CN116731982B - 一种鸭瘟疱疹病毒基因缺失减毒疫苗株及其构建方法和应用 - Google Patents
一种鸭瘟疱疹病毒基因缺失减毒疫苗株及其构建方法和应用 Download PDFInfo
- Publication number
- CN116731982B CN116731982B CN202310990050.8A CN202310990050A CN116731982B CN 116731982 B CN116731982 B CN 116731982B CN 202310990050 A CN202310990050 A CN 202310990050A CN 116731982 B CN116731982 B CN 116731982B
- Authority
- CN
- China
- Prior art keywords
- gene
- strain
- delta
- vac
- duck plague
- 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
- 241000272525 Anas platyrhynchos Species 0.000 title claims abstract description 83
- 206010035148 Plague Diseases 0.000 title claims abstract description 63
- 241000607479 Yersinia pestis Species 0.000 title claims abstract description 63
- 241001529453 unidentified herpesvirus Species 0.000 title claims abstract description 58
- 229940031567 attenuated vaccine Drugs 0.000 title claims abstract description 39
- 238000012224 gene deletion Methods 0.000 title claims abstract description 27
- 238000010276 construction Methods 0.000 title claims abstract description 13
- 229960005486 vaccine Drugs 0.000 claims abstract description 32
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 28
- 230000001018 virulence Effects 0.000 claims abstract description 17
- 208000015181 infectious disease Diseases 0.000 claims abstract description 13
- 230000002458 infectious effect Effects 0.000 claims abstract description 11
- 241000700605 Viruses Species 0.000 claims description 65
- 210000004027 cell Anatomy 0.000 claims description 54
- 101150042941 UL4 gene Proteins 0.000 claims description 21
- 101150072564 gE gene Proteins 0.000 claims description 16
- 101150003725 TK gene Proteins 0.000 claims description 15
- 101150030521 gI gene Proteins 0.000 claims description 15
- 210000001161 mammalian embryo Anatomy 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 14
- 241000588724 Escherichia coli Species 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 238000011081 inoculation Methods 0.000 claims description 10
- 238000004113 cell culture Methods 0.000 claims description 9
- 101150064645 gJ gene Proteins 0.000 claims description 9
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 8
- 239000012634 fragment Substances 0.000 claims description 8
- 101150020597 gG gene Proteins 0.000 claims description 8
- 238000011534 incubation Methods 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- 239000013612 plasmid Substances 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 238000000605 extraction Methods 0.000 claims description 6
- 210000002950 fibroblast Anatomy 0.000 claims description 6
- 238000012216 screening Methods 0.000 claims description 6
- 239000006285 cell suspension Substances 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 5
- 238000000338 in vitro Methods 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 241001289349 Exocoetidae Species 0.000 claims description 3
- 229940124841 Herpesvirus vaccine Drugs 0.000 claims description 3
- 229960000723 ampicillin Drugs 0.000 claims description 3
- 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 claims description 3
- 238000001638 lipofection Methods 0.000 claims description 3
- 230000006798 recombination Effects 0.000 claims description 3
- 238000005215 recombination Methods 0.000 claims description 3
- 230000001502 supplementing effect Effects 0.000 claims description 3
- 238000003780 insertion Methods 0.000 claims description 2
- 230000037431 insertion Effects 0.000 claims description 2
- 241001465754 Metazoa Species 0.000 abstract description 8
- 238000004321 preservation Methods 0.000 abstract description 5
- 230000007918 pathogenicity Effects 0.000 abstract description 4
- 238000010353 genetic engineering Methods 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 18
- 239000000047 product Substances 0.000 description 12
- 230000002238 attenuated effect Effects 0.000 description 10
- 241000272517 Anseriformes Species 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 241001492342 Anatid alphaherpesvirus 1 Species 0.000 description 8
- 238000010998 test method Methods 0.000 description 8
- 210000004436 artificial bacterial chromosome Anatomy 0.000 description 6
- 238000012217 deletion Methods 0.000 description 6
- 230000037430 deletion Effects 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- AFCARXCZXQIEQB-UHFFFAOYSA-N N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CCNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 AFCARXCZXQIEQB-UHFFFAOYSA-N 0.000 description 5
- 235000013601 eggs Nutrition 0.000 description 5
- 230000003902 lesion Effects 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 241000287828 Gallus gallus Species 0.000 description 4
- 235000013330 chicken meat Nutrition 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 238000002649 immunization Methods 0.000 description 4
- 230000003053 immunization Effects 0.000 description 4
- 244000144977 poultry Species 0.000 description 4
- 235000013594 poultry meat Nutrition 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000003053 toxin Substances 0.000 description 4
- 231100000765 toxin Toxicity 0.000 description 4
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 101100298998 Caenorhabditis elegans pbs-3 gene Proteins 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- 108010000912 Egg Proteins Proteins 0.000 description 2
- 102000002322 Egg Proteins Human genes 0.000 description 2
- 108060002716 Exonuclease Proteins 0.000 description 2
- 241000700586 Herpesviridae Species 0.000 description 2
- 238000002944 PCR assay Methods 0.000 description 2
- 108010019160 Pancreatin Proteins 0.000 description 2
- 206010044565 Tremor Diseases 0.000 description 2
- 210000001643 allantois Anatomy 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 210000003837 chick embryo Anatomy 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 210000003278 egg shell Anatomy 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000009585 enzyme analysis Methods 0.000 description 2
- 102000013165 exonuclease Human genes 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000003147 molecular marker Substances 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 150000007523 nucleic acids Chemical group 0.000 description 2
- 229940055695 pancreatin Drugs 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 238000004080 punching Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000010008 shearing Methods 0.000 description 2
- 239000010902 straw Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 210000001835 viscera Anatomy 0.000 description 2
- NOIIUHRQUVNIDD-UHFFFAOYSA-N 3-[[oxo(pyridin-4-yl)methyl]hydrazo]-N-(phenylmethyl)propanamide Chemical compound C=1C=CC=CC=1CNC(=O)CCNNC(=O)C1=CC=NC=C1 NOIIUHRQUVNIDD-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241000345397 Columbid alphaherpesvirus 1 Species 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 102100023933 Deoxyuridine 5'-triphosphate nucleotidohydrolase, mitochondrial Human genes 0.000 description 1
- 241001598169 Equid alphaherpesvirus 3 Species 0.000 description 1
- 101100226347 Escherichia phage lambda exo gene Proteins 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- 101100427969 Gallid herpesvirus 2 (strain GA) US639 gene Proteins 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 241000906682 Hemsleya Species 0.000 description 1
- 241000175212 Herpesvirales Species 0.000 description 1
- 208000032420 Latent Infection Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 231100000645 Reed–Muench method Toxicity 0.000 description 1
- 101150023763 UL12 gene Proteins 0.000 description 1
- 101150044932 UL2 gene Proteins 0.000 description 1
- 101150044021 UL41 gene Proteins 0.000 description 1
- 101150053996 UL47 gene Proteins 0.000 description 1
- 101150023587 US10 gene Proteins 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 208000022531 anorexia Diseases 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000005875 antibody response Effects 0.000 description 1
- 230000001174 ascending effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 210000004252 chorionic villi Anatomy 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 108010011219 dUTP pyrophosphatase Proteins 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 206010061428 decreased appetite Diseases 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000003412 degenerative effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 208000037771 disease arising from reactivation of latent virus Diseases 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 230000003628 erosive effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000012268 genome sequencing Methods 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 210000005210 lymphoid organ Anatomy 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 210000004738 parenchymal cell Anatomy 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000003660 reticulum Anatomy 0.000 description 1
- 238000011076 safety test Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- -1 such as the gC Proteins 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Classifications
-
- 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
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- 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
- A61P31/22—Antivirals for DNA viruses for herpes viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
-
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5254—Virus avirulent or attenuated
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16311—Mardivirus, e.g. Gallid herpesvirus 2, Marek-like viruses, turkey HV
- C12N2710/16321—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16311—Mardivirus, e.g. Gallid herpesvirus 2, Marek-like viruses, turkey HV
- C12N2710/16322—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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16311—Mardivirus, e.g. Gallid herpesvirus 2, Marek-like viruses, turkey HV
- C12N2710/16334—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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16311—Mardivirus, e.g. Gallid herpesvirus 2, Marek-like viruses, turkey HV
- C12N2710/16351—Methods of production or purification of viral material
- C12N2710/16352—Methods of production or purification of viral material relating to complementin g cells and packaging systems for producing virus or viral particles
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16311—Mardivirus, e.g. Gallid herpesvirus 2, Marek-like viruses, turkey HV
- C12N2710/16361—Methods of inactivation or attenuation
- C12N2710/16362—Methods of inactivation or attenuation by genetic engineering
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
-
- 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)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Virology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Animal Behavior & Ethology (AREA)
- Plant Pathology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Immunology (AREA)
- Pharmacology & Pharmacy (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本发明公开了一种鸭瘟疱疹病毒基因缺失减毒疫苗株及其构建方法和应用,涉及基因工程技术领域,该疫苗株是以鸭瘟疱疹病毒VAC疫苗株的感染性克隆为骨架载体,然后将骨架载体中的毒力基因敲除,得到两种减毒疫苗株,均保藏于中国典型培养物保藏中心,保藏单位地址:中国.武汉.武汉大学,保藏时间为2023年07月05日,保藏编号为CCTCC NO:V202340和CCTCC NO:V202339。本发明的疫苗株免疫保护率达到100%,本发明解决了现有疫苗株对非靶动物具有致病性、无法有效预防鸭瘟疱疹病毒强毒株的攻击,以及保护效果不佳的问题。
Description
技术领域
本发明涉及基因工程技术领域,具体涉及一种鸭瘟疱疹病毒基因缺失减毒疫苗株及其构建方法和应用。
背景技术
鸭瘟是由鸭瘟疱疹病毒(Duck enteritis virus,DEV)引起的鸭、鹅和其他雁形目禽类的一种急性、热性、败血性传染病,是国际兽医局(OIE)及我国规定的家禽B/二类传染病之一,该病毒主要侵害血管、组织出血、消化道黏膜糜烂、淋巴器官出现病变及实质器官有退行性的病变,感染机体后可广泛分布于全身各组织器官,造成严重的机体损伤,同时具有潜伏感染的特性,使机体长期带毒或不断向外界排毒,导致疾病再次发生。20世纪50年代以来,由于养殖技术落后,饲养数量和饲养密度的不断增加,以及各地区间频繁的活禽调运,导致DEV疫病愈发严重,鸭瘟疱疹病毒在国内相继爆发,流行十分广泛,给水禽业带来巨大的防控挑战。DEV属于疱疹病毒科(Herpesvirales)、α疱疹病毒亚科(Alphaherpesvirinae)、马立克病毒属的成员,与其同属的成员还有马立克氏病毒(MDV)、鸽疱疹病毒(PHV1)以及马疱疹病毒3型(EHV-3)等,DEV基因组为线状双股DNA,长约158kb,由一个长独特区(UL)和一个短独特区(US)以及US两侧的末端倒转重复(TR)与内部重复(IR)组成,细胞浆和细胞核间隙中病毒粒子呈球形,在细胞浆内质网小管系统中成熟病毒粒子直径较大,同时带有囊膜。
基因缺失减毒疫苗株主要是通过分子生物学的手段,对DEV基因组中毒力基因的若干碱基进行敲除,以达到缺失该基因的目的。DEV具有多个毒力相关基因,分别是UL区的gC、UL2、UL12、UL41、UL47、TK基因和US区的gI、gE、US1、US10基因等,这些基因共同调控DEV的毒力,缺失其中一个或多个基因联合缺失均可导致DEV毒力的减弱。
近年来,通过建立鸭瘟病毒基因组文库获取大量目的基因核酸序列,进而拼接获得鸭瘟病毒强毒株CHv和弱毒株VAC的全基因组序列,通过克隆至细菌人工染色体获得鸭瘟病毒欧洲强毒株2085的全基因组序列。1965年,中国将鸭瘟自然病毒通过鸭胚传至9代后,在鸡胚绒毛尿膜上连续传至20代,成功培育一弱毒株,命名为C-KCE,此毒株制成鸭瘟鸡胚化疫苗,该毒株免疫鸭只后安全性高。目前,已有多株鸭瘟病毒完成了基因组测序,如强毒株CHv、鸡胚化弱毒疫苗株C-KCE及分离弱毒疫苗株VAC等。鸭瘟疱疹病毒减毒株作为基因工程载体具备相当的优势:(1)减毒株对非靶动物不具有致病性,安全性很好;(2)基因组容量大:在DEV长达160 Kb的基因组中,含有多个非必需基因(如gI、gE、US5、TK、UL2和dUTPase等),在这些区域内可先后或同时插入和表达多种外源基因,而不显著影响其繁殖及免疫原性;(3)生产原材料来源方便,生产成本低:DEV疫苗可以接种SPF鸭胚成纤维细胞及BHK-21等传代细胞生产,原材料充足,生产工艺成熟,且DEV病毒滴度很容易达到免疫要求,大大降低了生产成本;(4)宿主范围广,各种类型的水禽、家禽等均可感染DEV,可研制针对不同动物的活载体疫苗。然而,现有的疫苗株存在对非靶动物具有致病性、无法有效预防鸭瘟疱疹病毒强毒株的攻击和保护效果不佳等缺陷,因此,迫切需要一种新的鸭瘟疱疹病毒基因缺失减毒疫苗株。
发明内容
为了解决上述技术问题,本发明的目的是提供一种鸭瘟疱疹病毒基因缺失减毒疫苗株及其构建方法和应用,以解决现有疫苗株对非靶动物具有致病性、无法有效预防鸭瘟疱疹病毒强毒株的攻击以及保护效果不佳的问题。
本发明解决上述技术问题的技术方案如下:提供一种鸭瘟疱疹病毒基因缺失减毒疫苗株,该疫苗株是以鸭瘟疱疹病毒VAC疫苗株的感染性克隆为骨架载体,然后将骨架载体中的毒力基因敲除,形成鸭瘟疱疹病毒基因缺失减毒疫苗株。
在上述技术方案的基础上,本发明还可以做如下改进:
进一步,该疫苗株是以鸭瘟疱疹病毒VAC疫苗株基因组,通过核酸外切酶联合Red/ET同源重组,直接克隆到大肠杆菌中的细菌人工染色体中,然后将骨架载体中毒力基因敲除,得到鸭瘟疱疹病毒基因缺失减毒疫苗株。
进一步,毒力基因为gI基因、gE基因、TK基因、gG基因和gJ基因。
进一步,毒力基因为gI基因、gE基因、TK基因和UL4基因。
进一步,gI基因缺失1-689位碱基,gE基因缺失544-754位碱基,TK基因缺失457-1017位碱基,gG基因缺失1145-1380位碱基,gJ基因缺失445-1183位碱基。
进一步,gI基因缺失1-689位碱基,gE基因缺失544-754位碱基,TK基因缺失457-1017位碱基,UL4基因缺失1-435位碱基。
进一步,gGgJ敲除后序列如SEQ ID No:1所示。
进一步,gIgE敲除后序列如SEQ ID No:2所示。
进一步,TK敲除后序列如SEQ ID No:3所示。
进一步,UL4敲除后序列如SEQ ID No:4所示。
进一步,敲除五个毒力基因得到的鸭瘟疱疹病毒基因缺失减毒疫苗株为DEV VAC/ΔgIgE-ΔTK-ΔgGgJ株,命名为鸭瘟疱疹病毒减毒疫苗株9C2。
进一步,鸭瘟疱疹病毒减毒疫苗株9C2现已于2023年07月05日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO: V202340,保藏地址为中国.武汉.武汉大学。
进一步,敲除四个毒力基因得到的鸭瘟疱疹病毒基因缺失减毒疫苗株为DEV VAC/ΔgIgE-ΔTK-ΔUL4株,命名为鸭瘟疱疹病毒减毒疫苗株9D1。
进一步,鸭瘟疱疹病毒减毒疫苗株9D1现已于2023年07月05日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO: V202339,保藏地址为中国.武汉.武汉大学。
本发明还提供上述鸭瘟疱疹病毒基因缺失减毒疫苗株的构建方法,包括以下步骤:
(1)BR322-amp-HA-BAC-RHA-gpt构建及筛选:高保真PCR制备DNA片段BR322-amp、同源臂LHA和RHA-gpt以及BAC,按照ExoCET方法所描述的方案用T4聚合酶孵育,将DNA混合物电孔插入大肠杆菌GB05-dir中,在含有氨苄青霉素抗性的LB板上筛选BR322-amp-HA-BAC-RHA-gpt;
(2)鸭瘟疱疹病毒的接种:将鸭瘟疱疹病毒VAC疫苗株接种到原代鸭胚成纤维细胞中,得到染毒DEF细胞;
(3)重组病毒的拯救:使用艾德莱BAC质粒提取试剂盒,从步骤(1)构建的BR322-amp-HA-BAC-RHA-gpt中提取基因组DNA,使用脂质体转染试剂盒,将基因组DNA转染到步骤(2)制得的染毒DEF细胞中,以拯救病毒,重组毒株标记为BAC-cm-DEV-VAC-gpt,即DEV VAC感染性克隆;
(4)将步骤(3)得到的DEV VAC感染性克隆采用Red/ET系统分别构建VAC/ΔTK-ΔgE/gI-ΔgG/gJ株和VAC/ΔTK-ΔgE/gI-ΔUL4株;
(5)分别将步骤(4)构建的VAC/ΔTK-ΔgE/gI-ΔgG/gJ株和VAC/ΔTK-ΔgE/gI-ΔUL4株中提取质粒DNA,转染DEF细胞,以拯救病毒,分别得到鸭瘟疱疹病毒基因缺失减毒疫苗株。
进一步,步骤(1)中,孵育具体方法为:基因组片段和载体的摩尔比为3:1,20μL反应由2 μL 10 × NE Buffer 2.1和0.13 μL 3 U μL−1 T4聚合酶组成,在PCR管中体外组装反应,热循环器中循环:25˚C 1 h,75˚C 20 min,50˚C 30 min,4˚C保存。
进一步,步骤(2)中,接种具体方法为:取冻存的鸭瘟疱疹病毒VAC疫苗株一支,加入DMEM基础培养基,混匀后得到疫苗毒;将密度为1.3×10^6cell/mL的新鲜原代DEF细胞,接种到6孔细胞培养板中,每板接种6孔,每孔接种1mL细胞悬液,用DMEM生长液补足每孔培养基体积到2mL,十字摇板分散细胞后,放入CO2细胞培养箱中,设定培养条件为5% CO2,37℃,培养12h;然后将疫苗毒接种到6孔细胞培养板中,每孔接种120μL,十字摇板分散细胞后,放入CO2细胞培养箱中,设定培养条件为5% CO2,37℃,继续培养12h。
进一步,步骤(2)中,原代鸭胚成纤维细胞通过以下方法制作:取10-12日龄发育良好的SPF鸭胚,气室向上置于蛋盘内,用碘酒擦拭鸭蛋表面,在气室端打一小孔,用无菌镊子剪去蛋壳并撕开尿囊膜,夹住鸭胚颈部,移置于一小培养皿中;去除鸭胚的头,四肢和内脏,用无菌剪刀剪碎胚体,转移组织碎块于三角锥瓶内,加入胰酶,热消化组织后用吸管移至离心管中离心,弃掉上清液,PBS重悬底部,将其过滤灭菌纱布,将过滤后的细胞悬液补加含有10%血清的细胞生长液,铺满至细胞瓶内,将细胞瓶置于5% CO2、37 ℃内静置培养24h。
本发明还提供上述鸭瘟疱疹病毒基因缺失减毒疫苗株在鸭瘟疱疹病毒的免疫保护中的应用。
进一步,鸭瘟疱疹病毒为鸭瘟疱疹病毒AV1221强毒株。
本发明具有以下有益效果:
1、本发明以鸭瘟疱疹病毒VAC疫苗株基因组通过核酸外切酶联合Red/ET同源重组,直接克隆到大肠杆菌中的细菌人工染色体(BAC)中,随后将骨架载体中的关键毒力基因敲除,从而获得鸭瘟疱疹病毒基因缺失减毒疫苗株,该鸭瘟疱疹病毒(Duck enteritisvirus,DEV)是gI、gE、TK、gG和gJ基因组合缺失株或gI、gE、TK和UL4基因组合缺失株,即DEVVAC/ΔgIgE-ΔTK-ΔgGgJ株或DEV VAC/ΔgIgE-ΔTK-ΔUL4株。本发明构建的鸭瘟疱疹病毒基因缺失株在DEF细胞上连续传代,具有很好的稳定性,不发生变异,为鸭瘟疱疹病毒基因缺失减毒疫苗的研制提供了安全的保障。
2、本发明的减毒疫苗株,可有效保护鸭抵抗鸭瘟疱疹病毒AV1221强毒株的攻击,免疫保护率达到100%,同时,对非靶动物具有良好的安全性,无副反应。
附图说明
图1为实施例1-2减毒疫苗株的酶切鉴定图;
图2为实施例1重组病毒遗传稳定性PCR鉴定图;
图3为实施例2重组病毒遗传稳定性PCR鉴定图;
图4为实施例1-2重组病毒PCR鉴定结果;
图5为实施例1-2重组病毒间接免疫荧光鉴定结果;
图6为实施例1-2重组病毒在DEF细胞中的一步生长曲线图;
图7为实施例1-2重组病毒的抗体消长规律图。
具体实施方式
以下结合附图对本发明的原理和特征进行描述,所举实例只用于解释本发明,并非用于限定本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
DEV疫苗株VAC株(Accession:EU082088.2)和DEV强毒株AV1221株购自国家兽医微生物菌(毒)种保藏中心;鸭必胜鸭瘟活疫苗购自哈药集团生物疫苗有限公司;携带表达质粒BR322-amp于本实验室保存,大肠杆菌GB05-dir、大肠杆菌GB08-red用于重组体的转化。
SPF鸭胚购自中国农科科学院哈尔滨兽医研究所,1日龄SPF鸡购自广东新兴大华农禽蛋有限公司SPF实验动物中心。
实施例1:五基因缺失减毒疫苗株的制备
一、DEV VAC感染性克隆的构建和验证
1、重组毒株BAC-cm-DEV-VAC-gpt的构建及筛选
(1)原代鸭胚成纤维细胞(DEF)的制作
取10-12日龄发育良好的SPF鸭胚,气室向上置于蛋盘内,用碘酒擦拭鸭蛋表面,在气室端打一小孔,用无菌镊子剪去蛋壳并撕开尿囊膜,夹住鸭胚颈部,移置于一小培养皿中;去除鸭胚的头,四肢和内脏,用无菌剪刀剪碎胚体,转移组织碎块于三角锥瓶内,加入胰酶若干,热消化组织后用吸管移至离心管中离心,弃掉上清液,PBS重悬底部,将其过滤灭菌纱布,将过滤后的细胞悬液补加含有10%血清的细胞生长液,铺满至细胞瓶内,将细胞瓶置于5% CO2、37 ℃内静置培养24h。
(2)BR322-amp-HA-BAC-RHA-gpt的构建及筛选
高保真PCR制备DNA片段BR322-amp、同源臂LHA和RHA-gpt以及BAC,按照ExoCET方法所描述的方案用T4聚合酶孵育,基因组片段和载体的摩尔比为3:1,20μL反应由2 μL 10× NE Buffer 2.1和0.13 μL 3 U μL−1 T4聚合酶组成,在PCR管中体外组装反应,热循环器中循环:25˚C 1 h,75˚C 20 min,50˚C 30 min,4˚C保存,将DNA混合物电孔插入大肠杆菌GB05-dir中,在含有15 μg/ml氨苄青霉素抗性的LB板上筛选BR322-amp-HA-BAC-RHA-gpt。
(3)鸭瘟疱疹病毒的接种
取冻存的鸭瘟疱疹病毒疫苗毒VAC株一支,加入DMEM基础培养基若干,混匀后备用。原代鸭胚成纤维细胞密度为1.3×10^6cell/mL,将新鲜原代DEF细胞,接种到6孔细胞培养板中,每板接种6孔,每孔接种1mL细胞悬液,用DMEM生长液补足每孔培养基体积到2mL,十字摇板分散细胞后放入CO2细胞培养箱中,设定培养条件为5% CO2,37℃,培养12h。将疫苗毒接种到6孔细胞培养板中,每孔接种120μL,十字摇板分散细胞后放入CO2细胞培养箱中,设定培养条件为5% CO2,37℃,继续培养12h。
(4)重组病毒的拯救
根据制造商的说明,使用艾德莱BAC质粒提取试剂盒从BR322-amp-HA-BAC-RHA-gpt中提取基因组DNA,使用Lipofectamine 3000脂质体转染试剂盒将其转染到染毒DEF细胞中以拯救病毒,重组毒株标记为BAC-cm-DEV-VAC-gpt,即为DEV VAC感染性克隆。
2、感染性克隆的分析验证
(1)重组病毒感染细胞总DNA提取及转化
根据制造商的说明,使用艾德莱BAC质粒提取试剂盒从染毒细胞中提取总DNA,将染毒细胞DNA转化至大肠杆菌工程菌株E.coli GB05red,将菌体悬液转移到氯霉素抗性LB平板,倒置于生化培养箱中,设置培养温度37℃,培养48h。
(2)限制性内切酶分析鉴定重组体
参照限制性内切酶酶切操作规程,20 μL反应由1μL PvuII、2μL Cutsmart buffer和2 μL DNA,ddH2O补齐至20μL体系组成,在PCR管中体外组装反应,通过PvuII限制性内切酶酶切分析验证克隆是否正确。
二、减毒疫苗株的构建及拯救
1、减毒疫苗株的构建
采用Red/ET系统构建VAC/ΔTK-ΔgE/gI-ΔgG/gJ株及VAC/ΔTK-ΔgE/gI-ΔUL4株。
在大肠杆菌GB08-red中进行Redαβ重组,缺失顺序依次为TK、gI/gE和gG/gJ基因,即先构建VAC/ΔTK株,然后在VAC/ΔTK株的基础上构建VAC/ΔTK-ΔgE/gI株,再在VAC/ΔTK-ΔgE/gI的基础上构建VAC/ΔTK-ΔgE/gI-ΔgG/gJ株(见图1),其是利用Red/ET重组工程技术对DEV VAC株感染性克隆进行修饰与拯救,获得重组毒株。
2、重组病毒的拯救
将DEF细胞传代于6孔细胞板中,细胞汇合度达70-80%时进行转染。在转染前30-60min,吸去细胞培养液,加入新鲜的不含抗生素的完全培养基2 mL。
取2-6 μg质粒DNA(重组毒株),加入到100 μL氯化钙溶液中,混匀,把DNA-氯化钙溶液加入到100 μLBBS溶液中,混匀,室温孵育10-20min,把DNA-氯化钙-BBS混合物均匀滴加到整个DEF细胞中,转染16h后,吸去磷酸钙沉淀,更换成含5%FBS的DMEM,将培养板继续放于37℃细胞培养48-72h,得到鸭瘟疱疹病毒基因缺失减毒疫苗株(DEV VAC/ΔgIgE-ΔTK-ΔgGgJ)。
实施例2:四基因缺失减毒疫苗株的制备
实施例1中的步骤二中的步骤1中,减毒疫苗株的构建,缺失顺序依次为TK、gI/gE、和UL4基因,即先构建VAC/ΔTK株,然后在VAC/ΔTK株的基础上构建VAC/ΔTK-ΔgE/gI株,再在VAC/ΔTK-ΔgE/gI的基础上构建VAC/ΔTK-ΔgE/gI-ΔUL4株(见图1),最终得到鸭瘟疱疹病毒基因缺失减毒疫苗株(DEV VAC/ΔgIgE-ΔTK-ΔUL4),其余同实施例1。
实施例3:重组病毒的传代与鉴定
一、重组病毒的传代
试验方法:将DEF细胞传代于六孔细胞板,待细胞汇合度达到90%时,弃掉原细胞培养液,并用PBS洗涤2次,重组病毒VAC/ΔgI/gE-ΔTK-ΔgG/gJ株及VAC/ΔgI/gE-ΔTK-ΔUL4株在DEF细胞上连续传代,选取第5代、10代、15代、20代、25代和30代病毒,利用病毒核酸DNA/RNA提取试剂盒提取重组病毒的基因组,使用鉴定引物BAC-DEV-F/R进行PCR扩增,引物序列如表1所示。
表1重组病毒遗传稳定性鉴定引物
试验结果:结果显示,使用鉴定引物BAC-DEV-1F/1R对重组病毒VAC/ΔgI/gE-ΔTK-ΔgG/gJ株进行鉴定时,其扩增产物大小为959bp(图2,图2中,M表示DNA分子标记(2000bp),P10表示第10代,P15表示第15代,P20表示第20代,P25表示第25代),使用鉴定引物BAC-DEV-1F/1R对重组病毒VAC/ΔgI/gE-ΔTK-ΔUL4株进行鉴定时,其扩增产物大小为449bp(图3,M表示DNA分子标记(2000bp),P10表示第10代,P15表示第15代,P20表示第20代,P25表示第25代),重组病毒能够在DEF细胞上稳定传代。
二、重组病毒的PCR鉴定
试验方法:利用DNA/RNA病毒核酸提取试剂盒提取重组病毒的基因组,根据病毒基因缺失位置,利用Oligo 7.0软件设计引物,然后以重组病毒的基因组为模板进行PCR扩增,核酸电泳及测序后进行比较与鉴定,引物序列如表2所示。
表2重组病毒PCR鉴定引物
试验结果:使用TK鉴定引物TK-delF/R对重组病毒进行鉴定时,亲本毒株VAC扩增产物大小为1330bp,VAC/ΔgIgE-ΔTK-ΔgGgJ减毒株扩增产物为796bp(图4中的1-2);使用gI/gE鉴定引物gIgE-delF/R对重组病毒进行鉴定时,野生型毒株VAC扩增产物大小为3046bp,DEV VAC/ΔgIgE-ΔTK-ΔgGgJ减毒株扩增产物为2146bp(图4中的3-4);使用gG/gJ鉴定引物gGgJ-delF/R对重组病毒进行鉴定时,野生型毒株VAC扩增产物大小为3359bp,VAC/ΔgIgE-ΔTK-ΔgGgJ减毒株扩增产物为2384bp(图4中的5-6);使用UL4鉴定引物UL4-delF/R对重组病毒进行鉴定时,野生型毒株VAC扩增产物大小为1126bp,VAC/ΔgIgE-ΔTK-ΔUL4减毒株扩增产物为691bp(图4中的7-8)。
三、重组病毒的间接免疫荧光鉴定
试验方法:将DEF细胞接种于48孔细胞板中,分为3组,每组重复6个孔,待细胞长满单层后,第一组以0.1MOI的剂量接种重组病毒DEV VAC/ΔgIgE-ΔTK-ΔgGgJ或DEV VAC/ΔgIgE-ΔTK-ΔUL4株,第2组接种亲本毒株DEV VAC株,第3组加入DMEM作为对照组(Mock),待80%的细胞出现病变时,弃去培养基,PBS洗涤3遍后,多聚甲醛4℃固定30min,然后用PBS洗涤3遍。随后3组细胞中的3个孔加入200uL gB单克隆抗体(1:500稀释),另外3个孔加入200uL gE单克隆抗体,37℃孵育1h;PBS洗涤3遍后,加入1mL羊抗鼠IgG(1:2000稀释),37℃孵育60min;PBS洗涤3遍后,置于荧光显微镜下观察。
试验结果:用gB单克隆抗体孵育后,接种重组病毒DEV VAC/ΔgIgE-ΔTK-ΔgGgJ株、DEV VAC/ΔgIgE-ΔTK-ΔUL4株或亲本毒株DEV VAC株的细胞均可观察到绿色荧光;然而用gE单克隆抗体孵育后,只有接种亲本毒株DEV VAC株的细胞可观察到绿色荧光(图5),表明重组病毒中gE基因已被有效缺失。
以上结果表明,本发明获得了TK、gE、gI、gG和gJ基因缺失的减毒株DEV VAC/ΔgIgE-ΔTK-ΔgGgJ株及TK、gI、gE和UL4基因缺失的减毒株DEV VAC/ΔgIgE-ΔTK-ΔUL4株。
实施例4:重组病毒的生长特性的测定
一、重组病毒遗传稳定性测定
试验方法:重组病毒DEV VAC/ΔgIgE-ΔTK-ΔgGgJ或DEV VAC/ΔgIgE-ΔTK-ΔUL4株在DEF细胞上连续传代,选取第10代、15代、20代、25代和30代病毒,使用鉴定引物TK-delF/R、gE/gI-delF/R 和gG/gJ-delF/R对缺失基因进行PCR扩增,并将PCR产物送至广州华大生物公司测序。
试验结果:DEV VAC/ΔgIgE-ΔTK-ΔgGgJ株在TK、gE、gI、gG、gJ或UL4基因缺失非常稳定。
二、重组病毒的一步生长曲线
试验方法:将病毒用生理盐水稀释,感染预先准备好的DEF细胞,重复3次,以0.1MOI的剂量感染细胞,分别于感染后12h、24h、36h、48h、60h、72h和96h各收获3孔细胞上清液,混合后分装,置于-80℃冻存。采用Reed-Muench法测定不同时间点病毒TCID50并绘制生长曲线。
试验结果:DEV VAC/ΔgIgE-ΔTK-ΔgGgJ株或DEV VAC/ΔgIgE-ΔTK-ΔUL4株生长曲线趋势与亲本病毒VAC株基本一致,接种后60h达到滴度峰值,病毒滴度分别为106.81TCID50/0.2mL和105.56TCID50/0.2mL(图6),重组病毒与亲本病毒VAC株滴度无显著差异(p>0.05)。
实施例5:重组病毒对非靶动物的安全性评价
试验方法:选取30只1日龄的鸭瘟疱疹病毒阴性SPF鸡,随机分为3组,每组10只雏鸡,其中一组的雏鸡腿肌接种DEV VAC/ΔgIgE-ΔTK-ΔgGgJ株(3×104.00ELD50/只),另一组的雏鸡腿肌接种DEV VAC株(3×104.00ELD50/只),第3组的雏鸡腿肌接种0.2mL的PBS作为对照组。接种后观察14天,每天观察和记录临床症状,试验过程中所有死亡鸡只立即进行剖检并观察大体病变。
试验结果:如表3所示,DEV VAC/ΔgIgE-ΔTK-ΔgGgJ株接种雏鸡后,雏鸡未出现打喷嚏、厌食、颤抖等发病症状,而DEV VAC接种雏鸡后则出现持续颤抖、死亡,表明缺失TK、gI、gE、gG和gJ基因或TK、gI、gE和UL4基因后的DEV VAC/ΔgIgE-ΔTK-ΔgGgJ及DEV VAC/ΔgIgE-ΔTK-ΔUL4已充分减毒,对雏鸡不致病。
表3重组病毒对雏鸡的安全性试验结果
表3 实验动物分组与接种毒株
实施例6:重组病毒的免疫保护力测定
试验方法:选取1日龄的鸭瘟疱疹病毒阴性麻鸭(10只),随机分为3组,每组10只麻鸭,实验鸭置于负压隔离器中饲养,自由饮水采食,3个实验组于7日龄接种重组病毒104.00DELD50/只,1组作为非免疫攻毒对照组接种无菌PBS,实验组2组、3组和非免疫攻毒对照组于14日龄接种强毒(AV1221)104.50DELD50/只。另外1组作为空白对照组。免疫鸭攻毒后每日观察并记录各组鸭只状态、发病和死亡情况。试验过程中所有死亡鸭只立即进行剖检并观察大体病变。具体分组与免疫、攻毒毒株见表4。
表4 实验动物分组与接种、攻毒毒株
结果见表5,由表5可知DEV VAC/ΔgIgE-ΔTK-ΔgGgJ及DEV VAC/ΔgIgE-ΔTK-ΔUL4疫苗株免疫靶动物(麻鸭)后,免疫保护率达到100%,发病率及死亡率均为0%,非免疫攻毒对照组发病率及死亡率为100%。
表5免疫和攻毒后各组发病率和死亡率
实验例7:重组病毒的抗体消长规律测定
试验方法:采集免疫前试验鸭血清以及免疫后每周采集一次试验鸭血清,所有血清样品均用鸭瘟疱疹病毒抗体检测试剂盒测定特异性抗体效价,按照试剂盒推荐的结果判定标准,比值>0.2判为阳性,结果如图7所示。
由图7可知,实验组重组抗体增长趋势与VAC组基本一致。空白对照组在实验全程中未检测到DEV特异性抗体效价阳性,攻毒组攻毒前未检测到抗体应答反应。各实验组在重组毒株接种后第7d,抗体水平出现上升趋势但抗体水平较低,接种后第14d均可检测出抗体阳性反应,攻毒后各实验组抗体水平快速上升。VAC/ΔgIgE-ΔTK-ΔgGgJ免疫组和VAC/ΔgIgE-ΔTK-ΔUL4免疫组攻毒后的抗体水平略高,无显著差异(P>0.05)。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (6)
1.一种鸭瘟疱疹病毒基因缺失减毒疫苗株,其特征在于,该疫苗株是以鸭瘟疱疹病毒VAC疫苗株的感染性克隆为骨架载体,然后将骨架载体中的毒力基因敲除,得到鸭瘟疱疹病毒基因缺失减毒疫苗株;所述毒力基因为gI基因、gE基因、TK基因、gG基因和gJ基因;或所述毒力基因为gI基因、gE基因、TK基因和UL4基因;
所述毒力基因为gI基因、gE基因、TK基因、gG基因和gJ基因时,gI基因缺失1-689位碱基,gE基因缺失544-754位碱基,TK基因缺失457-1017位碱基,gG基因缺失1145-1380位碱基,gJ基因缺失445-1183位碱基;
所述毒力基因为gI基因、gE基因、TK基因和UL4基因时,gI基因缺失1-689位碱基,gE基因缺失544-754位碱基,TK基因缺失457-1017位碱基,UL4基因缺失1-435位碱基。
2.权利要求1所述的鸭瘟疱疹病毒基因缺失减毒疫苗株的构建方法,其特征在于,包括以下步骤:
(1)BR322-amp-HA-BAC-RHA-gpt构建及筛选:高保真PCR制备DNA片段BR322-amp、同源臂LHA和RHA-gpt以及BAC,按照ExoCET方法所描述的方案用T4聚合酶孵育,将DNA混合物电孔插入大肠杆菌GB05-dir中,在含有氨苄青霉素抗性的LB板上筛选BR322-amp-HA-BAC-RHA-gpt;
(2)鸭瘟疱疹病毒的接种:将鸭瘟疱疹病毒VAC疫苗株接种到原代鸭胚成纤维细胞中,得到染毒DEF细胞;
(3)重组病毒的拯救:使用BAC质粒提取试剂盒,从步骤(1)构建的BR322-amp-HA-BAC-RHA-gpt中提取基因组DNA,使用脂质体转染试剂盒,将基因组DNA转染到步骤(2)制得的染毒DEF细胞中,以拯救病毒,重组毒株标记为BAC-cm-DEV-VAC-gpt,即DEV VAC感染性克隆;
(4)将步骤(3)得到的DEV VAC感染性克隆采用Red/ET重组分别构建VAC/ΔTK-ΔgE/gI-ΔgG/gJ株和VAC/ΔTK-ΔgE/gI-ΔUL4株;
(5)分别将步骤(4)构建的VAC/ΔTK-ΔgE/gI-ΔgG/gJ株和VAC/ΔTK-ΔgE/gI-ΔUL4株中提取质粒DNA,转染DEF细胞,以拯救病毒,分别得到鸭瘟疱疹病毒基因缺失减毒疫苗株。
3.根据权利要求2所述的鸭瘟疱疹病毒基因缺失减毒疫苗株的构建方法,其特征在于,步骤(1)中,孵育具体方法为:基因组片段和载体的摩尔比为3:1,20μL反应由2 μL 10 ×NE Buffer 2.1和0.13 μL 3 U μL−1 T4聚合酶组成,在PCR管中体外组装反应,热循环器中循环:25˚C 1 h,75˚C 20 min,50˚C 30 min,4˚C保存。
4.根据权利要求2所述的鸭瘟疱疹病毒基因缺失减毒疫苗株的构建方法,其特征在于,步骤(2)中,接种具体方法为:取冻存的鸭瘟疱疹病毒VAC疫苗株一支,加入DMEM基础培养基,混匀后得到疫苗毒;将密度为1.3×10^6 cell/mL的新鲜原代DEF细胞,接种到6孔细胞培养板中,每板接种6孔,每孔接种1mL细胞悬液,用DMEM生长液补足每孔培养基体积到2mL,十字摇板分散细胞后,放入CO2细胞培养箱中,设定培养条件为5% CO2,37℃,培养12h;然后将疫苗毒接种到6孔细胞培养板中,每孔接种120μL,十字摇板分散细胞后,放入CO2细胞培养箱中,设定培养条件为5% CO2,37℃,继续培养12h。
5.权利要求1所述的鸭瘟疱疹病毒基因缺失减毒疫苗株在制备鸭瘟疱疹病毒疫苗中的应用。
6.根据权利要求5所述的鸭瘟疱疹病毒基因缺失减毒疫苗株在制备鸭瘟疱疹病毒疫苗中的应用,其特征在于,所述鸭瘟疱疹病毒为鸭瘟疱疹病毒AV1221强毒株。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310990050.8A CN116731982B (zh) | 2023-08-08 | 2023-08-08 | 一种鸭瘟疱疹病毒基因缺失减毒疫苗株及其构建方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310990050.8A CN116731982B (zh) | 2023-08-08 | 2023-08-08 | 一种鸭瘟疱疹病毒基因缺失减毒疫苗株及其构建方法和应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN116731982A CN116731982A (zh) | 2023-09-12 |
CN116731982B true CN116731982B (zh) | 2023-10-20 |
Family
ID=87906282
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310990050.8A Active CN116731982B (zh) | 2023-08-08 | 2023-08-08 | 一种鸭瘟疱疹病毒基因缺失减毒疫苗株及其构建方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116731982B (zh) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109536463A (zh) * | 2018-12-26 | 2019-03-29 | 四川农业大学 | 鸭瘟病毒gE和gI双基因无痕缺失株DPV CHv-ΔgE+ΔgI及其构建方法 |
NL2025748B1 (en) * | 2020-06-03 | 2021-04-21 | Univ Sichuan Agricultural | Duck Plague Virus gE-gI Double Gene Markerless Deletion Strain DPV CHVAgE+ AgI and Construction Method Thereof |
-
2023
- 2023-08-08 CN CN202310990050.8A patent/CN116731982B/zh active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109536463A (zh) * | 2018-12-26 | 2019-03-29 | 四川农业大学 | 鸭瘟病毒gE和gI双基因无痕缺失株DPV CHv-ΔgE+ΔgI及其构建方法 |
NL2025748B1 (en) * | 2020-06-03 | 2021-04-21 | Univ Sichuan Agricultural | Duck Plague Virus gE-gI Double Gene Markerless Deletion Strain DPV CHVAgE+ AgI and Construction Method Thereof |
Non-Patent Citations (1)
Title |
---|
Evaluation of safety and immunogenicity of duck-plague virus gC/gE double gene deletion;Peilin Ruan等;《Front. Immunol.》;第13卷;全文 * |
Also Published As
Publication number | Publication date |
---|---|
CN116731982A (zh) | 2023-09-12 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN104862286B (zh) | 猪伪狂犬病病毒基因缺失株、疫苗组合物及其制备方法和应用 | |
CN109439634B (zh) | 伪狂犬病病毒基因工程弱毒疫苗株及其应用 | |
CN104877972B (zh) | 一种重组猪伪狂犬病毒gE/gI双基因缺失株及其应用 | |
CN104826103B (zh) | 一种猪伪狂犬病病毒疫苗 | |
CN104830810B (zh) | 一种重组猪伪狂犬病毒TK/gE/gI三基因缺失株 | |
CN102373180B (zh) | 表达禽流感病毒血凝素(ha)基因的重组鸭病毒性肠炎病毒疫苗株及其构建方法和应用 | |
CN102533674B (zh) | 表达禽流感病毒血凝素基因的重组鸭病毒性肠炎病毒疫苗株及其构建方法和应用 | |
CN110218706B (zh) | 表达h7n9亚型高致病性禽流感病毒ha蛋白的重组火鸡疱疹病毒的构建与应用 | |
CN107384874A (zh) | 伪狂犬病毒流行株gI/gE基因缺失突变株及构建和应用 | |
CN103981153A (zh) | 伪狂犬病病毒双荧光标记缺失病毒的构建 | |
CN100503816C (zh) | 一种重组伪狂犬病-猪繁殖与呼吸综合征基因工程毒株及应用 | |
CN102559610B (zh) | 表达禽流感病毒血凝素基因的重组鸭病毒性肠炎病毒疫苗株及其构建方法和应用 | |
CN105018433A (zh) | 猪伪狂犬病病毒基因缺失株、疫苗组合物及其制备方法和应用 | |
CN117417904A (zh) | 表达C型aMPV F蛋白和G蛋白的新城疫病毒载体疫苗株及其应用 | |
CN107142280A (zh) | 一种表达h9亚型禽流感病毒ha基因的重组火鸡疱疹病毒株 | |
EP4076516A1 (en) | Multivalent hvt vector vaccine | |
CN110016457B (zh) | 一株重组细粒棘球蚴Eg95基因的粗糙型布鲁氏菌及其疫苗生产方法 | |
CN107296956A (zh) | 一种基因重组活载体疫苗 | |
CN112063596A (zh) | 鸽副黏病毒1型ppmv-1/bj-c株及其应用 | |
CN111647568A (zh) | 鸡传染性法氏囊病病毒新型变异株反向遗传疫苗株及其应用 | |
CN108342367B (zh) | 一种重组马立克病毒毒株sca13株及其应用 | |
CN116731982B (zh) | 一种鸭瘟疱疹病毒基因缺失减毒疫苗株及其构建方法和应用 | |
CN110499296A (zh) | 一种耐热的血清8b型禽腺病毒基因工程疫苗候选株及其构建方法 | |
CN114395536B (zh) | 一种禽腺病毒4、8和11型三价疫苗及其制备方法和应用 | |
CN105385666B (zh) | 伪狂犬病病毒双荧光标记5基因缺失株的构建 |
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 |