CN110893234B - Triple subunit vaccine for canine distemper, canine parvovirus disease and rabies - Google Patents
Triple subunit vaccine for canine distemper, canine parvovirus disease and rabies Download PDFInfo
- Publication number
- CN110893234B CN110893234B CN201911242612.0A CN201911242612A CN110893234B CN 110893234 B CN110893234 B CN 110893234B CN 201911242612 A CN201911242612 A CN 201911242612A CN 110893234 B CN110893234 B CN 110893234B
- Authority
- CN
- China
- Prior art keywords
- protein
- recombinant
- canine
- virus
- rabies
- 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.)
- Active
Links
- 241000701931 Canine parvovirus Species 0.000 title claims abstract description 61
- 229940031626 subunit vaccine Drugs 0.000 title claims abstract description 34
- 201000010099 disease Diseases 0.000 title claims abstract description 29
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title claims abstract description 29
- 206010037742 Rabies Diseases 0.000 title claims abstract description 25
- 208000000655 Distemper Diseases 0.000 title claims abstract description 21
- 208000014058 canine distemper Diseases 0.000 title claims abstract description 21
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 52
- 101710081079 Minor spike protein H Proteins 0.000 claims abstract description 50
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 46
- 241000701447 unidentified baculovirus Species 0.000 claims abstract description 44
- 241000712083 Canine morbillivirus Species 0.000 claims abstract description 42
- 241000238631 Hexapoda Species 0.000 claims abstract description 32
- 241000711798 Rabies lyssavirus Species 0.000 claims abstract description 32
- 239000000427 antigen Substances 0.000 claims abstract description 27
- 102000036639 antigens Human genes 0.000 claims abstract description 27
- 108091007433 antigens Proteins 0.000 claims abstract description 27
- 230000014509 gene expression Effects 0.000 claims abstract description 26
- 101710169105 Minor spike protein Proteins 0.000 claims abstract description 25
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims abstract description 24
- 108091006027 G proteins Proteins 0.000 claims abstract description 22
- 102000030782 GTP binding Human genes 0.000 claims abstract description 22
- 108091000058 GTP-Binding Proteins 0.000 claims abstract description 22
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 8
- 108020004705 Codon Proteins 0.000 claims abstract description 7
- 238000005457 optimization Methods 0.000 claims abstract description 4
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims abstract description 3
- 229960005486 vaccine Drugs 0.000 claims description 31
- 239000002671 adjuvant Substances 0.000 claims description 6
- 229940024546 aluminum hydroxide gel Drugs 0.000 claims description 5
- SMYKVLBUSSNXMV-UHFFFAOYSA-K aluminum;trihydroxide;hydrate Chemical group O.[OH-].[OH-].[OH-].[Al+3] SMYKVLBUSSNXMV-UHFFFAOYSA-K 0.000 claims description 5
- 241000282472 Canis lupus familiaris Species 0.000 abstract description 30
- 208000015181 infectious disease Diseases 0.000 abstract description 22
- 230000003053 immunization Effects 0.000 abstract description 15
- 238000002649 immunization Methods 0.000 abstract description 15
- 241001465754 Metazoa Species 0.000 abstract description 8
- 239000002773 nucleotide Substances 0.000 abstract description 4
- 125000003729 nucleotide group Chemical group 0.000 abstract description 4
- 210000004027 cell Anatomy 0.000 description 37
- 241000700605 Viruses Species 0.000 description 23
- 239000000047 product Substances 0.000 description 19
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 17
- 229930004094 glycosylphosphatidylinositol Natural products 0.000 description 17
- 238000002360 preparation method Methods 0.000 description 13
- 238000001514 detection method Methods 0.000 description 12
- 238000005516 engineering process Methods 0.000 description 9
- 241000282465 Canis Species 0.000 description 8
- 238000010276 construction Methods 0.000 description 8
- 230000002708 enhancing effect Effects 0.000 description 7
- 230000003612 virological effect Effects 0.000 description 7
- 150000007523 nucleic acids Chemical class 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 5
- 108010076504 Protein Sorting Signals Proteins 0.000 description 5
- 101710172711 Structural protein Proteins 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 102000002702 GPI-Linked Proteins Human genes 0.000 description 4
- 108010043685 GPI-Linked Proteins Proteins 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 238000011031 large-scale manufacturing process Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 230000014616 translation Effects 0.000 description 4
- 208000035473 Communicable disease Diseases 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 238000001042 affinity chromatography Methods 0.000 description 3
- 125000003275 alpha amino acid group Chemical group 0.000 description 3
- 229940031567 attenuated vaccine Drugs 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 229940031551 inactivated vaccine Drugs 0.000 description 3
- 230000000415 inactivating effect Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000003472 neutralizing effect Effects 0.000 description 3
- 230000001681 protective effect Effects 0.000 description 3
- 230000009145 protein modification Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000004114 suspension culture Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- 108090000565 Capsid Proteins Proteins 0.000 description 2
- 102100023321 Ceruloplasmin Human genes 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 241000223259 Trichoderma Species 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 229960000074 biopharmaceutical Drugs 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 239000012539 chromatography resin Substances 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 235000005911 diet Nutrition 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000002550 fecal effect Effects 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 230000003340 mental effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 231100000915 pathological change Toxicity 0.000 description 2
- 230000036285 pathological change Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000013605 shuttle vector Substances 0.000 description 2
- 102000035160 transmembrane proteins Human genes 0.000 description 2
- 108091005703 transmembrane proteins Proteins 0.000 description 2
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 1
- NOWKCMXCCJGMRR-UHFFFAOYSA-N Aziridine Chemical compound C1CN1 NOWKCMXCCJGMRR-UHFFFAOYSA-N 0.000 description 1
- 206010005908 Body temperature conditions Diseases 0.000 description 1
- 241000701157 Canine mastadenovirus A Species 0.000 description 1
- 102100025680 Complement decay-accelerating factor Human genes 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 208000004232 Enteritis Diseases 0.000 description 1
- 101001065501 Escherichia phage MS2 Lysis protein Proteins 0.000 description 1
- 206010017472 Fumbling Diseases 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 101710154606 Hemagglutinin Proteins 0.000 description 1
- 101000856022 Homo sapiens Complement decay-accelerating factor Proteins 0.000 description 1
- 101001063392 Homo sapiens Lymphocyte function-associated antigen 3 Proteins 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 206010024769 Local reaction Diseases 0.000 description 1
- 102100030984 Lymphocyte function-associated antigen 3 Human genes 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 201000005505 Measles Diseases 0.000 description 1
- 108010036176 Melitten Proteins 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 102100035486 Nectin-4 Human genes 0.000 description 1
- 101710043865 Nectin-4 Proteins 0.000 description 1
- 208000005736 Nervous System Malformations Diseases 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 1
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 description 1
- 241000711504 Paramyxoviridae Species 0.000 description 1
- 208000002606 Paramyxoviridae Infections Diseases 0.000 description 1
- 241000701945 Parvoviridae Species 0.000 description 1
- 101710194807 Protective antigen Proteins 0.000 description 1
- 101710176177 Protein A56 Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000519995 Stachys sylvatica Species 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- 101150093578 VP2 gene Proteins 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 208000035472 Zoonoses Diseases 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- 238000004873 anchoring Methods 0.000 description 1
- 238000013320 baculovirus expression vector system Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000005757 colony formation Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 102000034356 gene-regulatory proteins Human genes 0.000 description 1
- 108091006104 gene-regulatory proteins Proteins 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 101150118163 h gene Proteins 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000000185 hemagglutinin Substances 0.000 description 1
- 230000002008 hemorrhagic effect Effects 0.000 description 1
- 230000004727 humoral immunity Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- VDXZNPDIRNWWCW-JFTDCZMZSA-N melittin Chemical group NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(N)=O)CC1=CNC2=CC=CC=C12 VDXZNPDIRNWWCW-JFTDCZMZSA-N 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000013190 sterility testing Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000000273 veterinary drug Substances 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
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
-
- 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
- A61P31/20—Antivirals for DNA 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
- A61K2039/70—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
- 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
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/18011—Paramyxoviridae
- C12N2760/18411—Morbillivirus, e.g. Measles virus, canine distemper
- C12N2760/18434—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
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/20011—Rhabdoviridae
- C12N2760/20111—Lyssavirus, e.g. rabies virus
- C12N2760/20134—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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Virology (AREA)
- Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- Organic Chemistry (AREA)
- Mycology (AREA)
- Immunology (AREA)
- Epidemiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Molecular Biology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Biotechnology (AREA)
- Engineering & Computer Science (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a triple subunit vaccine for canine distemper, canine parvovirus diseases and rabies, belonging to the field of biological products for animals. The subunit vaccine of the invention is prepared by taking recombinant canine distemper virus H protein, recombinant canine parvovirus VP2 protein and recombinant rabies virus G protein as antigens, respectively infecting insect cells by three recombinant baculoviruses, and purifying. The recombinant baculovirus carries elements for improving protein expression, and the nucleotide sequence of the recombinant baculovirus is SEQ ID NO:1, a step of; the recombinant canine distemper virus H protein, the recombinant canine parvovirus VP2 protein and the recombinant rabies virus G protein are all sequences after optimization of insect cell codons, and the nucleotide sequences are SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO:4. the subunit vaccine prepared by the invention can rapidly generate specific antibodies after immunization of dogs, and can be used for preventing infection of canine distemper virus, canine parvovirus and rabies virus of dogs.
Description
Technical Field
The invention discloses a triple subunit vaccine for canine distemper, canine parvovirus diseases and rabies, belonging to the field of biological products for animals.
Background
Canine distemper (canine distemper) is an acute, febrile and highly contagious infectious disease caused by canine distemper Virus (canine distemper Virus, CDV), often causes symptoms such as digestive tract, respiratory tract and nervous system abnormalities, and can cause immunosuppression of the body (MAGANGA G D, et al Molecular characterization ofcomplete genome ofa canine distemper Virus associated with fatal infection in dogs in Gabon, central Africa [ J ]. Virus Research,2018, 247:21-25.). The disease has strong infectivity, high morbidity and mortality rate, and is one of important infectious diseases which endanger the canine and economic animal raising industries. CDV, an important member of the measles genus of the paramyxoviridae family, is a single-stranded negative-strand, non-segmented RNA virus whose genome encodes 6 structural proteins (N, P, M, F, H, L in turn) and 2 non-structural proteins (C and V). Wherein the H gene has a full length of 1947nt and only contains one open reading frame, and the coded H protein is one of glycoprotein on the surface of CDV envelope and can interact with receptors on the surface of target cells (such as SLAM and nectin-4) to mediate virus invasion into a host; meanwhile, H protein has a plurality of neutralizing epitopes, and is a main antigen (HIRAMA K, et al, cytotoc T-lymphocyte activity specific for hemagglutinin (H) protein of canine distemper virus in dogs [ J ]. Journal ofVeterinary Medical Science,2003,65 (1): 109-112.) for inducing the organism to produce neutralizing antibodies.
Canine parvovirus disease is a virulent infectious disease caused by canine parvovirus (canine parvovirus, CPV) and characterized by severe vomiting, diarrhea, and stinking feces as the main characteristics, and is divided into hemorrhagic enteritis and non-suppurative (Zheng Yiting. Diagnosis and control of canine parvovirus [ J ]. Veterinary journal, 2017 (16): 171.). The disease is characterized by high incidence, strong infectivity, rapid disease course and high death rate of puppies, and is one of the most main infectious viral diseases of canine animals. CPV is a non-envelope single-strand negative-strand DNA virus of parvoviridae genus, the genome is 5.2kb in total length, and two non-structural proteins of NS1 and NS2 and three structural proteins of VP1, VP2 and VP3 are encoded. The viral epitopes are usually located on their capsid proteins, CPV viral capsids are composed of 60 structural proteins, including 53-54 VP2 proteins, accounting for 90% of the total capsid proteins, and play a decisive role in the antigenicity of CPV (Wei, et al, canine parvovirus VP2 gene comparison and typing studies [ J ]. Animal medicine progress, 2005,26 (5): 69-72.).
Rabies (Rabies) is an important zoonotic infectious disease caused by Rabies virus (Rabies), is popular worldwide, has a disease death rate of 100%, and is also an important public health problem in China. The RABV genome is about 12kb in length, and 5 genes encoding N, M, M2 and G, L proteins are sequentially arranged from the 3 'end to the 5' end, and non-coding interval sequences are further contained among the genes. Wherein, the G protein forms a virus spike on the envelope, is related to the pathogenicity of the virus, is a main antigen of RABV, stimulates the organism to generate humoral and cellular immunity, and is also a protective immunity antigen only inducing neutralizing antibodies (Xu Lan, etc. rabies glycoprotein research progress [ J ]. Chinese zoonotic school of medicine, 2006,22 (9): 876-879).
The existing vaccines for preventing the canine diseases are still traditional inactivated vaccines and attenuated vaccines, and have obvious difference between antigenicity and the existing epidemic strains; furthermore, the preparation process has the problems of biological safety hazard, insufficient antigen amount, high cost and the like. Therefore, it is urgent to develop a novel vaccine which is safe, efficient, matched with the antigenicity of epidemic strains and easy for mass production. The development of novel subunit vaccines based on recombinant protein genetic engineering techniques has become a hotspot and trend of current vaccinology. The subunit vaccine does not contain nucleic acid substances, does not have potential virus pathogenic genes, and has higher safety; the high-efficiency expression system (such as an insect baculovirus expression system) can ensure antigenicity of the recombinant protein and improve the yield of the recombinant protein, and avoid the problem of biological safety hazard in the traditional preparation process; in addition, the specific antibody can be rapidly generated after inoculation, and the generated immune response can be distinguished from wild virus infection, so that the control, purification and elimination of epidemic diseases are facilitated, and the method has important practical significance for guaranteeing the continuous healthy development of the canine districts.
Glycosyl Phosphatidylinositol (GPI) has been shown to be the only way to bind proteins to cell membranes, unlike the general lipid-modifying components, which are extremely complex in structure. GPI is a means by which proteins bind to Cell membranes, and engineered GPI-anchored proteins are a class of proteins that are anchored to the surface of eukaryotic Cell membranes via their carboxy-terminal GPI anchor structure (MEDOF M E, et al. Cell-surface engineering with GPI-anchored proteins [ J ]. Faseb Journal Official Publication ofthe Federation ofAmerican Societies for Experimental Biology,1996,10 (5): 574-86.). More than 100 GPI-anchored proteins have been discovered and it is increasingly recognized that GPI is a very popular form of surface protein anchoring in mammals, insects, protozoa, yeast. GPI-anchored proteins are a class of proteins that are anchored to the cell membrane surface by way of their carboxy-terminal (C-terminal) GPI structure but do not span their phospholipid bilayer structure, such as complement regulatory protein CD55, adhesion protein CD58, and can to some extent replace chimeric forms of transmembrane proteins, e.g., by concatenating the extracellular domain C-terminal of a transmembrane protein with the GPI-anchored N-terminal. There is no report of developing recombinant protein (subunit) vaccines using GPI strategy.
Disclosure of Invention
Technical problem
The invention aims at solving the problems that the existing vaccine for preventing the canine diseases is still a traditional inactivated vaccine and a traditional attenuated vaccine, and has obvious difference between antigenicity and the existing epidemic strain; in addition, the preparation process has the problems of biological safety hazard, insufficient antigen amount, high cost and the like, and develops a novel vaccine which is safe, efficient, matched with the antigenicity of epidemic strains, easy for large-scale production, and is used for preventing the infection of canine distemper virus, canine parvovirus and rabies virus of dogs.
Technical proposal
In order to achieve the above purpose, the following technical scheme is adopted:
a triple subunit vaccine for canine distemper, canine parvovirus disease and rabies, which is characterized in that: the vaccine comprises an antigen and an immune adjuvant, wherein the antigen is recombinant canine distemper virus H protein, recombinant canine parvovirus VP2 protein and recombinant rabies virus G protein, and the nucleotide sequences of the antigens after insect cell codon optimization are SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO:4, a step of; the immune adjuvant is aluminum hydroxide gel.
The triple subunit vaccine for canine distemper, canine parvovirus diseases and rabies is characterized in that: the protein mass ratio of the recombinant canine distemper virus H protein to the recombinant canine parvovirus VP2 protein to the recombinant rabies virus G protein is 1:1:1, the protein content of the recombinant canine distemper virus H protein in each vaccine is more than or equal to 10 mug, the protein content of the recombinant canine parvovirus VP2 protein is more than or equal to 10 mug, the protein content of the recombinant rabies virus G protein is more than or equal to 10 mug, and the volume ratio of the mixed antigen of the recombinant canine distemper virus H protein to the immune adjuvant is 3:2.
The triple subunit vaccine for canine distemper, canine parvovirus diseases and rabies is characterized in that: the recombinant canine distemper virus H protein, the recombinant canine parvovirus VP2 protein and the recombinant rabies virus G protein are prepared by respectively infecting insect cells with three recombinant baculoviruses and purifying, wherein the recombinant baculoviruses all carry elements for improving protein expression, and the nucleotide sequences of the recombinant baculoviruses are SEQ ID NO:1.
the triple subunit vaccine for canine distemper, canine parvovirus diseases and rabies can be applied to preventing canine distemper virus, canine parvovirus and rabies virus infection of dogs.
Advantageous effects
The invention aims at that the existing vaccine for preventing the canine diseases is still a traditional inactivated vaccine and a attenuated vaccine, and has obvious difference between antigenicity and the current epidemic strain; in addition, the preparation process has the problems of biological safety hazard, insufficient antigen amount, high cost and the like, and the developed novel vaccine which is safe, efficient, matched with the antigenicity of epidemic strains and easy for large-scale production of canine distemper, canine parvovirus diseases and rabies triple subunits is used for preventing the infection of canine distemper viruses, canine parvoviruses and rabies viruses. The subunit vaccine prepared by the invention can rapidly generate specific antibodies after immunization of dogs, and can be used for preventing infection of canine distemper virus, canine parvovirus and rabies virus of dogs.
The invention has the following characteristics and advantages:
1. the invention prepares recombinant canine distemper virus H protein, recombinant canine parvovirus VP2 protein and recombinant rabies virus G protein by using the protective antigen sequences of the current canine distemper virus, canine parvovirus and rabies virus epidemic strains through an insect-baculovirus expression system, adopts an affinity chromatography technology to purify the recombinant protein, saves the production cost, and is suitable for large-scale production. The subunit vaccine prepared by the invention does not contain nucleic acid substances, g (the existing commercial vaccine is provided with nucleic acid substances), has extremely high safety, can quickly generate specific antibodies by immunized animals, and can be used for preventing canine distemper virus, canine parvovirus and rabies virus infection of dogs.
2. The antigens involved in the canine distemper, canine parvovirus disease and rabies triple subunit vaccine are all based on the main protective immune antigens of the current epidemic strain. Wherein, the canine distemper virus antigen adopts an extracellular domain of Asia-1 type strain H protein, the canine parvovirus antigen adopts a CPV-2c type VP2 complete sequence, and the rabies virus antigen adopts a street virus G protein extracellular domain. However, the virulence of existing vaccines has been significantly antigenically different from currently prevalent strains. Thus, the antigens described in this patent are more advantageous in terms of antigen matching. Commercial vaccines are pure viruses-i.e., live viral antigens, but have been antigenically altered from the currently prevalent viruses.
3. The recombinant protein is prepared by purifying insect cells infected by recombinant baculovirus. All recombinant baculoviruses carry protein expression enhancing elements comprising Kozak sequences (enhancing translation efficiency of eukaryotic genes), melittin signal peptides (enhancing soluble expression of foreign proteins and protein modification), GPI sequences (enhancing recombinant protein expression). In addition, the nucleotide sequences for encoding the canine distemper virus H protein, the canine parvovirus VP2 protein and the rabies virus G protein are optimized by insect codons. Therefore, the recombinant protein disclosed in the patent has originality in optimizing the expression strategy. Commercial vaccines are produced by cell culture of virus (culture-virus-purification-emulsification-vaccine) in a manner quite different from the strategy of the present invention (different cell culture systems).
4. The insect cells are suspended High-Five insect cell lines with High-efficiency expression of exogenous proteins, the recombinant baculovirus is used for respectively infecting the corresponding cell lines, the infection period is 48-72 hours, the yield is 110-160mg/L, and the infection period of the conventional insect Sf9 cells is 72-96 hours, and the yield is 60-90mg/L, so that the insect cells can obviously shorten the infection period, improve the yield, reduce the production cost, and are suitable for large-scale production. The preparation mode of the commercial vaccine is completely different from the strategy of the invention, the commercial vaccine produces viruses, the invention produces proteins, and the produced proteins are high in quantity and safe.
5. The immunoprotection results of canine distemper virus, canine parvovirus and rabies virus showed that 5 dogs were challenged each 14 days after the last immunization, and the oral route was infected with 0.1mL (containing 1×10) 7 EID 50 Virus). The dogs survived within 15 days after challenge. The results show that dogs all die after challenge with the blank control group, and the subunit vaccine group and the commercial vaccine group of the present invention all survive.
Detailed Description
The patent project group discovers that the soluble expression of the exogenous protein can be promoted by carrying GPI motif through early research. However, the GPI strategy is adopted to express the foreign protein, but only one sequence is technically added, so that the GPI strategy is really adopted, and the foreign protein sequence needs to be analyzed (signal peptide, transmembrane domain and the like), such as the operation of removing the signal peptide, retaining the extracellular domain and the like, is required to be performed on the analyzed protein sequence, so that the normal function of the GPI motif can be ensured; if the whole protein sequence is directly used without analysis, there is a possibility that the protein is not expressed or the expression amount is not increased, and the soluble expression of the protein is reduced. Therefore, on one hand, the GPI strategy is adopted, and on the other hand, a specific sequence is required to be added, and more importantly, the protein sequence is subjected to professional predictive analysis and ingenious interception, so that the purpose of utilization can be achieved. Thus, by virtue of the novel function of the GPI motif, the recombinant protein is expressed and prepared into subunit vaccine, and a series of fumbling processes are required in order to be suitable for industrialization.
Preparation of recombinant proteins
1. Construction of shuttle vector pFastBac-GPI carrying high efficiency expression element
According to the requirements of gene expression regulation (aspects of gene transcription, translation, protein modification and the like), a high-efficiency expression element comprising a Kozak sequence (enhancing the translation efficiency of eukaryotic genes), a bee-signal peptide (enhancing the soluble expression and protein modification of exogenous proteins) and a GPI sequence (enhancing the expression quantity of recombinant proteins) is subjected to insect cell codon optimization, and the nucleotide sequence (300 nucleotides) is synthesized into SEQ ID NO:1 (synthesized by general biosystems (Anhui) Inc.), and then inserted into shuttle plasmid pFastBac1 TM (product of Siemens technologies Co.) a shuttle vector pFastBac-GPI carrying high-efficiency expression elements was constructed.
2. Construction of recombinant baculoviruses rBV-GPI-CDV-H and rBV-CDV-H
The amino acid sequence (Protein ID: AHY 03300.1) of the currently popular Asia CDV virulent strain H Protein is selected from the extracellular domain by online software (TMHMM Server, v.2.0http:// www.cbs.dtu.dk/services/TMHMM /), and the nucleic acid sequence optimized by insect cell codons is artificially synthesized as SEQ ID NO:2 (synthesized by general biosystems (Anhui) Inc.). Sal I and Kpn I restriction sites are respectively introduced at two ends of the artificially synthesized sequence. Subsequently, the synthetic sequence was compared with shuttle plasmids pFastBac-GPI and pFastBac1, respectively TM Respectively performing enzyme digestion on Sal I and Kpn I (product of Bao Ri doctor technology (Beijing)) and performing agarose gel electrophoresis identification, respectively recovering fragments by using a gel recovery kit (product of Tian Gen Biochemical technology (Beijing)) and performing incubation at 4 ℃ for 16 hours under the connection action of T4 ligase (product of Bao Ri doctor technology (Beijing)) to obtain recombinant shuttle plasmids pFastBao-GPI-CDV-H and pFastBac-CDV-H, wherein the E.coli DH5 alpha competence (product of Bao Ri doctor technology (Beijing)) is obtained. The recombinant shuttle plasmid was transformed into E.coli DH10Bac competent cells (product of Semer Feishul technology Co.) and subjected to homologous recombination, and subjected to a recombinant vector containing a three-antibody (kanamycin final concentration 100. Mu.g/ml, gentamicin)And (3) culturing and screening the medium (IPTG final concentration 40mg/ml, X-Gal final concentration 40mg/ml, which can judge whether homologous recombination occurs or not through colony formation color, wherein white colonies represent successful recombination and blue colonies do not succeed) for 48 hours under the condition of IPTG final concentration 50 mug/ml and tetracycline final concentration 70 mug/ml, and picking white spots to obtain recombinant bacmid-GPI-CDV-H and rbmid-CDV-H. The recombinant bacmid is transfected into Sf9 insect cells (product of Simer Feishier technology company) by a liposome mediated transfection method (X-tremeGENE HP DNAtransfection Reagent, product of Roche company) for 72 hours, after cytopathy, cell supernatant is collected to obtain first generation recombinant baculovirus, then the first generation recombinant baculovirus is continuously inoculated into Sf9 insect cells, second generation recombinant baculovirus is collected under the same culture condition, and the like, fourth generation recombinant baculovirus is collected and named rBV-GPI-CDV-H and rBV-CDV-H, and the cells are stored at the temperature of minus 20 ℃ for standby.
3. Construction of recombinant baculoviruses rBV-GPI-CPV-VP2 and rBV-CPV-VP2
The amino acid sequence of VP2 Protein of the currently popular CPV-2c strain (Protein ID: AKP 54259.1) is removed from the signal peptide sequence by on-line software (SignalP-5.0 http:// www.cbs.dtu.dk/services/SignalP /), and the nucleic acid sequence optimized by insect cell codons is artificially synthesized as SEQ ID NO:4 (synthesized by general biosystems (Anhui) Inc.). Referring to the construction mode of the recombinant baculovirus (see the specific embodiment of the invention, (one) preparation of recombinant protein, 2. Construction of recombinant baculovirus rBV-GPI-CDV-H and rBV-CDV-H section content), recombinant baculovirus rBV-GPI-CPV-VP2 and rBV-CPV-VP2 are constructed and obtained, and are preserved at the temperature of-20 ℃ for standby.
4. Construction of recombinant baculoviruses rBV-GPI-RABV-G and rBV-RABV-G
The amino acid sequence of VP2 Protein of the currently popular RABV street strain (Protein ID: ACB 38373.1) was removed from its signal peptide sequence by on-line software (SignalP-5.0 http:// www.cbs.dtu.dk/services/SignalP/and TMHMM Server, v.2.0http:// www.cbs.dtu.dk/services/TMHMM /) and its ectodomain sequence was selected. Referring to the construction mode of the recombinant baculovirus (see the specific embodiment of the invention, (one) preparation of recombinant protein, 2. Construction of recombinant baculovirus rBV-GPI-CDV-H and rBV-CDV-H section content), recombinant baculovirus rBV-GPI-RABV-G and rBV-RABV-G are constructed and obtained, and the recombinant baculovirus rBV-GPI-RABV-G is stored at the temperature of minus 20 ℃ for standby.
5. Functional verification of high-efficiency expression element (GPI motif)
The recombinant baculoviruses rBV-GPI-CDV-H, rBV-GPI-CPV-VP2, rBV-GPI-RABV-G and control recombinant baculoviruses rBV-CDV-H, rBV-CPV-VP2 and rBV-RABV-G carrying the high-efficiency expression element are respectively infected with Sf9 insect cells (product of Simer Feishan technology Co.) to carry out virus titer determination (the detection kit is BacPAK) TM Baculovirus Rapid Titer Kit from Bao Ri doctor Material technology (Beijing) Co., ltd.) and protein yield determination. Wherein the protein yield is determined by purifying the protein by affinity chromatography resin material (purchased from GE company in the United states) according to the volume of the collected liquid (taking 500mL as an example), diluting and preserving the protein by PBS, and determining the concentration of the protein by BCA protein quantitative detection kit (product of Semer Feishul technology Co., ltd.) according to the calculation formula of yield= [ concentration (mg/mL). Times.resuspension volume (mL)]Culture volume (L).
The results (see Table 1) show that recombinant baculoviruses carrying high-efficiency expression elements (rBV-GPI-CDV-H, rBV-GPI-CPV-VP2, rBV-GPI-RABV-G) showed a significant increase in protein yield (32 mg/L for recombinant canine distemper virus H protein to 60mg/L, 55mg/L for recombinant canine parvovirus VP2 protein to 90mg/L, and 47mg/L for recombinant rabies virus G protein to 70 mg/L) compared to the control (without high-efficiency expression elements) recombinant baculoviruses (rBV-CDV-H, rBV-CPV-VP2, rBV-RABV-G) without significant (no significant change in viral titer and infection cycle). This indicates that GPI motif has the function of improving protein yield and can efficiently express recombinant protein related to the invention.
TABLE 1 determination of viral titre, period of infection and protein production of recombinant baculovirus-infected Sf9 insect cells
6. Screening of insect cell lines
The recombinant baculoviruses rBV-GPI-CDV-H, rBV-GPI-CPV-VP2 and rBV-GPI-RABV-G were obtained by using Sf9 insect cells and High-Five insect cells (High Five insect cells (BTI-TN-5B 1-4) obtained from the carrier Trichoderma, which were isolated from the parent Trichoderma, and were used for recombinant protein expression using baculovirus expression vector system, which were capable of serum-free suspension culture, significantly saving the time and cost associated with culture acclimation in serum-free and/or suspension culture) for virus titer determination (the detection kit was BacPAK) TM Baculovirus Rapid Titer Kit available from Bao Ri doctor Material technology (Beijing), wherein the serum-free medium is Express Five TM SFM (product of Siemens technologies).
The results (see Table 2) show that the virus titers of recombinant baculovirus rBV-GPI-CDV-H, rBV-GPI-CPV-VP2, rBV-GPI-RABV-G, infected with Sf9 insect cells, were 2X 10, respectively 6 IFU/mL、1×10 7 IFU/mL、5×10 6 IFU/mL. The protein yields were 60mg/L, 90mg/L and 70mg/L, respectively.
The use of High-Five insect cells enables the viral titre of recombinant baculovirus rBV-GPI-CDV-H to be varied from 2X 10 6 IFU/mL is promoted to 3X 10 7 IFU/mL, the infection period is shortened from 96 hours to 56 hours, and the protein yield is improved from 60mg/L to 110mg/L; the virus titer of the recombinant baculovirus rBV-GPI-CPV-VP2 is changed from 1X 10 7 IFU/mL is promoted to 5X 10 8 IFU/mL, the infection period is shortened from 72 hours to 48 hours, and the protein yield is improved from 90mg/L to 160mg/L; the virus titer of the recombinant baculovirus rBV-GPI-RABV-G is changed from 5X 10 6 IFU/mL is promoted to 2X 10 8 IFU/mL, the infection period is shortened from 84 hours to 72 hours, and the protein yield is improved from 70mg/L to 120mg/L.
In conclusion, the adoption of the High-Five insect cells can obviously improve the virus titer and the protein yield of three recombinant baculoviruses and shorten the infection period.
TABLE 2 statistics of the difference between Sf9 and High-Five insect cells infected with recombinant baculovirus
7. Preparation of recombinant proteins
Expanding High-Five insect cells into 500mL suspension culture flask (product of Siemens technology Co.) respectively, shake culturing at 28deg.C at 120r/min, and culturing until cell density reaches 5×10 6 At each/mL, recombinant baculoviruses rBV-GPI-CDV-H, rBV-GPI-CPV-VP2 and rBV-GPI-RABV-G were used to infect cells at MOI=0.5 (multiplicity of infection), and the cells were further cultured for 48-72 hours, and the cell culture supernatant was collected and centrifuged at 8000r/min for 30 minutes to remove large cell debris. The inactivating agent BEI (BEI is named as a whole of two ethyleneimine, the principle is that the virus nucleic acid is destroyed without destroying protein, and the antigenicity of the virus is maintained, and the method is more advantageous than the traditional formaldehyde inactivating mode, and the method is purchased from Sigma company) and has the final concentration of 0.5 percent, and after 24 hours of action, the liquid is taken for inactivating test (whether the cells are diseased after 96 hours of cell inoculation are observed, the process is repeated three times, and the success of the inactivation is determined after no cytopathy appears). According to the volume of the collected liquid (taking 500mL as an example), the collected liquid is purified by an affinity chromatography resin material (purchased from United states GE company), and the obtained recombinant proteins are respectively named as recombinant canine distemper virus H protein, recombinant canine parvovirus VP2 protein and recombinant rabies virus G protein, and the protein concentration is measured by a BCA protein quantitative detection kit (product of Semer Feishan technology Co.) and stored at-70 ℃ for standby.
Preparation of (II) Canine distemper, canine parvovirus disease and rabies triple subunit vaccine
1. Subunit vaccine preparation
1.1 antigen preparation
And properly diluting the prepared recombinant canine distemper virus H protein, recombinant canine parvovirus VP2 protein and recombinant rabies virus G protein, setting the protein mass ratio of the recombinant canine distemper virus H protein to the recombinant canine parvovirus VP2 protein to the recombinant rabies virus G protein to be 1:1:1, and emulsifying the recombinant canine distemper virus H protein, the recombinant canine parvovirus VP2 protein and the recombinant rabies virus G protein with aluminum hydroxide gel.
1.2 vaccine formulation
Referring to the preparation steps of related vaccine in quality standard of veterinary biological products of the people's republic of China, mixed antigen and aluminum hydroxide gel (purchased from Nanjing Tianbang biotechnology Co., ltd.) are prepared according to a volume ratio of 3:2, wherein the aluminum hydroxide gel content in the vaccine is not more than 3.9mg/ml expressed by alumina, and then 1% of merthiolate solution is added to make the final concentration in the vaccine be 0.01%, and split charging is carried out after full stirring. The protein content of the recombinant canine distemper virus H protein in each vaccine is more than or equal to 10 mug, the protein content of the recombinant canine parvovirus VP2 protein is more than or equal to 10 mug, the protein content of the recombinant rabies virus G protein is more than or equal to 10 mug, and finally the obtained canine distemper, canine parvovirus disease and rabies triple subunit vaccine is subjected to the next-step finished product detection.
2. Subunit vaccine finished product detection
2.1 appearance of Properties
After standing, the upper layer is clear liquid, the lower layer is white sediment, and the mixture is uniformly mixed with shaking.
2.2 sterility testing
The sterility test is carried out according to the quality standard of biological products for animals in the people's republic of China, and the results are as follows: and (5) sterile growth.
2.3 Security detection
Weaning beagle dogs (purchased from Qingdao Bolong laboratory animals Co., ltd.) of 2 months of age were taken and randomly divided into 3 groups, 5 groups, the first group was not vaccinated (as a control), the second group was vaccinated with a single dose (4 mL/one), and the third group was vaccinated with a high dose (8 mL/one), and canine distemper, canine parvovirus disease, rabies triple subunit vaccines were vaccinated respectively. Each group was kept separate for 14 days and recorded for mental, dietary, fecal, body temperature conditions, and whether the inoculation sites were swollen, necrotic and systemic adverse effects. Cutting all dogs at 14 days for pathological detection, and recording the mental, body temperature, diet and fecal conditions of the immunized dogs at 14 days after immunization; whether the inoculation part has swelling, necrosis and other reactions; all dogs were dissected 14 days after immunization, observed and recorded for the presence of relevant pathological changes caused by canine distemper virus, canine parvovirus, rabies virus.
As a result, dogs survived entirely, and the injection site was examined by dissection without severe local reactions caused by vaccine injection, and without significant pathological changes in the individual tissue organs.
2.4 efficacy detection
10 weaned beagle dogs (purchased from Qingdaozu laboratory animal Co., ltd.) of 2 months old were taken, 1 part (4 mL/piece) of subunit vaccine was subcutaneously and intramuscularly injected, and serum was collected every 3 days for antibody titer determination (antibody detection kit was purchased from vincristocet biosciences Co.). The detection result of the antibody detection kit shows that the subunit vaccine group can be generated on the 3 rd day after immunization, and the immunization maintenance period is at least 4 months.
Application of (III) canine distemper, canine parvovirus disease and rabies triple subunit vaccine
1. Immunization protocol
45 weaned beagle dogs (purchased from Qingdao Bolong laboratory animals Co., ltd.) are used, 1 subunit vaccine (4 mL/piece) is injected subcutaneously and intramuscularly, and meanwhile, similar commercial vaccines (since commercial triple vaccine for canine distemper, canine parvovirus disease and rabies does not exist at present, the experiment adopts five-combined live vaccine for canine rabies, canine distemper, canine parainfluenza, canine adenovirus and canine parvovirus disease, purchased from five-star animal health care pharmaceutical factory in Jilin province) and 15 blanks are respectively established; the immunization was boosted once 14 days after immunization.
2. Toxicity attack protection test
2.1 immunoprotection against canine distemper virus
Each group of 5 dogs was challenged with Asia-1 strain (purchased from Walker (Jiangsu) biopharmaceutical Co., ltd.) of canine distemper virus 14 days after the final immunization, and 0.1mL (containing 1×10) of the oral route infection was obtained 7 EID 50 Virus). The dogs survived within 15 days after challenge. The results show that dogs all die after challenge with the blank control group, and the subunit vaccine group and the commercial vaccine group of the present invention all survive.
2.2 immunoprotection against canine parvovirus
14 days after the last immunization, 5 dogs were each individually treated with canine parvovirus strain CPV-2c (purchased from wawter(Jiangsu) biopharmaceutical Co., ltd.) for combating poison, oral route infection 0.1mL (containing 1X 10) 6 TCID 50 Virus). The dogs survived within 15 days after challenge. The results show that dogs all die after challenge with the blank control group, and the subunit vaccine group and the commercial vaccine group of the present invention all survive.
2.3 immunization protection against rabies virus for 14 days after the last immunization, 5 dogs were individually challenged with street strains of rabies virus (purchased from China veterinary drug administration), and oral route was infected with 0.1mL (containing 1×10) 6 TCID 50 Virus). The dogs survived within 15 days after challenge. The results show that dogs all die after challenge with the blank control group, and the subunit vaccine group and the commercial vaccine group of the present invention all survive.
The invention discloses a triple subunit vaccine for canine distemper, canine parvovirus diseases and rabies, belonging to the field of biological products for animals. The subunit vaccine is prepared by taking recombinant canine distemper virus H protein, recombinant canine parvovirus VP2 protein and recombinant rabies virus G protein as antigens based on main protective immune antigens of current epidemic strains, respectively infecting insect cells by three recombinant baculoviruses, and purifying. Wherein, all three recombinant baculoviruses carry elements for improving protein expression and nucleotide fragments for encoding canine distemper virus H protein, canine parvovirus VP2 protein and rabies virus G protein. In addition, the adoption of the High-Five insect cells can obviously improve the virus titer and the protein yield of three recombinant baculoviruses and shorten the infection period. The subunit vaccine prepared by the invention can rapidly generate specific antibodies after immunization of dogs, and can be used for preventing infection of canine distemper virus, canine parvovirus and rabies virus of dogs.
SEQ ID NO：1
SEQ ID NO：2
SEQ ID NO：3
SEQ ID NO：4
/>
Sequence listing
<110> academy of agricultural sciences in Jiangsu province
<120> a canine distemper, canine parvovirus disease, rabies triple subunit vaccine
<141> 2019-12-06
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 300
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
gccaccatga aattcttagt caacgttgcc cttgttttta tggtcgtata catttcttac 60
atctatgcgg atcgatgggg acatcaccat caccatcacg attacgatat cccaacgacc 120
gaaaacctgt attttcaggg caagggctct gtcgacgagt ctagagcctg cagtctcgag 180
ggtaccccaa ataaaggcag cggcaccacc agcggcacca cccgcctcct gagcggcatg 240
acctgcttca ccctgaccgg cctgctgggc accctggtga ccatgggcct gctgacctga 300
<210> 2
<211> 1647
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
cgatttcacc aagtatcaac tagcaatatg gaatttagca gattgctgaa agaggatatg 60
gagaaatcag aggccgtaca tcaccaagtc atagatgtct tgacaccgct cttcaaaatt 120
attggagatg agattggttt acggttgcca caaaaactaa acgagatcaa acaatttatc 180
ctccaaaaga caaacttctt caatccgaac agggagttcg acttccgcga tctccactgg 240
tgtattaacc cgcctagcaa gatcaaggtg aattttacta attactgtga tacagttggg 300
gtcaaaaaat ctattgcatc agcagcaaat cccatcattt tgtcagcact ctccggggcc 360
agaggtgaca tattcccgcc gtacagatgc agtggagcta ctacttcagt aggcagagta 420
ttccccctat ccgtatcatt atccatgtct ttgatatcaa gaacatcaga gataatcaat 480
atgctaaccg ctatctcaga cggagtgtat ggtaaaactt atttgctagt gcctgattat 540
attgaagggg agttcgactc gcaaaagatt cgagtctttg agatagggtt tatcaaacgg 600
tggctgaatg acatgccttt actccagaca accaactata tggtcctccc ggaaacttcc 660
aaagccaagg tatgtactat agcagtgggt gagctgacac tagcttcctt gtgtgtagat 720
gagagcactg tattgttata tcatgacagc aacggttcac aagatggtat cctagtagtg 780
acattgggaa tatttggggc aacacctatg gatcaagttg aagaggtgat acctatcgct 840
cacccatcag tggagagaat acatataaca aatcaccgtg ggttcataaa agactcaata 900
gtaacctgga tggtgcctgt attggtctct gagaaacaag aggagcaaaa aaactgtctg 960
gagtctgctt gtcacagaaa atcctaccct atgtgcaacc aaacgtcatg ggaacccttt 1020
ggaggagggc agttgccttc ttatgggcgg ttgacagtac ctctagatcc aagcattgac 1080
cttcaactta acatatcatt tacatatggt ccggttatac tgaacggaga cggtatggat 1140
tattatgaaa gcccactttt ggactccgga tggcttacca tacctcctaa gaacgggaca 1200
gtccttggat tgataaacaa agcaagtaga ggagaccagt tcactgtgac cccccatgtg 1260
ttgacatttg cgcccaggga atcaagtgga aattgttatt tacctattca aacatcccag 1320
attatggata aagatgtcct gactgagtcc aatttagtgg tgttacctac acagaatttt 1380
agatatgtca tagcaacata tgatatatcc cggggcgatc atgcaattgt ttattatgtt 1440
tatgacccaa tccggacgat ttcttataca tacccattta gactaactac caagggtaga 1500
cctgatttcc taaggattga atgttttgtg tgggatgacg atttgtggtg tcatcaattt 1560
taccgattcg aggctaacat cactaactct acaactagtg ttgagaattt agtacgtata 1620
agattctcat gtaaccgttc aaaacct 1647
<210> 3
<211> 1752
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
atgagtgatg gaggagttca accagacggt ggtcaacctg ctgtcagaaa tgaaagagct 60
acaggatctg ggaacgggtc tggaggcggg ggtggtggtg gttctggggg tgtggggatt 120
tctacgggta cttttaataa tcagacggaa ttcaaatttt tggaaaacgg atgggtggaa 180
atcacagcaa actcaagcag acttgtgcat ttaaatatgc cagaaagtga aaattataga 240
agagtggttg taaataattt ggataaaact gcagttaacg gaaacatggc tttagatgat 300
actcatgcac aaattgtaac accttggtca ttggttgatg caaatgcttg gggagtttgg 360
tttaatccag gagattggca actaattgtt aatactatga gtgagttgca tttagttagt 420
tttgaacaag aaatttttaa tgttgtttta aagactgttt cagaatctgc tactcagcca 480
ccaactaaag tttataataa tgatttaact gcatcattga tggttgcatt agatagtaat 540
aatactatgc catttactcc agcagctatg agatctgaga cattgggttt ttatccatgg 600
aaaccaacca taccaactcc atggagatat tattttcaat gggatagaac attaatacca 660
tctcatactg gaactagtgg cacaccaaca aatatatacc atggtacaga tccagatgat 720
gttcaatttt acactattga aaattctgtg ccagtacact tactaagaac aggtgatgaa 780
tttgctacag gaacatttta ttttgattgt aaaccatgca gactaacaca tacatggcaa 840
acaaatagag cattgggctt accaccattt ctaaattctt tgcctcaagc tgaaggaggt 900
actaactttg gttatatagg agttcaacaa gataaaagac gtggtgtaac tcaaatggga 960
aacacaaaca ttattactga agctactatt atgagaccag ctgaggttgg ttatagtgca 1020
ccatattatt cttttgaggc gtctacacaa gggccattta aaacacctat tgcagcagga 1080
cgggggggag cgcaaacaga tgaaaatcga gcagcagatg gtgatccaag atatgcattt 1140
ggtagacaac atggtcaaaa aactaccaca acaggagaaa cacctgagag atttacatat 1200
atagcacatc aagatacagg aagatatcca gaaggagatt ggattcaaaa tattaacttt 1260
aaccttcctg taacagaaga taatgtattg ctaccaacag atccaattgg aggtaaaaca 1320
ggaattaact atactaatat atttaatact tatggtcctt taactgcatt aaataatgta 1380
ccaccagttt atccaaatgg tcaaatttgg gataaagaat ttgatactga cttaaaacca 1440
agacttcatg taaatgcacc atttgtttgt caaaataatt gtcctggtca attatttgta 1500
aaagttgcac ctaatttaac aaatgaatat gatcctgatg catctgctaa tatgtcaaga 1560
attgtaactt actcagattt ttggtggaaa ggtaaattag tatttaaagc taaactaaga 1620
gcctctcata cttggaatcc aattcaacaa atgagtatca atgtagataa ccaatttaac 1680
tatgtaccaa gtaatattgg aggtatgaaa attgtatatg aaaaatctca actagcacct 1740
agaaaattat at 1752
<210> 4
<211> 1371
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
aaattcccca tttacacgat accagacaaa cttggtccat ggagtcccat cgatatacat 60
catctcagct gtccgaacaa tctggttgtg gaagatgaag gatgcaccaa tttgtctgga 120
ttttcctaca tggaactcaa agtgggatat agttcagcca taaaagtgaa cggattcacc 180
tgtacaggtg tggtgaccga agcagaaacc tacactaact ttgttggtta tgtcaccacc 240
acgtttaaga gaaagcactt tcgccctatg ccggatgcgt gcagagccgc atacaactgg 300
aagatggctg gtgaccccag gtatgaagag tctctgcaca atccgtaccc cgactaccac 360
tggctccgaa ctgtcaagac caccaaggag tcccttgtca tcatatctcc gagcgttaca 420
gatttggacc cgtacgataa atcccttcac tcgagagttt ttcctggcgg aaaatgctca 480
ggaataaccg tgtcctccac ttgctgctct actaaccacg actacaccat ctggatgcct 540
gaaaatccta gactgggaac ttcatgtgac attttcacca atagtagagg gaagagagca 600
tccaaagggg gcaagacttg tggatttgtg gatgaaagag gcttgtacaa gtctctaaag 660
ggagcatgca agctgaagct atgtggggtt ctaggactta gacttatgga cggaacatgg 720
gtcgcgattc agacatcaga tgagatcaaa tggtgctctc ctgatcagct agtaaatcta 780
cacgactttc actcggatga gattgaacat cttgttgtgg aggagttggt caaaaagagg 840
gaagagtgtc tggacgcact ggagactatt atgaccacaa agtccgtgag tttcagacgt 900
cttagtcatt tgaggaagct agtccctggg tttggaaagg cgtataccat attcaacaaa 960
accttgatgg aggctgatgc tcactacaaa tcaatccgga cttggaatga aatcatcccc 1020
tccaaagggt gtttaagagt tggggggaga tgtcatcctc atgtgaacgg ggtgttcttc 1080
aacggcatta tactgggtcc tgacggacat gttctaatcc cagagatgca gtcgtccctc 1140
ctccaccagc atatggaatt gttggagtcc tcagtcatcc ctttgatgca tcccttagca 1200
gacccgtcaa cagtctttaa agacggcgac gaggcagagg actttgttga ggttcacctt 1260
ccggatgtgc acaagcagat ctcgggggtt gatcttggtc tcccaaactg ggggaagtat 1320
gtgctgataa gtgcaggtgc tttgactgca ttaatgttga cgattttctt a 1371
Claims (1)
1. A triple subunit vaccine for canine distemper, canine parvovirus disease and rabies, which is characterized in that:
the vaccine comprises an antigen and an immune adjuvant, wherein the antigen is recombinant canine distemper virus H protein, recombinant canine parvovirus VP2 protein and recombinant rabies virus G protein, and the nucleotide sequences of the antigens after insect cell codon optimization are SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO:4, a step of; the immune adjuvant is aluminum hydroxide gel;
the protein mass ratio of the recombinant canine distemper virus H protein to the recombinant canine parvovirus VP2 protein to the recombinant rabies virus G protein is 1:1:1, the protein content of the recombinant canine distemper virus H protein in each vaccine is more than or equal to 10 mug, the protein content of the recombinant canine parvovirus VP2 protein is more than or equal to 10 mug, the protein content of the recombinant rabies virus G protein is more than or equal to 10 mug, and the volume ratio of the mixed antigen of the three to the immune adjuvant is 3:2;
the recombinant canine distemper virus H protein, the recombinant canine parvovirus VP2 protein and the recombinant rabies virus G protein are prepared by respectively infecting insect cells with three recombinant baculoviruses and purifying, wherein the recombinant baculoviruses all carry elements for improving protein expression, and the nucleotide sequences of the recombinant baculoviruses are SEQ ID NO:1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911242612.0A CN110893234B (en) | 2019-12-06 | 2019-12-06 | Triple subunit vaccine for canine distemper, canine parvovirus disease and rabies |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911242612.0A CN110893234B (en) | 2019-12-06 | 2019-12-06 | Triple subunit vaccine for canine distemper, canine parvovirus disease and rabies |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110893234A CN110893234A (en) | 2020-03-20 |
| CN110893234B true CN110893234B (en) | 2023-08-15 |
Family
ID=69787112
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911242612.0A Active CN110893234B (en) | 2019-12-06 | 2019-12-06 | Triple subunit vaccine for canine distemper, canine parvovirus disease and rabies |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110893234B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112501186B (en) * | 2020-11-26 | 2023-04-07 | 浙江鼎持生物制品有限公司 | Porcine circovirus 2 d-type CAP protein and application thereof in preparation of subunit vaccine |
| CN114470184A (en) * | 2021-12-30 | 2022-05-13 | 孙嘉玮 | Combined vaccine for tetanus, canine distemper and rabies for livestock |
| CN114921481B (en) * | 2022-02-25 | 2024-01-30 | 上海赛伦生物技术股份有限公司 | Rabies virus modified mRNA vaccine and preparation method thereof |
| CN114790229B (en) * | 2022-04-12 | 2024-07-12 | 北京慧德易科技有限责任公司 | Recombinant protein and preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107988174A (en) * | 2017-12-13 | 2018-05-04 | 新疆农业大学 | A kind of rabies-canine distemper-the strain of canine parvovirus gene recombined virus, construction method and its application |
| CN108704128A (en) * | 2018-05-15 | 2018-10-26 | 青岛农业大学 | A kind of canine distemper parvovirus bigeminy subunit vaccine |
-
2019
- 2019-12-06 CN CN201911242612.0A patent/CN110893234B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107988174A (en) * | 2017-12-13 | 2018-05-04 | 新疆农业大学 | A kind of rabies-canine distemper-the strain of canine parvovirus gene recombined virus, construction method and its application |
| CN108704128A (en) * | 2018-05-15 | 2018-10-26 | 青岛农业大学 | A kind of canine distemper parvovirus bigeminy subunit vaccine |
Non-Patent Citations (1)
| Title |
|---|
| 犬瘟热病毒、犬细小病毒及狂犬病毒三联多表位重组蛋白的构建及其免疫效果研究;李全;《中国博士学位论文全文数据库农业科技辑》;20170815(第8期);摘要 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110893234A (en) | 2020-03-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110893234B (en) | Triple subunit vaccine for canine distemper, canine parvovirus disease and rabies | |
| CN112876570B (en) | African swine fever virus vaccine and preparation method thereof | |
| CN112076315A (en) | Nano antigen particle fused with new coronavirus S protein and ferritin subunit, new coronavirus vaccine, and preparation method and application thereof | |
| CN110078802B (en) | Cat parvovirus VP2 protein and prepared virus-like particle | |
| CN102971417B (en) | Canine Parvovirus-2 correlated virus is separated from racoon | |
| KR101919002B1 (en) | Soluble Multi-Epitope Antigen of Foot-and-Mouth Disease Virus and Uses Thereof | |
| Di Martino et al. | Assembly of feline calicivirus-like particle and its immunogenicity | |
| WO2023050484A1 (en) | Expression vector, recombinant adeno-associated virus, and use thereof in preparation of 2019 novel coronavirus vaccine | |
| Casal | Use of parvovirus‐like particles for vaccination and induction of multiple immune responses | |
| KR101986071B1 (en) | Influenza h5 vaccines | |
| ES2213743T3 (en) | CORONAVIRUS CANINO SUBUNITY VACCINE. | |
| CN113862284B (en) | Gene, virus-like particle, vaccine and preparation and application for encoding recombinant avian influenza virus HA protein | |
| CN106754765B (en) | Newcastle disease virus-like particle, preparation method and application thereof | |
| CN113817753B (en) | Expression of SARS-CoV-2 fiber protein or its variant S Δ21 Construction and use of pseudotyped VSV viruses | |
| CN113896773B (en) | Recombinant FCV antigen and feline calicivirus genetic engineering subunit vaccine | |
| AU2020103776A4 (en) | Koi herpesvirus (khv) orf-149-based carbon nanotube supported nucleic acid vaccine and application thereof | |
| CN104403006B (en) | Mink Parvovirus virus-like particle and preparation method and application | |
| US6241989B1 (en) | Recombinant multivalent viral vaccine | |
| CN112442130A (en) | Self-assembly ferritin-based nano antigen particle, rabies vaccine prepared from same and application of self-assembly ferritin-based nano antigen particle | |
| CN110917343B (en) | Newcastle disease and infectious bursal disease bigeminal subunit vaccine | |
| CN117897170A (en) | FMDV virus-like particles with stabilizing mutations | |
| CN112439057B (en) | Self-assembly ferritin nano-antigen particle, swine fever vaccine prepared from same and application of swine fever vaccine | |
| CN111349621B (en) | Recombinant baculovirus and application thereof in preparation of newcastle disease virus-like particles | |
| CN107325188A (en) | The construction method of the Chinese hamster ovary celI strain of pig blood Albumin Fusion pig circular ring virus Cap2 albumen and its application | |
| WO2007104979A1 (en) | Virus-like particles of rift valley fever virus |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |