CN118127077A - 基于盖塔病毒骨架的嵌合甲病毒制备方法和应用 - Google Patents
基于盖塔病毒骨架的嵌合甲病毒制备方法和应用 Download PDFInfo
- Publication number
- CN118127077A CN118127077A CN202410557841.6A CN202410557841A CN118127077A CN 118127077 A CN118127077 A CN 118127077A CN 202410557841 A CN202410557841 A CN 202410557841A CN 118127077 A CN118127077 A CN 118127077A
- Authority
- CN
- China
- Prior art keywords
- sequence
- orf2
- virus
- plasmid
- alphavirus
- 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.)
- Granted
Links
- 241000710929 Alphavirus Species 0.000 title claims abstract description 59
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 101710159752 Poly(3-hydroxyalkanoate) polymerase subunit PhaE Proteins 0.000 claims abstract description 36
- 101710130262 Probable Vpr-like protein Proteins 0.000 claims abstract description 36
- 241000700605 Viruses Species 0.000 claims abstract description 34
- 239000013612 plasmid Substances 0.000 claims abstract description 32
- 230000002238 attenuated effect Effects 0.000 claims abstract description 24
- 101150009852 ORF2 gene Proteins 0.000 claims abstract description 19
- 239000013600 plasmid vector Substances 0.000 claims abstract description 16
- 101150044789 Cap gene Proteins 0.000 claims abstract description 14
- 239000002299 complementary DNA Substances 0.000 claims abstract description 10
- 241000288902 Lemur catta Species 0.000 claims abstract description 6
- 208000015181 infectious disease Diseases 0.000 claims abstract description 6
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 5
- 230000002458 infectious effect Effects 0.000 claims abstract description 4
- 239000012634 fragment Substances 0.000 claims description 26
- 229960005486 vaccine Drugs 0.000 claims description 20
- 101000977023 Azospirillum brasilense Uncharacterized 17.8 kDa protein in nodG 5'region Proteins 0.000 claims description 18
- 101000961984 Bacillus thuringiensis Uncharacterized 30.3 kDa protein Proteins 0.000 claims description 18
- 101000644901 Drosophila melanogaster Putative 115 kDa protein in type-1 retrotransposable element R1DM Proteins 0.000 claims description 18
- 101000747702 Enterobacteria phage N4 Uncharacterized protein Gp2 Proteins 0.000 claims description 18
- 101000758599 Escherichia coli Uncharacterized 14.7 kDa protein Proteins 0.000 claims description 18
- 101000768930 Lactococcus lactis subsp. cremoris Uncharacterized protein in pepC 5'region Proteins 0.000 claims description 18
- 101000976302 Leptospira interrogans Uncharacterized protein in sph 3'region Proteins 0.000 claims description 18
- 101000778886 Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai (strain 56601) Uncharacterized protein LA_2151 Proteins 0.000 claims description 18
- 101001121571 Rice tungro bacilliform virus (isolate Philippines) Protein P2 Proteins 0.000 claims description 18
- 101000818098 Spirochaeta aurantia Uncharacterized protein in trpE 3'region Proteins 0.000 claims description 18
- 101001026590 Streptomyces cinnamonensis Putative polyketide beta-ketoacyl synthase 2 Proteins 0.000 claims description 18
- 101000750896 Synechococcus elongatus (strain PCC 7942 / FACHB-805) Uncharacterized protein Synpcc7942_2318 Proteins 0.000 claims description 18
- 101000916321 Xenopus laevis Transposon TX1 uncharacterized 149 kDa protein Proteins 0.000 claims description 18
- 101000760088 Zymomonas mobilis subsp. mobilis (strain ATCC 10988 / DSM 424 / LMG 404 / NCIMB 8938 / NRRL B-806 / ZM1) 20.9 kDa protein Proteins 0.000 claims description 18
- 241000710961 Semliki Forest virus Species 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 7
- 238000012408 PCR amplification Methods 0.000 claims description 6
- 230000004927 fusion Effects 0.000 claims description 6
- 239000013598 vector Substances 0.000 claims description 6
- 150000001413 amino acids Chemical class 0.000 claims description 4
- 238000003776 cleavage reaction Methods 0.000 claims description 4
- 238000001976 enzyme digestion Methods 0.000 claims description 4
- 229940124590 live attenuated vaccine Drugs 0.000 claims description 4
- 229940023012 live-attenuated vaccine Drugs 0.000 claims description 4
- 230000007017 scission Effects 0.000 claims description 4
- 102000003960 Ligases Human genes 0.000 claims description 3
- 108090000364 Ligases Proteins 0.000 claims description 3
- 102000018120 Recombinases Human genes 0.000 claims description 3
- 108010091086 Recombinases Proteins 0.000 claims description 3
- 230000029087 digestion Effects 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 claims 1
- 241000699670 Mus sp. Species 0.000 abstract description 24
- 230000005847 immunogenicity Effects 0.000 abstract description 5
- 230000001018 virulence Effects 0.000 abstract description 4
- 230000004580 weight loss Effects 0.000 abstract description 4
- 210000004027 cell Anatomy 0.000 description 9
- 210000003501 vero cell Anatomy 0.000 description 9
- 241000608297 Getah virus Species 0.000 description 7
- 238000010276 construction Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 108700026244 Open Reading Frames Proteins 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 4
- 241001502567 Chikungunya virus Species 0.000 description 3
- 241000282414 Homo sapiens Species 0.000 description 3
- 238000000137 annealing Methods 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 231100000636 lethal dose Toxicity 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 238000012257 pre-denaturation Methods 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- 239000012228 culture supernatant Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000000120 cytopathologic effect Effects 0.000 description 2
- 230000002354 daily effect Effects 0.000 description 2
- 230000003203 everyday effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 230000007918 pathogenicity Effects 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000005180 public health Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 208000007887 Alphavirus Infections Diseases 0.000 description 1
- 101000666833 Autographa californica nuclear polyhedrosis virus Uncharacterized 20.8 kDa protein in FGF-VUBI intergenic region Proteins 0.000 description 1
- 101000977027 Azospirillum brasilense Uncharacterized protein in nodG 5'region Proteins 0.000 description 1
- 101000962005 Bacillus thuringiensis Uncharacterized 23.6 kDa protein Proteins 0.000 description 1
- 108010055991 BglII endonuclease Proteins 0.000 description 1
- 108010041986 DNA Vaccines Proteins 0.000 description 1
- 229940021995 DNA vaccine Drugs 0.000 description 1
- 241000255925 Diptera Species 0.000 description 1
- 101000785191 Drosophila melanogaster Uncharacterized 50 kDa protein in type I retrotransposable element R1DM Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 241000710945 Eastern equine encephalitis virus Species 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 101000747704 Enterobacteria phage N4 Uncharacterized protein Gp1 Proteins 0.000 description 1
- 101000861206 Enterococcus faecalis (strain ATCC 700802 / V583) Uncharacterized protein EF_A0048 Proteins 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 101000769180 Escherichia coli Uncharacterized 11.1 kDa protein Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000976301 Leptospira interrogans Uncharacterized 35 kDa protein in sph 3'region Proteins 0.000 description 1
- 101710127721 Membrane protein Proteins 0.000 description 1
- 101000658690 Neisseria meningitidis serogroup B Transposase for insertion sequence element IS1106 Proteins 0.000 description 1
- 239000012124 Opti-MEM Substances 0.000 description 1
- 240000000220 Panda oleosa Species 0.000 description 1
- 235000016496 Panda oleosa Nutrition 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 101000748660 Pseudomonas savastanoi Uncharacterized 21 kDa protein in iaaL 5'region Proteins 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 229940022005 RNA vaccine Drugs 0.000 description 1
- 101000584469 Rice tungro bacilliform virus (isolate Philippines) Protein P1 Proteins 0.000 description 1
- 101000818096 Spirochaeta aurantia Uncharacterized 15.5 kDa protein in trpE 3'region Proteins 0.000 description 1
- 101000766081 Streptomyces ambofaciens Uncharacterized HTH-type transcriptional regulator in unstable DNA locus Proteins 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 101000804403 Synechococcus elongatus (strain PCC 7942 / FACHB-805) Uncharacterized HIT-like protein Synpcc7942_1390 Proteins 0.000 description 1
- 101000750910 Synechococcus elongatus (strain PCC 7942 / FACHB-805) Uncharacterized HTH-type transcriptional regulator Synpcc7942_2319 Proteins 0.000 description 1
- 101000644897 Synechococcus sp. (strain ATCC 27264 / PCC 7002 / PR-6) Uncharacterized protein SYNPCC7002_B0001 Proteins 0.000 description 1
- 238000011053 TCID50 method Methods 0.000 description 1
- 241000710924 Togaviridae Species 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 241000710959 Venezuelan equine encephalitis virus Species 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 241000710951 Western equine encephalitis virus Species 0.000 description 1
- 101000916336 Xenopus laevis Transposon TX1 uncharacterized 82 kDa protein Proteins 0.000 description 1
- 101001000760 Zea mays Putative Pol polyprotein from transposon element Bs1 Proteins 0.000 description 1
- 101000678262 Zymomonas mobilis subsp. mobilis (strain ATCC 10988 / DSM 424 / LMG 404 / NCIMB 8938 / NRRL B-806 / ZM1) 65 kDa protein Proteins 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 206010014599 encephalitis Diseases 0.000 description 1
- 108010017299 endodeoxyribonuclease BSSHII Proteins 0.000 description 1
- 108010045673 endodeoxyribonuclease XBAI Proteins 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000004727 humoral immunity Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 229940031551 inactivated vaccine Drugs 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 108700021021 mRNA Vaccine Proteins 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 229940068196 placebo Drugs 0.000 description 1
- 239000000902 placebo Substances 0.000 description 1
- 238000012809 post-inoculation Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 230000001131 transforming 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
- 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
-
- 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
- A61P31/14—Antivirals for RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
-
- 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
- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/36011—Togaviridae
- C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
- C12N2770/36121—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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/36011—Togaviridae
- C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
- C12N2770/36122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/36011—Togaviridae
- C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
- C12N2770/36134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/36011—Togaviridae
- C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
- C12N2770/36161—Methods of inactivation or attenuation
- C12N2770/36162—Methods of inactivation or attenuation by genetic engineering
-
- 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)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Virology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Public Health (AREA)
- Immunology (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Mycology (AREA)
- Oncology (AREA)
- Epidemiology (AREA)
- Communicable Diseases (AREA)
- Gastroenterology & Hepatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (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
本发明公开了一种基于盖塔病毒骨架的嵌合甲病毒制备方法和应用；本发明先公开一种质粒载体，所述质粒载体以含有盖塔病毒基因组的感染性cDNA克隆为骨架质粒，再通过如下（A1）或（A2）任一方式进行构建：（A1）将盖塔病毒的ORF2基因序列替换成其他甲病毒ORF2基因；（A2）保留盖塔病毒Cap基因前123位氨基酸序列，而其余盖塔病毒ORF2基因序列替换为去除Cap基因N端前102~123位氨基酸的其他甲病毒ORF2基因序列。本发明还公开了前述基础上的嵌合甲病毒的制备方法、所述制备方法获得的嵌合甲病毒减毒株。所述嵌合甲病毒对小鼠的毒力显著减弱，不引起死亡及严重的体重下降，但保持了良好的免疫原性。
Description
技术领域
本发明涉及生物工程技术领域，尤其涉及一种基于盖塔病毒骨架的嵌合甲病毒制备方法和应用。
背景技术
甲病毒属病毒严重危害人类和养殖动物的健康，引起感染动物或人类出现发热、斑丘疹、关节炎或脑炎等症状。甲病毒在蚊子媒介和脊椎动物宿主（包括人类、非人类灵长动物、马科动物和猪等）之间传播，大多数甲病毒涉及城市循环或森林循环。在2006-2019年爆发的基孔肯尼亚病毒（Chikungunya virus，CHIKV）显示了甲病毒全球传播的能力。提示有必要对其他甲病毒进行系统性研究并开发相应的疫苗，以防止潜在的大流行风险。
甲病毒属于披膜病毒科甲病毒属，其基因组长约11.5 kb，有2个开放阅读框（Openreading frame，ORF），第一个开放阅读框（ORF1）编码nsP1-4蛋白，而第二个开放阅读框（ORF2）编码Cap、E3、E2、6K/TF和E1结构蛋白。E2和E1糖蛋白主要诱导产生中和抗体，因此基于这些蛋白的亚单位或VLP疫苗可以诱导良好的血清转换。对于甲病毒防控而言，体液免疫尤其重要，中和抗体已被证明在控制甲病毒感染中的关键作用。当前除了针对CHIKV的减毒活疫苗（Ixchiq）被FDA批准外，对于其他大多数甲病毒而言，仍没有有效的疫苗以应对潜在的公共卫生威胁。因此，开发一种通用减毒平台对于快速制备甲病毒候选减毒株至关重要且更具应用前景，这将对进一步控制甲病毒流行发挥关键的作用。
发明内容
本发明的目的在于解决现有技术中尚无有效的疫苗以应对大多数甲病毒导致的潜在公共卫生威胁，提供一种基于盖塔病毒骨架的嵌合甲病毒制备方法和应用。
为解决上述技术问题，本发明采用的技术方案如下：
第一方面，本发明保护一种嵌合甲病毒质粒载体，所述质粒载体以含有盖塔病毒（Getah Virus，GETV）基因组的感染性cDNA克隆质粒pSM-GETV-3ΔS2-CM1为骨架质粒，再通过如下（A1）或（A2）任一方式进行构建：
（A1）将盖塔病毒的ORF2基因序列替换成其他甲病毒的ORF2基因；
（A2）保留盖塔病毒Cap基因N端，而其余盖塔病毒ORF2基因序列替换为去除Cap基因N端氨基酸的其他甲病毒ORF2基因序列；
其中，所述pSM-GETV-3ΔS2-CM1的序列如SEQ ID NO:1所示。
本发明使用的骨架质粒pSM-GETV-3ΔS2-CM1为盖塔病毒减毒株的感染性cDNA克隆，以其为骨架生成的嵌合甲病毒其安全性更高，且不利于嵌合病毒逆转并恢复毒力。
优选地，所述嵌合甲病毒质粒载体分为两类，分别为pSM-3ΔS2/Alpha/ORF2（对应前述（A1）的构建方式）和pSM-3ΔS2-CM1/nsR/GE/Alpha（对应前述（A2）的构建方式），其中Alpha为其他甲病毒的替代名称。
优选地，所述其他甲病毒包括但不限于塞姆利基森林病毒、罗斯河病毒、基孔肯尼亚病毒、马亚罗病毒、阿尼昂-尼昂病毒、巴马森林病毒、委内瑞拉马脑炎病毒、东部马脑炎病毒、西部马脑炎病毒等。
优选地，所述盖塔病毒Cap基因N端为保留盖塔病毒Cap基因前123位氨基酸序列。
优选地，其余盖塔病毒ORF2基因序列替换为去除Cap基因N端前102~123位氨基酸的其他甲病毒ORF2基因序列。
优选地，本发明还提供了前文所述质粒载体pSM-3ΔS2/nsR/Alpha/ORF2和pSM-3ΔS2-CM1/nsR/GE/Alpha的制备方法，包括以下步骤：
S1、以塞姆利基森林病毒（Semliki Forest virus，SFV）作为模式病毒，具体而言，以SFV6（GenBank: KT009012.1）的cDNA为模板，以F1+R1引物扩增SFV的ORF2序列，以F2+R2、F3+R3引物分别以pSM-GETV-3ΔS2-CM1为模板扩增SgrAI和XbaI酶切位点内部的序列，以去除GETV的ORF2序列，扩增片段分别命名为EF1和EF2片段；以回收产物EF1、EF2和SFV ORF2为模板，以F2和R3为引物，通过融合PCR扩增得到含有SFV ORF2序列的EF全长片段。将pSM-GETV-3ΔS2-CM1质粒利用SgrAI和XbaI进行双酶切，然后利用同源重组酶将酶切的载体与EF全长片段连接，构建质粒pSM-3ΔS2/nsR/Alpha/ORF2；
S2、以SFV6的cDNA为模板，以F4+R4引物扩增片段CD1，以pSM-GETV-3ΔS2-CM1质粒为模板，以F5+R5引物扩增片段CD2；以回收产物CD1和CD2为模板，以F5和R4为引物，通过融合PCR扩增得到GETV/SFV嵌合Cap基因的CD全长片段；将pSM-3ΔS2/nsR/Alpha/ORF2质粒和CD全长片段分别利用BglII和BssHII进行双酶切，然后利用T4连接酶连接，构建质粒pSM-3ΔS2-CM1/nsR/GE/Alpha。
优选地，所述S1中，
所述F1序列如SEQ ID NO:2所示；
所述R1序列如SEQ ID NO:3所示；
所述F2序列如SEQ ID NO:4所示；
所述R2序列如SEQ ID NO:5所示；
所述F3序列如SEQ ID NO:6所示；
所述R3序列如SEQ ID NO:7所示。
优选地，所述S2中，
所述F4序列如SEQ ID NO:8所示；
所述R4序列如SEQ ID NO: 9所示；
所述F5序列如SEQ ID NO:10所示；
所述R5序列如SEQ ID NO:11所示。
优选地，所述PCR扩增的条件为：95℃预变性3 min；95℃变性15 s，58℃退火15 s，72℃延伸30 s/kb，共15个循环；72℃终延伸5 min。
第二方面，本发明保护一种嵌合甲病毒减毒株的制备方法，将前文所述的质粒载体转染至Vero或BHK-21细胞，获得嵌合甲病毒减毒株。
具体的，将前文所述的pSM-3ΔS2/nsR/Alpha/ORF2或pSM-3ΔS2-CM1/nsR/GE/Alpha质粒转染至Vero或BHK-21细胞，培养2-4天，每天观察细胞病变，当80%细胞出现病变时收集上清液，即获得嵌合甲病毒3ΔS2/nsR/Alpha/ORF2或3ΔS2-CM1/nsR/GE/Alpha毒株。
将上述方法制备得到的嵌合甲病毒减毒活疫苗候选株制备成嵌合甲病毒减毒活疫苗免疫易感动物或人群，可以为免疫个体带来安全且高效的免疫保护。
第三方面，本发明还保护前文所述的制备方法制备获得的嵌合甲病毒减毒株。
第四方面，本发明保护前文所述的嵌合甲病毒减毒株在制备疫苗中的应用。
优选地，所述疫苗为嵌合甲病毒减毒活疫苗。
第五方面，本发明保护一种疫苗，所述疫苗含有前文所述的嵌合甲病毒减毒株。
第六方面，本发明保护前文所述的质粒载体在制备疫苗中的应用。
优选地，所述疫苗为嵌合甲病毒减毒活疫苗。
本发明所具有的有益效果：
1）本发明将pSM-GETV-3ΔS2-CM1质粒的ORF2基因序列替换为其他甲病毒的ORF2基因序列，获得了嵌合甲病毒（3ΔS2/nsR/Alpha/ORF2或3ΔS2-CM1/nsR/GE/Alpha）。
2）本发明制备的嵌合甲病毒（以SFV为例）在Vero细胞上保持了较高的增殖能力，小鼠致病性实验显示2株嵌合毒株（3ΔS2/nsR/Alpha/ORF2或3ΔS2-CM1/nsR/GE/Alpha）对3周龄小鼠的致病性均显著降低，不会引起死亡及严重的体重下降，但保持了良好的免疫原性，可以为其提供良好的免疫保护，使免疫小鼠免受致死剂量的SFV攻毒。此外，嵌合病毒基于GETV弱毒株骨架，其安全性更高，毒力返强的风险较低。因此，基于盖塔病毒减毒株骨架的嵌合甲病毒设计策略是制备甲病毒减毒活疫苗的优秀方案。
3）本发明的质粒载体和嵌合病毒还可以用于制备表达外源基因的重组减毒株、灭活疫苗、DNA疫苗或RNA疫苗。
附图说明
图1为pSM-3ΔS2/nsR/SFV/ORF2或pSM-3ΔS2-CM1/nsR/GE/SFV的构建示意图。
图2为3ΔS2/nsR/SFV/ORF2或3ΔS2-CM1/nsR/GE/SFV的IFA鉴定。
图3为3ΔS2/nsR/SFV/ORF2或3ΔS2-CM1/nsR/GE/SFV在Vero细胞上的多步生长曲线。
图4为3ΔS2/nsR/SFV/ORF2或3ΔS2-CM1/nsR/GE/SFV攻毒后生存曲线。
图5为3ΔS2/nsR/SFV/ORF2或3ΔS2-CM1/nsR/GE/SFV攻毒小鼠14天的抗体检测。
图6为小鼠免疫嵌合病毒14天后攻毒亲本SFV的生存曲线。
具体实施方式
下面结合附图和具体实施例对本发明作进一步的说明。所用试剂或者仪器设备未注明生产厂商的，均视为可以通过市场购买的常规产品。
实施例1、构建质粒载体pSM-3ΔS2/nsR/SFV/ORF2
以塞姆利基森林病毒（Semliki Forest virus，SFV）作为模式病毒验证嵌合甲病毒的设计策略。基于已建立的盖塔病毒减毒株的全长cDNA克隆pSM-GETV-3ΔS2-CM1，使用SgrAI和XbaI酶切位点之间的片段进行ORF2基因序列的替换，引物如表1所示。具体而言，以SFV6的cDNA为模板，以表1中的F1 +R1引物扩增SFV的ORF2序列，以表1中F2+R2、F3+R3引物分别以pSM-GETV-3ΔS2-CM1为模板扩增SgrAI和XbaI酶切位点内部的序列，以去除GETV的ORF2序列，扩增片段分别命名为EF1和EF2片段。以回收产物EF1、EF2和SFV ORF2为模板，以F2和R3为引物，通过融合PCR扩增得到含有SFV ORF2序列的EF全长片段。将pSM-GETV-3ΔS2-CM1质粒利用SgrAI和XbaI进行双酶切，条件为：SgrAI内切酶和XbaI内切酶（Buffer为Cutsmart）37℃酶切1 h后回收载体片段，最后通过同源重组酶将载体与EF全长片段连接获得重组质粒，转化至DH10B感受态细胞后，挑菌至1.5 mL EP管中过夜培养，所述LB液体培养基为卡纳抗性。使用F2+R3引物进行菌液PCR鉴定。对于阳性菌液通过测序验证引入的突变是否正确，将该质粒命名为pSM-3ΔS2/nsR/SFV/ORF2（图1）。
其中，PCR扩增的条件为：95℃预变性3 min；95℃变性15 s，58℃退火15 s，72℃延伸30 s/kb，共15个循环；72℃终延伸5 min。
实施例2、构建质粒载体pSM-3ΔS2-CM1/nsR/GE/SFV
以SFV6的cDNA为模板，以表1中的F4+R4引物扩增片段CD1，以pSM-GETV-3ΔS2-CM1质粒为模板，以表1中的F5+R5引物扩增片段CD2。以回收产物CD1和CD2为模板，以F5和R4为引物，通过融合PCR扩增得到GETV/SFV嵌合Cap基因的CD全长片段。将pSM-3ΔS2/nsR/Alpha/ORF2质粒和CD全长片段分别利用BglII和BssHII进行双酶切，条件为：BglII内切酶（Buffer为3.1）37℃酶切30 min，然后回收片段，使用BssHII内切酶（Buffer为3.1）50℃酶切30 min后回收片段，最后通过T4连接酶过夜连接获得重组质粒，转化至DH10B感受态细胞后，挑菌至1.5 mL EP管中过夜培养，所述LB液体培养基为卡纳抗性。使用F5+R4引物进行菌液PCR鉴定。对于阳性菌液通过测序验证引入的突变是否正确，将该质粒命名为pSM-3ΔS2-CM1/nsR/GE/SFV（图1）。
其中，PCR扩增的条件为：95℃预变性3 min；95℃变性15 s，58℃退火15 s，72℃延伸30 s/kb，共15个循环；72℃终延伸5 min。
表1：实施例1和2中所使用的引物序列
实施例3、转染拯救嵌合甲病毒3ΔS2/nsR/SFV/ORF2或3ΔS2-CM1/nsR/GE/SFV
使用去内毒素质粒提取试剂盒提取pSM-3ΔS2/nsR/SFV/ORF2或pSM-3ΔS2-CM1/nsR/GE/SFV质粒，然后将2 ug的pSM-3ΔS2/nsR/SFV/ORF2或pSM-3ΔS2-CM1/nsR/GE/SFV加入100 uL Opti-MEM中，在加入4 uL Lip2000转染试剂，充分混匀，室温避光孵育15 min后，将转染复合物添加至35 mm皿的Vero或BHK-21细胞中，于转染后4 h换液。继续培养2-4天，每天观察病变情况。当观察到80%细胞病变时，立即置于-80℃冰箱冻融细胞，并离心后收集上清液作为P0代次种毒。将10-20 uL的P0代次3ΔS2/nsR/SFV/ORF2或3ΔS2-CM1/nsR/GE/SFV重新接种至Vero细胞扩繁病毒，36-48 h后收样，所收集的上清液作为P1代次病毒。使用P1代次病毒进行后续实验。
实施例4、嵌合甲病毒3ΔS2/nsR/SFV/ORF2或3ΔS2-CM1/nsR/GE/SFV的IFA鉴定
将3ΔS2/nsR/SFV/ORF2或3ΔS2-CM1/nsR/GE/SFV接种至96孔板中的Vero细胞进行IFA鉴定。具体而言：将MOI=0.01的3ΔS2/nsR/SFV/ORF2或3ΔS2-CM1/nsR/GE/SFV接种至96孔板的Vero细胞中，在感染8 h后，使用SFV小鼠多克隆抗体进行IFA鉴定。具体的，使用4%多聚甲醛固定感染的细胞15 min，然后使用0.1% Triton X-100进行透化10 min，使用PBST清洗3次；然后加入5%脱脂奶粉进行室温封闭1 h，使用PBST清洗3次；然后加入SFV小鼠多克隆抗体（1:200）室温孵育2 h，使用PBST清洗3次；然后加入FITC-goat anti-mouse抗体（1:1000）室温避光孵育1 h，使用PBST清洗3次后，加入DAPI（1:1000）室温避光孵育10 min，于倒置荧光显微镜观察绿色荧光。
结果显示，3ΔS2/nsR/SFV/ORF2或3ΔS2-CM1/nsR/GE/SFV可以被SFV小鼠多克隆抗体识别而显示出绿色荧光，而模拟感染的细胞则没有绿色荧光（图2）。说明3ΔS2/nsR/SFV/ORF2或3ΔS2-CM1/nsR/GE/SFV的抗原性没有被改变。
实施例5、3ΔS2/nsR/SFV/ORF2或3ΔS2-CM1/nsR/GE/SFV在Vero细胞的多步生长曲线
将3ΔS2/nsR/SFV/ORF2或3ΔS2-CM1/nsR/GE/SFV以MOI=0.1接种至24孔板的单层Vero细胞，在37℃孵育1 h后，弃去病毒液，并使用预冷的PBS洗涤3次，其后更换含2%血清的DMEM培养液放至培养箱继续培养。分别于接种后12、24、36和48 h收集培养上清，并通过TCID50法测定培养上清中的病毒滴度。结果显示，在Vero细胞上，3ΔS2/nsR/SFV/ORF2或3ΔS2-CM1/nsR/GE/SFV可以复制到较高滴度（图3）。
实施例6、3ΔS2/nsR/SFV/ORF2或3ΔS2-CM1/nsR/GE/SFV对3周龄ICR鼠的毒力验证
3周龄ICR鼠20只，每组5只饲养在单独的笼子中。将亲本SFV6、3ΔS2/nsR/SFV/ORF2、3ΔS2-CM1/nsR/GE/SFV稀释至105TCID50/mL，皮下接种100 uL稀释的病毒液。空白对照小鼠皮下接种100 uL无菌PBS。接种后每天观察并记录小鼠的死亡情况和体重变化。结果表明3ΔS2/nsR/SFV/ORF2、3ΔS2-CM1/nsR/GE/SFV接种3周龄ICR鼠不会引起死亡和明显的体重下降。而亲本SFV6毒株则引起小鼠快速死亡（图4）和体重下降。以上结果说明，3ΔS2/nsR/SFV/ORF2、3ΔS2-CM1/nsR/GE/SFV在易感的ICR鼠中具有高度的安全性。
实施例7、3ΔS2/nsR/SFV/ORF2或3ΔS2-CM1/nsR/GE/SFV的免疫原性验证
将实施例6中存活的3ΔS2/nsR/SFV/ORF2或3ΔS2-CM1/nsR/GE/SFV组小鼠以及空白对照组小鼠（每组5只）用于免疫原性验证。在初次感染14天后，收集不同组老鼠的血清测定针对SFV p62-E1的IgG抗体。随后，对3组老鼠分别皮下接种100 uL稀释的病毒液（含有104TCID50的SFV6）。接种后每天观察并记录小鼠的死亡情况和体重变化。结果表明3ΔS2/nsR/SFV/ORF2、3ΔS2-CM1/nsR/GE/SFV接种3周龄ICR鼠后产生了良好的血清转化（图5），可以保护其免受随后致死剂量的SFV6攻毒，而空白对照组小鼠则表现出快速的体重下降及死亡（图6）。以上结果说明，3ΔS2/nsR/SFV/ORF2、3ΔS2-CM1/nsR/GE/SFV在ICR鼠中具有高度的免疫原性。
综上所述，本发明制备的嵌合甲病毒3ΔS2/nsR/SFV/ORF2、3ΔS2-CM1/nsR/GE/SFV在Vero细胞上保持了较高的增殖能力，对ICR小鼠的毒力显著减弱，不引起任何临床症状和死亡，但保持了良好的免疫原性，可以保护免疫小鼠免受随后致死剂量的SFV6攻毒。因此，基于盖塔病毒弱毒株骨架的嵌合甲病毒设计策略是制备甲病毒减毒活疫苗的优秀方案，可以针对性地快速开发不同甲病毒的减毒活疫苗。
本发明的保护内容不局限于以上实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求为保护范围。
Claims (10)
1.一种质粒载体，其特征在于，所述质粒载体以含有盖塔病毒基因组的感染性cDNA克隆质粒pSM-GETV-3ΔS2-CM1为骨架质粒，再通过如下（A1）或（A2）任一方式进行构建：
（A1）将盖塔病毒的ORF2基因序列替换成其他甲病毒的ORF2基因；
（A2）保留盖塔病毒Cap基因前123位氨基酸序列，而其余盖塔病毒ORF2基因序列替换为去除Cap基因N端前102~123位氨基酸的其他甲病毒ORF2基因序列；
其中，所述质粒pSM-GETV-3ΔS2-CM1的序列如SEQ ID NO:1所示。
2.根据权利要求1所述的质粒载体，其特征在于，通过将盖塔病毒的ORF2基因序列替换成其他甲病毒ORF2基因，构建质粒载体的方法具体如下：以塞姆利基森林病毒的cDNA为模板，以F1和R1为引物扩增塞姆利基森林病毒的ORF2序列，以F2和R2为引物、F3和R3为引物分别以pSM-GETV-3ΔS2-CM1为模板扩增SgrAI和XbaI酶切位点内部的序列，以去除盖塔病毒的ORF2序列，扩增片段分别命名为EF1和EF2片段；以回收产物EF1、EF2和塞姆利基森林病毒的ORF2为模板，以F2和R3为引物，通过融合PCR扩增得到含有塞姆利基森林病毒ORF2序列的EF全长片段；将pSM-GETV-3ΔS2-CM1质粒利用SgrAI和XbaI进行双酶切并回收载体片段，然后利用同源重组酶将载体和EF全长片段连接，构建质粒pSM-3ΔS2/nsR/Alpha/ORF2；其中，
所述F1序列如SEQ ID NO:2所示；
所述R1序列如SEQ ID NO: 3所示；
所述F2序列如SEQ ID NO:4所示；
所述R2序列如SEQ ID NO:5所示；
所述F3序列如SEQ ID NO:6所示；
所述R3序列如SEQ ID NO: 7所示。
3.根据权利要求1所述的质粒载体，其特征在于，通过保留盖塔病毒Cap基因前123位氨基酸序列，而其余盖塔病毒ORF2基因序列替换为去除Cap基因N端前102~123位氨基酸的其他甲病毒ORF2基因序列，构建质粒载体的方法具体如下：
以塞姆利基森林病毒的cDNA为模板，以F4和R4为引物扩增片段CD1，以pSM-GETV-3ΔS2-CM1质粒为模板，以F5和R5为引物扩增片段CD2，以回收产物CD1和CD2为模板，以F5和R4为引物，通过融合PCR扩增得到盖塔病毒/塞姆利基森林病毒嵌合Cap基因的CD全长片段，将权利要求2所述的pSM-3ΔS2/nsR/Alpha/ORF2质粒和CD全长片段分别利用BglII和BssHII进行双酶切，然后利用T4连接酶连接，构建质粒pSM-3ΔS2-CM1/nsR/GE/Alpha；其中，
所述F4序列如SEQ ID NO:8所示；
所述R4序列如SEQ ID NO:9所示；
所述F5序列如SEQ ID NO:10所示
所述R5序列如SEQ ID NO:11所示。
4.权利要求1-3任一所述的质粒载体在制备疫苗中的应用。
5.根据权利要求4所述的应用，其特征在于，所述疫苗为嵌合甲病毒减毒活疫苗。
6.一种嵌合甲病毒减毒株的制备方法，其特征在于，将权利要求1-3所述的质粒载体转染至Vero或BHK-21细胞，获得嵌合甲病毒减毒株。
7.权利要求6所述的制备方法制备获得的嵌合甲病毒减毒株。
8.权利要求7所述的嵌合甲病毒减毒株在制备疫苗中的应用。
9.根据权利要求8所述的应用，其特征在于，所述疫苗为嵌合甲病毒减毒活疫苗。
10.一种疫苗，其特征在于，所述疫苗含有权利要求7所述的嵌合甲病毒减毒株。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410557841.6A CN118127077B (zh) | 2024-05-08 | 2024-05-08 | 基于盖塔病毒骨架的嵌合甲病毒制备方法和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410557841.6A CN118127077B (zh) | 2024-05-08 | 2024-05-08 | 基于盖塔病毒骨架的嵌合甲病毒制备方法和应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN118127077A true CN118127077A (zh) | 2024-06-04 |
| CN118127077B CN118127077B (zh) | 2024-07-16 |
Family
ID=91244370
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410557841.6A Active CN118127077B (zh) | 2024-05-08 | 2024-05-08 | 基于盖塔病毒骨架的嵌合甲病毒制备方法和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118127077B (zh) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050221493A1 (en) * | 2002-12-04 | 2005-10-06 | Crucell Holland B.V. | Recombinant virus production for the manufacturing of vaccines |
| WO2017172698A1 (en) * | 2016-03-31 | 2017-10-05 | Takeda Vaccines, Inc. | Live, attenuated alphavirus constructs and methods and uses thereof |
| US20210268098A1 (en) * | 2018-08-03 | 2021-09-02 | Uab Research Foundation | Methods and compositions for alphavirus vaccine |
| CN116334009A (zh) * | 2023-03-21 | 2023-06-27 | 广西大学 | 表达乙型脑炎病毒e蛋白的重组盖他病毒及其构建方法和疫苗应用 |
| CN116769735A (zh) * | 2023-06-07 | 2023-09-19 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | 一种甲病毒减毒株的构建方法及应用 |
-
2024
- 2024-05-08 CN CN202410557841.6A patent/CN118127077B/zh active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050221493A1 (en) * | 2002-12-04 | 2005-10-06 | Crucell Holland B.V. | Recombinant virus production for the manufacturing of vaccines |
| WO2017172698A1 (en) * | 2016-03-31 | 2017-10-05 | Takeda Vaccines, Inc. | Live, attenuated alphavirus constructs and methods and uses thereof |
| US20210268098A1 (en) * | 2018-08-03 | 2021-09-02 | Uab Research Foundation | Methods and compositions for alphavirus vaccine |
| CN116334009A (zh) * | 2023-03-21 | 2023-06-27 | 广西大学 | 表达乙型脑炎病毒e蛋白的重组盖他病毒及其构建方法和疫苗应用 |
| CN116769735A (zh) * | 2023-06-07 | 2023-09-19 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | 一种甲病毒减毒株的构建方法及应用 |
Non-Patent Citations (2)
| Title |
|---|
| ZHAI X等: "LDLR is used as a cell entry receptor by multiple alphaviruses", NAT COMMUN, vol. 15, no. 1, 20 January 2024 (2024-01-20), pages 1 - 16 * |
| 莫清荣等: "盖他病毒Cap和E2蛋白多克隆抗体的制备", 动物医学进展, vol. 43, no. 9, 7 September 2022 (2022-09-07), pages 44 - 49 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN118127077B (zh) | 2024-07-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Warzecha et al. | Oral immunogenicity of human papillomavirus-like particles expressed in potato | |
| Denis et al. | Immunogenicity of papaya mosaic virus-like particles fused to a hepatitis C virus epitope: evidence for the critical function of multimerization | |
| Santos et al. | Development of transgenic alfalfa plants containing the foot and mouth disease virus structural polyprotein gene P1 and its utilization as an experimental immunogen | |
| US20070128213A1 (en) | Novel plant virus particles and methods of inactivation thereof | |
| WO2021088825A1 (zh) | F基因型腮腺炎病毒减毒株及构建方法和应用 | |
| CN110156896B (zh) | 重组口蹄疫病毒样颗粒及其制备方法和应用 | |
| US20180371026A1 (en) | Feline calicivirus vaccine | |
| KR101608121B1 (ko) | 노다바이러스의 바이러스 유사입자의 생산방법, 이를 발현하는 효모 세포주 및 이를 포함하는 백신 조성물 | |
| CN104524564B (zh) | 一种鲫鱼疱疹病毒病复合疫苗制剂及制备方法和应用 | |
| CN101264323A (zh) | 呼吸道合胞病毒亚单位疫苗、制备方法和应用 | |
| EP1752468A1 (en) | Method for the production of empty viral capsids in yeasts, said capsids comprising proteins derived from pvp2 of the virus that causes infectious bursal disease (ibdv) | |
| CN113293148B (zh) | 一种h基因替换的嵌合麻疹减毒株的构建 | |
| CN116200347A (zh) | 一种gI、gE和TK三基因缺失株猫疱疹病毒疫苗及其应用 | |
| CN113388587A (zh) | 表达牛病毒性腹泻e2基因的重组牛结节疹病毒及其应用 | |
| CN102816246B (zh) | 一种人巨细胞病毒免疫原融合蛋白及其制备方法和用途 | |
| CN118127077B (zh) | 基于盖塔病毒骨架的嵌合甲病毒制备方法和应用 | |
| CN111996201B (zh) | 一种重组a型口蹄疫病毒vp1基因的塞内卡重组病毒、重组疫苗株及其制备方法和应用 | |
| CN110257428B (zh) | 一种表达猪圆环病毒3型orf2基因的重组腺病毒及制备方法与应用 | |
| CN102127554A (zh) | 一种日本脑炎颗粒疫苗及其制备方法和应用 | |
| CN116102660B (zh) | 一种猪细小病毒基因工程表位疫苗及其制备方法 | |
| Sharma et al. | Plant-based vaccines: potentiality against severe acute respiratory syndrome coronavirus 2 | |
| US20230203101A1 (en) | Coronavirus Disease 2019(COVID -19) Recombinant Spike Protein Forming Trimer, Method for Mass Producing Recombinant Spike Protein in Plants, and Method for Preparing Vaccine Composition on Basis Thereof | |
| CN118086395B (zh) | 一种盖塔病毒质粒载体、弱毒株及其制备方法和应用 | |
| CN105400799B (zh) | 一种乙脑/黄热嵌合病毒及其制备方法和应用 | |
| CN113416714A (zh) | 一种表达埃博拉gp蛋白的重组病毒及其制备方法和应用 |
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 |