Further, the expression vector in the step (1) is a pFastBacDual plasmid; the escherichia coli is TOP10 competent cells and/or DH10Bac competent cells.
Further, the culture in the step (2) is carried out until the cell viability is lower than 10%.
The invention also provides a subunit vaccine which is prepared by taking the immunogen composition as an active ingredient and adding an immune adjuvant.
Further, the hemagglutination titer of the porcine parvovirus VP2 protein in the vaccine is 2 10～14 Preferably 2 10 The method comprises the steps of carrying out a first treatment on the surface of the The hemagglutination titer of the PRME protein of the porcine Japanese encephalitis virus is 2 10～14 Preferably 2 10 。
Further, the mass ratio of the immunogen composition to the adjuvant is 1-9: 1.
further, the mass ratio of the immunogen composition to the adjuvant is 1:1.
still further, the adjuvant includes 15A adjuvant, ISA201 adjuvant or Gel02 adjuvant, preferably ISA201 adjuvant.
According to the invention, codon sequence optimization is carried out on parvovirus VP2 and Japanese encephalitis virus prME genes, and porcine parvovirus VP2 protein and porcine Japanese encephalitis virus prME protein expressed by specific gene sequences are used as antigens, so that the prepared bivalent subunit vaccine has good immune effect and high safety, and experiments prove that: the subunit vaccine prepared by the porcine parvovirus VP2 protein and the porcine Japanese encephalitis virus prME protein prepared by the invention and the ISA201 adjuvant can enable susceptible animals to generate higher PPV and JEV HI antibodies, has good immunogenicity and higher immunization efficacy than commercial vaccines, and has practical popularization and application values.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only.
Detailed Description
Example 1 preparation of porcine parvovirus VP2 protein
(1) Taking a gene fragment with a nucleotide sequence shown as SEQ ID NO.1, connecting the gene fragment with a pFastBacDual vector, transforming the gene fragment into TOP10 competent cells, transforming a recombinant expression donor plasmid pFastPPV-VP2 into DH10Bac competent cells, screening and identifying positive colonies by blue white spots, and transfecting successfully identified recombinant baculo into Sf9 cells to obtain recombinant baculovirus BacPPV-VP2;
(2) Recombinant baculovirus Bacppv-VP2 was used and inoculated at an inoculum size of MOI=0.1 to 1 to a cell density of 2×10 6 Performing target protein expression on cells/ml Highfive cells, reducing the survival rate of the cultured cells to below 10% after inoculating the virus, and collecting the supernatant to obtain the recombinant DNA;
wherein the nucleotide sequence of SEQ ID NO. 1:
ATGTCAGAAAACGTAGAACAGCATAACCCAATTAATGCCGGTACGGAGTTGTCGGCTACCGGAAACGAATCTGGAGGGGGTGGTGGCGGCGGCGGCGGTAGAGGAGCGGGCGGCGTAGGAGTCTCTACAGGAAGTTTTAACAATCAGACTGAGTTCCAGTATCTCGGCGAGGGTCTAGTACGAATTACAG CGCACGCGAGTCGGCTGATT CATTTAAATATGCCGGAGCACGAGACGTATAAGAGGATACACGTGCTTAATTCTGAAAGTGGAGTGGCAGGCCAAATGGTACAGGACGATGCACATACGCAAATGGTTACACCCTGGTCACTAATTGACGCAAACGCGTGGGGGGTCTGGTTTAATCCTGCAGATTGGCAATTGATATCGAACAACATGACAGAAATAAATCTTGTCTCGTTCGAACAGGAGATTTTTAACGTTGTCCTTAAGACTATAACAGAATCAGCTACCTCGCCCCCCACGAAAATATACAATAATGATTTAACGGCCAGTCTGATGGTGGCCTTAGACACGAACAATACTTTACCCTACACCCCTGCTGCGCCGCGGAGCGAAACCCTCGGTTTTTATCCGTGGCTGCCGACGAAACCAACGCAGTACCGCTATTATCTATCATGTACCCGGAACCTAAATCCACCTACTTACACTGGGCAGAGTCAACAGATTACAGATTCTATACAAACCGGCCTCCACAGCGACATCATGTTCTATACAATTGAGAATGCCGTGCCAATACATCTCCTGCGTACGGGGGATGAGTTTAGTACCGGAATTTACCACTTCGATACGAAGCCTCTTAAATTGACGCATTCCTGGCAGACCAACAGATCCCTCGGTCTTCCGCCCAAATTGCTGACTGAGCCCACGACAGAGGGGGATCAACACCCAGGGACGCTACCAGCTGCAAACACACGGAAGGGCTATCATCAAACTATCAATAACTCTTATACAGAAGCAACAGCTATACGTCCCGCCCAAGTTGGCTACAATACACCGAACATGAACTTTGAATACTCCAATGGTGGACCTTTCCTGACGCCGATCGTTCCCACTGCAGATACACAGTACAACGATGATGAACCAAATGGAGCCATCAGGTTCACTATGGATTATCAGCACGGACATTTAACTACAAGCAGCCAAGAGCTAGAGCGCTATACTTTTAACCCCCAATCGAAATGTGGAAGAGCTCCCAAACAGCAATTCAACCAACAAGCGCCTTTAAACTTGGAAAATACTAATAATGGTACCTTGCTTCCGTCCGACCCGATCGGAGGTAAACCTAATATGCATTTCATGAACACCTTAAATACGTACGGGCCGTTGACTGCGCTTAACAATACGGCCCCTGTTTTTCCCAACGGTCAGATCTGGGACAAAGAGCTGGACACCGACCTAAAGCCACGACTCCATGTCACGGCTCCTTTCGTGTGCAAAAACAATCCACCAGGGCAACTCTTCGTAAAGATTGCGCCTAACCTCACCGACGATTTTAACGCCGACTCCCCGCAACAACCGCGTATCATCACTTACAGTAATTTCTGGTGGAAGGGGACGTTGACCTTTACCGCTAAGATGCGCAGCTCCAATATGTGGAATCCCATCCAGCAGCACACTACTACAGCAGAAAACATTGGTAATTACATCCCTACCAACATAGGGGGTATTAAAATGTTTCCAGAATATTCTCAGCTTATTCCTAGGAAGTTATATTGA
amino acid sequence of porcine parvovirus VP2 protein (SEQ ID NO. 3):
MSENVEQHNPINAGTELSATGNESGGGGGGGGGRGAGGVGVSTGSFNNQTEFQYLGEGLVRITAHASRLIHLNMPEHETYKRIHVLNSESGVAGQMVQDDAHTQMVTPWSLIDANAWGVWFNPADWQLISNNMTEINLVSFEQEIFNVVLKTITESATSPPTKIYNNDLTASLMVALDTNNTLPYTPAAPRSETLGFYPWLPTKPTQYRYYLSCTRNLNPPTYTGQSQQITDSIQTGLHSDIMFYTIENAVPIHLLRTGDEFSTGIYHFDTKPLKLTHSWQTNRSLGLPPKLLTEPTTEGDQHPGTLPAANTRKGYHQTINNSYTEATAIRPAQVGYNTPNMNFEYSNGGPFLTPIVPTADTQYNDDEPNGAIRFTMDYQHGHLTTSSQELERYTFNPQSKCGRAPKQQFNQQAPLNLENTNNGTLLPSDPIGGKPNMHFMNTLNTYGPLTALNNTAPVFPNGQIWDKELDTDLKPRLHVTAPFVCKNNPPGQLFVKIAPNLTDDFNADSPQQPRIITYSNFWWKGTLTFTAKMRSSNMWNPIQQHTTTAENIGNYIPTNIGGIKMFPEYSQLIPRKLY
example 2 preparation of PRME protein from porcine Japanese encephalitis Virus
(1) Taking a gene fragment with a nucleotide sequence shown as SEQ ID NO.2, connecting the gene fragment with a pFastBacDual vector, transforming the gene fragment into TOP10 competent cells, transforming a recombinant expression donor plasmid pFastJEV-preME into DH10Bac competent cells, screening and identifying positive colonies by blue white spots, and transfecting successfully identified recombinant bacJEV-prME into Sf9 cells to obtain recombinant baculovirus BacJEV-prME;
(2) Recombinant baculovirus BacJEV-prME was used and inoculated at an inoculum size of MOI=0.1 to 1 to a cell density of 2×10 6 The cells/ml Highfive cells express the target protein,when the survival rate of the cultured cells subjected to the inoculation is reduced to below 10 percent, collecting the supernatant to obtain the strain;
wherein the nucleotide sequence of SEQ ID NO. 2:
ATGAAGCTGTGCATCCTGCTGGCTGTGGTGGCTTTCGTGGGCCTGTCCCTGGGCATGAAGCTGTCCAACTTCCAAGGCAAGCTGCTGATGACCATCAACAACACCGACATCGCTGACGTGATCGTGATCCCCACCTCCAAGGGCGAGAACAGATGCTGGGTGAGAGCTATCGACGTGGGCTACATGTGCGAGGACACCATCACCTACGAGTGCCCCAAGCTGACTATGGGCAACGACCCCGAGGACGTGGACTGCTGGTGCGACAACCAAGAGGTGTACGTGCAGTACGGCAGATGCACTAGAACTAGACACTCCAAGAgaAGCAGAAGATCCGTGTCCGTGCAGACCCACGGCGAGTCCTCCCTGGTGAACAAGAAGGAGGCTTGGCTGGACTCCACCAAGGCTACTAGATACCTGATGAAGACCGAGAACTGGATCATCAGAAACCCCGGCTACGCTTTTCTGGCTGCCGTGCTGGGCTGGATGCTGGGCTCCAACAACGGTCAGAGAGTGGTGTTCACCATCCTGCTCCTGCTGGTGGCTCCCGCTTACTCCTTCAACTGCCTGGGCATGGGCAACAGAGACTTCATCGAGGGCGCTTCCGGCGCTACCTGGGTGGACCTGGTGCTGGAGGGCGACTCCTGCCTGACCATCATGGCTAACGACAAGCCCACCCTGGACGTGAGAATGATCAACATCGAGGCTTCCCAACTGGCTGAGGTGAGATCCTACTGCTACCACGCTTCCGTGACCGACATCTCCACCGTGGCTAGATGCCCCACCACCGGCGAGGCTCACAACGAGAAGAGAGCTGACTCCTCCTACGTGTGCAAGCAAGGCTTCACCGACAGAGGCTGGGGCAACGGCTGCGGCTTCTTCGGCAAGGGCTCCATCGACACCTGCGCTAAGTTCTCCTGCACCTCCAAGGCTATCGGCAGAACCATTCAGCCCGAGAACATCAAGTACAAGGTGGGCATCTTCGTGCACGGCACCACTACCTCCGAGAACCACGGCAACTACTCCGCTCAAGTGGGCGCTTCCCAAGCTGCTAAGTTCACCGTGACCCCCAACGCTCCCTCCGTGGCTCTGAAGCTGGGCGACTACGGCGAGGTGACCCTGGACTGCGAGCCTAGATCCGGCCTGAACACCGAGGCTTTCTACGTGATGACCGTGGGCTCCAAGTCCTTCCTGGTGCACAGAGAGTGGTTCCACGACCTGGCTCTGCCCTGGACCTCCCCCTCCTCCACCGCTTGGAGAAACAGAGAGCTGCTGATGGAGTTCGAGGGCGCTCACGCTACCAAGCAGTCCGTGGTGGCTCTGGGCTCCCAAGAGGGCGGCCTGCACCACGCTCTGGCTGGCGCTATCGTGGTGGAGTACAGCTCCTCCGTGATGCTGACCTCCGGCCACCTGAAGTGCAGACTGAAGATGGACAAGCTGGCTCTGAAGGGCACCACCTACGGCATGTGCACCGAGAAGTTCTCCTTCGCTAAGAACCCCGTGGACACCGGCCACGGCACCGTGGTGATCGAGCTGTCCTACTCCGGCTCCGACGGCCCCTGCAAGATCCCCATCGTGTCCGTGGCTTCCCTGAACGACATGACCCCCGTGGGCAGACTGGTGACCGTGAACCCCTTCGTGGCTACTTCCTCCGCTAACTCCAAGGTGCTGGTGGAGATGGAGCCCCCCTTCGGCGACTCCTACATCGTGGTGGGCAGAGGCGACAAGCAGATCAACCACCACTGGCACAAGGCTGGCTCCACCCTGGGCAAGGCTTTCTCCACCACCCTGAAGGGCGCTCAGAGACTGGCTGCTCTGGGCGACACCGCTTGGGACTTCGGCTCCATCGGCGGCGTGTTCAACTCCATCGGCAGAGCTGTGCACCAAGTGTTCGGCGACGCTTTCAGAACCCTGTTCGGCGGCATGTCCTGGATCACCCAAGGCCTGATGGGCGCTCTGCTCCTGTGGATGGGCGTGAACGCTAGAGACAGATCCATCGCTCTGGCTTTCCTGGCTACCGGCGGCGTGCTGGTGTTTCTCGCTACCAACGTGCACGCTTAA
amino acid sequence of prME protein of porcine encephalitis b virus (SEQ ID No. 4):
MKLCILLAVVAFVGLSLGMKLSNFQGKLLMTINNTDIADVIVIPTSKGENRCWVRAIDVGYMCEDTITYECPKLTMGNDPEDVDCWCDNQEVYVQYGRCTRTRHSKRSRRSVSVQTHGESSLVNKKEAWLDSTKATRYLMKTENWIIRNPGYAFLAAVLGWMLGSNNGQRVVFTILLLLVAPAYSFNCLGMGNRDFIEGASGATWVDLVLEGDSCLTIMANDKPTLDVRMINIEASQLAEVRSYCYHASVTDISTVARCPTTGEAHNEKRADSSYVCKQGFTDRGWGNGCGFFGKGSIDTCAKFSCTSKAIGRTIQPENIKYKVGIFVHGTTTSENHGNYSAQVGASQAAKFTVTPNAPSVALKLGDYGEVTLDCEPRSGLNTEAFYVMTVGSKSFLVHREWFHDLALPWTSPSSTAWRNRELLMEFEGAHATKQSVVALGSQEGGLHHALAGAIVVEYSSSVMLTSGHLKCRLKMDKLALKGTTYGMCTEKFSFAKNPVDTGHGTVVIELSYSGSDGPCKIPIVSVASLNDMTPVGRLVTVNPFVATSSANSKVLVEMEPPFGDSYIVVGRGDKQINHHWHKAGSTLGKAFSTTLKGAQRLAALGDTAWDFGSIGGVFNSIGRAVHQVFGDAFRTLFGGMSWITQGLMGALLLWMGVNARDRSIALAFLATGGVLVFLATNVHA
example 3 preparation of porcine parvovirus and Japanese encephalitis bigeminal subunit vaccine
Blood clot removal potency 2 10 Porcine parvovirus VP2 protein prepared in example 1 and hemagglutination potency 2 10 The PRME protein of the porcine Japanese encephalitis virus prepared in example 2 is mixed with an ISA201 adjuvant in a mass ratio of 1:1.
The beneficial effects of the invention are further illustrated by the following test examples:
test example 1
1 construction of the virus seed corresponding codon-optimized gene sequences (SEQ ID NO.1 and SEQ ID NO. 2) were synthesized from the GeneBank parvovirus VP2 protein sequence and the Japanese encephalitis preME protein sequence, respectively, and the sequences were synthesized by commercial companies. The synthetic genes are respectively connected with pFastBacDual vectors and transformed into TOP10 competent cells, the expression donor plasmids pFastPPV-VP2 and pFastJEV-preME with correct sequencing (the sequencing result is 100% consistent with the gene sequence) and transformed into DH10Bac competent cells are respectively connected with pFastBacDual vectors, blue white spots are screened and positive colonies are identified, and then successfully identified recombinant bacmid is transfected into Sf9 cells for packaging expression, so that recombinant baculoviruses BacPPV-VP2 and BacJEV-prME are obtained.
2 antigen reproduction at an inoculum size of MOI of 0.1, recombinant baculoviruses BacPPV-VP2 and BacJEV-prME were inoculated to a cell density of 2X 10, respectively 6 The cells/ml Highfive cells are used for expressing target proteins, the cell viability of the virus-inoculated culture cells is reduced to below 10%, and the supernatant is collected. Detection of the hemagglutination titre of the expressed PPV-VP2 antigen by the Hemagglutination Assay (HA) of 2 19 . HA detection JEV-prME antigen hemagglutination titre of 2 18 . The antigens are all preserved at the temperature of 2-8 ℃ for standby.
3 adjuvant screening
3.1 preparation of vaccine the porcine parvovirus and porcine Japanese encephalitis virus bigeminal subunit vaccine was prepared with 15A, ISA and Gel02 adjuvants, respectively, so that each adjuvant preparation vaccine contained the same amount of antigen. Specific formulation information is shown in table 1 below.
Table 1 vaccine formulation and immunization information
3.2 efficacy test screening 12 SPF-grade guinea pigs were randomly divided into 4 groups of 3, each guinea pig of groups 1-3 was vaccinated by intramuscular injection of one adjuvant to the leg, each 0.5ml, and group 4 was a non-immunized control group. All guinea pigs were bled for serum isolation for PPV HI antibody and JEV HI antibody assays 28 days after vaccine immunization. The results show that: vaccine preparation by three adjuvants the immunized guinea pigs all produced higher PPV and JEV HI antibodies, and were significantly higher than the existing vaccine quality standards (PPV HI antibody 1:64, JEV HI antibody 1:20). The HI antibody detection results are shown in Table 2.
TABLE 2 PPV HI antibody levels for each vaccine immunization group
4 preparing two-joint vaccine and single vaccine with different antigen contents by ISA201 adjuvant for compatibility research, and respectively immunizing guinea pigs and vaccineSerum was collected 28 days after immunization, and PPV HI antibodies and JEV HI antibodies were detected after vaccine immunization. Vaccine antigen content and immune group were prepared as shown in table 3. The results show that: containing 2 10 HA-titer PPV-VP2 protein and 2-containing 10 PPV HI antibody generated after the guinea pigs are immunized by the bigeminal vaccine of the JEV-prME protein with the HA titer is consistent with PPV HI generated by single vaccine of PPV-VP2 with the same antigen content to be 1:4096; the JEV HI antibody produced was also associated with the antibody containing 2 10 The antibody generated by immunization of JEV-prME single vaccine with HA titer is consistent, which is 1:2580; the method shows that the immune efficacy generated by the preparation of the combined vaccine after mixing the two antigens is not lower than that of the single vaccine, and the compatibility of the two antigens is good. The PPV antibody and the JEV antibody which are produced after the PPV-VP2 protein and the JEV-prME protein are added to prepare the vaccine for immunization are not obviously improved. The results of the immunization of each vaccine to produce antibodies are shown in Table 4.
Table 3 vaccine preparation and immunization
TABLE 4 different antigen content and Single shoot immune antibody detection results
Preparation of 15A, ISA and Gel02 adjuvants for 5 safety inspection containing 2 10 HA-titer PPV-VP2 protein and 2-containing 10 The bivalent subunit vaccine of the JEV-prME protein with the HA titer is 10 in each case by intraperitoneal injection of Balb/C mice, each of which is 0.5ml, 10 in each case as a non-immune control group, and the continuous observation is carried out for 10 days. The results show that: the three adjuvants are used for preparing vaccine to immunize the injection site of the mice, so that the vaccine is well absorbed, the spirit and ingestion of an immune group are not abnormal, and the weight gain is basically consistent with that of a control group. The security inspection results are shown in Table 5.
TABLE 5 safety inspection
6 efficacy assay preparation of 2-containing using ISA201 adjuvant 10 HA-titer PPV-VP2 protein and 2-containing 10 The bivalent subunit vaccine of JEV-prME protein with HA titer is injected into the neck muscle to immunize 5 heads of healthy piglets with PPV and JEV HI antibodies of 3-4 weeks old less than 1:8, and meanwhile, 3 heads of a non-immunized control group and 2.0 ml/head are established. PPV and JEV HI antibody titers were collected 14, 21 and 28 days post immunization of piglets. The results show that: after immunization of the vaccine, the piglets have normal mental appetite, the vaccine injection part has good absorption and no stress. The stepwise rise of PPV and JEV antibodies was significantly higher than in the control group at 14, 21 and 28 days post vaccine immunization, and the antibody detection results are shown in table 6.
TABLE 6 vaccine immunization against PPV and JEV HI antibody levels
7 quality study of similar products
Preparation of adjuvant containing 2 for intensive study of ISA201 10 HA-titer PPV-VP2 protein and 2-containing 10 The immune efficacy of the bigeminal subunit vaccine of the JEV-prME protein with the HA titer on animal pigs is screened, and 20 healthy piglets with 3-4 weeks of age PPV and JEV HI antibodies less than 1:8 are randomly divided into 4 groups, and 5 healthy piglets are selected from each group. The vaccine of each immune product of the first group is 2ml respectively, the vaccine of each immune porcine parvovirus commercialized inactivated vaccine of the second group, the vaccine of the third group is the vaccine of the immune porcine Japanese encephalitis commercialized live vaccine, all the vaccine is injected into the neck muscle, and the fourth group is a negative control group. Serum was collected 28 days after the immunization and isolated for PPV and JEV HI antibody titers. The results show that: the titer of PPV HI antibodies of the test vaccine is not lower than 1:128, the average value of PPV HI antibodies of an immune group of a commercial inactivated vaccine for porcine parvovirus disease is 1:84, and the immune efficacy is lower than that of the vaccine. The mean value of JEV HI antibodies in a commercial live vaccine immune group for Japanese encephalitis is 1:64, and the vaccine of the JEV HI antibody reaches 1:168. The results of the antibody detection are shown in Table 7.
TABLE 7 levels of PPV and JEV HI antibodies for each vaccine
In conclusion, the invention expresses subunit protein to prepare vaccine, solves the problem that porcine parvovirus is easy to cause environmental pollution, and cuts off the way of the porcine Japanese encephalitis attacking human health; the constructed PPV-VP2 virus-like particle HAs high titer, and the HA titer reaches 2 19 The method comprises the steps of carrying out a first treatment on the surface of the The constructed JEV-prME virus-like particle HAs high titer, and the HA titer reaches 2 18 . The vaccine has good immunogenicity, and can induce the susceptible animals to generate higher levels of PPV and JEV HI antibodies.

Claims (10)

1. An immunogenic composition comprising a hemagglutination assay having a cost effectiveness of 2 10～18 ：2 10～18 Is composed of porcine parvovirus VP2 protein and porcine Japanese encephalitis virus prME protein;
the porcine parvovirus VP2 protein is a protein coded by a nucleotide sequence shown in SEQ ID NO. 1;
the porcine Japanese encephalitis virus premE protein is a protein coded by a nucleotide sequence shown in SEQ ID NO. 2.
2. The immunogenic composition of claim 1, wherein: the hemagglutination efficiency of the porcine parvovirus VP2 protein and the porcine Japanese encephalitis virus prME protein is 2 10～14 ：2 10～14 Preferably 2 10 ：2 10 。
3. The immunogenic composition of claim 1, wherein: the amino acid sequence of the porcine parvovirus VP2 protein is shown as SEQ ID NO. 3; the amino acid sequence of the porcine Japanese encephalitis virus preME protein is shown as SEQ ID NO. 4.
4. A method of preparing an immunogenic composition according to any one of claims 1 to 3, characterized in that: it comprises the following steps:
(1) Respectively taking SEQ ID NO:1 and SEQ ID NO:2, connecting the gene segment of the nucleotide sequence shown in the formula 2 with an expression vector, introducing escherichia coli, extracting recombinant bacmid, and transfecting the recombinant bacmid into insect cells sf9 for culture to obtain recombinant baculovirus;
(2) Respectively taking the recombinant baculovirus obtained in the step 2), inoculating the recombinant baculovirus into insect cells Highfive, culturing, and collecting porcine parvovirus VP2 protein and porcine Japanese encephalitis virus prME protein;
(3) Mixing the porcine parvovirus VP2 protein obtained in the step 2) and the porcine Japanese encephalitis virus prME protein according to a proportion.
5. The method of manufacturing according to claim 4, wherein: the expression vector in the step (1) is a pFastBacDual plasmid; the escherichia coli is TOP10 competent cells and/or DH10Bac competent cells.
6. The method of manufacturing according to claim 4, wherein: culturing in the step (2) until the cell viability is lower than 10%.
7. A bivalent subunit vaccine, characterized in that: a vaccine comprising the immunogenic composition of claim 1 as an active ingredient, together with an immunoadjuvant.
8. The subunit vaccine of claim 6 wherein: the hemagglutination titer of the porcine parvovirus VP2 protein in the vaccine is 2 10～14 Preferably 2 10 The method comprises the steps of carrying out a first treatment on the surface of the The hemagglutination titer of the PRME protein of the porcine Japanese encephalitis virus is 2 10～14 Preferably 2 10 。
9. The subunit vaccine of claim 7 wherein: the mass ratio of the immunogen composition to the adjuvant is 1-9: 1, preferably, the mass ratio is 1:1.
10. the subunit vaccine of any one of claims 7-9, wherein: the adjuvant is 15A adjuvant, ISA201 adjuvant and/or Gel02 adjuvant, preferably ISA201 adjuvant.
CN202210896463.5A 2022-07-27 2022-07-27 Preparation method and application of porcine parvovirus and epidemic encephalitis B bigeminal subunit vaccine Pending CN117462665A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210896463.5A CN117462665A (en) 2022-07-27 2022-07-27 Preparation method and application of porcine parvovirus and epidemic encephalitis B bigeminal subunit vaccine

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210896463.5A CN117462665A (en) 2022-07-27 2022-07-27 Preparation method and application of porcine parvovirus and epidemic encephalitis B bigeminal subunit vaccine

Publications (1)

Publication Number Publication Date
CN117462665A true CN117462665A (en) 2024-01-30

Family

ID=89631729

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210896463.5A Pending CN117462665A (en) 2022-07-27 2022-07-27 Preparation method and application of porcine parvovirus and epidemic encephalitis B bigeminal subunit vaccine

Country Status (1)

Country Link
CN (1) CN117462665A (en)

Similar Documents

Publication Publication Date Title
JP6475741B2 (en) Herpesvirus subunit vaccine and its production method and application
CN104248762B (en) A kind of pig epidemic diarrhea vaccine combination and its preparation method and application
CN110869047A (en) Seneca virus a immunogenic compositions and methods thereof
JP2014516041A (en) Combination vaccine for the prevention of swine virus infection
US20190225654A1 (en) Methods and compositions for dengue virus vaccines
JP7303306B2 (en) Foot-and-mouth disease virus-like particle antigen, vaccine composition thereof, preparation method and application
CN111233984B (en) O-type foot-and-mouth disease virus-like particle antigen, vaccine composition thereof, preparation method and application
US3155589A (en) Parenteral extravascular injectable vaccines for simultaneous immunization of canidae against rabies, canine distemper, and infectious canine hepatitis
Pearson et al. Genetically engineered multi‐component virus‐like particles as veterinary vaccines
CN110559434B (en) Avian influenza virus-like particle vaccine, and preparation method and application thereof
CN113563432B (en) Foot-and-mouth disease virus-like particle antigen, vaccine composition, preparation method and application thereof
JP2021501757A (en) Rabies virus vaccine
CN110559433B (en) Avian influenza virus-like particle vaccine, and preparation method and application thereof
CN107338227B (en) Bovine parainfluenza virus PBIV3-B strain and application thereof
KR102365045B1 (en) Suspension Cell Culture Adapted Vaccine Strain Derived from Foot-and-mouth disease virus of O/ME-SA/Ind-2001 Lineage and Method of Preparing the Same
CN117462665A (en) Preparation method and application of porcine parvovirus and epidemic encephalitis B bigeminal subunit vaccine
CN111840533B (en) A-type foot-and-mouth disease virus-like particle antigen, vaccine composition thereof, preparation method and application
KR102365464B1 (en) Development of recombinant subunit Zika virus vaccine and preparing method thereof
KR101609946B1 (en) Foot and mouth disease vectored vaccine
CN110917343B (en) Newcastle disease and infectious bursal disease bigeminal subunit vaccine
CN109880839B (en) Preparation method of swine mycoplasma pneumonia and porcine circovirus bivalent genetic engineering vaccine
CN116390753A (en) Canine parvovirus
KR100622399B1 (en) Vaccine Comprising the Mixture of Inactivated TGEV and PEDV, and Preparation Method thereof
JP3043353B2 (en) vaccine
CN116102660B (en) Porcine parvovirus gene engineering epitope vaccine and preparation method thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination