CN111793651A - Lentiviral expression vector for COVID-19 drug screening and construction method thereof - Google Patents
Lentiviral expression vector for COVID-19 drug screening and construction method thereof Download PDFInfo
- Publication number
- CN111793651A CN111793651A CN202010700324.1A CN202010700324A CN111793651A CN 111793651 A CN111793651 A CN 111793651A CN 202010700324 A CN202010700324 A CN 202010700324A CN 111793651 A CN111793651 A CN 111793651A
- Authority
- CN
- China
- Prior art keywords
- ncovn
- puro
- pcw
- fragment
- expression vector
- 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
- 239000013604 expression vector Substances 0.000 title claims abstract description 14
- 238000007877 drug screening Methods 0.000 title claims abstract description 11
- 208000025721 COVID-19 Diseases 0.000 title claims abstract description 10
- 238000010276 construction Methods 0.000 title claims abstract description 10
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 17
- 239000013598 vector Substances 0.000 claims abstract description 8
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 claims abstract description 6
- 241000713666 Lentivirus Species 0.000 claims abstract description 5
- 239000002299 complementary DNA Substances 0.000 claims abstract description 5
- 108700002099 Coronavirus Nucleocapsid Proteins Proteins 0.000 claims abstract description 3
- 229950010131 puromycin Drugs 0.000 claims abstract description 3
- 239000012634 fragment Substances 0.000 claims description 26
- 239000013612 plasmid Substances 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 10
- 108090000790 Enzymes Proteins 0.000 claims description 7
- 102000004190 Enzymes Human genes 0.000 claims description 7
- 108090001074 Nucleocapsid Proteins Proteins 0.000 claims description 7
- 238000001976 enzyme digestion Methods 0.000 claims description 7
- 108091008146 restriction endonucleases Proteins 0.000 claims description 4
- 230000003321 amplification Effects 0.000 claims description 3
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 3
- 102000003960 Ligases Human genes 0.000 claims description 2
- 108090000364 Ligases Proteins 0.000 claims description 2
- 101710141454 Nucleoprotein Proteins 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 claims description 2
- 238000003752 polymerase chain reaction Methods 0.000 claims description 2
- 238000004064 recycling Methods 0.000 claims 1
- 230000004907 flux Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 13
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 241000315672 SARS coronavirus Species 0.000 description 6
- 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 5
- 239000001963 growth medium Substances 0.000 description 4
- 238000003125 immunofluorescent labeling Methods 0.000 description 4
- 241001678559 COVID-19 virus Species 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 101150063416 add gene Proteins 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- 239000013504 Triton X-100 Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 239000012103 Alexa Fluor 488 Substances 0.000 description 1
- 241000008904 Betacoronavirus Species 0.000 description 1
- 241000711573 Coronaviridae Species 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 101710091045 Envelope protein Proteins 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 208000025370 Middle East respiratory syndrome Diseases 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 229940096437 Protein S Drugs 0.000 description 1
- 101710188315 Protein X Proteins 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 101710198474 Spike protein Proteins 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 102100021696 Syncytin-1 Human genes 0.000 description 1
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 1
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- XMEVHPAGJVLHIG-FMZCEJRJSA-N chembl454950 Chemical compound [Cl-].C1=CC=C2[C@](O)(C)[C@H]3C[C@H]4[C@H]([NH+](C)C)C(O)=C(C(N)=O)C(=O)[C@@]4(O)C(O)=C3C(=O)C2=C1O XMEVHPAGJVLHIG-FMZCEJRJSA-N 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000003950 pathogenic mechanism Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 229960004989 tetracycline hydrochloride Drugs 0.000 description 1
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
- 230000003612 virological effect Effects 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
- C12N15/86—Viral vectors
-
- 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
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
-
- 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
- C12N2510/00—Genetically modified cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Virology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Developmental Biology & Embryology (AREA)
- Transplantation (AREA)
- Cell Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a construction method of a lentivirus expression vector for COVID-19 drug screening, which takes pCW-Cas9-Blast as a skeleton vector, subclones nCoVN cDNA into the skeleton vector, and forms a lentivirus expression vector pCW-Puro-T2A-nCoVN of a novel coronavirus nucleocapsid protein; wherein the nCoVN cDNA carries a 6 x his tag and a puromycin resistance gene. The constructed lentiviral expression vector for COVID-19 drug screening has low requirements on experimental environment, can be operated in a conventional laboratory, can be operated at high flux, and can be constructed and obtained in large quantities only in 2 days.
Description
Technical Field
The invention relates to an iPS-based lentiviral expression vector for COVID-19 drug screening and a construction method thereof.
Background
SARS-CoV-2 is a beta coronavirus, and has genome identity of 79% with SARS-CoV, and its pathogenesis is similar to that of SARS-CoV. Many current studies on SARS-CoV-2 rely on prior work with SARS-CoV. By referring to the pathogenic mechanism of SARS and MERS, and selecting suitable candidate drug for detection, the risk of long development period, high cost and the like of new drugs can be greatly reduced. Therefore, the construction of a rapid and accurate novel pneumonia drug screening tool has important significance for epidemic situation prevention and control work under the current severe situation. The conventional cell level research method for COVID-19 drug screening is to infect mammalian cells cultured in vitro with SARS-CoV-2 virus strain, but the method relates to experiments such as culture of virus strain, and has the advantages of safety risk, high requirement on experimental environment and no contribution to high-throughput screening.
The structural analysis of SARS-CoV genome shows that its coding protein mainly includes spike protein (S), nucleocapsid protein (N), membrane protein (M), envelope protein (E) and so on. Wherein the nucleocapsid protein (N) is the main structural protein of SARS-CoV, can wrap the viral genome into a helical Ribocapsid (RNP), and plays an important role in the self-assembly of the virus. Research shows that the nucleocapsid protein can regulate the division and proliferation of cells, inhibit the generation of I-type interferon and participate in signal pathways such as NF-K B, AP-1, TGF-beta and the like. In addition, the nucleocapsid protein has the characteristic of high conservation compared with other SARS-CoV proteins. Thus, the nucleocapsid protein is an ideal tool for coronavirus research.
Disclosure of Invention
The invention aims to solve the technical problem of providing a lentiviral expression vector for COVID-19 drug screening and a construction method thereof.
Specifically, the construction method of the lentiviral expression vector is that pCW-Cas9-Blast (Addgene, 83481) is used as a skeleton vector, nCoVN cDNA (with a 6 x his label and a puromycin resistance gene) is subcloned into the skeleton vector to form the lentiviral expression vector pCW-Puro-T2A-nCoVN of the novel coronavirus nucleocapsid protein; the recombinant plasmid is transferred into a human induced pluripotent stem cell by using a lentivirus transfection technology, and a lentivirus vector capable of highly expressing the nCoVN gene is determined and constructed by detecting the nCoVN mRNA expression level and immunofluorescence staining.
The constructed lentiviral expression vector for COVID-19 drug screening has low requirements on experimental environment, can be operated in a conventional laboratory, can be operated at high flux, and can be constructed and obtained in large quantities only in 2 days.
Drawings
FIG. 1 is a diagram showing the electrophoresis of the Puro-T2A fragment and nCoVN fragment after enzyme digestion, wherein: m: DNA Marker; 1: Puro-T2A; 2: nCoVN;
FIG. 2 is a schematic structural diagram of a recombinant plasmid vector pCW-Puro-T2A-nCoVN;
FIG. 3 is Dox-induced expression of nCoVN gene in iPS-nCoVN;
FIG. 4 shows immunofluorescence staining, in which 6 XHis Tag is anti-6 XHis Tag (green) antibody to detect nCoVN protein expression in cells, and DAPI is staining of nuclei with DAPI (blue) at a scale of 10 um.
Detailed Description
The invention will be further described with reference to the following description of the drawings and specific examples, which should not be construed as limiting the invention. Modifications and substitutions of the methods, procedures, and conditions of the present invention can be made without departing from the spirit and substance of the invention. Unless otherwise indicated, the experimental procedures used in the examples are all conventional procedures and techniques well known to those skilled in the art, and reagents or materials are all commercially available.
Example 1
Construction of Lentiviral expression vector pCW-Puro-T2A-nCoVN
The commercial plasmids pCW-Cas9 (Addge, 50661), pCW-Cas9-Blast (Addge, 83481) and pUC57 (from GeneMedi) containing the nucleocapsid protein (N) sequence. After the respective plasmids were subjected to amplification culture, plasmids were extracted.
Primers (synthesized by Shanghai Bioengineering Co., Ltd.) were designed with the following sequences:
CoVN-6His-NheI-F：ttgctagccatcaccatcaccatcacatgtctgataatggacc；
CoVN-BamHI-R：agggatccttaggcctgagttgagtcagcactgc；
Puro-NheI-F：ttgctagcatggccaccgagtacaa；
T2A-AvrII-R：cctaggtgggccaggattct。
firstly, amplifying a Puro-T2A fragment with NheI and AvrII enzyme cutting sites at two ends from a pCW-Cas9 plasmid; and amplifying the full-length sequence of the N protein gene to obtain an nCoVN fragment, wherein the N end of the nCoVN fragment is provided with a 6 × his tag and an NheI enzyme cutting site, and the other end of the nCoVN fragment is provided with a BamHI enzyme cutting site.
The PCR reaction system is as follows:
the PCR amplification procedure is as follows:
the enzymes used below were all purchased from Thermo Fisher, inc. The obtained Puro-T2A fragment was digested with restriction enzymes NheI and AvrII, and the pCW-Cas9-Blast plasmid and the amplified nCoVN fragment were digested with restriction enzymes NheI and BamHI, respectively. After enzyme digestion, a double enzyme digestion product of Puro-T2A gene fragment and nCoVN gene fragment is obtained (shown in figure 1), the pCW-Cas9-Blast plasmid is divided into two fragments of about 7.4kb and 4.3kb, and the 7.4kb fragment is recovered.
The Puro-T2A and nCoVN gene fragments were ligated to a 7.4kb pCW-Cas9-Blast plasmid fragment using T4 ligase to obtain plasmid pCW-Puro-T2A-nCoVN containing the Puro-T2A-nCoVN gene. (see FIG. 2)
Example 2
Obtaining iPS-nCoVN cell strain
2.1 Lentiviral packaging
2.1.1 inoculating HEK293T cells into six-well plates, culturing with DMEM culture solution containing 10% fetal bovine serum, and transfecting when the cell confluency reaches 70% -80%.
2.1.2 pCW-Puro-T2A-nCoVN, pVSVg (Addgene), psPAX2(Addgene) were co-transfected into HEK293T cells following the procedures described in the EZ Trans instructions.
2.1.3 at 37 ℃ 5% CO2After 24 hours of culture in the incubator, the culture medium containing the EZ Trans-DNA complex was removed and replaced with a culture medium of D10 (DMEM culture medium + 10% fetal bovine serum + 1% BSA), and the culture was continued for 3 days, and the culture medium was collected every day.
2.1.4 taking the culture solution, centrifuging at 3000rpm and 4 ℃ for 10min, and removing cell residues. Concentrating virus by conventional sucrose concentration method, and storing at-80 deg.C.
2.1.5 transfection into iPS cells. And (3) carrying out transfection when the confluence degree of the iPS cells reaches 70-80%, wherein the multiplicity of infection (MOI) is 0.3-0.5.
Example 3
Dox induces expression of nCoVN
3.1 addition of tetracycline hydrochloride Dox (purchased from Sigma under code No. D9891) to the medium at a final concentration of 2. mu.g/ml induced nCoVN expression (see FIG. 3), and an equivalent amount of DMSO was added to the control group.
3.2 detection of nCoVN mRNA expression levels: total RNA was extracted using UNLQ-10 column Trizol Total RNA isolation kit (Sangon Biotech, B511321-0100) and then treated with DNase I (Sangon Biotech, B618252) for 30 min. mRNA was Reverse transcribed using an iScript Reverse Transcription Supermix (Bio-Rad, 1708841). The use of Pikoreal-time PCR system (Thermo Fisher) and Sso AdvancedTMUniversal
Green SuperMix (BioRad, 1725271) was used for quantitative polymerase chain reaction. Primers were designed with GAPDH as the internal reference, and the primer sequences for detecting nCoVN expression levels in iPSC and iPSC-nCoVN (DMSO control, Dox induction) were as follows:
nCoVN-RT-F：CATTGGCATGGAAGTCACAC；
nCoVN-RT-R：TCTGCGGTAAGGCTTGAGTT；
GAPDH-RT-F：TGGGTGTGAACCATGAGAAG；
GAPDH-RT-R：GTGTCGCTGTTGAAGTCAGA。
3.3 immunofluorescence staining: standing with 4% paraformaldehyde fixing solution at room temperature for 20min to fix cells, and washing with PBS for 3 times; soaking in 0.25% Triton X-100 (PBS) at room temperature for 20min, and washing with PBS for 2min for 3 times; dripping 10% normal goat serum (PBS preparation), sealing at 37 deg.C for 1 hr; the blocking solution was aspirated off, sufficient diluted primary antibody 6 × His Tag, 1:250, mouse, (Sangon Biotech, D191001) was added and incubated overnight at 4 ℃; the cells were washed 3 times with 0.1% Triton X-100 (PBS) and incubated with a second antibody Alexa Fluor488 goat anti-mouse for 1 hour at 37 ℃; adding DAPI (4', 6-diamidino-2-phenylindole, 1 mu g/ml) dropwise, incubating for 5min in dark, staining cell nucleus, and washing redundant DAPI with PBS; sealing the plate by using a sealing liquid containing an anti-fluorescence quenching agent, and observing and acquiring an image under a fluorescence microscope, wherein the image is shown in figure 4; the results show that the expression level of nCoVN in the Dox-induced group is 267-fold higher than that of the DMSO control group. immunofluorescence staining with a 6 × His tag (green) was evident in iPSC-nCoVN cells. The result shows that the invention successfully constructs the lentiviral vector capable of expressing the nCoVN gene at high level.
The embodiments described above are presented to enable those skilled in the art to make and use the invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the embodiments described herein, and those skilled in the art should make improvements and modifications to the present invention based on the disclosure of the present invention within the protection scope of the present invention.
Claims (3)
1. A construction method of a lentivirus expression vector for COVID-19 drug screening is characterized in that,
the method is characterized in that pCW-Cas9-Blast is used as a skeleton vector, nCoVN cDNA is subcloned into the skeleton vector to form a novel coronavirus nucleocapsid protein lentiviral expression vector pCW-Puro-T2A-nCoVN;
wherein the nCoVN cDNA carries a 6 x his tag and a puromycin resistance gene.
2. The construction method according to claim 1, comprising the steps of:
(1) amplifying a Puro-T2A fragment with NheI and AvrII enzyme cutting sites at two ends from a pCW-Cas9 plasmid; amplifying the full-length sequence of the N protein gene of the pUC57 plasmid containing the nucleocapsid protein sequence to obtain an nCoVN fragment, wherein the N end of the nCoVN fragment is provided with a 6 x his label and an NheI enzyme cutting site, and the other end of the nCoVN fragment is provided with a BamHI enzyme cutting site;
(2) carrying out enzyme digestion on the obtained Puro-T2A fragment by using restriction enzymes NheI and AvrII, and simultaneously carrying out enzyme digestion on the pCW-Cas9-Blast plasmid and the amplified nCoVN fragment by using restriction enzymes NheI and BamHI respectively;
(3) after enzyme digestion, obtaining double enzyme digestion products of Puro-T2A gene fragments and nCoVN gene fragments, dividing pCW-Cas9-Blast plasmid into two fragments of about 7.4kb and 4.3kb, and recycling the 7.4kb fragment;
(4) the Puro-T2A and nCoVN gene fragments are connected to a 7.4kb pCW-Cas9-Blast plasmid fragment by using T4 ligase to obtain a plasmid pCW-Puro-T2A-nCoVN containing the Puro-T2A-nCoVN gene;
wherein, in the step (1), the two amplified PCR reaction systems are:
in the step (1), the two PCR amplification procedures for amplification are as follows:
the primer sequences used for amplification were as follows:
CoVN-6His-NheI-F：ttgctagccatcaccatcaccatcacatgtctgataatggacc；
CoVN-BamHI-R：agggatccttaggcctgagttgagtcagcactgc；
Puro-NheI-F：ttgctagcatggccaccgagtacaa；
T2A-AvrII-R：cctaggtgggccaggattct。
3. the lentiviral expression vector for COVID-19 drug screening constructed according to the method of claim 1 or 2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010700324.1A CN111793651A (en) | 2020-07-17 | 2020-07-17 | Lentiviral expression vector for COVID-19 drug screening and construction method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010700324.1A CN111793651A (en) | 2020-07-17 | 2020-07-17 | Lentiviral expression vector for COVID-19 drug screening and construction method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111793651A true CN111793651A (en) | 2020-10-20 |
Family
ID=72808162
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010700324.1A Pending CN111793651A (en) | 2020-07-17 | 2020-07-17 | Lentiviral expression vector for COVID-19 drug screening and construction method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111793651A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108779447A (en) * | 2015-09-28 | 2018-11-09 | 天普大学-联邦高等教育系统 | The method and composition of RNA guiding treatments for HIV infection |
| CN111239392A (en) * | 2020-02-26 | 2020-06-05 | 浙江诺迦生物科技有限公司 | Novel coronavirus pneumonia (COVID-19) serological diagnosis kit |
| GB202005985D0 (en) * | 2020-04-23 | 2020-06-10 | Univ Johannesburg Witwatersrand | Sars-Cov-2 Diagnostic control compositions |
| CN111378785A (en) * | 2020-03-12 | 2020-07-07 | 仁宽(上海)生物科技有限公司 | Pseudo virus standard substance for nucleic acid diagnosis of novel coronavirus 2019-nCov and application thereof |
-
2020
- 2020-07-17 CN CN202010700324.1A patent/CN111793651A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108779447A (en) * | 2015-09-28 | 2018-11-09 | 天普大学-联邦高等教育系统 | The method and composition of RNA guiding treatments for HIV infection |
| CN111239392A (en) * | 2020-02-26 | 2020-06-05 | 浙江诺迦生物科技有限公司 | Novel coronavirus pneumonia (COVID-19) serological diagnosis kit |
| CN111378785A (en) * | 2020-03-12 | 2020-07-07 | 仁宽(上海)生物科技有限公司 | Pseudo virus standard substance for nucleic acid diagnosis of novel coronavirus 2019-nCov and application thereof |
| GB202005985D0 (en) * | 2020-04-23 | 2020-06-10 | Univ Johannesburg Witwatersrand | Sars-Cov-2 Diagnostic control compositions |
Non-Patent Citations (3)
| Title |
|---|
| DEGUO WANG: "One-pot Detection of COVID-19 with Real-time Reverse-transcription Loop-mediated Isothermal Amplification (RT-LAMP) Assay and Visual RT-LAMP Assay" * |
| ZEBIN LIN等: "The Nucleocapsid Protein of SARS-CoV-2 Abolished Pluripotency in Human Induced Pluripotent Stem Cells" * |
| 秦小波等: "SAR-CoV-2核衣壳蛋白预测与原核重组表达应用" * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ikegami et al. | Rescue of infectious rift valley fever virus entirely from cDNA, analysis of virus lacking the NSs gene, and expression of a foreign gene | |
| Plante et al. | Spike mutation D614G alters SARS-CoV-2 fitness and neutralization susceptibility | |
| Cornu et al. | Characterization of the arenavirus RING finger Z protein regions required for Z-mediated inhibition of viral RNA synthesis | |
| Bullido et al. | Influenza A virus NEP (NS2 protein) downregulates RNA synthesis of model template RNAs | |
| Wong-Ho et al. | Unconventional sequence requirement for viral late gene core promoters of murine gammaherpesvirus 68 | |
| Pickar et al. | Roles of serine and threonine residues of mumps virus P protein in viral transcription and replication | |
| Wang et al. | Determination of lentiviral infectious titer by a novel droplet digital PCR method | |
| CN106755089B (en) | Cell line for expressing goat lymphocyte activating molecules and construction method and application thereof | |
| Wang et al. | Susceptibility to SARS-CoV-2 of cell lines and substrates commonly used to diagnose and isolate influenza and other viruses | |
| Dang et al. | Marek’s disease virus type 1 encoded analog of miR-155 promotes proliferation of chicken embryo fibroblast and DF-1 cells by targeting hnRNPAB | |
| Kondo et al. | Recognition of a novel stage of betaherpesvirus latency in human herpesvirus 6 | |
| Li et al. | IRF1 up-regulates isg15 gene expression in dsRNA stimulation or CSFV infection by targeting nucleotides− 487 to− 325 in the 5′ flanking region | |
| Galvis et al. | Conformational changes in the 5′ end of the HIV-1 genome dependent on the debranching enzyme DBR1 during early stages of infection | |
| Chen et al. | Neuropilin-1 facilitates pseudorabies virus replication and viral glycoprotein B promotes its degradation in a furin-dependent manner | |
| Yin et al. | Fowl adenovirus serotype 4 uses gga-miR-181a-5p expression to facilitate viral replication via targeting of STING | |
| CN111793651A (en) | Lentiviral expression vector for COVID-19 drug screening and construction method thereof | |
| CN110129272B (en) | PK-15 cell strain for stably expressing MAP3K8 protein and construction and application thereof | |
| Murakami et al. | High-level expression of exogenous genes by replication-competent retrovirus vectors with an internal ribosomal entry site | |
| Murphy et al. | Identification of sendai virus L protein amino acid residues affecting viral mRNA cap methylation | |
| US8906386B2 (en) | Mutant proteins of the F protein of PIV-5 and PIV-2 | |
| CN109722465B (en) | HIV drug resistance detection vector and construction method | |
| US11105805B2 (en) | Method for detecting HIV-1 co-receptor tropism | |
| Sleman et al. | Human Cytomegalovirus UL24 and UL43 Cooperate to Modulate the Expression of Immunoregulatory UL16 Binding Protein 1 | |
| Feng et al. | A DNA-based non-infectious replicon system to study SARS-CoV-2 RNA synthesis | |
| Hou et al. | Two polypyrimidine tracts in intron 4 of the major immediate early gene are critical for gene expression switching from IE1 to IE2 and for replication of human cytomegalovirus |
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: 20201020 |
|
| RJ01 | Rejection of invention patent application after publication |