CN113584080A - 一种Nluc标记的重组猪δ冠状病毒感染性克隆质粒的构建及其应用 - Google Patents
一种Nluc标记的重组猪δ冠状病毒感染性克隆质粒的构建及其应用 Download PDFInfo
- Publication number
- CN113584080A CN113584080A CN202110779553.1A CN202110779553A CN113584080A CN 113584080 A CN113584080 A CN 113584080A CN 202110779553 A CN202110779553 A CN 202110779553A CN 113584080 A CN113584080 A CN 113584080A
- Authority
- CN
- China
- Prior art keywords
- nluc
- gene
- delta
- delta coronavirus
- plasmid
- 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.)
- Pending
Links
- 239000013612 plasmid Substances 0.000 title claims abstract description 46
- 208000015181 infectious disease Diseases 0.000 title claims abstract description 32
- 230000002458 infectious effect Effects 0.000 title claims abstract description 24
- 241001361508 Porcine deltacoronavirus Species 0.000 title claims description 68
- 238000010276 construction Methods 0.000 title claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 74
- 241000700605 Viruses Species 0.000 claims abstract description 34
- 108091033409 CRISPR Proteins 0.000 claims abstract description 21
- 241001461743 Deltacoronavirus Species 0.000 claims abstract description 18
- 239000003814 drug Substances 0.000 claims abstract description 10
- 230000006801 homologous recombination Effects 0.000 claims abstract description 10
- 238000002744 homologous recombination Methods 0.000 claims abstract description 10
- 238000012216 screening Methods 0.000 claims abstract description 10
- 238000010354 CRISPR gene editing Methods 0.000 claims abstract description 8
- 229940079593 drug Drugs 0.000 claims abstract description 7
- 239000012634 fragment Substances 0.000 claims description 32
- 238000011144 upstream manufacturing Methods 0.000 claims description 22
- 241000282898 Sus scrofa Species 0.000 claims description 15
- 238000006243 chemical reaction Methods 0.000 claims description 15
- 238000000338 in vitro Methods 0.000 claims description 15
- 238000013518 transcription Methods 0.000 claims description 15
- 230000035897 transcription Effects 0.000 claims description 15
- 239000002299 complementary DNA Substances 0.000 claims description 13
- 230000004927 fusion Effects 0.000 claims description 13
- 239000013598 vector Substances 0.000 claims description 13
- 230000003321 amplification Effects 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 9
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 9
- 238000007857 nested PCR Methods 0.000 claims description 7
- 238000003776 cleavage reaction Methods 0.000 claims description 6
- 239000011535 reaction buffer Substances 0.000 claims description 6
- 238000012408 PCR amplification Methods 0.000 claims description 5
- 108091027544 Subgenomic mRNA Proteins 0.000 claims description 5
- 230000007017 scission Effects 0.000 claims description 5
- 238000012163 sequencing technique Methods 0.000 claims description 5
- 230000008685 targeting Effects 0.000 claims description 5
- 238000010367 cloning Methods 0.000 claims description 4
- 238000005520 cutting process Methods 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- GYKXKOROZFUMIC-UHFFFAOYSA-N 4-[(4-carbamoylphenyl)carbamoylamino]benzenesulfonyl chloride Chemical compound C1=CC(C(=O)N)=CC=C1NC(=O)NC1=CC=C(S(Cl)(=O)=O)C=C1 GYKXKOROZFUMIC-UHFFFAOYSA-N 0.000 claims description 3
- 239000013613 expression plasmid Substances 0.000 claims description 3
- 102000006382 Ribonucleases Human genes 0.000 claims description 2
- 108010083644 Ribonucleases Proteins 0.000 claims description 2
- 238000001890 transfection Methods 0.000 claims description 2
- 230000001131 transforming effect Effects 0.000 claims description 2
- 238000002474 experimental method Methods 0.000 abstract description 12
- 238000005516 engineering process Methods 0.000 abstract description 4
- 238000011160 research Methods 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 33
- 108020004414 DNA Proteins 0.000 description 17
- 239000000047 product Substances 0.000 description 14
- 230000000694 effects Effects 0.000 description 10
- 108060001084 Luciferase Proteins 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 239000005089 Luciferase Substances 0.000 description 7
- 241000711573 Coronaviridae Species 0.000 description 6
- 101710141454 Nucleoprotein Proteins 0.000 description 6
- XASIMHXSUQUHLV-UHFFFAOYSA-N camostat Chemical compound C1=CC(CC(=O)OCC(=O)N(C)C)=CC=C1OC(=O)C1=CC=C(N=C(N)N)C=C1 XASIMHXSUQUHLV-UHFFFAOYSA-N 0.000 description 5
- 229960000772 camostat Drugs 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000003443 antiviral agent Substances 0.000 description 4
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 230000010076 replication Effects 0.000 description 4
- 230000029812 viral genome replication Effects 0.000 description 4
- 108700008625 Reporter Genes Proteins 0.000 description 3
- QNVSXXGDAPORNA-UHFFFAOYSA-N Resveratrol Natural products OC1=CC=CC(C=CC=2C=C(O)C(O)=CC=2)=C1 QNVSXXGDAPORNA-UHFFFAOYSA-N 0.000 description 3
- LUKBXSAWLPMMSZ-OWOJBTEDSA-N Trans-resveratrol Chemical compound C1=CC(O)=CC=C1\C=C\C1=CC(O)=CC(O)=C1 LUKBXSAWLPMMSZ-OWOJBTEDSA-N 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 239000012154 double-distilled water Substances 0.000 description 3
- XGVJWXAYKUHDOO-UHFFFAOYSA-N galanthidine Natural products C1CN2CC3=CC=4OCOC=4C=C3C3C2C1=CC(O)C3O XGVJWXAYKUHDOO-UHFFFAOYSA-N 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- XGVJWXAYKUHDOO-DANNLKNASA-N lycorine Chemical compound C1CN2CC3=CC=4OCOC=4C=C3[C@H]3[C@H]2C1=C[C@H](O)[C@H]3O XGVJWXAYKUHDOO-DANNLKNASA-N 0.000 description 3
- KQAOMBGKIWRWNA-UHFFFAOYSA-N lycorine Natural products OC1C=C2CCN3C2C(C1O)c4cc5OCOc5cc34 KQAOMBGKIWRWNA-UHFFFAOYSA-N 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 108010054624 red fluorescent protein Proteins 0.000 description 3
- 229940016667 resveratrol Drugs 0.000 description 3
- 235000021283 resveratrol Nutrition 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 108050009160 DNA polymerase 1 Proteins 0.000 description 2
- 206010012735 Diarrhoea Diseases 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241001135549 Porcine epidemic diarrhea virus Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 101710137500 T7 RNA polymerase Proteins 0.000 description 2
- 241000711484 Transmissible gastroenteritis virus Species 0.000 description 2
- 208000036142 Viral infection Diseases 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 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 2
- 229960005091 chloramphenicol Drugs 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000007877 drug screening Methods 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 239000005457 ice water Substances 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000012257 pre-denaturation Methods 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- LUKBXSAWLPMMSZ-UHFFFAOYSA-N resveratrol Chemical compound C1=CC(O)=CC=C1C=CC1=CC(O)=CC(O)=C1 LUKBXSAWLPMMSZ-UHFFFAOYSA-N 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 230000009261 transgenic effect Effects 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000112287 Bat coronavirus Species 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
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 208000005156 Dehydration Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 241000709661 Enterovirus Species 0.000 description 1
- 101150066002 GFP gene Proteins 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108090001074 Nucleocapsid Proteins Proteins 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 244000025272 Persea americana Species 0.000 description 1
- 235000008673 Persea americana Nutrition 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 108091028733 RNTP Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000193996 Streptococcus pyogenes Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 230000005574 cross-species transmission Effects 0.000 description 1
- 230000000120 cytopathologic effect Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000001476 gene delivery Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 108040007771 luciferin monooxygenase activity proteins Proteins 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 208000026775 severe diarrhea Diseases 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Images
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
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
-
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
-
- 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
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
-
- 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
- C12N2770/20011—Coronaviridae
- C12N2770/20021—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
- C12N2770/20011—Coronaviridae
- C12N2770/20022—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
- C12N2770/20011—Coronaviridae
- C12N2770/20051—Methods of production or purification of viral material
- C12N2770/20052—Methods of production or purification of viral material relating to complementing 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Public Health (AREA)
- Immunology (AREA)
- Animal Behavior & Ethology (AREA)
- Plant Pathology (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Analytical Chemistry (AREA)
- Mycology (AREA)
- Toxicology (AREA)
- Epidemiology (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)
Abstract
本发明公开了一种Nluc标记的重组猪δ冠状病毒感染性克隆质粒构建及其应用,针对猪δ冠状病毒,采用BAC系统和CRISPR/Cas9技术相结合的方式,基于已构建成功的PDCoV感染性克隆质粒,先通过CRISPR/Cas9切割PDCoV感染性克隆质粒,再通过同源重组方式获得Nluc基因替换NS6基因的重组质粒,转染细胞后拯救出稳定表达Nluc的重组病毒,通过药物实验证实该重组病毒可为抗猪δ冠状病毒药物的筛选研究提供非常有价值的技术工具。
Description
技术领域
本发明属于动物传染病防治和分子生物学领域。具体涉及一种Nluc标记的重组猪δ冠状病毒感染性克隆质粒的构建及其应用。
背景技术
猪δ冠状病毒(PDCoV)属于套式病毒目冠状病毒科δ冠状病毒属,是一种有囊膜、基因组为不分节段的单股正链RNA病毒。2012年,香港学者Woo等首次在病料中检测到猪δ冠状病毒(Woo P.C.Y,Discovery of seven novel Mammalian and avian coronaviruses inthe genus deltacoronavirus supports bat coronaviruses as the gene source ofalphacoronavirus and betacoronavirus and avian coronaviruses as the genesource of gammacoronavirus and deltacoronavirus.J.Virol.2012,86:3995-4008)。2014年,PDCoV在美国猪场爆发,研究人员从腹泻猪的粪便和肠道中首次分离出猪δ冠状病毒,并发现了PDCoV在美国多个州广泛流行。随后,韩国、泰国、老挝、越南、日本等多个国家报道了PDCoV的感染(Saeng-Chuto K et al,Different Lineage of PorcineDeltacoronavirus in Thailand,Vietnam and Lao PDR in 2015.Transbound EmergDis.2017,64(1):3-10;Suzuki T et al,Genetic characterization and pathogenicityof Japanese porcine deltacoronavirus.2018,61:176-182)。本课题组分析了2004~2014年间我国多个省份规模化猪场的粪便样品,首次证实我国规模化猪场内存在PDCoV,从河南省的仔猪腹泻内容物中分离获得PDCoV CHN-HN-2014株,动物实验证实PDCoV感染可引起仔猪严重腹泻、呕吐、脱水(Dong et al,Isolation,genomic characterization,andpathogenicity of a Chinese porcine deltacoronavirus strain CHN-HN-2014.VetMicrobiol.2016,196:98-106)。最近的研究发现犊牛和鸡对PDCoV易感,体外细胞实验证实PDCoV也可感染人源细胞,提示PDCoV存在跨种传播的可能性(Jung et al,Calves aresusceptible to infection with the newly emerged porcine deltacoronavirus,butnot with the swine enteric alphacoronavirus,porcine epidemic diarrheavirus.2017,162:2357-2362;Li et al,Broad receptor engagement of an emergingglobal coronavirus may potentiate its diverse cross-speciestransmissibility.2018,115:E5135-E5143)。此外,PDCoV常与猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)及其他病毒混合感染,给猪肠道病毒的防控带来了更大的挑战,严重影响了养猪业的发展。
PDCoV作为一种新发的猪冠状病毒,目前还没有商业化疫苗、特效治疗药物。冠状病毒反向遗传学操作系统可定向对靶基因进行改造(突变、缺失)或插入外源基因获得重组病毒,为新型疫苗的研发和抗病毒药物的研发提供技术平台。许多研究已经报道通过病毒感染性克隆引入外源基因(包括绿色荧光蛋白GFP基因、红色荧光蛋白RFP基因,以及荧光素酶蛋白基因)构建重组病毒,为抗病毒药物的筛选提供良好的技术平台。冠状病毒辅助蛋白具有种特异性,不同冠状病毒编码不同数量、大小的辅助蛋白,这些辅助蛋白是病毒复制非必需的,因此,可以直接缺失或者引入外源基因构建重组病毒。目前已经确定PDCoV辅助蛋白NS6是病毒复制非必需的,可作为一个非常合适的靶基因被外源基因进行替换。已有研究利用体外cDNA连接的方法构建PDCoV的反向遗传操作系统,用绿色荧光蛋白EGFP基因替换NS6基因,获得表达EGFP蛋白的重组PDCoV病毒(Deng X et al,Development andutilization of an infectious clone for porcine deltacoronavirus strain USA/IL/2014/026.Virology.2021,553:35-45)。然而,通过体外多个片段连接和RNA转录的方式,存在耗时长、效率低等缺点;而BAC系统不涉及到体外连接和转录,且重组病毒的拯救效率高,加上成熟的CRISPR/Cas9技术,可高效的改造重组质粒,该系统具有更强的优势。目前,还没有很好的抗PDCoV药物筛选的技术平台,EGFP作为外源基因引入,存在蛋白较大,灵敏性不够高等缺点。基于上述的缺陷和不足,本课题组利用前期构建的猪δ冠状病毒感染性克隆质粒,我们采用BAC系统和CRISPR/Cas9技术,构建稳定表达Nluc基因的重组PDCoV病毒,为抗PDCoV药物的筛选提供非常有价值的技术平台。
发明内容
本发明的目的在于针对猪δ冠状病毒,采用BAC系统和CRISPR/Cas9技术相结合的方式,基于已构建成功的PDCoV感染性克隆质粒,先通过CRISPR/Cas9切割PDCoV感染性克隆质粒,再通过同源重组方式获得Nluc基因替换NS6基因的重组质粒,转染细胞后拯救出表达Nluc的重组病毒,通过药物实验证实该重组病毒可为抗猪δ冠状病毒药物的筛选研究提供非常有价值的技术工具。
本发明的技术方案如下:
一种Nluc标记的重组猪δ冠状病毒感染性克隆质粒构建方法,包括以下步骤:
(1)设计两条靶向猪δ冠状病毒NS6基因的sgRNA引物,分别通过与引物scaffoldoligo进行重叠延伸PCR扩增,获得猪δ冠状病毒sgRNA-△NS6a、sgRNA-△NS6b的转录模板,经体外转录获得sgRNA-△NS6a和sgRNA-△NS6b,利用CRISPR/Cas9系统,通过sgRNA-△NS6a和sgRNA-△NS6b体外切割含有猪δ冠状病毒全长cDNA的BAC质粒,经试剂盒回收获得线性化BAC载体;
(2)以含有猪δ冠状病毒全长cDNA的BAC质粒为模板,设计引物分别扩增猪δ冠状病毒NS6基因上游片段和下游片段;以Nluc表达质粒为模板,设计引物扩增Nluc基因片段;将所述NS6基因上、下游片段和Nluc基因片段进行重叠延伸PCR扩增,获得含有猪δ冠状病毒NS6基因上、下游序列以及Nluc基因序列的融合片段,该融合片段与上述线性化BAC载体上、下游各含有20bp的同源臂;
(3)将步骤(1)的线性化BAC载体与步骤(2)的融合片段进行同源重组,获得Nluc标记的重组猪δ冠状病毒感染性克隆质粒;
(4)将重组产物转化至化学感受态细胞中,利用PCR筛选阳性克隆,将测序正确的阳性克隆扩大培养后大量提取质粒。
优选地,所述猪δ冠状病毒为CHN-HN-2014株,但本发明所针对的猪δ冠状病毒不局限于CHN-HN-2014株。
优选地,所述两条靶向猪δ冠状病毒NS6基因的sgRNA引物分别为:
sgPDCoV-△NS6a:SEQ ID NO:1;
sgPDCoV-△NS6b:SEQ ID NO:2。
优选地,步骤(1)中所述的体外切割,其反应体系为:2.5μg含有猪δ冠状病毒全长cDNA的BAC质粒,2μL Cas9 nuclease,1μ1sgRNA-△NS6a,1μl sgRNA-△NS6b,5μL 10×reaction buffer,补加RNase水至总体积为50μL。
所述scaffold oligo引物序列如SEQ ID NO:3所示。
步骤(2)中,用于分别扩增猪δ冠状病毒NS6基因上游序列和下游序列的引物分别为:
PDCoV-NS6-upF:SEQ ID NO:4;
PDCoV-NS6-upR:SEQ ID NO:5;
PDCoV-NS6-downF:SEQ ID NO:6;
PDCoV-NS6-downR:SEQ ID NO:7。
步骤(2)中,用于扩增Nluc基因片段的引物为:
Nluc-F:SEQ ID NO:8;
Nluc-R:SEQ ID NO:9。
优选地,步骤(2)中,在进行重叠延伸PCR扩增时,所述NS6基因上、下游片段和Nluc基因片段的摩尔量比为2:2:1。
优选地,步骤(3)中,在进行同源重组时,所述线性化BAC载体与融合片段的摩尔量比为1:4。
所述质粒转染细胞后获得的重组病毒rCHN-HN-2014-△NS6-Nluc不能表达NS6,能表达Nluc蛋白。本发明选择生物发光报告基因Nluc作为外源报告基因,其具有分子量小、热稳定性高、发光强度高、自发光背景低、光信号亮等突出的特性,针对猪δ冠状病毒具有很高的检测灵敏性,是一种相比GFP基因、RFP基因和EGFP基因性能更好的报告基因,本发明构建的感染性克隆质粒和表达Nluc的重组病毒在筛选抗猪δ冠状病毒药物中具有更好的应用前景。本发明还具有重组效率高等优点。
附图说明
图1:猪δ冠状病毒重组质粒pBAC-CHN-HN-2014-△NS6-Nluc的构建示意图。
图2:sgRNA-△NS6a和sgRNA-△NS6b的电泳图。
图3:体外切割后的pBAC-CHN-HN-2014电泳图。
图4:IFA鉴定拯救病毒rCHN-HN-2014-△NS6-Nluc的图片。附图标记说明:图4中的A图:正常未感染病毒的LLC-PK1细胞在倒置显微镜下的图片;图4中的B图:CHN-HN-2014感染的LLC-PK1细胞在倒置荧光显微镜下的图片;图4中的C图:rCHN-HN-2014感染的LLC-PK1细胞在倒置显微镜下的图片;图4中的D图:rCHN-HN-2014-△NS6-Nluc感染的LLC-PK1细胞在倒置荧光显微镜下的图片。
图5:Western blot鉴定拯救病毒rCHN-HN-2014-△NS6-Nluc的图片。
图6:荧光素酶活性检测分析拯救病毒rCHN-HN-2014-△NS6-Nluc感染条件下Nluc表达的图片。
图7:rCHN-HN-2014株与rCHN-HN-2014-△NS6-Nluc株体外一步生长曲线比较。
图8:rCHN-HN-2014与rCHN-HN-2014-△NS6-Nluc在LLC-PK1细胞上形成的空斑表型比较。
图9:不同浓度的盐酸石蒜碱(Lycorine),甲磺酸卡莫司他(Camostat mesilate)以及白藜芦醇(Resveratrol)对rCHN-HN-2014-△NS6-Nluc复制的影响。
具体实施方式
下面结合具体实施例对本发明进行详细描述,但本发明并不仅限于以下实施例,实施例中未详细描述的试验方法均为本领域常规方法。
实施例:
1.主要试验材料、载体、质粒、试剂与相关培养基或溶液的配方:
PDCoV CHN-HN-2014株(GenBank登录号:KT336560):由华中农业大学农业微生物学国家重点实验室病毒分室分离鉴定(Dong N,Fang L,Yang H,Liu H,Du T,Fang P,WangD,Chen H,Xiao S.Isolation,genomic characterization,and pathogenicity of aChinese porcine deltacoronavirus strain CHN-HN-2014.Vet Microbiol.2016,196:98-106)。
pBAC-CHN-HN-2014为含PDCoV CHN-HN-2014株全长cDNA的感染性克隆,转染LLC-PK1细胞可拯救出重组PDCoV(命名为rCHN-HN-2014),为华中农业大学农业微生物学国家重点实验室病毒分室构建保存,申请人已将该质粒的构建方法于2021年2月4日申请了一项中国发明专利,申请号为202110154305.8。
包含Nluc的质粒:由华中农业大学农业微生物学国家重点实验室病毒分室保存。
LLC-PK1细胞:购自American Type Culture Collection(ATCC)。
氯霉素(货号:BS049B):购自Biosharp公司。
各种限制性内切酶:购自New England Biolabs公司。
MEM培养基,T4 DNA ligase(货号:15224025),Lipofectamine 3000(货号:L3000015),pJET1.2/blunt vector均购自美国Thermofisher Scientific公司。FastPfu DNA Polymerase购自TransGen Biotech公司。TranscriptorReverse Transcriptase(货号:39083420),
Cycle Pure Kit(货号:D6492-02),Gel Extraction Kit(货号:D2500-02):购自美国Omega Bio-tek公司。
Plasmid DNA purification Kit(货号:740410.50):购自宝日生物技术有限公司。
Cas9 nuclease,S.Pyogenes(货号:M0386S),T7 RNA聚合酶(货号:M0251S):均购自英国NEB公司。
DH10B化学感受态细胞:购自上海超研生物技术有限公司。
LB培养基的配制:称取5g蛋白胨,2.5g酵母提取物,5g NaCl于1L的锥形瓶中,加ddH2O定容至500mL,121℃高压灭菌20min。
2.重组质粒pBAC-CHN-HN-2014-△NS6-Nluc的构建
2.1PDCoV CHN-HN-2014株sgRNA-△NS6a及sgRNA-△NS6b的制备:设计两条靶向PDCoV CHN-HN-2014株NS6基因的sgRNA引物(sgPDCoV-△NS6a、sgPDCoV-△NS6b),利用引物对sgPDCoV-△NS6a和scaffold oligo及引物对sgPDCoV-△NS6b和scaffold oligo分别进行重叠延伸PCR扩增。反应体系如下:2×Taq Master Mix 50μL,上下游引物各25μL,总体积为100μL。反应条件如下:95℃预变性3min;95℃30s,58℃30s,72℃20s;30个循环后,72℃延伸5min。反应结束后,按照说明书用Cycle Pure Kit进行回收,回收产物即为PDCoV CHN-HN-2014株sgRNA-△NS6a及sgRNA-△NS6b的转录模板。
以上述回收产物为模板,利用T7体外转录试剂盒进行体外转录,转录体系与条件如下:1μL 10×reaction buffer,0.2μL rNTPs,200ng的回收产物,1μL T7 RNApolymerase,补加无RNase水至10μL,混匀后于37℃反应4h。反应完成后,即获得sgRNA-△NS6a及sgRNA-△NS6b(图2),及时用于后续实验或保存于-80℃备用。
2.2pBAC-CHN-HN-2014的切割与回收:用NEB公司的Cas9核酸酶对重组质粒pBAC-CHN-HN-2014进行切割,切割反应体系和条件如下:2.5μg pBAC-CHN-HN-2014,2μL Cas9nuclease,1μ1sgRNA-△NS6a,1μl sgRNA-△NS6b,5μL 10×reaction buffer,补加无RNase水至总体积为50μL,混匀后,37℃孵育3h。按照说明书用Cycle Pure Kit对切割产物进行回收,获得线性化的载体。取5μL回收产物经1%琼脂糖凝胶电泳以检测切割效果(图3)。
2.3置换模板基因(含有PDCoV CHN-HN-2014NS6基因上游片段,NS6基因下游片段的基因序列和Nluc基因片段)的扩增:以pBAC-CHN-HN-2014质粒为模板,用引物对PDCoV-NS6-upF和PDCoV-NS6-upR及引物对PDCoV-NS6-downF和PDCoV-NS6-downR分别扩增PDCoVCHN-HN-2014株NS6基因上游序列和下游序列。同时,以实验室保存的Nluc表达质粒为模板,Nluc-F和Nluc-R为引物对进行PCR扩增,获得Nluc基因片段。反应体系如下:10μL 5×PCRreaction buffer,4μL dNTPs,10μM上下游引物,cDNA模板1μL,FastPfu DNAPolymerase 1μL,加ddH2O至总体积为50μL。反应条件如下:95℃2min;95℃20s;95℃20s,58℃20s,72℃1min;30个循环后,72℃延伸5min。PCR扩增完成后,将扩增产物经1%琼脂糖凝胶电泳,按照说明书用Gel Extraction Kit进行回收,再将回收产物(PDCoV-NS6-up,PDCoV-NS6-down和Nluc)进行重叠延伸PCR反应。反应体系如下:10μL5×PCR reactionbuffer,4μL dNTPs,10μM上下游引物(PDCoV-NS6-upF,PDCoV-NS6-downR),cDNA模板(PDCoV-NS6-up,PDCoV-NS6-down,Nluc),FastPfu DNA Polymerase1μL,加ddH2O至总体积为50μL。反应条件如下:95℃2min;95℃20s;95℃20s,58℃20s,72℃2min;35个循环后,72℃延伸5min。我们尝试不同摩尔量比例的3种cDNA模板进行重叠延伸PCR反应,发现比例为2:2:1能够产生丰度较高,特异性最强的融合片段。按照说明书用GelExtraction Kit进行回收,将回收产物克隆至pJET1.2/blunt vector进行测序分析,保证序列的准确性。
2.4置换模板与线性化的pBAC-CHN-HN-2014进行同源重组连接:将步骤(2.2)切割后回收的线性化pBAC-CHN-HN-2014与步骤(2.3)测序正确的融合PCR回收产物构建同源重组体系,体系及条件如下:pBAC-CHN-HN-2014,融合PCR产物,2μL 5×In-Fusion EnzymePremix,总体积为10μL,轻轻混匀后,50℃反应20min。在该实验过程中,我们也尝试了pBAC-CHN-HN-2014,融合PCR产物不同的比例进行同源重组,包括1:1、1:2、1:3、1:4、1:5、1:6、1:7、1:8、1:9、1:10,结果发现1:4的比例同源重组后产生的阳性克隆率最高,可达到80%,而其他比例都不到50%。这个实验的载体大小约为32kb,片段大小约为1.6kb,因此,载体和片段大小与之接近的可以采用1:4的比例进行同源重组,可获得较高的阳性率。
2.5连接产物的转化:取5μL步骤(2.4)的连接产物缓慢加入100μL DH10B化学感受态细胞中,轻轻混匀后,在冰水浴中静置30min。42℃热激60s后,立即放置冰水浴中2min,加600μL LB培养基,37℃180rpm复苏1h。复苏完成后于5000rpm离心5min,弃掉500μL LB培养基,用剩余的LB培养基重悬感受态细胞,涂布于含有25μg/mL氯霉素的LB固体平板。10min后将平板倒置于37℃培养箱中继续培养15h。
2.6阳性克隆的筛选:挑取6-8个菌落于含有1mL LB的细菌瓶中,37℃,225rpm培养8h后,利用引物PDCoV-NS6-upF和PDCoV-NS6-downR,PCR扩增插入Nluc基因的片段进行鉴定。PCR条件如下:2×Taq Master Mix 12.5μL,上下游引物各10μM,1μL菌液,补加ddH2O至总体积为25μL。反应条件如下:95℃预变性3min;95℃30s,58℃30s,72℃2min;25个循环后,72℃延伸5min。反应结束后,取8μL用1%琼脂糖凝胶进行电泳以鉴定阳性克隆,将鉴定为阳性克隆的菌夜送武汉擎科生物技术有限公司测序,阳性克隆质粒命名为pBAC-CHN-HN-2014-△NS6-Nluc。
2.7pBAC-CHN-HN-2014-△NS6-Nluc质粒的提取:将步骤(2.6)鉴定为阳性的菌落扩大培养后,按照说明书用Plasmid DNA purification Kit大量提取质粒pBAC-CHN-HN-2014-△NS6-Nluc。最后一步用100μL ddH2O洗脱,-20℃保存备用。
2.8重组病毒rCHN-HN-2014-△NS6-Nluc的拯救:按照说明书用Lipofectamine3000将3μg重组质粒pBAC-CHN-HN-2014-△NS6-Nluc转染至80%融合度的LLC-PK1细胞。转染4h后,将培养基换成含有10μg/mL胰酶的无血清DMEM,继续培养,至细胞80%以上病变时,收获细胞培养物,于-70℃冻融2次,2000rpm离心5min,取上清再接种LLC-PK1细胞进行传代培养。
结果:cDNA克隆pBAC-CHN-HN-2014-△NS6-Nluc转染LLC-PK1细胞后60h,细胞出现明显的病变,主要表现为细胞变圆,聚集,脱落,与野生型PDCoV株所致的细胞病变一致,说明病毒拯救成功,将其命名为rCHN-HN-2014-△NS6-Nluc。间接免疫光实验结果,发现利用针对PDCoV N蛋白的单克隆抗体作为一抗进行的实验,所有病毒感染的细胞都能在倒置荧光显微镜下观察到特异性红色荧光,而利用针对NS6单克隆体作为一抗进行的实验,发现野生型病毒和拯救的rCHN-HN-2014感染的细胞有特异性的红色荧光,而rCHN-HN-2014-△NS6-Nluc感染的细胞没有红色荧光,整个实验中对照的未感染细胞没有特异性荧光(图4)。进一步对病毒感染的细胞进行Western blot分析,与IFA结果一致,利用PDCoV N蛋白单克隆抗体作为一抗进行实验,发现野生型PDCoV CHN-HN-2014、rCHN-HN-2014以及rCHN-HN-2014-△NS6-Nluc组都能检测到N蛋白的表达,利用NS6蛋白单克隆抗体作为一抗进行实验,仅仅野生型PDCoV CHN-HN-2014、rCHN-HN-2014能表达NS6蛋白(图5)。为了进一步鉴定Nluc在病毒感染条件下正常表达,将rCHN-HN-2014-△NS6-Nluc感染LLC-PK1细胞,不同时间点收样(3,6,9,12hpi),检测荧光素酶活性。发现荧光素酶活性随着病毒感染时间的延长显著增加,呈时间依赖性,这种增加趋势与病毒N蛋白的表达水平的趋势一致(图6),表明重组病毒表达了Nluc。这些结果证实重组病毒rCHN-HN-2014-△NS6-Nluc拯救成功。同时,进一步对重组病毒rCHN-HN-2014和rCHN-HN-2014-△NS6-Nluc进行体外生物学特性分析,发现两种病毒都能正常复制,在感染后36h达到病毒复制的最高水平,但rCHN-HN-2014-△NS6-Nluc复制水平整体较rCHN-HN-2014的低(图7),形成的空斑也稍小(图8)。将获得的病毒在LLC-PK1细胞上连续传5代,能稳定地观察到细胞病变,Nluc基因也能稳定存在。说明重组病毒rCHN-HN-2014-△NS6-Nluc具有稳定的增殖能力。随后,进一步选择已报道的几种药物,包括盐酸石蒜碱(Lycorine),甲磺酸卡莫司他(Camostat mesilate)以及白藜芦醇(Resveratrol),分析它们对表达Nluc的重组病毒复制影响,鉴定其作为抗病毒药物筛选平台的可行性。将不同浓度的药物处理LLC-PK1细胞,再感染重组病毒rCHN-HN-2014-△NS6-Nluc,12h后收样,检测荧光素酶活性和N蛋白表达,结果表明只有Lycorine显著抑制病毒的增殖,呈剂量依赖性,荧光素酶活性值与N蛋白的表达趋势一致,最高浓度药物组几乎检测不到N蛋白,但仍能检测到荧光素酶活性(图9A),其他两种药物甲磺酸卡莫司他(图9B)和白藜芦醇(图9C)对Nluc报告病毒复制没有显著的抑制作用。这些结果表明荧光素酶活性检测能灵敏地反应病毒真实复制水平,可作为抗病毒药物的筛选工具。
本发明提供了一种Nluc基因标记的PDCoV CHN-HN-2014株感染性克隆质粒以及重组病毒rCHN-HN-2014-△NS6-Nluc。该荧光素酶标记性蛋白的检测具有十分灵敏、稳定、高效的优势,为快速筛选有效抗PDCoV的药物提供非常有价值的技术工具。
序列表说明:
SEQ ID NO:1:sgPDCoV-△NS6a引物序列,用于扩增sgRNA-△NS6a的转录模板(120bp);
SEQ ID NO:2:sgPDCoV-△NS6b引物序列,用于扩增sgRNA-△NS6b的转录模板(120bp);
SEQ ID NO:3:scaffold oligo引物序列,用于扩增sgRNA-△NS6a、sgRNA-△NS6b的转录模板;
SEQ ID NO:4:PDCoV-NS6-upF引物序列,用于扩增543bp的NS6基因上游片段;
SEQ ID NO:5:PDCoV-NS6-upR引物序列,用于扩增543bp的NS6基因上游片段;
SEQ ID NO:6:PDCoV-NS6-downF引物序列,用于扩增596bp的NS6基因下游片段;
SEQ ID NO:7:PDCoV-NS6-downR引物序列,用于扩增596bp的NS6基因下游片段;
SEQ ID NO:8:Nluc-F引物序列,用于扩增556bp的Nluc基因片段;
SEQ ID NO:9:Nluc-R引物序列,用于扩增556bp的Nluc基因片段;
SEQ ID NO:10:sgRNA-△NS6a的转录模板基因序列,共120bp。
SEQ ID NO:11:sgRNA-△NS6b的转录模板基因序列,共120bp。
SEQ ID NO:12:NS6基因上游序列,共543bp。
SEQ ID NO:13:Nluc基因序列,共556bp。
SEQ ID NO:14:NS6基因下游序列,共596bp。
SEQ ID NO:15:含有NS6基因上、下游序列以及Nluc基因序列的融合片段,共1616bp。
序列表
<110> 华中农业大学
<120> 一种Nluc标记的重组猪δ冠状病毒感染性克隆质粒的构建及其应用
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ttctaatacg actcactata ggtccaaatg gtcaccacta gttttagagc taga 54
<210> 2
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ttctaatacg actcactata ggtcttacga cgtaccgcag gttttagagc taga 54
<210> 3
<211> 80
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
aaaagcaccg actcggtgcc actttttcaa gttgataacg gactagcctt attttaactt 60
gctatttcta gctctaaaac 80
<210> 4
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gctccaaccc ttcaccctag 20
<210> 5
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atcttcgagt gtgaagacca ttacatatac ttatacaggc g 41
<210> 6
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gaacgcattc tggcgtaaag ttttgacacc aatctatc 38
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ttgggtctta cgacgtaccg 20
<210> 8
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
cgcctgtata agtatatgta atggtcttca cactcgaaga t 41
<210> 9
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gatagattgg tgtcaaaact ttacgccaga atgcgttc 38
<210> 10
<211> 120
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ttctaatacg actcactata ggtccaaatg gtcaccacta gttttagagc tagaaatagc 60
aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 120
<210> 11
<211> 120
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ttctaatacg actcactata ggtcttacga cgtaccgcag gttttagagc tagaaatagc 60
aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 120
<210> 12
<211> 543
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
gctccaaccc ttcaccctag tggtgaccat ttggaccgca gttgacagat catctaagaa 60
ggacgcagtt ttcattgtgt ccataatttt tgccgtactg accttcatat cctgggccaa 120
gtactggtat gactcaattc gtttattaat gaaaaccaga tctgcatggg cactctcacc 180
tgagagtaga ctccttgcag ggattatgga tccaatgggt acatggaggt gcattcccat 240
cgaccacatg gctccaattc tcacaccagt cgttaagcat ggcaagctca agctacatgg 300
gcaagagctg gccaatggca tatcagtcag aaatccgcca caggatatgg tgatagtgtc 360
accaagtgac acctttcact acacttttaa gaaacctgtg gaatcaaaca acgatccaga 420
attcgctgtt ctgatatacc agggtgaccg cgcttcaaac gctggacttc acaccataac 480
cacttcaaag gccggtgacg ctcgcctgta taagtatatg taatggtctt cacactcgaa 540
gat 543
<210> 13
<211> 556
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
cgcctgtata agtatatgta atggtcttca cactcgaaga tttcgttggg gactggcgac 60
agacagccgg ctacaacctg gaccaagtcc ttgaacaggg aggtgtgtcc agtttgtttc 120
agaatctcgg ggtgtccgta actccgatcc aaaggattgt cctgagcggt gaaaatgggc 180
tgaagatcga catccatgtc atcatcccgt atgaaggtct gagcggcgac caaatgggcc 240
agatcgaaaa aatttttaag gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc 300
tgcactatgg cacactggta atcgacgggg ttacgccgaa catgatcgac tatttcggac 360
ggccgtatga aggcatcgcc gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt 420
ggaacggcaa caaaattatc gacgagcgcc tgatcaaccc cgacggctcc ctgctgttcc 480
gagtaaccat caacggagtg accggctggc ggctgtgcga acgcattctg gcgtaaagtt 540
ttgacaccaa tctatc 556
<210> 14
<211> 596
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
gaacgcattc tggcgtaaag ttttgacacc aatctatcat ggctgcacca gtagtcccta 60
ctactgacgc gtcttggttt caggtgctca aagctcaaaa caaaaaagcc attcatcctc 120
agtttcgtgg caatggagtt ccgcttaact ccgccatcaa acccgttgaa aatcatggct 180
actggctgcg ttacaccaga caaaagccag gtggtactcc gattcctcca tcctatgcct 240
tttattatac tggcacaggt cccagaggaa atcttaagta tggtgaactc cctcctaatg 300
ataccccagc aaccactcgt gttacttggg ttaagggttc gggagctgac acttctatta 360
agcctcatgt tgccaaacgc aaccccaaca atcctaaaca tcagctgcta cctctccgat 420
tcccaaccgg agatggccca gctcaaggtt tcagagttga ccccttcaac gctagaggaa 480
gacctcagga gcgtggaggt ggcccaagat ctcaatctgt taactccaga ggcacaggca 540
atcagcctag gaaacgcgac caatctgcac ccgctgcggt acgtcgtaag acccaa 596
<210> 15
<211> 1616
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
gctccaaccc ttcaccctag tggtgaccat ttggaccgca gttgacagat catctaagaa 60
ggacgcagtt ttcattgtgt ccataatttt tgccgtactg accttcatat cctgggccaa 120
gtactggtat gactcaattc gtttattaat gaaaaccaga tctgcatggg cactctcacc 180
tgagagtaga ctccttgcag ggattatgga tccaatgggt acatggaggt gcattcccat 240
cgaccacatg gctccaattc tcacaccagt cgttaagcat ggcaagctca agctacatgg 300
gcaagagctg gccaatggca tatcagtcag aaatccgcca caggatatgg tgatagtgtc 360
accaagtgac acctttcact acacttttaa gaaacctgtg gaatcaaaca acgatccaga 420
attcgctgtt ctgatatacc agggtgaccg cgcttcaaac gctggacttc acaccataac 480
cacttcaaag gccggtgacg ctcgcctgta taagtatatg taatggtctt cacactcgaa 540
gatttcgttg gggactggcg acagacagcc ggctacaacc tggaccaagt ccttgaacag 600
ggaggtgtgt ccagtttgtt tcagaatctc ggggtgtccg taactccgat ccaaaggatt 660
gtcctgagcg gtgaaaatgg gctgaagatc gacatccatg tcatcatccc gtatgaaggt 720
ctgagcggcg accaaatggg ccagatcgaa aaaattttta aggtggtgta ccctgtggat 780
gatcatcact ttaaggtgat cctgcactat ggcacactgg taatcgacgg ggttacgccg 840
aacatgatcg actatttcgg acggccgtat gaaggcatcg ccgtgttcga cggcaaaaag 900
atcactgtaa cagggaccct gtggaacggc aacaaaatta tcgacgagcg cctgatcaac 960
cccgacggct ccctgctgtt ccgagtaacc atcaacggag tgaccggctg gcggctgtgc 1020
gaacgcattc tggcgtaaag ttttgacacc aatctatcat ggctgcacca gtagtcccta 1080
ctactgacgc gtcttggttt caggtgctca aagctcaaaa caaaaaagcc attcatcctc 1140
agtttcgtgg caatggagtt ccgcttaact ccgccatcaa acccgttgaa aatcatggct 1200
actggctgcg ttacaccaga caaaagccag gtggtactcc gattcctcca tcctatgcct 1260
tttattatac tggcacaggt cccagaggaa atcttaagta tggtgaactc cctcctaatg 1320
ataccccagc aaccactcgt gttacttggg ttaagggttc gggagctgac acttctatta 1380
agcctcatgt tgccaaacgc aaccccaaca atcctaaaca tcagctgcta cctctccgat 1440
tcccaaccgg agatggccca gctcaaggtt tcagagttga ccccttcaac gctagaggaa 1500
gacctcagga gcgtggaggt ggcccaagat ctcaatctgt taactccaga ggcacaggca 1560
atcagcctag gaaacgcgac caatctgcac ccgctgcggt acgtcgtaag acccaa 1616
Claims (10)
1.一种Nluc标记的重组猪δ冠状病毒感染性克隆质粒构建方法,其特征在于包括以下步骤:
(1)设计两条靶向猪δ冠状病毒NS6基因的sgRNA引物,分别通过与引物scaffold oligo进行重叠延伸PCR扩增,获得猪δ冠状病毒sgRNA-△NS6a、sgRNA-△NS6b的转录模板,经体外转录获得sgRNA-△NS6a和sgRNA-△NS6b,利用CRISPR/Cas9系统体外切割含有猪δ冠状病毒全长cDNA的BAC质粒,经试剂盒回收获得线性化BAC载体;
(2)以含有猪δ冠状病毒全长cDNA的BAC质粒为模板,设计引物分别扩增猪δ冠状病毒NS6基因上游片段和下游片段;以Nluc表达质粒为模板,设计引物扩增Nluc基因片段;将所述NS6基因上、下游片段和Nluc基因片段进行重叠延伸PCR扩增,获得含有猪δ冠状病毒NS6基因上、下游序列以及Nluc基因序列的融合片段,该融合片段与上述线性化BAC载体上、下游各含有20bp的同源臂;
(3)将步骤(1)的线性化BAC载体与步骤(2)的融合片段进行同源重组,获得Nluc标记的重组猪δ冠状病毒感染性克隆质粒;
(4)将重组产物转化至化学感受态细胞中,利用PCR筛选阳性克隆,将测序正确的阳性克隆扩大培养后大量提取质粒。
2.如权利要求1所述Nluc标记的重组猪δ冠状病毒感染性克隆质粒构建方法,其特征在于:所述猪δ冠状病毒为CHN-HN-2014株。
3.如权利要求1所述Nluc标记的重组猪δ冠状病毒感染性克隆质粒构建方法,其特征在于,所述两条靶向猪δ冠状病毒NS6基因的sgRNA引物分别为:
sgPDCoV-△NS6a:SEQ ID NO:1;
sgPDCoV-△NS6b:SEQ ID NO:2。
4.如权利要求1所述Nluc标记的重组猪δ冠状病毒感染性克隆质粒构建方法,其特征在于,步骤(1)中所述的体外切割,其反应体系为:2.5μg含有猪δ冠状病毒全长cDNA的BAC质粒,2μL Cas9 nuclease,1μ1sgRNA-△NS6a,1μl sgRNA-△NS6b,5μL 10×reactionbuffer,补加RNase水至总体积为50μL。
5.如权利要求1所述Nluc标记的重组猪δ冠状病毒感染性克隆质粒构建方法,其特征在于,步骤(2)中,用于分别扩增猪δ冠状病毒NS6基因上游序列和下游序列的引物分别为:
PDCoV-NS6-upF:SEQ ID NO:4;
PDCoV-NS6-upR:SEQ ID NO:5;
PDCoV-NS6-downF:SEQ ID NO:6;
PDCoV-NS6-downR:SEQ ID NO:7。
6.如权利要求1所述Nluc标记的重组猪δ冠状病毒感染性克隆质粒构建方法,其特征在于:步骤(2)中,在进行重叠延伸PCR扩增时,所述NS6基因上、下游片段和Nluc基因片段的摩尔量比为2:2:1。
7.如权利要求1所述Nluc标记的重组猪δ冠状病毒感染性克隆质粒构建方法,其特征在于:步骤(3)中,在进行同源重组时,所述线性化BAC载体与融合片段的摩尔量比为1:4。
8.Nluc标记的重组猪δ冠状病毒感染性克隆质粒转染细胞后获得的重组病毒,其特征在于:所述Nluc标记的重组猪δ冠状病毒感染性克隆质粒是按权利要求1-8任一项所述方法构建的。
9.权利要求8所述的重组病毒在筛选抗猪δ冠状病毒药物中的应用。
10.盐酸石蒜碱在制备抗猪δ冠状病毒药物中的用途。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110779553.1A CN113584080A (zh) | 2021-07-09 | 2021-07-09 | 一种Nluc标记的重组猪δ冠状病毒感染性克隆质粒的构建及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110779553.1A CN113584080A (zh) | 2021-07-09 | 2021-07-09 | 一种Nluc标记的重组猪δ冠状病毒感染性克隆质粒的构建及其应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN113584080A true CN113584080A (zh) | 2021-11-02 |
Family
ID=78246882
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110779553.1A Pending CN113584080A (zh) | 2021-07-09 | 2021-07-09 | 一种Nluc标记的重组猪δ冠状病毒感染性克隆质粒的构建及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113584080A (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116445528A (zh) * | 2023-04-14 | 2023-07-18 | 扬州大学 | 重组猪流行性腹泻病毒感染性克隆的构建方法及其感染性克隆和应用 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108478571A (zh) * | 2018-05-04 | 2018-09-04 | 中国疾病预防控制中心病毒病预防控制所 | 石蒜碱在制备广谱抗冠状病毒药物中的应用 |
CN110331171A (zh) * | 2019-07-11 | 2019-10-15 | 华中农业大学 | 一种高效快速构建猪肠道冠状病毒重组病毒的方法 |
CN112852873A (zh) * | 2021-02-04 | 2021-05-28 | 华中农业大学 | 一种猪δ冠状病毒感染性克隆质粒的构建方法 |
CN113967211A (zh) * | 2021-12-15 | 2022-01-25 | 山东达因海洋生物制药股份有限公司 | 盐酸石蒜碱硫酯在制备抗冠状病毒药物中的应用 |
-
2021
- 2021-07-09 CN CN202110779553.1A patent/CN113584080A/zh active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108478571A (zh) * | 2018-05-04 | 2018-09-04 | 中国疾病预防控制中心病毒病预防控制所 | 石蒜碱在制备广谱抗冠状病毒药物中的应用 |
CN110331171A (zh) * | 2019-07-11 | 2019-10-15 | 华中农业大学 | 一种高效快速构建猪肠道冠状病毒重组病毒的方法 |
CN112852873A (zh) * | 2021-02-04 | 2021-05-28 | 华中农业大学 | 一种猪δ冠状病毒感染性克隆质粒的构建方法 |
CN113967211A (zh) * | 2021-12-15 | 2022-01-25 | 山东达因海洋生物制药股份有限公司 | 盐酸石蒜碱硫酯在制备抗冠状病毒药物中的应用 |
Non-Patent Citations (2)
Title |
---|
AITOR NOGALES等: "A Novel Fluorescent and Bioluminescent Bireporter Influenza A Virus To Evaluate Viral Infections", 《J VIROL》 * |
MENGJIA ZHANG等: "Genetic manipulation of porcine deltacoronavirus reveals insights into NS6 and NS7 functions: a novel strategy for vaccine design", 《EMERG MICROBES INFECT》 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116445528A (zh) * | 2023-04-14 | 2023-07-18 | 扬州大学 | 重组猪流行性腹泻病毒感染性克隆的构建方法及其感染性克隆和应用 |
CN117286162A (zh) * | 2023-04-14 | 2023-12-26 | 扬州大学 | 重组猪δ冠状病毒感染性克隆及其构建方法和应用 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN110079541B (zh) | 一种构建冠状病毒感染性克隆的方法及其应用 | |
CN112852873B (zh) | 一种猪δ冠状病毒感染性克隆质粒的构建方法 | |
CN110468155B (zh) | 一种用于拯救猪肠道甲型冠状病毒的系统、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 | |
CN101979598B (zh) | 一种携带荧光素酶报告基因的hsv-1 bac系统的构建方法 | |
CN113584080A (zh) | 一种Nluc标记的重组猪δ冠状病毒感染性克隆质粒的构建及其应用 | |
CN106939320B (zh) | 一种伪狂犬病毒js-2012株感染性克隆质粒、构建方法与应用 | |
CN111334528B (zh) | 水貂肠炎细小病毒全基因组感染性克隆及其构建方法和应用 | |
CN105713866A (zh) | 人巨细胞病毒感染性克隆及其构建方法和应用 | |
CN111690669A (zh) | Sva 3c蛋白在促进猪源病毒复制中的应用 | |
CN116463297A (zh) | 一种表达鸡传染性贫血病病毒vp1蛋白的重组血清4型禽腺病毒及其制备方法 | |
CN113862226B (zh) | 一种Dicer基因敲除的BHK-21细胞系 | |
CN110904056B (zh) | 一种传染性支气管炎病毒rH120-YZS1Δ5a及其构建方法和应用 | |
CN114214338A (zh) | 猪源piv5全长感染性克隆及其制备方法和应用 | |
CN113416713A (zh) | 一种重组腺病毒的构建及其应用 | |
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 | |
CN113564165B (zh) | 一种胞内编辑伪狂犬病毒关键基因的细胞株及其构建方法和应用 | |
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 | |
CN114480378B (zh) | 一种致鸭短喙与侏儒综合征的新型鹅细小病毒sd株全长感染性克隆的构建方法及应用 | |
CN116200348A (zh) | 一种适应Vero细胞的柯萨奇病毒A10型毒株的构建方法及其应用 | |
CN115725622A (zh) | 一株表达asfv p30蛋白的重组gi型日本脑炎病毒及其构建方法与应用 | |
Luo et al. | Construction and Characterization of an Infectious Clone of Chikungunya Virus Strain 37997 | |
CN116804210A (zh) | 一种肠道病毒载体及其应用 | |
CN116855459A (zh) | 一种基于CRISPR-Cas9技术的表达新城疫病毒La Sota毒株HN蛋白的重组血清4型禽腺病毒及其制备方法 | |
CN116121451A (zh) | 一种猪繁殖与呼吸综合征通用型rt-raa荧光型检测方法 |
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 | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20211102 |
|
RJ01 | Rejection of invention patent application after publication |