CN114774418B - shRNA分子及其在敲低TM9SF2基因表达中的应用 - Google Patents
shRNA分子及其在敲低TM9SF2基因表达中的应用 Download PDFInfo
- Publication number
- CN114774418B CN114774418B CN202210411689.1A CN202210411689A CN114774418B CN 114774418 B CN114774418 B CN 114774418B CN 202210411689 A CN202210411689 A CN 202210411689A CN 114774418 B CN114774418 B CN 114774418B
- Authority
- CN
- China
- Prior art keywords
- tm9sf2
- shrna
- cells
- cell
- 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
- 108091027967 Small hairpin RNA Proteins 0.000 title claims abstract description 55
- 239000004055 small Interfering RNA Substances 0.000 title claims abstract description 54
- 230000014509 gene expression Effects 0.000 title abstract description 21
- 101150030386 TM9SF2 gene Proteins 0.000 title abstract description 12
- 241000700605 Viruses Species 0.000 claims abstract description 13
- 208000015181 infectious disease Diseases 0.000 claims description 13
- 241001115402 Ebolavirus Species 0.000 claims description 10
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 3
- 230000002401 inhibitory effect Effects 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 24
- 108090000623 proteins and genes Proteins 0.000 abstract description 24
- 102000004169 proteins and genes Human genes 0.000 abstract description 21
- 238000010361 transduction Methods 0.000 abstract description 5
- 230000026683 transduction Effects 0.000 abstract description 5
- 108020004999 messenger RNA Proteins 0.000 abstract description 4
- 101000798727 Homo sapiens Transmembrane 9 superfamily member 2 Proteins 0.000 abstract description 2
- 208000037273 Pathologic Processes Diseases 0.000 abstract description 2
- 230000009054 pathological process Effects 0.000 abstract description 2
- 230000035790 physiological processes and functions Effects 0.000 abstract description 2
- 102100032503 Transmembrane 9 superfamily member 2 Human genes 0.000 abstract 1
- 230000002194 synthesizing effect Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 93
- 238000012216 screening Methods 0.000 description 18
- 238000011529 RT qPCR Methods 0.000 description 16
- 238000003197 gene knockdown Methods 0.000 description 15
- 238000000034 method Methods 0.000 description 15
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 13
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 12
- 238000001262 western blot Methods 0.000 description 9
- 230000009385 viral infection Effects 0.000 description 8
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 7
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 230000003834 intracellular effect Effects 0.000 description 7
- 108060001084 Luciferase Proteins 0.000 description 6
- 239000005089 Luciferase Substances 0.000 description 6
- 238000004113 cell culture Methods 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 238000013461 design Methods 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 230000001404 mediated effect Effects 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 238000003259 recombinant expression Methods 0.000 description 5
- 230000008685 targeting Effects 0.000 description 5
- 239000013598 vector Substances 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 4
- 229920002971 Heparan sulfate Polymers 0.000 description 4
- 230000004663 cell proliferation Effects 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 238000013518 transcription Methods 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- 238000001712 DNA sequencing Methods 0.000 description 3
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 3
- 101000798726 Homo sapiens Transmembrane 9 superfamily member 4 Proteins 0.000 description 3
- 241000713666 Lentivirus Species 0.000 description 3
- 206010057249 Phagocytosis Diseases 0.000 description 3
- 108700008625 Reporter Genes Proteins 0.000 description 3
- 102100032466 Transmembrane 9 superfamily member 4 Human genes 0.000 description 3
- 208000036142 Viral infection Diseases 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 230000008782 phagocytosis Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000012096 transfection reagent Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 241000219195 Arabidopsis thaliana Species 0.000 description 2
- 101100369980 Arabidopsis thaliana TMN1 gene Proteins 0.000 description 2
- 231100000699 Bacterial toxin Toxicity 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 108091033409 CRISPR Proteins 0.000 description 2
- 238000010354 CRISPR gene editing Methods 0.000 description 2
- 208000005623 Carcinogenesis Diseases 0.000 description 2
- 241000252212 Danio rerio Species 0.000 description 2
- 101710091045 Envelope protein Proteins 0.000 description 2
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- 101000798731 Homo sapiens Transmembrane 9 superfamily member 3 Proteins 0.000 description 2
- 101710188315 Protein X Proteins 0.000 description 2
- 101100369981 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) EMP70 gene Proteins 0.000 description 2
- 102100021696 Syncytin-1 Human genes 0.000 description 2
- 239000006180 TBST buffer Substances 0.000 description 2
- 102100032504 Transmembrane 9 superfamily member 3 Human genes 0.000 description 2
- 239000000688 bacterial toxin Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000036952 cancer formation Effects 0.000 description 2
- 231100000504 carcinogenesis Toxicity 0.000 description 2
- 230000021164 cell adhesion Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 210000001163 endosome Anatomy 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- 150000002339 glycosphingolipids Chemical class 0.000 description 2
- 210000002288 golgi apparatus Anatomy 0.000 description 2
- 206010022000 influenza Diseases 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 230000004807 localization Effects 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 244000000010 microbial pathogen Species 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 238000010206 sensitivity analysis Methods 0.000 description 2
- 230000004946 small molecule transport Effects 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- 241000712461 unidentified influenza virus Species 0.000 description 2
- 230000007502 viral entry Effects 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 208000030507 AIDS Diseases 0.000 description 1
- 241000224489 Amoeba Species 0.000 description 1
- 101000702760 Arabidopsis thaliana Cytosolic sulfotransferase 12 Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 description 1
- 208000004293 Chikungunya Fever Diseases 0.000 description 1
- 241001502567 Chikungunya virus Species 0.000 description 1
- 206010067256 Chikungunya virus infection Diseases 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 241001454374 Drosophila <fruit fly, subgenus> Species 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 102100031497 Heparan sulfate N-sulfotransferase 1 Human genes 0.000 description 1
- 101000588589 Homo sapiens Heparan sulfate N-sulfotransferase 1 Proteins 0.000 description 1
- 101000798717 Homo sapiens Transmembrane 9 superfamily member 1 Proteins 0.000 description 1
- GRRNUXAQVGOGFE-UHFFFAOYSA-N Hygromycin-B Natural products OC1C(NC)CC(N)C(O)C1OC1C2OC3(C(C(O)C(O)C(C(N)CO)O3)O)OC2C(O)C(CO)O1 GRRNUXAQVGOGFE-UHFFFAOYSA-N 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 102000004310 Ion Channels Human genes 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001477931 Mythimna unipuncta Species 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 102100033543 Proton-transporting V-type ATPase complex assembly regulator TMEM9 Human genes 0.000 description 1
- 101710183261 Proton-transporting V-type ATPase complex assembly regulator TMEM9 Proteins 0.000 description 1
- 241001112090 Pseudovirus Species 0.000 description 1
- 108010039491 Ricin Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 108010079723 Shiga Toxin Proteins 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- -1 TM9SF2 Proteins 0.000 description 1
- 101150118462 TMN3 gene Proteins 0.000 description 1
- 102100032463 Transmembrane 9 superfamily member 1 Human genes 0.000 description 1
- 241000700618 Vaccinia virus Species 0.000 description 1
- 206010046865 Vaccinia virus infection Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 108091036078 conserved sequence Proteins 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 102000046442 human TM9SF2 Human genes 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- GRRNUXAQVGOGFE-NZSRVPFOSA-N hygromycin B Chemical compound O[C@@H]1[C@@H](NC)C[C@@H](N)[C@H](O)[C@H]1O[C@H]1[C@H]2O[C@@]3([C@@H]([C@@H](O)[C@@H](O)[C@@H](C(N)CO)O3)O)O[C@H]2[C@@H](O)[C@@H](CO)O1 GRRNUXAQVGOGFE-NZSRVPFOSA-N 0.000 description 1
- 229940097277 hygromycin b Drugs 0.000 description 1
- 230000009610 hypersensitivity Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000008558 metabolic pathway by substance Effects 0.000 description 1
- 206010061289 metastatic neoplasm Diseases 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000000242 pagocytic effect Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000003950 pathogenic mechanism Effects 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000005670 sulfation reaction Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000009220 viral host cell interaction Effects 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
-
- 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
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
-
- 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
- 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
- C12N5/0684—Cells of the urinary tract or kidneys
- C12N5/0686—Kidney 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
- 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
- C12N5/0688—Cells from the lungs or the respiratory tract
-
- 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
- C12N5/0693—Tumour cells; Cancer 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
-
- 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
- C12N2320/00—Applications; Uses
- C12N2320/30—Special therapeutic applications
- C12N2320/32—Special delivery means, e.g. tissue-specific
-
- 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
- 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)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Cell Biology (AREA)
- Virology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Urology & Nephrology (AREA)
- Oncology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Pulmonology (AREA)
- Communicable Diseases (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明涉及一种shRNA分子及其在敲低TM9SF2基因表达中的应用。本发明通过分析TM9SF2基因序列,确定特异性shRNA的靶向序列并合成shRNA,利用慢病毒转导***构建稳定表达shRNA的细胞系,并在mRNA和蛋白层面检测特异性shRNA的干扰效果,为深入研究TM9SF2在细胞生理或病理过程中的作用提供了细胞模型。
Description
技术领域
本发明涉及分子生物技术领域,特别是涉及一种shRNA分子及其在在敲低TM9SF2基因表达中的应用。
背景技术
九次跨膜超家族蛋白(Transmembrane 9superfamily protein,TM9SF)又称为nonaspanins蛋白,是一组进化高度保守且包含9个跨膜结构域的蛋白。目前发现该家族蛋白在细菌、酵母、果蝇、哺乳动物等多个物种均有表达,其中人TM9SF家族包括4个成员即TM9SF1、TM9SF2、TM9SF3和TM9SF4,在酿酒酵母(Saccharomyces cerevisiae)中发现3个成员TMN1、TMN1和TMN3,果蝇体内发现TM9SF2、TM9SF3、TM9SF4三个成员。该类蛋白在人体组织和多种细胞系广泛表达,与细胞免疫、细胞黏附、吞噬、内质网应激、肿瘤发生及病原微生物感染与致病等多种生理或病理反应密切相关。目前,关于该蛋白家族的***研究较少,且多数研究主要集中于模式生物如阿米巴变形虫细胞粘菌(Dictyosteliumdiscoideum)、果蝇(Drosophila)、斑马鱼(zebrafish)、拟南芥(Arabidopsis thaliana)、酿酒酵母(Saccharomyces cerevisiae)、大鼠(Rat)。
TM9SF2(Transmembrane 9Superfamily Member 2)又称p76跨膜蛋白,从酵母到植物和人类都具有高度保守的序列和结构,表明其在细胞生理功能方面发挥重要作用。人TM9SF2蛋白主要定位于细胞内体,可能扮演离子通道或小分子转运载体的角色。另外,近期一些研究发现,TM9SF2在某些病原微生物感染和致病过程中发挥重要作用,如痘苗病毒、基孔肯雅病毒、肠出血性大肠杆菌以及细菌毒素如志贺毒素和蓖麻毒素。而且,TM9SF2的单核苷酸多态性分析发现,其与获得性免疫缺陷综合征即艾滋病的病程存在某种相关性,但遗憾的是这种相关性仍然没有被揭示。CRISPR-Cas9介导的全基因组筛选实验发现,TM9SF2是腺相关病毒(Adeno-associated virus,AAV)基因转导的关键因子。
然而,目前缺乏一种显著敲低TM9SF2的细胞系以针对该类蛋白的功能及机制进行深入研究。
发明内容
本发明的目的在于提供一种可显著敲低TM9SF2基因表达的shRNA分子及其在敲低TM9SF2基因表达中的应用。
本发明提供了一种shRNA分子,所述shRNA分子的核苷酸序列如SEQ ID NO:1或SEQID NO:2所示。
本发明提供了一种DNA片段,所述DNA片段编码权利要求1所述的shRNA分子。
本发明提供了一种重组表达载体,所述重组表达载体含有编码如上所述的shRNA分子的核苷酸序列。
在一些实施例中,所述重组表达载体为慢病毒载体。
在一些实施例中,所述重组表达载体基于psi-LVRU6MH载体构建得到。
本发明提供了如上所述的shRNA分子、DNA片段或重组表达载体在敲低TM9SF2基因表达中的应用。
本发明提供了一种宿主细胞,其基因组中含有如上所述的DNA片段。
在一些实施例中,所述宿主细胞为A549细胞或293T细胞。
本发明提供了一种TM9SF2基因敲低细胞系的制备方法,包括以下步骤:在目的细胞中外源表达如上所述的shRNA分子。
本发明提供了如上所述的shRNA分子、DNA片段或重组表达载体在制备用于抑制病毒感染的产品中的应用,所述病毒为埃博拉病毒。
本发明通过TM9SF2基因序列分析,根据shRNA设计的原则确定靶向序列并合成shRNA,构建到含U6启动子的shRNA表达慢病毒载体pLVRU6H上并进行DNA测序鉴定;然后利用慢病毒包装***制备携带shRNA的VSV-G蛋白包裹的假型慢病毒,感染靶细胞48h时收集细胞总RNA,通过RT-qPCR方法确定特异性shRNA的干扰效率;进而利用潮霉素压力多轮筛选获得细胞克隆,扩大培养后提取细胞总RNA和总蛋白,分别通过RT-qPCR、Western blot进行鉴定。结果显示,其中靶向2个位点的TM9SF2 shRNA的干扰效果最好,筛选获得两株分别靶向TM9SF2不同位点的A549细胞系,其中1株在RNA和蛋白水平的敲低效果最好。本研究首次成功筛选获得TM9SF2稳定敲低的A549细胞系,且TM9SF2的敲低对A549细胞活性无显著影响,为其功能研究奠定了细胞基础。
附图说明
图1为本发明一实施例的qRT-PCR和Western blot检测TM9SF2在细胞内固有表达情况;其中,A为qRT-PCR检测293T、A549和THP-1细胞内TM9SF2 mRNA的相对表达分析,以293T细胞作为参照,B为Western blot分析293T、A549和THP-1细胞内TM9SF2蛋白的表达;
图2为本发明一实施例的TM9SF2 shRNA的设计与干扰表达效果分析;其中,A为TM9SF2 shRNA设计与靶向位点分析,B为qRT-PCR检测shRNA靶向干扰A549内TM9SF2的转录水平;
图3为本发明一实施例的TM9SF2 shRNA稳定表达细胞系的鉴定;其中,A为PCR鉴定TM9SF2转录水平,B为qRT-PCR检测shRNA靶向干扰A549内TM9SF2的转录水平,C为Westernblot分析细胞内TM9SF2的敲低情况;
图4为本发明一实施例的A549-TM9SF2KD细胞活性分析;
图5为本发明一实施例的含萤光素酶报告基因的EBOV GP包裹的VSV或HIV假型病毒感染A549-TM9SF2KD细胞后48h细胞内萤光素酶活性检测结果;其中,A为EBOV GP包裹的VSV假型病毒,B为EBOV GP包裹的VSV或HIV假型病毒。
具体实施方式
为了更加简洁明了的展示本发明的技术方案、目的和优点,下面结合具体实施例及其附图对本发明做进一步的详细描述。可以理解,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
一、材料与方法
1.1细胞和主要试剂
293T、A549和THP-1细胞购自中国科学院细胞库;PEIpro转染试剂购自polyplus-transfection公司;PrimeScriptTM RT Master Mix(RR036A)为TaKaRa产品;GoTaq qPCRMaster Mix(A6002)购自Promega公司;潮霉素(Hygromycin B solution)购自invivogen公司;青-链霉素溶液(10000U/mL青霉素,10000μg/mL链霉素)为Gibco产品;南美胎牛血清(FBS),细胞培养基(DMEM)购自Hyclone公司;Anti-TM9SF2抗体(ab121227)购自abcam公司;GAPDH抗体(10494-1-AP)购自proteintech公司;HRP-山羊抗兔IgG为碧云天生物技术有限公司产品;ECL化学发光超敏显色试剂盒(36208ES60)购自上海翊圣生物科技有限公司。
1.2A549细胞对潮霉素的敏感性分析
按照1×105个/孔将A549细胞接种于12孔细胞培养板,37℃、5%CO2恒温培养箱中培养。培养12h后,在细胞培养液中加入潮霉素,使其终浓度为50、100、200、400μg/mL。每天通过镜下观察记录不同浓度的潮霉素对细胞状态的影响,从而确定最适浓度。
1.3shRNA合成及其表达质粒构建
根据shRNA设计的原则,选择3条TM9SF2基因的靶序列作为干扰位点(shRNA#1:GCTGCTAAACTTGAACCGAAA,shRNA#2:GCTAGATATAATCAGATGGAC,shRNA#3:GGTCACACCAGATGTATTACA),合成shRNA序列,并构建到psi-LVRU6H载体(GeneCopoeia)上(该部分工作委托广州易锦生物技术有限公司进行),DNA测序确定shRNA重组质粒pLV-U6-TM9SF2 shRNA。
shRNA#1:GCTGCTAAACTTGAACCGAAATCAAGAGTTTCGGTTCAAGTTTAGCAGC;
shRNA#2:GCTAGATATAATCAGATGGACTCAAGAGGTCCATCTGATTATATCTAGC;
shRNA#3:GGTCACACCAGATGTATTACATCAAGAGTGTAATACATCTGGTGTGACC。
1.4慢病毒介导的细胞转导
接种适量的293T细胞至6孔细胞培养板中,于37℃、5%CO2环境中培养至融合度约70-80%。将shRNA携带的慢病毒质粒pLV-U6-TM9SF2 shRNA或空质粒载体pLVRU6H与pCMV-dR8.2 dVpr、pCMV-VSV-G按质量比为2:1:1混合,然后参照PEIpro转染试剂的使用说明,将质粒混合物与转染试剂混合,孵育15min后加入到细胞培养板中,转染后48h收集细胞培养上清,低速离心去除细胞碎片,0.45μm过滤,收集上清即为病毒悬液。然后,取一定量的病毒悬液感染提前准备好的A549单层细胞,感染后48h收集部分RNA和蛋白样品(用于初步鉴定),其余部分加入确定浓度的潮霉素进行筛选。
1.5shRNA稳定细胞系的筛选与鉴定
经过多次潮霉素压力筛选获得细胞克隆,然后进行扩大培养,获得稳定传代的细胞系,收集细胞总RNA和总蛋白,利用RT-qPCR和Western blot方法进行鉴定。
1.6RT-qPCR
Trizol法提取细胞总RNA,定量后利用PrimeScript RT Master Mix参照说明书将RNA反转录为cDNA。然后用GoTaq qPCR试剂盒和StepOnePlus实时荧光定量PCR***对TM9SF2进行相对定量分析,以GAPDH作为内部参照,引物见表1。
表1实时荧光定量PCR引物
1.7Western blot
收集细胞,用一定量的RIPA裂解液裂解细胞,蛋白定量后制备样品,进行SDS-PAGE凝胶电泳分离,然后将蛋白样品转印到硝酸纤维素膜上,10%脱脂乳-TBST封闭1h,接着与TM9SF2抗体(200倍稀释)4℃摇床孵育过夜,TBST洗膜3次,然后与HRP-山羊抗兔IgG(5000倍稀释)室温孵育1h,充分洗涤后进行化学发光法显迹。
1.8细胞活性试验
TM9SF2敲低细胞及A549-LVRU6H对照细胞,经细胞计数接种至96孔板(1×104个/孔)中,分别在培养24h、48h和72h时检测细胞活性。即每孔加入培养基总体积10%的CCKSolution,继续培养1.5h,酶标仪测定450nm处的吸光值。
1.9细胞系功能验证
按照1×104个/孔分别接种A549-TM9SF2KD#3和A549-LVRU6H对照细胞至96孔板,置细胞培养箱培养12h,然后用一定剂量的含萤光素酶报告基因的埃博拉病毒(EBOV)囊膜蛋白(GP)包裹的假型水疱性口炎病毒(VSV)或慢病毒(HIV)分别感染A549-TM9SF2KD#3和A549-LVRU6H对照细胞,感染后48h弃掉培养液,无菌的PBS清洗细胞,加入100μL裂解液,室温静置10min使细胞充***解,然后将细胞裂解物转移至不透明的96孔板,加入100μL萤光素酶检测底物,进行萤光活性分析,从而判定病毒感染情况。
二、结果与讨论
2.1TM9SF2在细胞内的固有表达分析
通过提取A549、THP-1、293T细胞的RNA和蛋白,分别通过qRT-PCR和Western blot方法检测细胞内TM9SF2的固有表达情况。qRT-PCR结果显示,THP-1细胞内的TM9SF2的转录本稍高于293T和A549细胞(图1A);蛋白水平检测发现,TM9SF2在3种细胞内存在不同程度的固有表达,THP-1细胞内固有表达水平高于A549和293T细胞(图1B)。另外,TM9SF2在A549和THP-1细胞内存在两个不同分子量的条带约75kDa和56kDa,但在293T内表现出分子量约56kDa的条带。表明,TM9SF2在细胞内呈现不同的表达形式。
2.2TM9SF2干扰靶点的选取、shRNA合成与表达质粒构建
本研究根据shRNA设计的规律和原则,在TM9SF2 mRNA上选择3个靶向干扰位点(图2A),合成并构建到携带U6启动子的shRNA表达慢病毒载体pLVRU6H上,并通过DNA测序进行确定。然后利用慢病毒包装***包装VSV囊膜蛋白形成的假型病毒感染A549细胞,感染后48h收集细胞提取RNA,取相同量的RNA进行反转录-定量PCR(qRT-PCR)分析TM9SF2转录本的变化,结果显示shRNA#1和shRNA#3显著下调TM9SF2转录水平(图2B)。
2.3A549对潮霉素的敏感性分析
分别以终浓度50、100、200、400μg/mL的潮霉素处理A549细胞,处理后每天观察细胞状态,实验结果显示,潮霉素的工作浓度为400、200、100μg/mL时,处理后1-3天细胞均相继出现死亡。相比较400、200μg/mL浓度,100μg/mL细胞发生死亡需要时间过长。200μg/mL浓度即可满足试验需求,因此,选取200μg/mL的潮霉素作为A549细胞系筛选浓度。
2.4TM9SF2 shRNA稳定细胞系的筛选与鉴定
根据上述结果,本研究选择TM9SF2 shRNA#1和shRNA#3表达慢病毒质粒,通过假型慢病毒感染A549细胞,感染48h后消化细胞,传代并加入潮霉素(Hygromycin)至工作浓度为200μg/mL进行筛选。通过多轮筛选,出现细胞集落,然后扩大培养,收集细胞,通过PCR、qRT-PCR和Western blot检测细胞内TM9SF2的转录和表达情况。PCR结果显示,与无效对照shRNA细胞相比,筛选获得的A549-TM9SF2KD#3细胞内TM9SF2的转录本明显降低,而A549-TM9SF2KD#1不显著(图3A);qRT-PCR结果显示,A549-TM9SF2KD#1和A549-TM9SF2KD#3细胞内的TM9SF2 mRNA均显著下调,但A549-TM9SF2KD#3更显著(图3B)。综合数据,本研究选择A549-TM9SF2KD#3作为后续的研究对象和TM9SF2敲低细胞模型。Western blot结果显示,A549-TM9SF2KD#3内TM9SF2蛋白显著降低(图3C),表明,成功筛选获得TM9SF2稳定敲低A549细胞。
2.5TM9SF2敲低对A549细胞增殖的影响
进而,利用CCK-8方法检测筛选获得A549-TM9SF2KD细胞活性,从而分析TM9SF2敲低对细胞增殖和细胞活性的影响。结果显示,TM9SF2敲低细胞与对照细胞的细胞活性无显著差异(图4),表明TM9SF2对A549细胞增殖无显著影响,可作为TM9SF2敲低细胞模型进行后续研究。
2.6TM9SF2有助于EBOV GP介导的病毒进入和感染
为了分析TM9SF2对病毒感染的影响,我们利用含萤光素酶报告基因的EBOV GP包裹的VSV或HIV假型病毒(EBOVpv-VSV,EBOVpv-HIV)感染A549-TM9SF2KD细胞,感染后48h细胞内萤光素酶活性检测结果显示(图5),TM9SF2 KD细胞内萤光强度显著低于对照细胞A549-LVRU6H(即Control),表明TM9SF2在EBOV GP蛋白介导的病毒进入和感染中具有促进作用。
三、讨论
跨膜蛋白9(TM9)是一组以包含9个跨膜结构域的进化高度保守的蛋白。其中,TM9SF2从酵母、果蝇到人类在小分子转运、物质代谢、细胞黏附和吞噬、生长发育、肿瘤发生发展以及某些细菌毒素致病和病毒感染等生理和病理过程发挥重要作用。在人类细胞中,TM9SF4是TM9SF2的一个非常重要的同系物,在细胞吞噬中功能比较保守,有助于增强转移性肿瘤细胞的吞噬活性。另外,TM9SF2定位于细胞内体和高尔基体,可能影响内小体的成熟,并参与鞘糖脂调节和内体转运。另外,近年来,基于全基因组功能筛选发现,TM9SF2亦在某些病毒感染过程中发挥重要作用。
Tanaka等在探索与基孔肯尼亚病毒感染有关的宿主因子时发现,TM9SF2对硫酸乙酰肝素(heparan sulfate,HS)的N-硫酸化过程中至关重要,而HS是CHIKV感染的重要受体,TM9SF2通过调节N-去乙酰化酶/磺基转移酶1(NDST1)的正确定位和稳定性发挥作用,当TM9SF2缺失时,NDST1就不能正确定位且稳定性较差,从而导致CHIKV的受体HS数量减少、活性降低,病毒的感染性也随之下降。简而言之,TM9SF2在CHIKV感染过程中具有促进作用。然而,Heaton等利用CRISPR全基因组筛选技术(CRISPR SAM genome-wide screening)进行的人或禽流感病毒通用宿主限制因子的筛选研究中发现过表达TM9SF2能够抑制流感病毒的感染,提示该分子在甲型流感病毒感染中可能发挥负调控作用,但具体作用机制未被解析。2019年,Luteijn等研究确定TM9SF2参与硫酸乙酰肝素的表达,从而有助于痘苗病毒感染。另有研究发现,TM9SF2还是介导腺相关病毒转导的重要因子之一,可能与其定位于高尔基体并参与鞘糖脂调节和内体转运的功能有关。然而,其在病毒-宿主细胞互作中作用分子机制仍不清楚。
为此,我们结合前期在病毒-细胞互作中研究基础,开展了相关研究。首先分析了A549、THP-1和293T细胞中TM9SF2的固有表达情况,发现其在细胞内存在两种表达形式,但目前不清楚发挥功能的是何种形式,结合我们课题的研究目的,以A549为细胞模型采用shRNA技术筛选稳定敲低或敲除的细胞系。进而,基于TM9SF2的基因序列设计了特异性的shRNA,利用慢病毒转导和抗生素压力筛选标记,筛选获得了两株shRNA介导的TM9SF2稳定敲低的A549细胞系。应用RT-qPCR和Western blot方法对其敲低水平进行了鉴定,结果获得一株敲低效果显著的细胞系。通过细胞的增殖或活性实验确定TM9SF2基因的敲低不显著影响A549细胞的增殖或活性。本发明首次获得了TM9SF2稳定敲低的细胞系。目前,国内外对于TM9SF2在病毒致病机制中的研究较少,其在病毒感染过程中的调控作用尚不明了,本研究必将在病毒-细胞互作研究领域起到积极作用。
以上所述实施例仅表达了本发明的几种实施方式,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,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
序列表
<110> 申请人:深圳市人民医院
<120> shRNA分子及其在敲低TM9SF2基因表达中的应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 49
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 1
gcugcuaaac uugaaccgaa aucaagaguu ucgguucaag uuuagcagc 49
<210> 2
<211> 49
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 2
ggucacacca gauguauuac aucaagagug uaauacaucu ggugugacc 49
Claims (1)
1.shRNA分子在制备用于抑制病毒感染的产品中的应用,其特征在于,所述病毒为埃博拉病毒;
所述shRNA分子的核苷酸序列如SEQ ID NO:1或SEQ ID NO:2所示。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210411689.1A CN114774418B (zh) | 2022-04-19 | 2022-04-19 | shRNA分子及其在敲低TM9SF2基因表达中的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210411689.1A CN114774418B (zh) | 2022-04-19 | 2022-04-19 | shRNA分子及其在敲低TM9SF2基因表达中的应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114774418A CN114774418A (zh) | 2022-07-22 |
CN114774418B true CN114774418B (zh) | 2023-11-14 |
Family
ID=82431124
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210411689.1A Active CN114774418B (zh) | 2022-04-19 | 2022-04-19 | shRNA分子及其在敲低TM9SF2基因表达中的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114774418B (zh) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102574889A (zh) * | 2009-08-17 | 2012-07-11 | 诺克斯科技有限公司 | arNOX跨膜蛋白超家族9(TM9SF)的克隆与表达及其方法和应用 |
CN108603188A (zh) * | 2015-11-24 | 2018-09-28 | 联邦科学技术研究组织 | 在细胞培养物中产生病毒 |
CN108884461A (zh) * | 2015-11-24 | 2018-11-23 | 联邦科学技术研究组织 | 在禽蛋中产生病毒 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10633662B2 (en) * | 2015-11-10 | 2020-04-28 | The Board Of Trustees Of The Leland Stanford Junior University | Methods and compositions for modulating AAV infection |
-
2022
- 2022-04-19 CN CN202210411689.1A patent/CN114774418B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102574889A (zh) * | 2009-08-17 | 2012-07-11 | 诺克斯科技有限公司 | arNOX跨膜蛋白超家族9(TM9SF)的克隆与表达及其方法和应用 |
CN108603188A (zh) * | 2015-11-24 | 2018-09-28 | 联邦科学技术研究组织 | 在细胞培养物中产生病毒 |
CN108884461A (zh) * | 2015-11-24 | 2018-11-23 | 联邦科学技术研究组织 | 在禽蛋中产生病毒 |
Non-Patent Citations (2)
Title |
---|
Homo sapiens transmembrane 9 superfamily member 2 (TM9SF2), mRNA;Li Q 等;NCBI Reference Sequence: NM_004800.3;序列 * |
Transposon mutagenesis screen in mice identifies TM9SF2 as a novel colorectal cancer oncogene;Christopher R. Clark等;SCIENTIFIC REPORTS;15327 * |
Also Published As
Publication number | Publication date |
---|---|
CN114774418A (zh) | 2022-07-22 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Cameron et al. | Epstein-Barr virus latent membrane protein 1 induces cellular MicroRNA miR-146a, a modulator of lymphocyte signaling pathways | |
Yang et al. | CircRNA_100876 promote proliferation and metastasis of breast cancer cells through adsorbing microRNA-361-3p in a sponge form. | |
CN112126647B (zh) | 一种流感病毒的环状rna疫苗 | |
Zhang et al. | GADD45β, an anti-tumor gene, inhibits avian leukosis virus subgroup J replication in chickens | |
JP2020005657A (ja) | 癌の将来の発生についての早期マーカーならびに癌の治療および予防のための標的としてのTTV miRNA配列 | |
US20210322455A1 (en) | Methods and compositions for increasing the suppressive function of regulatory t-cells (tregs) | |
Yao et al. | PAd-shRNA-PTN reduces pleiotrophin of pancreatic cancer cells and inhibits neurite outgrowth of DRG | |
CN114774418B (zh) | shRNA分子及其在敲低TM9SF2基因表达中的应用 | |
US10457938B2 (en) | TTV miRNA sequences as an early marker for the future development of cancer and as a target for cancer treatment and prevention | |
CN112656806A (zh) | 多配体蛋白聚糖4的siRNA序列在抑制犬瘟热病毒复制中的应用 | |
CN114672460B (zh) | 一种靶向cd44的异质型cic细胞模型的制备方法及应用 | |
CN102660777A (zh) | 白叶枯病菌胁迫的普通野生稻ssh文库的构建方法 | |
CN109055429B (zh) | 一种靶向RunX2基因的小鼠成骨样细胞慢病毒载体及其构建方法 | |
CN110893240B (zh) | Nme2基因在抑制禽呼肠病毒复制中的应用 | |
CN113493770A (zh) | 人源glut5稳定转染细胞株及其在筛选glut5抑制剂中的应用 | |
CN112011628A (zh) | 一种与湖羊肌肉细胞增殖相关的LncRNA标志物及其检测引物和应用 | |
Liu et al. | Identification of genes regulated by nanog which is involved in ES cells pluripotency and early differentiation | |
CN117159748B (zh) | Tmprss12基因在制备预防或治疗新型冠状病毒感染药物的应用 | |
CN117343932B (zh) | 靶向抑制MS4A7基因表达的siRNA及其应用 | |
Nishimori et al. | Upregulation of host genes during disease progression in bovine leukemia virus infection is independent of overexpression of viral transcriptional regulators in vitro | |
CN111304201B (zh) | 一种降低乙型脑炎脑病毒感染的siRNA及其应用 | |
CN110093348B (zh) | 增强小鼠NPFFR2基因表达的shRNA | |
CN103255137B (zh) | 一种检测SKA1基因表达的方法及其siRNA的用途 | |
CN116832162A (zh) | Cd244在作为抗非洲猪瘟的基因编辑靶点中的应用 | |
CN108619489B (zh) | Id1蛋白和bmp4蛋白在制备抗***药物中的应用以及抗***药物 |
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 |