CN117126818B - 利用ABE构建gE基因缺失PRV毒株的方法和应用 - Google Patents
利用ABE构建gE基因缺失PRV毒株的方法和应用 Download PDFInfo
- Publication number
- CN117126818B CN117126818B CN202311385572.1A CN202311385572A CN117126818B CN 117126818 B CN117126818 B CN 117126818B CN 202311385572 A CN202311385572 A CN 202311385572A CN 117126818 B CN117126818 B CN 117126818B
- Authority
- CN
- China
- Prior art keywords
- gene
- sgrna
- prv
- fragments
- virus
- 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
- 101150072564 gE gene Proteins 0.000 title claims abstract description 31
- 238000000034 method Methods 0.000 title claims abstract description 17
- 238000012224 gene deletion Methods 0.000 title abstract description 8
- 108091027544 Subgenomic mRNA Proteins 0.000 claims abstract description 25
- 239000012634 fragment Substances 0.000 claims abstract description 23
- 239000013612 plasmid Substances 0.000 claims abstract description 23
- 108020004414 DNA Proteins 0.000 claims abstract description 17
- 241000700605 Viruses Species 0.000 claims abstract description 17
- 239000000047 product Substances 0.000 claims abstract description 15
- 238000001890 transfection Methods 0.000 claims abstract description 13
- 102000053602 DNA Human genes 0.000 claims abstract description 12
- 108091034117 Oligonucleotide Proteins 0.000 claims abstract description 11
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000007788 liquid Substances 0.000 claims abstract description 10
- 241000894006 Bacteria Species 0.000 claims abstract description 9
- 108091081024 Start codon Proteins 0.000 claims abstract description 8
- 238000001976 enzyme digestion Methods 0.000 claims abstract description 8
- 108091008146 restriction endonucleases Proteins 0.000 claims abstract description 8
- 239000006228 supernatant Substances 0.000 claims abstract description 6
- 230000003321 amplification Effects 0.000 claims abstract description 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 4
- 238000012216 screening Methods 0.000 claims abstract description 4
- 238000005119 centrifugation Methods 0.000 claims abstract description 3
- 241000701093 Suid alphaherpesvirus 1 Species 0.000 claims description 30
- 108090000623 proteins and genes Proteins 0.000 claims description 22
- 238000000137 annealing Methods 0.000 claims description 10
- 229930024421 Adenine Natural products 0.000 claims description 9
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims description 9
- 229960000643 adenine Drugs 0.000 claims description 9
- 239000013598 vector Substances 0.000 claims description 9
- 238000010276 construction Methods 0.000 claims description 7
- 238000004321 preservation Methods 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 5
- 239000013604 expression vector Substances 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 5
- 239000012091 fetal bovine serum Substances 0.000 claims description 4
- 208000009305 pseudorabies Diseases 0.000 claims description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 3
- 238000007747 plating Methods 0.000 claims description 3
- 230000002194 synthesizing effect Effects 0.000 claims description 3
- 229960005486 vaccine Drugs 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 claims description 2
- 230000003698 anagen phase Effects 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 6
- 230000033228 biological regulation Effects 0.000 abstract description 2
- 239000011248 coating agent Substances 0.000 abstract 1
- 238000000576 coating method Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 15
- 238000012163 sequencing technique Methods 0.000 description 14
- 108091033409 CRISPR Proteins 0.000 description 10
- 230000035772 mutation Effects 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 6
- 238000010354 CRISPR gene editing Methods 0.000 description 4
- 241000282887 Suidae Species 0.000 description 4
- 238000009395 breeding Methods 0.000 description 4
- 230000001488 breeding effect Effects 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 230000003612 virological effect Effects 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 239000012154 double-distilled water Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000030279 gene silencing Effects 0.000 description 3
- 238000010362 genome editing Methods 0.000 description 3
- 230000003902 lesion Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 229920000936 Agarose Polymers 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 206010060860 Neurological symptom Diseases 0.000 description 2
- 238000010459 TALEN Methods 0.000 description 2
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 description 2
- 108010067390 Viral Proteins Proteins 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000026731 phosphorylation Effects 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 230000001018 virulence Effects 0.000 description 2
- 238000001262 western blot Methods 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
- 102000006267 AMP Deaminase Human genes 0.000 description 1
- 108700016228 AMP deaminases Proteins 0.000 description 1
- 108010052875 Adenine deaminase Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 208000000059 Dyspnea Diseases 0.000 description 1
- 206010013975 Dyspnoeas Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 206010020741 Hyperpyrexia Diseases 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 102000014736 Notch Human genes 0.000 description 1
- 108010070047 Notch Receptors Proteins 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 206010000210 abortion Diseases 0.000 description 1
- 231100000176 abortion Toxicity 0.000 description 1
- 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 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 230000000981 bystander Effects 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 101150038500 cas9 gene Proteins 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000009615 deamination Effects 0.000 description 1
- 238000006481 deamination reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000005782 double-strand break Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000012173 estrus Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000037440 gene silencing effect Effects 0.000 description 1
- 238000012226 gene silencing method Methods 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000009438 off-target cleavage Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000032696 parturition Effects 0.000 description 1
- 230000007505 plaque formation Effects 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 208000002254 stillbirth Diseases 0.000 description 1
- 231100000537 stillbirth Toxicity 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000005723 virus inoculator Substances 0.000 description 1
- 230000005727 virus proliferation Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
-
- 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
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
- C12N2710/16721—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
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
- C12N2710/16722—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
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
- C12N2710/16734—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
- C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
- C12N2710/16761—Methods of inactivation or attenuation
- C12N2710/16762—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
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- 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)
- Virology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (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)
Abstract
本发明公开了利用ABE构建gE基因缺失PRV毒株的方法和应用,涉及生物技术领域,选择PRV病毒中gE基因的起始密码子作为靶标位点,设计sgRNA序列,根据sgRNA序列分别合成单链寡核苷酸,将单链寡核苷酸退火获得带粘性末端的双链DNA片段,将载体经限制性内切酶酶切后回收片段,再与带粘性末端的双链DNA片段连接,获得连接产物,再转化至感受态细胞中,涂板筛选培养,挑阳性菌扩大培养,对阳性菌液抽提质粒;将质粒转染,转染24h后,吸取病毒液,离心收集上清液。本发明的PRV毒株不存在结构变异,仅是修改少量ATG的中A或T的碱基,就能够实现gE表达的终止,而且该序列潜在的调控作用受到的影响最少。
Description
技术领域
本发明涉及生物技术领域,具体涉及利用ABE构建gE基因缺失PRV毒株的方法和应用。
背景技术
伪狂犬病是一种伪狂犬病病毒(PRV)感染引起的高度接触性、败血性及烈性传染病,各年龄段的猪均易感,妊娠母猪感染后可引发流产、死胎、木乃伊胎或产弱胎;仔猪感染后可引起高热、神经症状及呼吸困难,新生仔猪感染后多表现为神经症状;种猪感染后则造成种猪不育,母猪不发情,配种困难,公猪睾丸肿胀萎缩,丧失种用能力。PRV为双股DNA病毒,基因组全长约150kb,平均G+C含量高达74%,由独特长区段UL、独特短区段US及位于两侧的末端重复序列(TR)和内部重复序列(IR)组成。研究发现gE是主要毒力基因,但不是病毒增殖所必需的,因此沉默上述毒力基因,可以降低病毒的毒性,但不影响免疫原性,可作为疫苗的候选株,用于防控和净化伪狂犬病。
早期的基因编辑技术常利用DNA同源重组原理,包括ZFNs和TALENs,但因其设计复杂、敲除效率低、脱靶严重、成本高、可操作性差等缺点,发展受到了限制。CRISPR/Cas9系统自发现以来由于其高效、方便和应用范围广等特点,成为最受欢迎的基因改造工具,实现了基因的成功敲除。然而与ZFNs和TALENs等技术类似,CRISPR/Cas9实现基因编辑依赖于DNA双链断裂的产生,触发修复,造成非预期的碱基突变或脱靶切割及基因组的结构变异。为了克服这一不足,单碱基编辑技术的出现为实现精确高效的碱基转换带来了希望。
单碱基编辑技术是利用无核酸酶切割活性或切割单链产生缺口活性的改造型Cas蛋白和脱氨酶进行融合,依靠sgRNA准确地将脱氨酶锚定到靶位点,进行碱基脱氨,从而实现单碱基的精确编辑,极大提高了碱基编辑的精确度和效率。其中,tRNA腺苷酸脱氨酶(TadA)与nCas9融合,开发获得可将腺嘌呤精准转换成鸟嘌呤的新型单碱基转化系统(Adenine base editor,ABE),经过7轮改造,筛选到效率较高的ABE版本ABE7.10,其有效编辑窗口在sgRNA的第4-7位。继续改进ABE7.10的脱氨酶组分,从而产生了目前编辑效率较高的ABE版本:ABE8e。与ABE7.10相比,ABE8e包含8个额外的突变,可将活性提高590倍,且与各种Cas9或Cas12同源物配对时,可以大大提高编辑效率。自ABE工具被开发后,研究证实单碱基编辑器可在多个物种中进行高效率工作,如细菌、植物及哺乳动物,但目前在病毒基因组改造的应用未见报道。
现有的ZFNs、TALENs耗时耗力使用昂贵,CRISPR/Cas9效率低,筛选困难,同时因为基因组的断裂造成结构变异的产生,而其他的碱基编辑工具虽然可以实现沉默的目的,但产生的旁观者效应较多,同时受PAM限制,有些区域无法编辑。
发明内容
本发明的目的在于至少解决现有技术中存在的技术问题之一,提供利用腺嘌呤碱基编辑器构建gE基因缺失PRV毒株的方法和应用。
本发明的技术解决方案如下:
一种gE基因缺失的PRV毒株,于2023年6月30日,保藏在中国典型培养物保藏中心(CCTCC),保藏地址为中国湖北省武汉市武汉大学,保藏编号CCTCC NO:V202323,命名为:猪伪狂犬病毒PRV-ΔgE-ABE,Porcine pseudorabies virus PRV-ΔgE-ABE,所述gE基因缺失的PRV毒株利用腺嘌呤碱基编辑器构建得到。
优选地,在野生型PRV编码gE基因的第2位碱基T突变成C,突变后的gE基因如SEQID NO.1所示。
利用ABE构建gE基因缺失PRV毒株的方法,包括以下步骤:
S1:构建sgRNA骨架质粒;
选择PRV病毒中gE基因的起始密码子作为靶标位点,利用腺嘌呤碱基编辑器设计一条sgRNA序列,根据sgRNA序列分别合成单链寡核苷酸,将单链寡核苷酸退火获得带粘性末端的双链DNA片段,将pU6-sgRNA-Puro-2A-EGFP载体经限制性内切酶酶切后回收片段,再与上述带粘性末端的双链DNA 片段连接,获得连接产物,即sgRNA表达载体;
将连接产物转化至感受态细胞中,涂板筛选培养,挑阳性菌扩大培养,对阳性菌液抽提质粒;
S2:质粒转染;
将质粒转染至Vero81细胞中,转染后,吸取病毒液,离心收集上清液。
作为本发明的优选方案,sgRNA序列如SEQ ID NO.2所示。
作为本发明的优选方案,限制性内切酶采用BbsⅠ酶。
作为本发明的优选方案,转染前,将Vero81细胞在含有胎牛血清的培养液中进行培养,培养至对数生长期。
作为本发明的优选方案,单链寡核苷酸的序列如下:
NG-ABE8e-gE-F: CACCGGCCGCATGGTCTCAACCCC;
NG-ABE8e-gE-R: AAACGGGGTTGAGACCATGCGGCC。
作为本发明的优选方案, NG-ABE8e为表达nCas9蛋白的质粒。
本发明还公开了gE基因缺失的PRV毒株,采用如上任一构建方法制得。
本发明还公开了一种gE基因缺失的PRV毒株在制备伪狂犬病疫苗上的应用。
本发明的有益效果是:
本发明采用ABE方法,参考图1,修改起始密码子ATG,可以从源头阻断蛋白的翻译,能够作为全新的基因沉默技术,补充现有技术中编辑位点的限制。运用该技术制备的gE基因缺失的PRV毒株不存在结构变异,只是修改少量ATG的中A或T的碱基,就能够实现gE表达的终止,而且该序列潜在的调控作用受到的影响最少。
本发明效率高,对病毒基因的改变小,不引起病毒基因组出现大的结构变异,成本低。
本发明的一种gE基因缺失的PRV毒株,于2023年6月30日,保藏在中国典型培养物保藏中心(CCTCC),保藏地址为中国湖北省武汉市武汉大学,保藏编号CCTCC NO:V202323,命名为:猪伪狂犬病毒PRV-ΔgE-ABE,Porcine pseudorabies virus PRV-ΔgE-ABE。
附图说明
图1为本发明构建gE基因缺失的PRV毒株的原理流程图;
图2为本发明gE基因首个起始密码子突变后PCR产物核酸电泳凝胶图;其中,泳道M为DL2000 Marker,泳道1为测序用目的条带;
图3为本发明编辑病毒gE基因测序图谱;其中,下划线为预期突变基因,三角形为突变位点,箭头为突变后结果;
图4为传代10次后PRV-ΔgE测序图谱;
图5为PRV-ΔgE株gE蛋白表达验证结果图。
具体实施方式
以下以具体实施例对本发明的技术方案作进一步说明。
下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂。
NG-ABE8e载体:Addgene,货号:138491;
NG-ABE8e载体中,由CMV启动子启动相关蛋白的表达;
单碱基编辑器NG-ABE8e表达用于点突变的腺嘌呤脱氨酶;
sgRNA骨架质粒包括结合靶序列的结合区和结合cas9蛋白的结合区。
一、构建sgRNA骨架质粒
本实施例选择gE基因的起始密码子作为靶标位点;
设计一条sgRNA,其识别序列为:
NG-ABE8e-gE: GGCCGCATGGTCTCAACCCC(SEQ ID NO.2);
根据设计出的序列分别合成单链寡核苷酸,单链寡核苷酸的序列如下:
NG-ABE8e-gE-F: CACCGGCCGCATGGTCTCAACCCC(SEQ ID NO.3);
NG-ABE8e-gE-R: AAACGGGGTTGAGACCATGCGGCC(SEQ ID NO.4);
将上述NG-ABE8e-gE-F和NG-ABE8e-gE-R退火获得带粘性末端的双链DNA片段;
将pU6-sgRNA-Puro-2A-EGFP载体经BbsⅠ酶切后回收片段,再与上述带粘性末端的双链DNA 片段连接,获得sgRNA表达载体;
单碱基编辑器NG-ABE8e包括Cas9(N),在本实施例中,具体可以为表达nCas9蛋白的质粒。
(1)构建双链sgRNA
下表为磷酸化退火的体系;
表1为磷酸化退火的体系
试剂 | 体系(μl) |
正向单链(100μM) | 1 |
反向单链(100μM) | 1 |
10×T4 DNA 连接缓冲液(NEB) | 1 |
T4 PNK(NEB) | 1 |
双蒸水 | 补充至10μl |
梯度退火程序如表2所示:
表2为梯度退火程序
温度 | 时间 | 梯度 |
37℃ | 30min | —— |
95℃ | 5min | —— |
95-85℃ | 1min | -5℃ |
85-75℃ | 1min | -5℃ |
75-65℃ | 1min | -5℃ |
65-55℃ | 1min | -5℃ |
55-45℃ | 1min | -5℃ |
45-35℃ | 1min | -5℃ |
35-25℃ | 1min | -5℃ |
4℃ | Hold | —— |
(2)构建表达线性化骨架载体质粒
下表(表3)为PX459载体的酶切体系。
表3为PX459载体的酶切体系
试剂 | 体积(μL) |
10×NEB | 5 |
BbsⅠ限制性内切酶 | 1 |
PX459载体(2ug) | 3.5 |
双蒸水 | 40.5 |
总计 | 50 |
反应程序:上述酶切体系置于37℃水浴反应2h,得到酶切产物。
将上述酶切产物进行琼脂糖凝胶电泳检测,电泳结束后,胶回收目的条带。
(3)构建表达sgRNA的骨架质粒
下表(表4)为连接反应体系。
表4为连接反应体系
试剂 | 体积(μL) |
10×T4 DNA连接缓冲液 | 1 |
T4 DNA 连接酶(NEB) | 1 |
线性化PX459载体(50ng) | 2 |
稀释200倍的gE-sgRNA退火产物 | 1 |
双蒸水 | 5 |
总计 | 10 |
连接产物通过常规方法转化至Top10感受态细胞中,取适量菌液涂布于含氨苄青霉素的LB平板,待平板表面液体蒸发,倒置于37℃温箱过夜培养16-18h,观察转化情况,挑取5个单克隆菌落,震荡培养后测序,进一步选取测序正确的阳性菌落克隆扩大培养,抽提用于细胞转染的无内毒质粒,置于-20℃保存。
二、质粒转染
(1)转染前一天,用胰蛋白酶消化生长良好的Vero81细胞,将1×106细胞转接至六孔板,加入2mL含10%(v/v)胎牛血清的DMEM培养液(均购自Gibco),37℃、5%(v/v)CO2培养至对数生长期。
(2)参照Lipofectamine3000官方说明书进行细胞转染,转染的DNA 总量为3μg,包含病毒基因组DNA和NG-ABE8e质粒、sgRNA表达载体质粒,转染质量各1μg。
(3)转染后24h,使用显微镜观察有无PRV特征性病变,待观察到病变后,将六孔板置于-80℃-常温冻融三次,收取病毒液,8000rpm离心5分钟,吸取上清液,取一部分提取基因测序。根据表5合成检测引物,并使用相对应引物扩增目的基因,程序如表6,扩增时采用引物对中Tm值最低的温度作为PCR反应的退火温度,根据所用聚合酶的特性将延伸时间设为7s。琼脂糖凝胶电泳分析PCR产物结果,切取鉴定成功的目的片段,送上海生工生物技术有限公司测序。
表 5 PCR检测引物的合成序列
表 6 PCR程序
温度(℃) | 时间 | ||
预变性 | 98 | 30 s | |
变性 | 98 | 10 s | 35 循环 |
退火 | 57 | 5 s | 35循环 |
延伸 | 72 | 7 s | 35循环 |
总延伸 | 72 | 1 min | |
短期储存 | 4 | 30min |
结果:使用gE-T-F、gE-T-R作为上下游引物,进行PCR检测,获得符合预期大小的目的条带,PCR产物凝胶电泳结果如图2。
(4)测序成功后将测序结果与野生型PRV基因进行比对,获得详细的突变信息。
测序结果如图3,对比结果显示将碱基T成功突变成碱基C,仅还有少量T残余,突变后的gE基因如SEQ ID NO.1所示。
上述获得的gE基因缺失的PRV毒株,于2023年6月30日,保藏在中国典型培养物保藏中心,保藏编号CCTCC NO:V202323,命名为:猪伪狂犬病毒PRV-ΔgE-ABE,Porcinepseudorabies virus PRV-ΔgE-ABE。
三、蚀斑纯化突变毒株
(1)病毒上清液按十倍比连续稀释10至106倍,将不同稀释度的病毒液接种于长满Vero81单层细胞的12孔板中,每孔接种200μL,每半小时摇晃一次细胞板,孵育2h,吸弃病毒液,用无血清的DMEM培养基洗涤细胞表面3次备用。
(2)将高压灭菌的2% (w/v)低培点琼脂糖微波加热溶解后,冷却至37℃左右与等体积含4%FBS的2×DMEM培养基充分混匀并迅速加入12孔板中,每孔加入1mL,室温条件下放置约0.5h,待培养基自然凝固后倒置放入二氧化碳细胞培养箱中进行培养。
(3)孵育48h,观察细胞病变及蚀斑形成情况,用10μL小枪头尽可能在低稀释度的孔内随机挑取多个单一蚀斑,将其分别吹入200μL的DMEM培养基,混匀后接种于24孔板内的Vero81细胞进行扩培。
(4)待细胞出现大量病变后,观察并收集病毒液、提取病毒DNA,利用引物扩增目的片段,根据相应的测序结果随后进行下一轮的纯化。
(5)纯化后病毒经过10次连续传代,测序验证突变基因得稳定性,收取病毒液。
结果:传代10次后的PRV-ΔgE-ABE基因测序结果如图4,根据测序结果可初步判断,gE基因的单碱基突变遗传较为稳定。
四、基因沉默的病毒蛋白表达情况分析
(1)Western blot检测目的基因的表达是否沉默
取纯化后病毒接入Vero81细胞中,在接毒后24h收取病毒蛋白,进行Westernblot,检测gE基因的表达。
(2)实验结果见图5,结果表明,与野生型毒株相比,突变后的gE基因没有检测到该蛋白的表达,可见突变该起始密码子可有效抑制gE蛋白的表达。
(3)上述结果表明,利用NG-ABE碱基编辑器突变该基因的起始密码子后,病毒中gE的表达会受到抑制,从而达到较好的基因沉默的效果。
以上所述实施例仅表达了本发明的优选实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,可根据以上描述的技术方案以及构思,还可以做出其他各种相应的改变以及变形,而所有的这些改变以及形变都应该属于本发明权利要求的保护范围之内。
Claims (5)
1.一种gE基因缺失的PRV毒株,其特征在于,保藏在中国典型培养物保藏中心,保藏编号CCTCC NO:V202323,所述gE基因缺失的PRV毒株利用腺嘌呤碱基编辑器构建得到;将野生型PRV编码gE基因的第2位碱基因由T突变成C,突变后的gE基因如SEQ ID NO.1所示;
包括以下步骤的构建方法:
S1:构建sgRNA骨架质粒;
选择PRV病毒中gE基因的起始密码子作为靶标位点,利用腺嘌呤碱基编辑器设计一条sgRNA序列,根据sgRNA序列分别合成单链寡核苷酸,将单链寡核苷酸退火获得带粘性末端的双链DNA片段,将pU6-sgRNA-Puro-2A-EGFP载体经限制性内切酶酶切后回收片段,再与上述带粘性末端的双链DNA 片段连接,获得连接产物,即sgRNA表达载体;
将连接产物转化至感受态细胞中,涂板筛选培养,挑阳性菌扩大培养,对阳性菌液抽提质粒;
S2:质粒转染;
将质粒转染至Vero81细胞中,转染后,吸取病毒液,离心收集上清液;
所述腺嘌呤碱基编辑器为NG-ABE8e;
所述sgRNA序列如SEQ ID NO.2所示。
2.利用ABE构建如权利要求1所述的gE基因缺失的PRV毒株的方法,其特征在于,包括以下步骤:
S1:构建sgRNA骨架质粒;
选择PRV病毒中gE基因的起始密码子作为靶标位点,利用腺嘌呤碱基编辑器设计一条sgRNA序列,根据sgRNA序列分别合成单链寡核苷酸,将单链寡核苷酸退火获得带粘性末端的双链DNA片段,将pU6-sgRNA-Puro-2A-EGFP载体经限制性内切酶酶切后回收片段,再与上述带粘性末端的双链DNA 片段连接,获得连接产物,即sgRNA表达载体;
将连接产物转化至感受态细胞中,涂板筛选培养,挑阳性菌扩大培养,对阳性菌液抽提质粒;
S2:质粒转染;
将质粒转染至Vero81细胞中,转染后,吸取病毒液,离心收集上清液。
3.根据权利要求2所述的方法,其特征在于,所述限制性内切酶采用BbsⅠ酶。
4.根据权利要求2所述的方法,其特征在于,所述转染前,将Vero81细胞在含有胎牛血清的培养液中进行培养,培养至对数生长期。
5.一种如权利要求1所述的gE基因缺失的PRV毒株在制备伪狂犬病疫苗上的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311385572.1A CN117126818B (zh) | 2023-10-25 | 2023-10-25 | 利用ABE构建gE基因缺失PRV毒株的方法和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202311385572.1A CN117126818B (zh) | 2023-10-25 | 2023-10-25 | 利用ABE构建gE基因缺失PRV毒株的方法和应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN117126818A CN117126818A (zh) | 2023-11-28 |
CN117126818B true CN117126818B (zh) | 2024-02-02 |
Family
ID=88860364
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202311385572.1A Active CN117126818B (zh) | 2023-10-25 | 2023-10-25 | 利用ABE构建gE基因缺失PRV毒株的方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN117126818B (zh) |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104928261A (zh) * | 2015-07-03 | 2015-09-23 | 江苏省农业科学院 | 伪狂犬病病毒la-a株、构建方法及其应用 |
WO2016026264A1 (zh) * | 2014-08-22 | 2016-02-25 | 普莱柯生物工程股份有限公司 | 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 |
CN107129999A (zh) * | 2017-05-09 | 2017-09-05 | 福建省农业科学院畜牧兽医研究所 | 利用稳转CRISPR/Cas9系统对病毒基因组进行靶向编辑的方法 |
CN109182282A (zh) * | 2018-08-20 | 2019-01-11 | 北京康谷生物科技有限公司 | 猪伪狂犬病病毒gE/gI双基因缺失疫苗株及其构建方法和应用 |
CN109706185A (zh) * | 2019-02-01 | 2019-05-03 | 国家卫生计生委科学技术研究所 | 基于碱基编辑系统突变起始密码子实现基因敲除的方法及应用 |
CN110846285A (zh) * | 2018-08-20 | 2020-02-28 | 上海市农业科学院 | 伪狂犬病毒基因缺失株、猪伪狂犬病灭活疫苗及其制备方法和应用 |
CN112280753A (zh) * | 2020-10-23 | 2021-01-29 | 武汉科前生物股份有限公司 | 一种伪狂犬病病毒TK、gE、gI和gG基因缺失株及其制备方法和应用 |
CN114561429A (zh) * | 2022-03-22 | 2022-05-31 | 绍兴市妇幼保健院 | 一种基于碱基编辑atg起始密码子抑制hbv表面抗原的治疗方法 |
-
2023
- 2023-10-25 CN CN202311385572.1A patent/CN117126818B/zh active Active
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2016026264A1 (zh) * | 2014-08-22 | 2016-02-25 | 普莱柯生物工程股份有限公司 | 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 |
CN104928261A (zh) * | 2015-07-03 | 2015-09-23 | 江苏省农业科学院 | 伪狂犬病病毒la-a株、构建方法及其应用 |
CN107129999A (zh) * | 2017-05-09 | 2017-09-05 | 福建省农业科学院畜牧兽医研究所 | 利用稳转CRISPR/Cas9系统对病毒基因组进行靶向编辑的方法 |
CN109182282A (zh) * | 2018-08-20 | 2019-01-11 | 北京康谷生物科技有限公司 | 猪伪狂犬病病毒gE/gI双基因缺失疫苗株及其构建方法和应用 |
CN110846285A (zh) * | 2018-08-20 | 2020-02-28 | 上海市农业科学院 | 伪狂犬病毒基因缺失株、猪伪狂犬病灭活疫苗及其制备方法和应用 |
CN109706185A (zh) * | 2019-02-01 | 2019-05-03 | 国家卫生计生委科学技术研究所 | 基于碱基编辑系统突变起始密码子实现基因敲除的方法及应用 |
CN112280753A (zh) * | 2020-10-23 | 2021-01-29 | 武汉科前生物股份有限公司 | 一种伪狂犬病病毒TK、gE、gI和gG基因缺失株及其制备方法和应用 |
CN114561429A (zh) * | 2022-03-22 | 2022-05-31 | 绍兴市妇幼保健院 | 一种基于碱基编辑atg起始密码子抑制hbv表面抗原的治疗方法 |
Non-Patent Citations (3)
Title |
---|
"Application of Gene Editing Technology in Resistance Breeding of Livestock";Sutian Wang et al.;《Life》;第12卷;第1-17页 * |
"基于 CRISPR/Cas9 技术快速构建PRV gE 基因缺失病毒";金铭等;《中国动物检疫》;第37卷(第5期);第87-93页 * |
"猪CD163基因的单碱基编辑研究";赵为民等;《畜牧兽医学报》;第53卷(第4期);第1041-1050页 * |
Also Published As
Publication number | Publication date |
---|---|
CN117126818A (zh) | 2023-11-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN112080521B (zh) | 一种表达外源蛋白的重组伪狂犬病毒载体构建及重组伪狂犬病毒制备方法 | |
CN109880851B (zh) | 用于富集CRISPR/Cas9介导的同源重组修复细胞的筛选报告载体及筛选方法 | |
CN111849979B (zh) | 一种靶向敲除RPSA基因的sgRNA及RPSA基因敲除细胞系的构建方法 | |
CN113215193B (zh) | 小分子化合物提高基因敲除和碱基编辑系统活性的方法及其应用方法 | |
CN106987560B (zh) | Rk-13细胞hbb基因敲除稳定株的构建方法 | |
US11866703B2 (en) | Method for knocking out N-myristoyltransferase (NMT) gene from Eimeria tenella | |
CN111979264B (zh) | 基于CRISPR/Cas9系统对博落回PDS基因编辑体系的构建方法及应用 | |
CN114058619B (zh) | Riplet敲除细胞系的构建及作为小核糖核酸病毒科病毒疫苗生产细胞系的应用 | |
CN117126818B (zh) | 利用ABE构建gE基因缺失PRV毒株的方法和应用 | |
CN112126652A (zh) | 水稻OsAUX3基因在调控水稻种子粒长中的应用 | |
CN111676198A (zh) | 快速构建鸭坦布苏病毒反向遗传株的方法 | |
CN114107304B (zh) | 一种表达α毒素蛋白和荧光标签蛋白的重组球虫载体及其检测方法 | |
CN111925449B (zh) | 一种表达鸡vp2和鸡gal-1融合蛋白的重组cho细胞株及其构建方法和应用 | |
CN117106736B (zh) | 利用cbe构建三基因缺失prv毒株的方法和应用 | |
CN117417905A (zh) | 一种基因缺失的减毒非洲猪瘟病毒毒株及其构建方法和应用 | |
CN108754019B (zh) | 一种猪流行性腹泻病毒orf1基因全序列的扩增方法 | |
CN112553200A (zh) | 一种用于敲除猪ugt2c1基因的打靶载体制备方法及其应用 | |
CN108754616B (zh) | 伪狂犬病病毒基因组Fosmid文库、构建方法及其在构建突变体中的应用 | |
CN117431223A (zh) | 利用腺嘌呤碱基编辑器制备gE和gI双基因缺失PRV毒株的方法及应用 | |
CN111117974B (zh) | 一种可视化绿色荧光猪伪狂犬病毒及其构建方法 | |
CN111849990B (zh) | Orf016基因缺失型山羊痘病毒株及其制备方法和应用 | |
CN114875046B (zh) | 一种丝状真菌复制子 | |
CN114606207B (zh) | 一种不依赖胰酶的猪流行性腹泻病毒变异毒株及其构建与应用 | |
CN115948466B (zh) | Tollip敲除细胞系的构建及作为小RNA病毒科病毒疫苗生产细胞系的应用 | |
CN108384764B (zh) | 一种传染性脾肾坏死病毒orf069基因缺失株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 |