CN109021115B - Porcine circovirus trivalent subunit vaccine - Google Patents
Porcine circovirus trivalent subunit vaccine Download PDFInfo
- Publication number
- CN109021115B CN109021115B CN201810905436.3A CN201810905436A CN109021115B CN 109021115 B CN109021115 B CN 109021115B CN 201810905436 A CN201810905436 A CN 201810905436A CN 109021115 B CN109021115 B CN 109021115B
- Authority
- CN
- China
- Prior art keywords
- porcine circovirus
- vaccine
- subunit vaccine
- trivalent subunit
- days
- 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
- 241000202347 Porcine circovirus Species 0.000 title claims abstract description 74
- 229940031626 subunit vaccine Drugs 0.000 title claims abstract description 42
- 229960005486 vaccine Drugs 0.000 claims abstract description 38
- 238000002360 preparation method Methods 0.000 claims abstract description 11
- 239000013598 vector Substances 0.000 claims abstract description 10
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 14
- 201000010099 disease Diseases 0.000 claims description 13
- 239000002773 nucleotide Substances 0.000 claims description 11
- 125000003729 nucleotide group Chemical group 0.000 claims description 11
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 10
- 108020004707 nucleic acids Proteins 0.000 claims description 4
- 102000039446 nucleic acids Human genes 0.000 claims description 4
- 150000007523 nucleic acids Chemical class 0.000 claims description 4
- 239000003937 drug carrier Substances 0.000 claims description 3
- 108020001507 fusion proteins Proteins 0.000 claims 4
- 102000037865 fusion proteins Human genes 0.000 claims 4
- 241001673669 Porcine circovirus 2 Species 0.000 abstract description 28
- 238000000034 method Methods 0.000 abstract description 25
- 108090000623 proteins and genes Proteins 0.000 abstract description 25
- 241001465754 Metazoa Species 0.000 abstract description 19
- 102000004169 proteins and genes Human genes 0.000 abstract description 17
- 238000000855 fermentation Methods 0.000 abstract description 14
- 230000004151 fermentation Effects 0.000 abstract description 14
- 210000001744 T-lymphocyte Anatomy 0.000 abstract description 11
- 210000003719 b-lymphocyte Anatomy 0.000 abstract description 11
- 238000000746 purification Methods 0.000 abstract description 8
- 230000005847 immunogenicity Effects 0.000 abstract description 6
- 241000588724 Escherichia coli Species 0.000 abstract description 5
- 230000008569 process Effects 0.000 abstract description 5
- 230000024932 T cell mediated immunity Effects 0.000 abstract description 3
- 230000004727 humoral immunity Effects 0.000 abstract description 3
- 241001432873 Porcine circovirus 3 Species 0.000 abstract 1
- 238000002474 experimental method Methods 0.000 abstract 1
- 230000003053 immunization Effects 0.000 description 19
- 238000002649 immunization Methods 0.000 description 16
- 108090000765 processed proteins & peptides Proteins 0.000 description 14
- 238000012360 testing method Methods 0.000 description 13
- 229920001184 polypeptide Polymers 0.000 description 12
- 102000004196 processed proteins & peptides Human genes 0.000 description 12
- 238000001514 detection method Methods 0.000 description 11
- 241000699670 Mus sp. Species 0.000 description 10
- 239000000427 antigen Substances 0.000 description 10
- 239000013612 plasmid Substances 0.000 description 10
- 238000002965 ELISA Methods 0.000 description 9
- 241000928435 Porcine circovirus 1 Species 0.000 description 9
- 241000282887 Suidae Species 0.000 description 9
- 239000012634 fragment Substances 0.000 description 9
- 102000036639 antigens Human genes 0.000 description 8
- 108091007433 antigens Proteins 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 238000010171 animal model Methods 0.000 description 7
- 210000001165 lymph node Anatomy 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 6
- 241000700605 Viruses Species 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 230000034994 death Effects 0.000 description 6
- 231100000517 death Toxicity 0.000 description 6
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 210000003000 inclusion body Anatomy 0.000 description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 108090001074 Nucleocapsid Proteins Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000002159 abnormal effect Effects 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 4
- 230000036760 body temperature Effects 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 239000013604 expression vector Substances 0.000 description 4
- 238000010353 genetic engineering Methods 0.000 description 4
- 230000036449 good health Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 231100000915 pathological change Toxicity 0.000 description 4
- 230000036285 pathological change Effects 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 241001533384 Circovirus Species 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000001976 enzyme digestion Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 230000001575 pathological effect Effects 0.000 description 3
- 238000004153 renaturation Methods 0.000 description 3
- 230000001850 reproductive effect Effects 0.000 description 3
- 208000023504 respiratory system disease Diseases 0.000 description 3
- 238000011076 safety test Methods 0.000 description 3
- 238000009781 safety test method Methods 0.000 description 3
- 239000011265 semifinished product Substances 0.000 description 3
- 208000011580 syndromic disease Diseases 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 231100000041 toxicology testing Toxicity 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 2
- 201000004624 Dermatitis Diseases 0.000 description 2
- 241000588914 Enterobacter Species 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- 230000036528 appetite Effects 0.000 description 2
- 235000019789 appetite Nutrition 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000003862 health status Effects 0.000 description 2
- 239000000568 immunological adjuvant Substances 0.000 description 2
- 230000006698 induction 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
- 210000003205 muscle Anatomy 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 230000007943 positive regulation of appetite Effects 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 239000012474 protein marker Substances 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 101150044789 Cap gene Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 108020004638 Circular DNA Proteins 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 241000450599 DNA viruses Species 0.000 description 1
- 241001664991 Duck circovirus Species 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 241000672609 Escherichia coli BL21 Species 0.000 description 1
- 206010055690 Foetal death Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 206010023232 Joint swelling Diseases 0.000 description 1
- 206010024642 Listless Diseases 0.000 description 1
- 239000006142 Luria-Bertani Agar Substances 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 206010029164 Nephrotic syndrome Diseases 0.000 description 1
- 206010067482 No adverse event Diseases 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 241001135989 Porcine reproductive and respiratory syndrome virus Species 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 108091034057 RNA (poly(A)) Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 208000010399 Wasting Syndrome Diseases 0.000 description 1
- 241000607734 Yersinia <bacteria> Species 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 208000022531 anorexia Diseases 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 210000000436 anus Anatomy 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 238000011888 autopsy Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000009529 body temperature measurement Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 206010061428 decreased appetite Diseases 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000035622 drinking Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000004945 emulsification Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000028996 humoral immune response Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000000091 immunopotentiator Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 239000002085 irritant Substances 0.000 description 1
- 231100000021 irritant Toxicity 0.000 description 1
- 208000017169 kidney disease Diseases 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 208000017971 listlessness Diseases 0.000 description 1
- 230000001926 lymphatic effect Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000003340 mental effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000000926 neurological effect Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000008807 pathological lesion Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000002516 postimmunization Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 239000012521 purified sample Substances 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 235000015096 spirit Nutrition 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 230000002485 urinary effect Effects 0.000 description 1
- 230000005570 vertical transmission Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- 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
- A61K2039/70—Multivalent vaccine
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/10011—Circoviridae
- C12N2750/10022—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
- C12N2750/10011—Circoviridae
- C12N2750/10034—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)
- Virology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Mycology (AREA)
- Genetics & Genomics (AREA)
- Epidemiology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention relates to preparation and application of a porcine circovirus trivalent subunit vaccine. The porcine circovirus 3 type Cap protein, the screened B cells and T cell epitopes of the porcine circovirus 2 type and 1 type Cap proteins are used as a vaccine frame structure of the vaccine, are connected by a flexible linker, are cloned into a pRSETB vector and then are transformed into escherichia coli, and the porcine circovirus trivalent subunit vaccine with ideal immunogenicity is obtained through processes of fermentation, purification, preparation and the like. Animal experiments show that the porcine circovirus trivalent subunit vaccine not only has good safety, but also can stimulate effective humoral immunity and cellular immune response.
Description
Technical Field
The invention belongs to the field of biotechnology and genetic engineering, and mainly relates to preparation and application of a porcine circovirus trivalent subunit vaccine. Specifically, by utilizing a gene recombination technology, the porcine circovirus type 3 Cap protein, B cells and T cell epitopes of screened porcine circovirus type 2 and type 1 Cap proteins are connected in series and cloned into a vector, host bacteria are transformed, and a porcine circovirus trivalent subunit vaccine and application of the vaccine in preventing various diseases caused by the porcine circovirus are obtained through processes such as fermentation, purification, preparation and the like.
Background
Porcine Circovirus (PCV) is a single-stranded circular DNA virus with a genome length of about 1.7kb, one of the smallest animal DNA viruses. Two types of PCV have been identified, circovirus type 1 (PCV1) and circovirus type 2 (PCV 2). PCV1 was first identified in 1974 as a contaminant in PK cell cultures, which is only non-pathogenic to pigs. PCV2 was first reported in 1998 to cause Porcine circovirus-associated diseases (PCVAD) in pigs under clinical conditions, mainly causing piglet multisystemic wasting syndrome, pneumonia, Porcine dermatitis and nephropathy end-syndrome and reproductive disorders, mainly manifested as respiratory, urinary, intestinal, lymphatic, cardiovascular, neurological, reproductive and cutaneous dysfunction, which is one of the most devastating diseases affecting the live pig industry and causes significant economic losses worldwide.
In 2016, a new porcine circovirus, porcine circovirus type 3 (PCV3), was discovered. The first recorded event occurred in pigs of 2 to 3 weeks and 9 to 10 weeks in the united states with multisystemic inflammation and cardiac pathology of undetermined etiology, clinically manifested by infected piglet wasting, joint swelling, respiratory disease, sow reproductive failure, and the like. PCV3 was also detected in sows with Porcine Dermatitis and Nephrotic Syndrome (PDNS) and in mummy fetuses aborted from these sows. In addition, PCV3 was found in 45 of 48 archived PDNS tissue samples, and PCV3 was detected by real-time pcr (qpcr) in 34 of 271 samples collected from respiratory disease cases. In china PCV3 was detected in piglets with respiratory disease and fever, which were also negative for PCV2, porcine reproductive and respiratory syndrome virus and the yersinia virus. Furthermore, epidemiological studies in brazil, polish, germany, korea, thailand, uk, and other countries have also found porcine circovirus type 3. PCV3 was detected from different porcine tissues, and positive detection of PCV3 in dead fetus and semen samples indicated that PCV3 was at risk of vertical transmission.
Genomic sequence analysis found that the PCV3 genome comprises 2000 bases, has a similar genomic structure to PCV1 and PCV2, and mainly encodes two genes, namely Cap and Rep. The Cap gene encodes 214 amino acid residues, 19-20 amino acid residues are reduced compared with PCV2, and in addition, the similarity of PCV2 and duck circovirus is only 36-37%.
Summary of The Invention
The invention takes the common conserved Cap protein of different epidemic strains of the porcine circovirus type 3, the B cell epitope of the porcine circovirus type 2 and the T cell epitope of the porcine circovirus type 1 as vaccine structures, and after the vaccine structures are expressed in escherichia coli, the porcine circovirus trivalent subunit vaccine with ideal immunogenicity is obtained through processes of protein purification, preparation and the like. The vaccine can induce effective humoral immunity and cellular immune response after immunizing target animals.
One of the purposes of the invention is to provide a new porcine circovirus trivalent subunit vaccine polypeptide capable of stimulating humoral immunity and cellular immunity and a vaccine composition thereof, wherein the vaccine composition can be used for preventing related diseases caused by the porcine circovirus; the second purpose of the invention is to provide a construction and obtaining method of the porcine circovirus trivalent subunit vaccine; the third purpose of the invention is to provide a genetic engineering strain capable of expressing the porcine circovirus trivalent subunit vaccine; the fourth purpose of the invention is to provide a preparation method of the porcine circovirus trivalent subunit vaccine; the fifth purpose of the invention is to provide the application of the circovirus trivalent subunit vaccine in preventing porcine circovirus disease.
In a first aspect, the invention provides a recombinant porcine circovirus trivalent subunit vaccine polypeptide for prophylaxis and compositions thereof. The porcine circovirus type-3 virus epitope peptide contains common conserved Cap proteins of different epidemic strains of the porcine circovirus type-3 virus, B cell epitopes of the porcine circovirus type-2 virus and T cell epitopes of the porcine circovirus type-1 virus. B cell epitope, T cell epitope refers to a part of polypeptide in the main nucleocapsid protein with immunogenicity or equivalent thereof with basically the same immunogenicity function. The tandem connection can be carried out by a genetic engineering method or artificial synthesis, and the recombinant porcine circovirus trivalent subunit vaccine contains non-immune active substances besides the main nucleocapsid protein epitope. The non-immune active substance is a connecting part of each polypeptide, has no nucleocapsid protein antigen immunogenicity, also has no adjuvant activity, and mainly comprises a linker peptide, a chemical modification part, an N-terminal signal peptide, a C-terminal polyadenylic acid and the like. The pharmaceutically acceptable salt is non-toxic, irritant and allergic, and is suitable for human or animal tissues. Inactive substances and pharmaceutically acceptable salts are well known to those skilled in the art.
Preferably, the major nucleocapsid protein antigen and the epitope sequence thereof in the recombinant porcine circovirus trivalent subunit vaccine polypeptide of the first aspect of the invention are respectively selected from the group consisting of consensus conserved Cap proteins of different circulating strains of porcine circovirus type 3, B-cell epitopes of porcine circovirus type 2 and T-cell epitopes of porcine circovirus type 1.
Preferably, the amino acid sequence of the recombinant porcine circovirus trivalent subunit vaccine polypeptide of the first aspect of the invention is as follows:
(1) consensus conserved Cap protein of different circulating strains of porcine circovirus type 3(SEQ ID No. 4):
(2) the B cell epitope 1 amino acid sequence of porcine circovirus type 2 is Be1(SEQ ID No. 6): VDMMRFNINDFLPPGGGSNP, respectively;
(3) the amino acid sequence of the B cell epitope 2 of porcine circovirus type 2 is Be2(SEQ ID No. 8): VPFEYYRIRKVKVEFWPCSPITQGDRGV, respectively;
(4) the B cell epitope 3 amino acid sequence of porcine circovirus type 2 is Be3(SEQ ID No. 10):
(5) the B cell epitope 4 amino acid sequence of porcine circovirus type 2 is Be4(SEQ ID No. 12): TMYVQFREFNLKDPPLNP, respectively;
(6) the T cell epitope 1 amino acid sequence of porcine circovirus type 1 is Te1(SEQ ID No. 14): PYLAHPAFRNRYRWRRKTGIFNGSGEFVLTIK
(7) The T cell epitope 2 amino acid sequence of porcine circovirus type 1 is Te2(SEQ ID No. 16):
(8) the T cell epitope 3 amino acid sequence of porcine circovirus type 1 is Te3(SEQ ID No. 18):
(9) the T cell epitope 4 amino acid sequence of porcine circovirus type 1 is Te4(SEQ ID No. 20):
based on the vaccine elicited immune response mechanism and the biochemical characteristics of the vaccine, the better peptide fragment combination sequence is as follows: PCV3 Cap-PCV2 Be1-PCV2 Be2-PCV2 Be3-PCV2 Be4-PCV1 Te1-PCV1 Te2-PCV1 Te3-PCV1 Te 4.
Therefore, the amino acid sequence of the polypeptide of the recombinant porcine circovirus trivalent subunit vaccine is as follows:
in a second aspect, the present invention provides a nucleotide molecule encoding a recombinant porcine circovirus trivalent subunit vaccine polypeptide according to the first aspect of the invention. The nucleotide can be in an RNA form or a DNA form, a multi-antigen epitope tandem sequence is synthesized in an artificial synthesis mode, then the multi-antigen epitope tandem sequence is cloned into a vector after being connected through genetic engineering operation, and the vector is transformed into escherichia coli, and the recombinant porcine circovirus trivalent subunit vaccine polypeptide is obtained after screening, fermentation and purification. The nucleic acid may be subjected to conventional molecular biological procedures in the present invention, such as: PCR, restriction enzyme digestion, connection and the like, and enzyme digestion sites are added to the 5 'end and the 3' end of the nucleic acid design. Preferably, the nucleotide sequence of the present invention is as follows:
note: within the box are cleavage sites.
In a third aspect, the present invention provides a vector comprising, in addition to the nucleotide molecule encoding a recombinant porcine circovirus trivalent subunit vaccine according to the second aspect of the invention, expression control elements required for expression (transcription and translation) in prokaryotic cells operably linked to the nucleotide sequence. The most basic expression control elements include promoters, transcription terminators, enhancers, selectable markers, and the like, which are well known in the art. pRSETB is preferably used as an expression vector for a target gene in the present invention.
In a fourth aspect, the present invention provides a host cell comprising a vector according to the third aspect of the invention. The host cell is transformed or transfected with a gene sequence containing the coding protein, and then the gene sequence is detected to have good heredity and expression stability, so that the gene sequence can be used for producing the required recombinant porcine circovirus trivalent subunit vaccine antigen through fermentation expression. In the present invention, Escherichia coli BL21(DE3, Plys) is preferred as a host bacterium for expression of a target protein.
In a fifth aspect, the present invention provides a method for preparing a recombinant porcine circovirus trivalent subunit vaccine, comprising the steps of: the engineering bacteria are fermented to express the recombinant porcine circovirus trivalent subunit vaccine polypeptide, and the required recombinant porcine circovirus trivalent subunit vaccine is obtained through a crude purification and fine purification process and a subsequent vaccine preparation process. The methods involved therein include, but are not limited to, cell disruption, inclusion body washing, centrifugation, denaturation, affinity chromatography, hydrophobic chromatography, anion exchange chromatography, reverse phase chromatography, renaturation, emulsification and the like.
In a sixth aspect, the present invention provides a vaccine for preventing a trivalent subunit of a recombinant porcine circovirus, comprising a polypeptide according to the first aspect of the invention and a pharmaceutically acceptable carrier. The recombinant porcine circovirus trivalent subunit vaccine can prevent syndromes caused by porcine circovirus. The pharmaceutically acceptable carrier is an immunopotentiator or an immunologic adjuvant, and preferably the immunologic adjuvant is a water-in-oil-in-water adjuvant.
In a seventh aspect, the present invention provides the use of the recombinant trivalent subunit porcine circovirus vaccine of the sixth aspect. The vaccine can be administered to the animal intramuscularly, intradermally or subcutaneously in an effective amount, preferably intramuscularly, to produce a humoral and cellular immune response sufficiently effective (see examples five, six, seven, eight, nine, ten, eleven, twelve), stimulate the production of IgG, induce IFN γ production, provide antiviral activity, reduce pathological reactions, and protect the animal from attack by porcine circovirus. Furthermore, in an embodiment of the present invention, the recombinant porcine circovirus trivalent subunit vaccine of the present invention is shown to be safe by laboratory safety testing of the vaccine (see example four).
In addition, it is noted that other aspects of the invention having substantial characteristics will be apparent to those skilled in the art based on the disclosure in the context of the present application. Further, the present invention also uses publications, the entire contents of which are incorporated herein by reference.
Drawings
The following drawings are included to illustrate specific embodiments of the invention and are not intended to limit the scope of the invention as defined by the claims. FIG. 1 is a diagram of the construction of recombinant porcine circovirus trivalent subunit vaccine expression plasmid pRSETB-PCV (3/2/1); FIG. 2 is an enzyme-cleaved diagram of pRSETB-PCV (3/2/1) vector plasmid, wherein lane 1 is a DNA marker, the molecular weights thereof are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom, lane 2 is a blank control, lane 3 is a complete plasmid, and lane 4 is an enzyme-cleaved plasmid; FIG. 3 is a SDS-PAGE detection chart, wherein lane 1 is an un-induced control sample, lane 2 is a protein Marker, 175kDa, 80kDa, 58kDa, 46kDa, 30kDa, 25kDa, 17kDa and 7kDa are shown from top to bottom, lane 3 is an induced sample, and the arrow indicates the expressed target protein; FIG. 4 is a Western-blot detection chart, in which lane 1 is a pre-stained marker, and 99kDa, 70kDa, 43kDa, 31kDa, 24kDa and 15kDa are shown from top to bottom, lane 2 is a negative control, and lane 3 is a target protein. FIG. 5 is an SDS-PAGE pattern of fermentation samples: wherein, the lane 1 is a protein Marker, 175KDa, 80KDa, 58KDa, 46KDa, 30KDa, 25KDa, 17KDa and 7KDa are sequentially arranged from top to bottom, the lane 2 is an un-induced control, the lane 3 is a positive control, and the lane 4 is a fermentation induced sample; FIG. 6 is a Western-blot detection chart of the fermentation sample, wherein lane 1 is a pre-stained marker, and 99KDa, 70KDa, 43KDa, 31KDa, 24KDa and 15KDa are sequentially arranged from top to bottom, lane 2 is a negative control, lane 3 is a positive control lane, and lane 4 is a fermentation purified sample. FIG. 7 shows the result of detecting PCV1 IgG antibody by ELISA method; FIG. 8 shows the detection of vaccine PCV2 by ELISA method; FIG. 9 shows the result of detecting PCV3 IgG antibody by ELISA method; FIG. 10 results of IFN-gamma detection in serum of immunized animals.
Detailed Description
The specific test method descriptions set forth in the examples are illustrative only and are intended to be illustrative of the invention in detail, and are not intended to limit the scope of the invention.
Example a design concept of a recombinant porcine circovirus trivalent subunit vaccine protein
The invention analyzes the common conserved Cap protein of different epidemic strains of the current porcine circovirus type 3, the B cell epitope of the porcine circovirus type 2 and the T cell epitope of the porcine circovirus type 1. The designed epitopes are expressed in the enterobacter coli after being connected in series, and the recombinant porcine circovirus trivalent subunit vaccine with ideal immunogenicity is obtained through processes of fermentation, purification, preparation and the like. The vaccine prepared by the invention can effectively prevent the syndrome caused by the porcine circovirus. Comprehensively analyzing the genome sequences, antigen structures and epidemiological research progress of domestic porcine circovirus type 3, type 2 and type 1 epidemic strains, optimally designing a recombinant porcine circovirus trivalent subunit vaccine, finally determining one section of common conserved Cap protein, 4 sections of PCV 2B cell epitopes and 4 sections of PCV 1T cell antigen epitopes of different epidemic strains of the porcine circovirus type 3, and connecting all epitopes in series to form the framework structure of the vaccine. The overall structure of the vaccine is as follows: PCV3 Cap-PCV2 Be1-PCV2 Be2-PCV2 Be3-PCV2 Be4-PCV1 Te1-PCV1 Te2-PCV1 Te3-PCV1 Te 4.
EXAMPLE two construction of E.coli expression vectors and expression strains
1, synthesizing the designed polypeptide coding nucleotide by Shanghai handsome biotechnology company, designing restriction sites of BamH I (5 'end) and HindIII (3' end) at two ends of a nucleotide fragment respectively, cloning the synthesized fragment to a pMD18T vector respectively, and confirming that the inserted gene fragment is consistent with the designed sequence by sequence determination (see a sequence table). The recombinant plasmids were designated pMD18T-PCV (3/2/1), respectively. The plasmid is cut by corresponding restriction enzyme, the plasmid pRSETB of Invitrogen is selected as the escherichia coli expression vector, and the same restriction enzyme is used for cutting under the conditions that: mu.l of plasmid was added to 10. mu.l of the reaction system, and 5 units of restriction enzyme (New England Biolabs) were added thereto, followed by 1. mu.l of 10 Xbuffer, completion with deionized water, and digestion at 37 ℃ for 1.5 hours. After completion of the digestion, the reaction was stopped by adding 1. mu.l of 200mM EDTA. In 1% agarose gel electrophoresis, electrophoresis is carried out for 30 minutes. The 2.85kb pRSETB plasmid and the 1614bp PCV (3/2/1) fragment were excised under an ultraviolet lamp and gel recovered according to the instructions of the Qiagen gel recovery kit. According to the carrier: the polyepitope nucleotide fragment is mixed with an expression vector independently according to the proportion of the fragment 1: 2-3, a reaction system is 15 mu l, the polyepitope nucleotide fragment is connected by T4 DNA ligase, the polyepitope nucleotide fragment is connected overnight at 16 ℃, and the obtained recombinant plasmid is named as pRSETB-PCV (3/2/1) (shown in figure 1) and is transformed competent Enterobacter coli BL21(DE3) pLysS.
2, transformation: placing pRSETB-PCV (3/2/1) on ice to melt, adding 2 mu l of connecting reaction solution, mixing uniformly again, carrying out ice-water bath for 30 minutes at 42 ℃ for 30 seconds, then quickly placing the solution back into an ice bath for 1.5 minutes, adding 1mL of LB culture solution, carrying out static culture for 1 hour at 37 ℃, centrifuging at the low temperature of 4000g for 10 seconds, removing supernatant, and suspending the bacteria by using the culture medium of 200 mu lLB; and uniformly coating the bacterial liquid on an LB agar culture plate containing 100 mu g/mL ampicillin, and inversely placing the plate in a 37 ℃ incubator for culturing for 12-16 hours until the bacterial liquid is cloned.
3, identification: picking single clone on the plate to LB culture medium, shaking culturing for 12 hours at 37 ℃ and 200rpm, extracting plasmid, performing double enzyme digestion by using endonuclease BamH I and HindIII respectively, and cutting out clone of corresponding gene size fragment of porcine circovirus trivalent subunit vaccine, wherein 1614bp can be preliminarily determined as positive clone (shown in figure 2); the positive clones were subjected to DNA sequencing to further verify their correctness (see sequence listing).
4 inducing expression. The positive clone is cultured overnight, the positive clone is transferred according to the ratio of 1: 100 in the next morning, after 3 hours of culture, 0.1mM IPTG is added, and the culture is continued for 4 hours to prepare a sample; the expression of the target protein is detected by conventional SDS-PAGE, and a specific band is seen as a correct clone at 65KD (see figure 3); taking correct clone, amplifying and culturing, after SDS-PAGE confirms that the expression is correct, further confirming the expression accuracy by using conventional Western-blot (see figure 4); after the construction and identification procedures, the selected positive clone can be used as engineering bacteria to establish an original seed bank, and the strain is named as pRSETB-PCV (3/2/1)/BL21(DE3, Plys).
EXAMPLE three fermentation, purification and preparation of engineering bacteria
1 fermentation production strain pRSETB-PCV (3/2/1)/BL21(DE3, Plys) was inoculated into 2mL of LB liquid medium (containing 100. mu.g/mL of ampicillin), and cultured at 37 ℃ for 12 hours with shaking at 200 rpm. Inoculating into shake flask at ratio of 1: 100, shaking at 37 deg.C, culturing to OD600 ═ 3, and inoculating into fermenter at ratio of 10%. The fermentation medium is semisynthetic medium, and is prepared from distilled water. Correcting the dissolved oxygen and pH value electrodes, starting the tank body for stirring at the rotation speed of 300rpm, sterilizing the tank body on line, and calibrating the pH and the zero point of dissolved Oxygen (OD) when the temperature of the culture solution in the tank is reduced to 37.0 ℃. The fermentation temperature is 37.0 +/-0.1 ℃, the dissolved oxygen is controlled to be about 40%, the pH is controlled to be 7.0, 500mL of fed-batch materials are fed when the OD600 of the inoculated cultured thalli is 1.0-1.2, IPTG (the final concentration is 0.2mM) is added 1 hour after the fed-batch materials are fed for induction and expression, the fermentation is finished after 5 hours of continuous induction, and samples are taken for SDS-PAGE to identify the expression condition (see figure 5).
2 purification the collected cells were suspended in an inclusion-body wash solution I (1% Triton X-100, 20mM Tris-cl pH8.0), sonicated, and sonicated at 2000W for 1 hour. The inclusion bodies were collected by centrifugation at 12000rpm at 4 ℃ and suspended twice by ultrasonic washing with inclusion body wash solution II (1% DOC, 4M urea, 20mM Tris-cl pH8.0), and collected by centrifugation at a second low temperature. The inclusion body precipitate was mixed with 8M urea, 0.3% β -ME, 20mM Tris-cl (pH 8.00), stirred at room temperature for 4 hours, centrifuged at 8000rpm for 30min, and the precipitate was discarded. The denatured protein was diluted 1: 100, and the renaturation solution was renatured with Tris (pH8.0) buffer by adding 0.3M arginine at 4 ℃ under stirring for 24 hours. The renaturation solution is equilibrated on an affinity chromatography column by using 20mM phosphate buffer solution with the pH value of 8.0, 0.5M sodium chloride and 20mM imidazole, and is eluted by using 20mM phosphate buffer solution with the pH value of 8.0, 0.5M sodium chloride and 0.5M imidazole; thus obtaining the semi-finished product stock solution of the recombined porcine circovirus trivalent subunit vaccine, and taking the semi-finished product stock solution to perform western-blot identification (see figure 6).
3 preparation the purified semi-finished product was diluted to 300. mu.g/mL with sterile PBS, and sterilized for 15 minutes in 206 oil adjuvant at 121 ℃. The adjuvant and the vaccine are prepared according to the proportion of 1: 1, slowly stirred at the speed of 80-100 r/min, uniformly mixed, sterilized and subpackaged, and then the mixture can be used for immunization.
Example four recombinant porcine circovirus trivalent subunit vaccine safety test
1 Material
1.1 vaccine: the recombinant porcine circovirus trivalent subunit vaccine is provided by Qingdao Mingqin biological research and development center and has the batch numbers of 201701, 201702 and 201703.
1.2 test animals: 18-22g Balb/C mice were purchased from Beijing Huafukang. Healthy piglets of 20-30 days old (detecting PCV1, PCV2 and PCV3 antibody negatives by an ELISA method, and detecting PCV1, PCV2 and PCV3 antigen negatives by a PCR method) are provided by Guangdong Yongshun medicine.
2 method
2.1 safety of vaccines against white mice
18-22g Balb/C mice are immunized by each batch of vaccine with 5 mice, three batches of vaccine are used for immunizing 15 mice, and the immunization method comprises the following steps: each mouse was injected subcutaneously with 0.5ml, 2 blank controls were set, and the health status of the mice was observed for 10 consecutive days.
2.2 safety of vaccines against piglets
Healthy piglets of 20-30 days old are selected, the porcine circovirus trivalent subunit vaccine is recombined, 5 piglets in each batch are selected, three piglets in all are selected, and 15 piglets are immunized. The immunization method comprises the following steps: 2ml of injection is injected into the retroauricular muscle of each pig, 5 blank controls are set at the same time, and the clinical observation lasts for 14 days.
3 results
3.1 safety testing of vaccines against white mice
The results are shown in table 1, after immunization, all mice had no abnormal appetite, mental status and health status, consistent with the blank control group, no death occurred, and it was found that the recombinant porcine circovirus trivalent subunit vaccine was safe for white mice, as shown in table 1.
Table 1 safety test results of vaccines against mice
| Group of | Number of animals | Body temperature | Appetite stimulation | Spirit of the invention | Health condition | Number of deaths |
| 201701 | 5 are | Is normal | Is normal | Is normal | |
0 |
| 201702 | 5 are | Is normal | Is normal | Is normal | |
0 |
| 201703 | 5 are | Is normal | Is normal | Is normal | |
0 |
| Control | 2 are | Is normal | Is normal | Is normal | |
0 |
3.2 safety testing of vaccines on piglets
The results are shown in table 2, all immunized piglets are normal in body temperature, spirit and appetite within 14 days of the whole test observation period, no clinical abnormal phenomenon occurs, and 15 piglets are healthy and alive after the test is finished. The 5 piglets in the blank control group also have no adverse reaction. This indicates that the recombinant porcine circovirus trivalent subunit vaccine is safe for piglets.
TABLE 2 safety test results of vaccines on piglets
| Group of | Number of animals | Body temperature | Appetite stimulation | Spirit of the invention | Abnormal situation | Number of deaths |
| 201701 | 5 heads | Is normal | Is normal | Is normal | Is free of | 0 |
| 201702 | 5 heads | Is normal | Is normal | Is normal | Is free of | 0 |
| 201703 | 5 heads | Is normal | Is normal | Is normal | Is free of | 0 |
| Control | 5 heads | Is normal | Is normal | Is normal | Is free of | 0 |
Example five recombinant porcine circovirus trivalent subunit vaccine efficacy test animal grouping, immunization and evaluation method
1 test animal
Healthy piglets of 20-30 days old (antibody negative of PCV1, PCV2 and PCV3 detected by an ELISA method and antigen negative of PCV1, PCV2 and PCV3 detected by a PCR method) are subjected to environmental adaptation for one week before the test is started.
2 vaccine
The recombinant porcine circovirus trivalent subunit vaccine is provided by Qingdao Mingqin biological research and development center and has the batch numbers of 201701, 201702 and 201703.
3 test design and method
3.1, dividing the 20-30-day-old healthy piglets into 3 groups by using 45 heads (detecting the negative of PCV1, PCV2 and PCV3 antibodies by an ELISA method and detecting the negative of PCV1, PCV2 and PCV3 antigens by a PCR method), wherein the first group is an immune challenge group with 30 heads, each batch of vaccine is used for immunizing 10 pigs, the second group is a non-immune challenge group with 10 heads, and the third group is a blank control group (non-immune and non-challenge) with 5 heads. Blood is collected from the test animals for standby use 0 day, 7 days, 14 days, 21 days and 28 days after immunization. Randomly selecting 5 pigs for each immunization group 28 days after immunization, and inoculating 2ml of porcine circovirus type 2 GD-SG-09 strain (the virus content is 1.0 × 10) to each pig by nasal drip and intramuscular injection7.0TCID50Perml), inoculating porcine circovirus type 3 to the remaining 5 pigs in each batch, and injecting the porcine circovirus type 3 SD-HZ-17 strain (the virus content is 1.0 multiplied by 10) into each pig through nasal drip and muscle injection5.0TCID50In ml). After attacking, the piglets in the test are observed for ingestion, drinking, spirits, excrement and death, and the anal temperature is detected and recorded. And performing a caesarean examination on dead piglets. And (4) killing all piglets 28 days after the toxin is attacked, taking inguinal lymph nodes and mesenteric lymph nodes, and carrying out pathological section detection.
3.2 detecting the antibody titer and IFN gamma, collecting the serum of the immune group and the control group at 0 day, 7 days, 14 days, 21 days and 28 days after the immunity, and detecting the immune antibody IgG and the IFN gamma concentration by an ELISA method;
3.3 recording the anal temperature every day from two days before the anal temperature counteraction to 14 days after the anal temperature counteraction;
3.5 protective rate of counteracting toxic substances
3.5.1 clinical observation result of disease onset standard is abnormal and continuously exceeds three days, or the anus temperature is continuously exceeded 40 ℃ for three days, the disease is judged to be the onset of disease, death is judged to be the onset of disease, and obvious pathological changes can be seen through the autopsy and judged to be the onset of disease.
3.5.2 judging that the standard experimental pig challenge protection index is less than 80% and is unprotected, and the standard experimental pig challenge protection index is greater than or equal to 80% and is protected, and then calculating the experimental pig challenge protection rate according to the following formula: the protection rate of the experimental pig against the toxic materials is the number of the experimental pigs with the protection/the total number of the experimental pigs.
Example six serum evaluations: IgG antibody titer detection
Serum antibody IgG is detected by ELISA method at 0, 7, 14, 21 and 28 days after the vaccine of the experimental animal is immunized. The PCV1, PCV2 and PCV3 IgG change rules are shown in the figures 7, 8 and 9. Three batches of vaccines were free of antibody detection at 0 and 7 days post immunization. Low levels of antibody were detected 14 days after immunization. At 28 days after immunization, higher titers of PCV1, PCV2 and PCV3 antibody titers in peripheral blood can be detected, and compared with a non-immune non-attack control group, the differences are all significant (p < 0.05).
Example seven immune animal IFN gamma concentration detection
The serum of the experimental animals is collected by the conventional method at 0 day, 7 days, 14 days, 21 days and 28 days after immunization, and the IFN gamma concentration of the collected serum is detected according to the specification of the goat anti-pig IFN gamma ELISA detection kit. The result shows that after the vaccine is immunized, the IFN gamma concentration continuously rises along with the increase of the immunization time, and the concentration is higher 28 days after the immunization; the IFN γ concentration in the test pigs was consistently low in the control group that was not immune to non-challenge (see FIG. 10).
Example eight post-challenge clinical observations and anal temperature measurements
The anal temperature change was measured from two days before challenge to 14 days after challenge. The body temperature of all immune animals in the group of the porcine circovirus type 2 GD-SG-09 strain immunization challenge is not increased. The non-attack control group has 2 subjects with normal temperature continuously for three days, 4 test animals with temperature over 40 ℃ and 3 subjects with continuous three days over 40 ℃ in the non-attack control group.
All experimental animals of the vaccine immunization group do not have obvious clinical symptoms in the whole challenge period, and the non-immune challenge group gradually shows clinical apparent symptoms such as anorexia, listlessness, poor activity and the like from 3 days after challenge.
Example nine lymph node pathological section
The slicing results show that: no obvious lymph node pathological lesion is found in all immune animals of the porcine circovirus type 2 GD-SG-09 strain immune challenge group; in the non-immune control group, 4 experimental animals showed obvious pathological changes of lymph nodes. 1 immune animal of the porcine circovirus type 3 SD-HZ-17 immune challenge group has obvious pathological changes of lymph nodes; in the non-immune control group, 5 experimental animals showed obvious pathological changes of lymph nodes.
EXAMPLE twelve vaccine protection Rate determinations
The results of animal challenge tests are summarized in table 3, and no piglet death occurs in the whole experimental process. According to the disease incidence and judgment standards, the protection rates of the experimental animals of the vaccine immunization groups of the three batches are 5/5 for the porcine circovirus type 2 GD-SG-09 strain, the protection rates of the experimental animals for the porcine circovirus type 3 SD-HZ-17 strain are 5/5, 4/5 and 5/5 respectively, and the disease incidence of the two non-immune control groups is 4/5 and 5/5 respectively.
TABLE 3 test results and decisions on counteracting and attacking
Claims (6)
1. A porcine circovirus trivalent subunit vaccine fusion protein has an amino acid sequence of SEQ ID No. 2.
2. A nucleic acid molecule encoding the fusion protein of claim 1, wherein the specific nucleotide sequence is SEQ ID No. 1.
3. A vector comprising the nucleic acid molecule of claim 2.
4. A host cell comprising the vector of claim 3.
5. A vaccine for preventing porcine circovirus-associated disease comprising the fusion protein of claim 1 and a pharmaceutically acceptable carrier.
6. Use of the fusion protein of claim 1 in the preparation of a porcine circovirus trivalent subunit vaccine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810905436.3A CN109021115B (en) | 2018-08-06 | 2018-08-06 | Porcine circovirus trivalent subunit vaccine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810905436.3A CN109021115B (en) | 2018-08-06 | 2018-08-06 | Porcine circovirus trivalent subunit vaccine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109021115A CN109021115A (en) | 2018-12-18 |
| CN109021115B true CN109021115B (en) | 2021-05-04 |
Family
ID=64632619
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810905436.3A Active CN109021115B (en) | 2018-08-06 | 2018-08-06 | Porcine circovirus trivalent subunit vaccine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109021115B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114409741B (en) * | 2021-12-02 | 2023-04-21 | 吉林大学 | PCV2, PCV3 and PCV4 triple subunit vaccine, and preparation method and application thereof |
| CN117143888A (en) * | 2023-08-29 | 2023-12-01 | 上海杰威医药科技有限公司 | Porcine circovirus 2a, 2b and 2d trivalent virus-like particle vaccine and preparation method and application thereof |
| CN117843814A (en) * | 2024-01-11 | 2024-04-09 | 广东光峰生物技术有限公司 | Duck circovirus subunit vaccine, and preparation method and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101151329B1 (en) * | 2004-12-06 | 2012-07-19 | 주식회사 코미팜 | Recombinant protein, vaccine and prevention method for pmws using the same |
| JP2013188211A (en) * | 1997-12-11 | 2013-09-26 | Univ Of Saskatchewan | Postweaning multisystemic wasting syndrome virus from pig |
| CN106279431A (en) * | 2016-07-13 | 2017-01-04 | 青岛明勤生物科技有限公司 | A kind of pig circular ring virus subunit inactivated vaccine |
| CN108159409A (en) * | 2017-12-25 | 2018-06-15 | 南京大爻网络科技有限公司 | A kind of 3 type Cap protein vaccine of pig circular ring virus and its preparation method and application |
| CN108359677A (en) * | 2018-02-01 | 2018-08-03 | 福建农林大学 | The method for improving porcine circovirus 2 type and 3 type Cap protein expressing in series efficiency |
-
2018
- 2018-08-06 CN CN201810905436.3A patent/CN109021115B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013188211A (en) * | 1997-12-11 | 2013-09-26 | Univ Of Saskatchewan | Postweaning multisystemic wasting syndrome virus from pig |
| KR101151329B1 (en) * | 2004-12-06 | 2012-07-19 | 주식회사 코미팜 | Recombinant protein, vaccine and prevention method for pmws using the same |
| CN106279431A (en) * | 2016-07-13 | 2017-01-04 | 青岛明勤生物科技有限公司 | A kind of pig circular ring virus subunit inactivated vaccine |
| CN108159409A (en) * | 2017-12-25 | 2018-06-15 | 南京大爻网络科技有限公司 | A kind of 3 type Cap protein vaccine of pig circular ring virus and its preparation method and application |
| CN108359677A (en) * | 2018-02-01 | 2018-08-03 | 福建农林大学 | The method for improving porcine circovirus 2 type and 3 type Cap protein expressing in series efficiency |
Non-Patent Citations (2)
| Title |
|---|
| Protective efficacy of a virus-vectored multi-component vaccine against porcine reproductive and respiratory syndrome virus, porcine circovirus type 2 and swine influenza virus;Debin Tian等;《J Gen Virol》;20171231;第98卷(第12期);第3026-3036页 * |
| 猪圆环病毒2型Cap蛋白B细胞表位的预测及鉴定;宋瑞雪等;《中国优秀硕士学位论文全文数据库 农业科技辑》;20171115(第11期);D050-10 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109021115A (en) | 2018-12-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108586618B (en) | Preparation and application of porcine epidemic diarrhea subunit vaccine | |
| KR20190110605A (en) | Swine Coronavirus Vaccine | |
| CN109021115B (en) | Porcine circovirus trivalent subunit vaccine | |
| Pilehchian et al. | Fusion of Clostridium perfringens type D and B epsilon and beta toxin genes and it’s cloning in E. coli | |
| CN104628865B (en) | A kind of pseudo- mad dog epitope polypeptide recombinant vaccine | |
| CN115427071A (en) | Compositions comprising LTB and pathogenic antigens and uses thereof | |
| CN104628871B (en) | A kind of preparation for recombinating bursal disease protein engineering vaccine | |
| CN110452889B (en) | Construction method and primary application of recombinant bovine enterovirus expressing BVDV-E0 | |
| CN109053896B (en) | Porcine circovirus bivalent gene engineering vaccine | |
| CN112812193A (en) | Recombinant protein vaccine of norovirus GII.4 type and enterovirus 71 type | |
| CN110891597B (en) | Composition for preventing and treating poultry synovium infection | |
| CN113940993B (en) | Perch rhabdovirus G2-2M subunit vaccine and preparation method thereof | |
| CN114437237B (en) | Staphylococcus aureus TRAP targeting recombinant protein antigen and application thereof | |
| CN114058634B (en) | Chicken bursa synovialis mycoplasma gene engineering subunit vaccine | |
| CN111925449B (en) | Recombinant CHO cell strain expressing chicken VP2 and chicken GAL-1 fusion protein and construction method and application thereof | |
| CN112279925B (en) | Fusion protein, canine toxoplasma subunit vaccine and vaccine composition thereof | |
| CN110092840B (en) | Chicken infectious laryngotracheitis and egg drop syndrome bigeminal multi-epitope vaccine | |
| CN112159480B (en) | Chicken infectious bursal disease virus multi-antigen epitope protein and application thereof | |
| CN110066827B (en) | Recombinant baculovirus transfer vector containing porcine pseudorabies virus gB protein gene, recombinant baculovirus, preparation method and application | |
| CN114437236A (en) | Recombinant African swine fever virus multi-epitope fusion protein, preparation and application thereof | |
| CN113855795A (en) | Avian hepatitis E virus ORF2 subunit vaccine | |
| CN111560386A (en) | Soluble porcine circovirus type 2 Cap protein and application thereof | |
| CN107827986B (en) | Pig O/Mya98 and O/PanAsia type foot-and-mouth disease gene engineering inactivated vaccine | |
| CN113861277A (en) | Bovine rotavirus recombinant VP8 protein and application thereof | |
| CN114045298B (en) | Chicken bursa mycoplasma subunit vaccine and preparation method and application 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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240204 Address after: Room 303, Building 16, No.1 Jinhui Road, Lanwan Entrepreneurship Park, High tech Zone, Qingdao City, Shandong Province, 266111 Patentee after: Qingdao Mingqin Technology Co.,Ltd. Country or region after: China Address before: Room 302, East Unit, High Level Talent Entrepreneurship Center, No. 153 Zhuzhou Road, Laoshan District, Qingdao City, Shandong Province, 266061 Patentee before: QINGDAO MINGQIN BIOTECHNOLOGY CO.,LTD. Country or region before: China |
|
| TR01 | Transfer of patent right |