CN115341010A - 参与黄曲霉毒素b1毒性效应的靶点及其应用 - Google Patents
参与黄曲霉毒素b1毒性效应的靶点及其应用 Download PDFInfo
- Publication number
- CN115341010A CN115341010A CN202110525917.3A CN202110525917A CN115341010A CN 115341010 A CN115341010 A CN 115341010A CN 202110525917 A CN202110525917 A CN 202110525917A CN 115341010 A CN115341010 A CN 115341010A
- Authority
- CN
- China
- Prior art keywords
- bach1
- aflatoxin
- afb1
- cell
- cells
- 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
- OQIQSTLJSLGHID-WNWIJWBNSA-N aflatoxin B1 Chemical compound C=1([C@@H]2C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O OQIQSTLJSLGHID-WNWIJWBNSA-N 0.000 title claims abstract description 86
- 229930020125 aflatoxin-B1 Natural products 0.000 title claims abstract description 86
- 239000002115 aflatoxin B1 Substances 0.000 title claims abstract description 16
- 231100000331 toxic Toxicity 0.000 title abstract description 24
- 230000002588 toxic effect Effects 0.000 title abstract description 16
- 101000894871 Homo sapiens Transcription regulator protein BACH1 Proteins 0.000 claims abstract description 77
- 101000848171 Homo sapiens Fanconi anemia group J protein Proteins 0.000 claims abstract description 76
- 108091033409 CRISPR Proteins 0.000 claims abstract description 47
- XWIYFDMXXLINPU-UHFFFAOYSA-N Aflatoxin G Chemical class O=C1OCCC2=C1C(=O)OC1=C2C(OC)=CC2=C1C1C=COC1O2 XWIYFDMXXLINPU-UHFFFAOYSA-N 0.000 claims abstract description 18
- 102100034212 AFG1-like ATPase Human genes 0.000 claims abstract description 13
- 101000780581 Homo sapiens AFG1-like ATPase Proteins 0.000 claims abstract description 13
- MJBWDEQAUQTVKK-IAGOWNOFSA-N aflatoxin M1 Chemical compound C=1([C@]2(O)C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O MJBWDEQAUQTVKK-IAGOWNOFSA-N 0.000 claims abstract description 7
- 102100034553 Fanconi anemia group J protein Human genes 0.000 claims description 74
- 108091027544 Subgenomic mRNA Proteins 0.000 claims description 34
- 108090000623 proteins and genes Proteins 0.000 claims description 29
- 231100000419 toxicity Toxicity 0.000 claims description 11
- 230000001988 toxicity Effects 0.000 claims description 11
- 101150000392 BACH1 gene Proteins 0.000 claims description 10
- 101100165126 Homo sapiens BACH1 gene Proteins 0.000 claims description 10
- 239000003814 drug Substances 0.000 claims description 10
- 102000004169 proteins and genes Human genes 0.000 claims description 10
- -1 ENSSSCG00000006349 Proteins 0.000 claims description 8
- 230000008685 targeting Effects 0.000 claims description 5
- 229940079593 drug Drugs 0.000 claims description 4
- 102100036774 Afamin Human genes 0.000 claims description 3
- 102100022649 Homeobox protein Hox-A6 Human genes 0.000 claims description 3
- 101000928239 Homo sapiens Afamin Proteins 0.000 claims description 3
- 101001045083 Homo sapiens Homeobox protein Hox-A6 Proteins 0.000 claims description 3
- 101001120086 Homo sapiens P2Y purinoceptor 12 Proteins 0.000 claims description 3
- 101001074444 Homo sapiens Polycystin-1 Proteins 0.000 claims description 3
- 101000596084 Homo sapiens TATA box-binding protein-associated factor RNA polymerase I subunit C Proteins 0.000 claims description 3
- 102100026171 P2Y purinoceptor 12 Human genes 0.000 claims description 3
- 102100036143 Polycystin-1 Human genes 0.000 claims description 3
- 102100035213 TATA box-binding protein-associated factor RNA polymerase I subunit C Human genes 0.000 claims description 3
- 238000010362 genome editing Methods 0.000 claims description 3
- 102100031500 Beta-1,4-glucuronyltransferase 1 Human genes 0.000 claims description 2
- 102100024466 Beta-defensin 124 Human genes 0.000 claims description 2
- 102100029348 CDGSH iron-sulfur domain-containing protein 2 Human genes 0.000 claims description 2
- 102100034946 Coiled-coil domain-containing protein 169 Human genes 0.000 claims description 2
- 102100034952 Coiled-coil domain-containing protein 66 Human genes 0.000 claims description 2
- 102100030376 Ermin Human genes 0.000 claims description 2
- 102100039207 Exportin-T Human genes 0.000 claims description 2
- 108010009306 Forkhead Box Protein O1 Proteins 0.000 claims description 2
- 102100023367 Forkhead box protein N4 Human genes 0.000 claims description 2
- 102100035427 Forkhead box protein O1 Human genes 0.000 claims description 2
- 108010021779 GATA5 Transcription Factor Proteins 0.000 claims description 2
- 102000008412 GATA5 Transcription Factor Human genes 0.000 claims description 2
- 102100022123 Hepatocyte nuclear factor 1-beta Human genes 0.000 claims description 2
- 101000874516 Homo sapiens Acetylgalactosaminyl-O-glycosyl-glycoprotein beta-1,3-N-acetylglucosaminyltransferase Proteins 0.000 claims description 2
- 101000729794 Homo sapiens Beta-1,4-glucuronyltransferase 1 Proteins 0.000 claims description 2
- 101000832283 Homo sapiens Beta-defensin 124 Proteins 0.000 claims description 2
- 101000989662 Homo sapiens CDGSH iron-sulfur domain-containing protein 2 Proteins 0.000 claims description 2
- 101000946663 Homo sapiens Coiled-coil domain-containing protein 169 Proteins 0.000 claims description 2
- 101000946606 Homo sapiens Coiled-coil domain-containing protein 66 Proteins 0.000 claims description 2
- 101001063322 Homo sapiens Ermin Proteins 0.000 claims description 2
- 101000745703 Homo sapiens Exportin-T Proteins 0.000 claims description 2
- 101000907587 Homo sapiens Forkhead box protein N4 Proteins 0.000 claims description 2
- 101001045758 Homo sapiens Hepatocyte nuclear factor 1-beta Proteins 0.000 claims description 2
- 101001078211 Homo sapiens Izumo sperm-egg fusion protein 2 Proteins 0.000 claims description 2
- 101001011887 Homo sapiens Matrix metalloproteinase-17 Proteins 0.000 claims description 2
- 101000707599 Homo sapiens Rhophilin-1 Proteins 0.000 claims description 2
- 101000979912 Homo sapiens Sphingomyelin phosphodiesterase 2 Proteins 0.000 claims description 2
- 101000702545 Homo sapiens Transcription activator BRG1 Proteins 0.000 claims description 2
- 101001072037 Homo sapiens cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A Proteins 0.000 claims description 2
- 102100025318 Izumo sperm-egg fusion protein 2 Human genes 0.000 claims description 2
- 102100030219 Matrix metalloproteinase-17 Human genes 0.000 claims description 2
- 102100031363 Rhophilin-1 Human genes 0.000 claims description 2
- 102100024550 Sphingomyelin phosphodiesterase 2 Human genes 0.000 claims description 2
- 102100031027 Transcription activator BRG1 Human genes 0.000 claims description 2
- 108010005656 Ubiquitin Thiolesterase Proteins 0.000 claims description 2
- 102000005918 Ubiquitin Thiolesterase Human genes 0.000 claims description 2
- 102100036377 cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A Human genes 0.000 claims description 2
- 239000003112 inhibitor Substances 0.000 claims description 2
- 102100022910 ADP-ribosylation factor-like protein 15 Human genes 0.000 claims 1
- 102100040022 Eukaryotic translation initiation factor 4 gamma 3 Human genes 0.000 claims 1
- 102100040669 F-box only protein 32 Human genes 0.000 claims 1
- 101000974504 Homo sapiens ADP-ribosylation factor-like protein 15 Proteins 0.000 claims 1
- 101001034840 Homo sapiens Eukaryotic translation initiation factor 4 gamma 3 Proteins 0.000 claims 1
- 101000892323 Homo sapiens F-box only protein 32 Proteins 0.000 claims 1
- 101001086424 Homo sapiens Olfactory receptor 1I1 Proteins 0.000 claims 1
- 101000613810 Homo sapiens Osteocrin Proteins 0.000 claims 1
- 101000620584 Homo sapiens Ras-related protein Rab-15 Proteins 0.000 claims 1
- 101000671853 Homo sapiens Ubiquitin domain-containing protein 1 Proteins 0.000 claims 1
- 101000743587 Homo sapiens Vacuolar protein sorting-associated protein 26A Proteins 0.000 claims 1
- 102100032720 Olfactory receptor 1I1 Human genes 0.000 claims 1
- 102100040556 Osteocrin Human genes 0.000 claims 1
- 102000028676 Rab15 Human genes 0.000 claims 1
- 102100040340 Ubiquitin domain-containing protein 1 Human genes 0.000 claims 1
- 102100038398 Vacuolar protein sorting-associated protein 26A Human genes 0.000 claims 1
- 101100449517 Arabidopsis thaliana GRH1 gene Proteins 0.000 abstract description 71
- 101100434479 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) AFB1 gene Proteins 0.000 abstract description 71
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 abstract description 26
- 238000010354 CRISPR gene editing Methods 0.000 abstract description 23
- 238000002474 experimental method Methods 0.000 abstract description 13
- 238000005516 engineering process Methods 0.000 abstract description 10
- 239000007800 oxidant agent Substances 0.000 abstract description 8
- 229930195730 Aflatoxin Natural products 0.000 abstract description 7
- 239000005409 aflatoxin Substances 0.000 abstract description 7
- 238000012917 library technology Methods 0.000 abstract description 6
- 230000001590 oxidative effect Effects 0.000 abstract description 6
- 231100000433 cytotoxic Toxicity 0.000 abstract description 2
- 230000004907 flux Effects 0.000 abstract description 2
- 102100021268 Transcription regulator protein BACH1 Human genes 0.000 abstract 2
- 230000001472 cytotoxic effect Effects 0.000 abstract 1
- 230000030833 cell death Effects 0.000 description 27
- 239000001963 growth medium Substances 0.000 description 21
- 238000012163 sequencing technique Methods 0.000 description 20
- 238000012216 screening Methods 0.000 description 17
- 239000002609 medium Substances 0.000 description 14
- 238000000034 method Methods 0.000 description 12
- 238000004113 cell culture Methods 0.000 description 11
- 241000713666 Lentivirus Species 0.000 description 10
- 238000001514 detection method Methods 0.000 description 10
- 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 10
- 108020004414 DNA Proteins 0.000 description 9
- 241000282414 Homo sapiens Species 0.000 description 9
- 238000012408 PCR amplification Methods 0.000 description 9
- 239000013604 expression vector Substances 0.000 description 9
- 238000012258 culturing Methods 0.000 description 8
- 238000010276 construction Methods 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 6
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 6
- 101000600434 Homo sapiens Putative uncharacterized protein encoded by MIR7-3HG Proteins 0.000 description 6
- 102100037401 Putative uncharacterized protein encoded by MIR7-3HG Human genes 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000012165 high-throughput sequencing Methods 0.000 description 6
- 244000144972 livestock Species 0.000 description 6
- 238000002835 absorbance Methods 0.000 description 5
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 5
- 238000013537 high throughput screening Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 244000144977 poultry Species 0.000 description 5
- 235000013594 poultry meat Nutrition 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 230000004663 cell proliferation Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000012217 deletion Methods 0.000 description 4
- 230000037430 deletion Effects 0.000 description 4
- 230000036541 health Effects 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 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 description 4
- 239000003642 reactive oxygen metabolite Substances 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 102100031780 Endonuclease Human genes 0.000 description 3
- 108010042407 Endonucleases Proteins 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000001976 enzyme digestion Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 230000004792 oxidative damage Effects 0.000 description 3
- 238000004806 packaging method and process Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 241000710842 Japanese encephalitis virus Species 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 238000003766 bioinformatics method Methods 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 238000001784 detoxification Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 229950010131 puromycin Drugs 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 102100034213 ATPase family protein 2 homolog Human genes 0.000 description 1
- 101100434480 Arabidopsis thaliana AFB2 gene Proteins 0.000 description 1
- 241000228197 Aspergillus flavus Species 0.000 description 1
- 241000228230 Aspergillus parasiticus Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 206010014596 Encephalitis Japanese B Diseases 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 101000780587 Homo sapiens ATPase family protein 2 homolog Proteins 0.000 description 1
- 201000005807 Japanese encephalitis Diseases 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 206010034133 Pathogen resistance Diseases 0.000 description 1
- 108091046869 Telomeric non-coding RNA Proteins 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- WWSYXEZEXMQWHT-WNWIJWBNSA-N aflatoxin B2 Chemical compound C=1([C@@H]2CCO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O WWSYXEZEXMQWHT-WNWIJWBNSA-N 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000001668 ameliorated effect Effects 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000001147 anti-toxic effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- RQNWIZPPADIBDY-UHFFFAOYSA-N arsenic atom Chemical compound [As] RQNWIZPPADIBDY-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000000120 cytopathologic effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- OEBRKCOSUFCWJD-UHFFFAOYSA-N dichlorvos Chemical compound COP(=O)(OC)OC=C(Cl)Cl OEBRKCOSUFCWJD-UHFFFAOYSA-N 0.000 description 1
- 229950001327 dichlorvos Drugs 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000010201 enrichment analysis Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 231100000221 frame shift mutation induction Toxicity 0.000 description 1
- 230000037433 frameshift Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 102000052162 human BACH1 Human genes 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 208000021601 lentivirus infection Diseases 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 230000007110 pathogen host interaction Effects 0.000 description 1
- 230000003950 pathogenic mechanism Effects 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- NNFCIKHAZHQZJG-UHFFFAOYSA-N potassium cyanide Chemical compound [K+].N#[C-] NNFCIKHAZHQZJG-UHFFFAOYSA-N 0.000 description 1
- 235000013613 poultry product Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000011027 product recovery Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 231100000378 teratogenic Toxicity 0.000 description 1
- 230000003390 teratogenic effect Effects 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/02—Antidotes
-
- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- 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
- 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
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Public Health (AREA)
- Biotechnology (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Epidemiology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Marine Sciences & Fisheries (AREA)
- Virology (AREA)
- General Chemical & Material Sciences (AREA)
Abstract
本发明公开了一类参与黄曲霉毒素B1毒性效应的靶点及其应用,利用CRISPR/Cas9敲除文库技术,高通量筛选参与黄曲霉毒素B1(AFB1)毒性效应的靶点,实验证明,利用CRISPR/Cas9技术敲除候选靶点,能显著抑制AFB1诱导细胞毒性效应,特别是构建了关键靶点BACH1纯合敲除单克隆细胞系,并通过实验证明BACH1敲除后可显著提高宿主细胞对黄曲霉毒素B1或黄曲霉毒素衍生物AFG1、AFM1与强氧化剂(双氧水)的抗性。总之,本发明所提供的靶点可作为防治黄曲霉毒素或强氧化剂诱导机体损伤的分子靶标。
Description
技术领域
本发明属于生物技术领域,具体涉及利用猪全基因组CRISPR/Cas9敲除文库技术,高通量筛选并鉴定到一类参与黄曲霉毒素B1毒性效应的靶点及其应用。
背景技术
黄曲霉毒素(Aflatoxin,AFT)主要是黄曲霉和寄生曲霉的次生代谢产物,具有极强的致畸致癌毒性。它的毒性相当于氰化钾的10倍,敌敌畏100倍,砒霜的68倍。AFT主要包含AFB1、AFB2、AFG1和AFG2等衍生物,其中黄曲霉毒素B1(AflatoxinB1,AFB1)最为普遍,且毒性最强。目前,仍能从牛奶、坚果、饲料等中检测出AFB1,甚至有超标的情况,对人类健康和畜牧业构成严重的威胁。
AFB1在饲料原料(玉米、大豆)及成品中广泛存在,具有极强的毒性,不仅直接影响畜禽本身健康,造成巨大经济损失和降低动物福利,而且也能转移至畜禽产品(肉、蛋、奶)中,通过食物链间接威胁人类健康。虽然,综合利用各种AFB1检测技术和脱毒技术可从来源处控制AFB1,保证食品、饲料的安全,但畜禽依旧处于AFB1急慢性暴露危险中,如饲粮在储存过程中发霉和料槽未清洗干净等。目前,对于AFB1的防治,一方面可从源头上抑制真菌产生AFB1,另一方面可利用多种先进的AFB1检测技术,控制AFB1污染的食品、饲粮进入市场,甚至可使用生物脱毒等技术对污染的饲料进行脱毒。然而,无论是人类还是动物,仍无法完全避免AFB1的慢性暴露,严重威胁人类健康,影响畜禽生产性能、繁殖性能。
尽管对AFB1的防治已有一些措施,但是由于AFB1致病的机制尚不清楚,目前缺乏有效的治疗药物,尤其缺乏有效分子靶点。因此,本领域迫切需要开发新的高效抵抗AFB1毒性效应的有效药物靶点。近年来,全基因组CRISPR文库筛选技术在高通量鉴定病原-宿主互作因子上具备巨大的优势,已被广泛应用于各种病原抗性/易感因子的筛选与鉴定。申请人在前期已设计并构建了猪全基因组CRISPR/Cas9敲除文库,并用该文库筛选鉴定了一系列参与日本乙型脑炎(JEV)的关键宿主因子,为猪抗JEV育种与JEV的治疗提供了新的靶标(Zhao et al.,2020)。
因此,本发明利用了猪全基因组CRISPR/Cas9敲除文库策略,高通量筛选到一批参与AFB1毒性效应的靶点。这些靶点可为制备抵抗AFB1毒性效应的畜禽提供新靶标,同时也为AFB1毒性效应的防治提供了新的药物靶点。特别是其中关键靶点BACH1敲除后,不仅可显著缓解AFB1诱导的细胞死亡,也能缓解黄曲霉毒素的其它衍生物(如AFM1和AFG1)诱导的细胞死亡,表明该靶点对黄曲霉毒素的抗性具有一定的广谱作用。
发明内容
本发明的目的是基于猪的全基因组CRISPR/Cas9敲除文库技术,高通量筛选并鉴定到一类参与AFB1毒性效应的靶点,筛选的靶点可用于制备抗黄曲霉毒素B1或黄曲霉毒素衍生物AFG1、AFB1毒性的药物或基因编辑细胞。
为了实现上述目的,本发明采用以下技术方案:
本发明基于猪的全基因组CRISPR/Cas9敲除文库技术,高通量筛选并鉴定到一类参与黄曲霉毒素B1(AFB1)毒性效应的靶点,所述的靶点包括:BACH1,AFM,FOXO1,UCHL1,DEFB124,ENSSSCG00000006349,PKD1,HOXA6,RHPN1,P2RY12,HNF1B,CCDC169,ENSSSCG00000000674,B3GNT6,ENSSSCG00000020901,ENSSSCG00000000264,ERMN,TAF1C,MMP17,FOXN4,ENSSSCG00000026076,ENSSSCG00000009848,ENSSSCG00000023332,SMPD2,CISD2,ENSSSCG00000012735,ENSSSCG00000028077,FBXO32,ENSSSCG00000009706,PDE10A,SMARCA4,ENSSSCG00000007897,ENSSSCG00000026405,XPOT,IZUMO2,GATA5,CCDC66,ENSSSCG00000027964,ENSSSCG00000014233,UBTD1,ENSSSCG00000014579,OSTN,ENSSSCG00000011811,VPS26A,ENSSSCG00000027396,ENSSSCG00000013098,ARL15,EIF4G3,RAB15,ENSSSCG00000023036,OR1I1,这类靶点可用于制备抗黄曲霉毒素B1毒性的药物。
优选地,BACH1基因作为靶点用于制备抗黄曲霉毒素B1(AFB1)或黄曲霉毒素衍生物AFG1、AFB1或强氧化剂(双氧水)的毒性的药物。在本发明的具体实施例中,BACH1纯合敲除单克隆细胞株可抵抗AFB1、黄曲霉毒素衍生物AFG1、AFB1或双氧水诱导的细胞死亡。
上述用于防治黄曲霉毒素B1或黄曲霉毒素衍生物AFG1、AFM1毒性的药物为BACH1基因或其编码蛋白的靶向抑制剂,优选地,所述药物包括Cas9蛋白和靶向BACH1基因的sgRNA。
BACH1基因作为靶点在制备抗黄曲霉毒素B1或黄曲霉毒素衍生物AFG1、AFM1毒性的基因编辑细胞中的应用。在本发明的具体实施例中,提供了BACH1纯合敲除单克隆细胞系,制备方法如下:
(1)挑选靶向BACH1基因的sgRNA,然后将sgRNA序列构建到慢病毒表达载体上;
(2)通过慢病毒包装及感染方法,将目的sgRNA表达载体导入到PK-15-Cas9细胞系中,利用流式细胞仪分选单克隆细胞;再通过TA克隆测序检测和Western Blot技术检测BACH1蛋白表达情况,以挑选出BACH1蛋白缺失的细胞株。
本发明的技术方案具有如下有益效果:
1.本发明首次提供了一种基于全基因组CRISPR/Cas9敲除文库技术高通量筛选参与AFB1毒性效应的靶点的策略;
2.本发明首次提供了一类参与AFB1毒性效应的靶点,所提供的靶点为畜禽的抗病育种研究提供材料,同时也为AFB1的防治提供药物靶点;
3.本发明提供的候选靶点对黄曲霉毒素的其它衍生物(如AFM1、AFG1等)同样具有潜在的应用价值;
4.本发明提供关键候选靶点BACH1敲除单克隆细胞株,并表明BACH1敲除后可同时抵抗AFB1毒性效应与强氧化剂(双氧水)诱导的氧化损伤。
附图说明
图1显示了基于猪全基因组CRISPR/Cas9敲除文库策略筛选参与AFB1毒性效应的靶点的流程图。大致流程为:构建猪的全基因组sgRNA质粒文库并包装慢病毒,其中sgRNA数量为85674条。慢病毒感染稳定表达Cas9的PK-15细胞(PK-15-Cas9);感染2天后,通过流式细胞仪筛选GFP阳性细胞,继续培养7天获得突变体细胞库后,取部分细胞提取基因组DNA,取一定量细胞用AFB1处理,取存活细胞并进行连续3轮不同剂量的AFB1处理实验,分别提取各轮存活细胞基因组DNA,进行PCR扩增和制备DNA测序文库,对高通量测序数据进行生物信息学分析,鉴定富集的sgRNA及靶基因功能分析。
图2显示了通过生物信息学技术分析CRISPR/Cas9文库筛选结果。A.第2轮与第3轮AFB1筛选结果中排名前0.1%的基因的韦恩图;B.第3轮AFB1处理后sgRNA读数排名前100所对应的靶点。
图3显示了关键靶点BACH1敲除单克隆细胞系的构建及鉴定。A.BACH1敲除单克隆细胞进行单克隆测序鉴定;B.BACH1敲除单克隆细胞系的敲除模式图;C.Western blot鉴定BACH1敲除结果;D.EdU细胞增殖实验检测PK-15-Cas9细胞系(对照)与BACH1敲除细胞系活性;E.EdU细胞增殖实验检测量化结果。
图4显示了BACH1敲除后能显著抑制AFB1和强氧化剂诱导的细胞死亡。A.BACH1敲除后抵抗AFB1诱导的细胞死亡;B.CCK8增殖实验检测AFB1处理野生型与敲除细胞系后的IC50;C.BACH1敲除后显著抵抗强氧化剂(双氧水)诱导的细胞死亡;D.CCK8增殖实验检测双氧水处理野生型与敲除细胞系后的IC50;E.双氧水处理细胞后的活性氧(ROS)水平检测。
图5显示了BACH1敲除后显著抑制其它黄曲霉毒素衍生物诱导的细胞死亡。A.BACH1敲除后缓解AFM1诱导的细胞死亡;B.BACH1敲除后缓解AFG1诱导的细胞死亡;
图6显示了人肝癌细胞(Huh7)-BACH1敲除细胞库显著缓解AFB1诱导的细胞死亡。A.T7核酸内切酶鉴定结果;B.Western blot鉴定结果;C.Huh7-BAHC1敲除细胞库缓解AFB1诱导的细胞死亡。
具体实施方式
本发明人首次利用CRISPR/Cas9敲除文库技术,高通量筛选参与AFB1毒性效应的靶点;实验证明,利用CRISPR/Cas9技术敲除候选靶点,能显著抑制AFB1诱导细胞毒性效应。特别的,构建了关键候选靶点BACH1纯合敲除单克隆细胞系,并通过实验证明,BACH1敲除后可显著提高宿主细胞对AFB1(及AFM1与AFG1)与强氧化剂的抗性。
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件,例如《分子克隆:实验室手册》(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。除非另外说明,否则百分比和份数按重量计算。
实施例1利用猪的全基因组CRISPR/Cas9敲除文库高通量筛选参与AFB1毒性效应的关键候选靶点
1.1全基因组CRISPR/Cas9敲除文库高通量筛选流程
为了采用全基因组无偏策略筛选参与AFB1毒性效应的关键靶点,制定的实验流程如图1所示,包括步骤:
(1)利用本实验室前期制备的猪全基因组CRISPR/Cas9文库质粒,包装文库慢病毒(Zhao et al.,2020);
(2)文库慢病毒感染PK-15-Cas9细胞系,构建突变体细胞库,备用;
(3)使用一定剂量的AFB1分别处理未经任何处理的PK-15-Cas9(对照组)和突变体细胞库,持续观察细胞病变,待对照组细胞全部死亡后,收集存活细胞;对于上一轮获得的存活细胞,进行第2轮、第3轮的AFB1处理实验;
(4)对第1轮、第2轮及第3轮AFB1处理后收集的存活细胞,提取基因组DNA,进行PCR扩增和构建高通量测序文库,随后通过生物信息学分析鉴定富集的sgRNA;
(5)对筛选到的sgRNA对应的靶基因进行功能研究。
值得指出的是,本实施例中采用的猪的CRISPR/Cas9敲除文库,由本实验室前期研究开发并大量制备,共含85674条sgRNAs,包含针对17743个蛋白编码基因,11053个lncRNA和551个miRNA的sgRNAs,以及1000个阴性对照sgRNAs。
1.2 AFB1处理PK-15突变体细胞库存活细胞收集,制备测序文库与上机测序
依据1.1所述文库筛选实验流程,首先用0.2μg/mL AFB1分别处理PK-15-Cas9(对照组)和全基因组突变体细胞库。AFB1处理7天后,可观察到AFB1处理的PK-15-Cas9细胞全部死亡,而同样处理的全基因组突变体细胞有少量细胞存活。更换新鲜的含10%胎牛血清的培养基扩大培养,再分别进行第2轮(1μg/mL),第3轮AFB1处理(6μg/mL),其中AFB1的剂量逐轮增加。最后分别收集每轮处理后的存活细胞,提取基因组DNA并进行PCR扩增,制备测序文库并上机进行高通量测序。
对收集的细胞,参考DNA提取试剂盒(血液/细胞/组织基因组DNA提取试剂盒(天根#DP304))的说明书提取细胞基因组DNA。其中PCR扩增引物对的序列为:Lib-cell-F:GTGGAAAGGACGAAACACCG和Lib-cell-R:GCGGTACCTCTAGAGCCATT。
PCR扩增反应体系为:
对PCR产物进行琼脂糖凝胶电泳检测,确定为扩增阳性带后,使用PCR产物纯化试剂盒MinElute PCR Purification Kit(QIAGEN)进行纯化。将制备好的PCR纯化产物送公司构建测序文库并进行高通量测序。
实施例2利用生物信息学技术分析CRISPR/Cas9文库筛选结果
2.1对高通量测序数据进行生物信息学分析
设定每轮AFB1筛选样品的测序数排名前0.03%的sgRNA所对应的基因为候选基因,除去各轮冗余基因外,一共富集到51个参与AFB1毒性效应的靶点(表1),将这些靶点作为参与AFB1毒性效应的候选靶点。
接着利用Venny2.1在线工具对第2轮与第3轮AFB1筛选结果中排名前0.03%(Top20)的基因进行韦恩图分析(如图2A),发现两轮筛选结果中共有的基因有15个(重叠率为35.7%)。其中基因重叠率不是特别高,主要原因可能是采用AFB1剂量逐轮递增的筛选方式,选择压在逐轮增加以加强阳性靶点的富集,最终导致每轮筛选的靶点在排名上呈现出差异。特别的,在第3轮筛选过程中,使用高剂量的AFB1(6μg/mL)进行筛选,可显著富集参与AFB1毒性效应的靶点。另外,第2轮特有的基因有14个(33.3%),第3轮特有的基因有13个(31%)。第3轮筛选结果中特有的基因比较多,可能是有些靶点对高剂量的AFB1更为敏感,在高剂量处理时被显著富集。接着,对第3轮高通量测序数据进行sgRNA富集分析,并对排名靠前的基因进行标记,发现其中BACH1、AFM、TAF1C、PKD1、HOXA6、P2RY12等基因在各轮筛选结果中都有显著富集,将其作为参与AFB1毒性效应的关键候选靶点。挑选候选靶点并设计sgRNA,构建候选基因敲除细胞库进行验证,发现BACH1敲除后,对AFB1的抗性最强,因此确定该靶点为关键靶点,进一步研究。
在表1中,列举了基于各轮不同剂量的AFB1处理宿主细胞后,筛选存活细胞获得的sgRNA及对应的靶基因。所筛选到的候选基因,可作为抑制AFB1毒性效应的重要分子靶标。
表1.利用CRISPR/Cas9敲除文库鉴定排名前0.03%的sgRNA及靶基因
上表中sgRNA的序列参考Zhao et al.,2020文章中表述。
实施例3利用CRISPR/Cas9技术构建BACH1敲除单克隆细胞株
3.1获得BACH1敲除细胞库
(1)构建靶向BACH1的sgRNA表达载体
从猪全基因组CRISPR/Cas9敲除文库中选取靶向BACH1的sgRNA序列,如下:
表2.sgRNA序列及sgRNA表达载体构建所用的引物对
sgRNA表达载体构建流程为:sgRNA退火:稀释sgRNA正反向引物浓度至10μM;吸取正反向引物各5μL,混匀;PCR仪上95℃,10min;65℃,1h。然后与酶切线性化的sgRNA-Cas9表达载体(Lenti-sgRNA-GFP)进行连接。
sgRNA表达载体酶切体系:
组份 | 反应体积 |
Lenti-sgRNA-GFP | 1μg |
10x Cutsmart buffer | 5μL |
BsmBI | 1μL |
补水至 | 50μL |
配置好酶切反应液后,置于37℃,金属浴2h酶切;随后对酶切产物进行凝胶电泳与纯化回收,再与退火的sgRNA引物对进行连接。
sgRNA表达载体连接体系:
组份 | 反应体积 |
Ligation Mix(Takara) | 5μL |
线性化的慢病毒表达质粒 | 50ng |
退火的sgRNA引物 | 1ng |
补水至 | 10μL |
置于16℃金属浴连接20min,然后进行转化;第二天挑选单克隆菌株,200rpm,37℃摇床摇菌,取部分送擎科测序鉴定。最后将测序鉴定正确的菌液,扩大摇菌后,使用去内毒素质粒提取试剂盒提取质粒备用。
(2)包装sgRNA表达质粒慢病毒并构建BACH1敲除单克隆细胞
慢病毒包装的实验步骤为:1)取新复苏的HEK293T细胞,培养3代左右进行转染,在转染前一天细胞接种至6孔板细胞培养皿;2)待细胞达到80~90%汇合度时进行转染,转染前弃除旧培养基,PBS冲洗一遍,加入1mL新鲜2%FBS DMEM培养基(无抗生素),置于培养箱中继续培养;3)一个6孔板的1个孔的细胞需要转染质粒总量为2.5μg,用100μL JetPrimeBuffer稀释质粒DNA(PMD2.G:PSPAX:目的=1:2:3),混匀;4)加入6μL JetPrime(PolyPlus,#B180306),混匀;5)室温放置10min,小心加入细胞培养基中,轻轻摇匀;6)转染后4-6h,补加1mL含有1%双抗,2%FBS的DMEM培养基,混匀继续培养;7)24h后再补加1mL含有1%双抗,2%FBS的DMEM培养基;8)60h后收集全部上清液至5mL离心管中,用0.45μm滤器过滤;9)过滤后的上清液置于-80℃保存备用。
慢病毒感染PK-15-Cas9细胞系构建BACH1敲除细胞系,实验步骤为:1)在感染前一天,将PK-15-Cas9细胞铺于6孔板细胞培养皿;2)待细胞汇合至30%-40%时,弃除旧培养基,PBS冲洗一遍,加入1ml 10%FBA DMEM培养基,然后取1ml解冻的sgRNA慢病毒加入培养皿中,继续培养;3)24h后弃除旧培养基,PBS冲洗一遍,加入2ml 10%FBA DMEM培养基继续培养;4)72h后,观察荧光,此时的细胞作为BACH1敲除细胞库,备用。
3.2挑选单克隆细胞株并鉴定
对3.1所述步骤中构建的BACH1敲除细胞库,进一步培养后,使用流式细胞仪分选单个的细胞于96孔板细胞培养皿,继续培养,待细胞长成较大的细胞团后,用胰酶消化,取部分细胞铺于24孔板细胞培养皿继续培养,剩余部分细胞提取基因组DNA,进行PCR扩增并测序鉴定编辑情况。对挑选的单克隆细胞株进行鉴定,实验步骤如下:
(1)BACH1敲除细胞系测序鉴定
1)裂解法提取基因组DNA:微量细胞中加入10μl细胞裂解液(含蛋白酶K),混匀,PCR:65℃10min,95℃60min。
2)PCR扩增并测序鉴定单克隆细胞敲除情况:首先在BACH1的sgRNA序列两端设计PCR扩增引物对,如下:
名称 | 引物序列(5’-3’) |
BACH1-sgR-seq-F | AGTAAGGACAATGTGGACGAAG |
BACH1-sgR-seq-R | ATTTGGGGCATAAAGACGG |
PCR退火温度为60℃,对PCR产物进行产物回收并测序;挑选测序结果中未出现双峰,但又已发生插入或缺失(Indel)的单克隆细胞株,进一步进行TA克隆测序,挑选数个单克隆进行测序鉴定,鉴定其是否为纯合编辑细胞株。
所挑选的单克隆细胞株的测序结果显示,该单克隆的TA克隆测序结果无双峰且显示一种突变情况(如图3A)。具体的,发生了移码突变,在第208位氨基酸位置突变成了终止密码子(如图3B),最终导致BACH1蛋白不能表达。
(2)Westen blot鉴定BACH1蛋白表达水平
提取PK-15-Cas9和上述测序所鉴定的BACH1纯合敲除单克隆细胞株的总蛋白,使用BCA法测定蛋白浓度,对各组蛋白进行等量化,然后蛋白变性,取等量蛋白上样电泳,剩余变性后的蛋白置于-20℃保存。结果表明敲除单克隆细胞株的BACH1蛋白完全缺失(如图3C),充分说明通过CRISPR/Cas9技术成功缺失了宿主细胞的BACH1蛋白。
3.3 BACH1纯合敲除单克隆细胞株活性鉴定
使用碧云天BeyoClickTMEdU-555细胞增殖检测试剂盒,检测上述鉴定好的BACH1敲除单克隆细胞株和PK-15-Cas9细胞的细胞活性,两类细胞系各设置3组重复(如图3D),并对检测结果进行量化,对其细胞活性进行比较(图3E)。EdU细胞增殖实验检测结果表明,BACH1敲除细胞系的细胞活性与对照组PK-15-Cas9细胞系相当,因此成功构建了BACH1敲除细胞模型,可用于后续研究。
实施例4.BACH1敲除后显著抑制AFB1和双氧水诱导的细胞死亡
4.1 BACH1纯合敲除单克隆细胞株可抵抗AFB1诱导的细胞死亡
提前将BACH1敲除细胞株与PK-15-Cas9细胞(对照组)铺于6孔板细胞培养皿,待细胞汇合至60%左右,弃去旧的培养基,PBS洗一遍后,加入2mL 10%FBS DMEM(含双抗)培养基,其中含2μg/mL AFB1,处理48h后,弃去旧培养基,PBS洗一遍,加入1mL PBS后,拍照记录(如图4A)。发现同样剂量的AFB1处理后,BACH1敲除细胞系几乎可完全抵抗AFB1诱导的细胞死亡,而PK-15-Cas9(对照组)细胞在AFB1处理后基本死完,表明敲除BACH1后可显著抵抗AFB1诱导的细胞死亡。
4.2 AFB1诱导的细胞死亡的IC50的测定
进一步验证BACH1敲除后对AFB1入侵宿主细胞的保护作用,测定了AFB1诱导细胞死亡的IC50,IC50测定实验步骤为:
(1)提前一天将PK-15-Cas9细胞与BACH1敲除细胞株铺于96孔板细胞培养皿中(104cells/well);
(2)待细胞汇合至60%左右时,弃去旧培养基,PBS洗一遍,更换含不同剂量AFB1的生长培养基(100μL/well)继续培养,AFB1剂量梯度为:0,0.5,1,2,4,6,8,10,12,16,20μg/mL;
(3)继续培养36h后,弃去旧培养基,更换CCK8溶液(CCK8:10%FBS培养基=1:10),100μL/well,置于培养箱中孵育40min后使用酶标仪测定450nm的吸光度,计算AFB1的IC50。
结果如图4B所示,AFB1处理PK-15-Cas9细胞(对照组)的IC50=1.215μg/mL,而BACH1敲除细胞系的IC50=10.12μg/mL,因此可知宿主细胞敲除BACH1后,对于AFB1的抗性可提高近10倍。而且,在低剂量的AFB1处理下,BACH1敲除细胞株基本上不受AFB1毒性效应影响。
4.3 BACH1敲除细胞株可抵抗双氧水诱导的细胞死亡
提前一天将BACH1敲除细胞株和PK-15-Cas9细胞(对照组)铺于12孔板细胞培养皿中,待细胞汇合至60%左右时,弃去旧培养基,PBS洗一遍,更换含有1.6mM双氧水的正常生长培养基继续培养,12h后观察细胞状态,使用PI稀释液(PI染料:正常培养基=1:1000)孵育,每孔500μL PI稀释液,拍照记录(如图4C)。
检测结果显示,敲除BACH1可以显著缓解双氧水诱导的细胞死亡,表明BACH1参与氧化损伤过程,敲除BACH1可以显著增强宿主细胞的抗氧化能力。
4.4 BACH1敲除后显著抑制细胞活性氧的产生
进一步探究BACH1敲除细胞株的抗氧化能力,对细胞活性氧(Reactive oxygenspecies,ROS)水平进行检测。实验前一天将BACH1敲除细胞株和PK-15-Cas9细胞(对照组)铺于12孔板细胞培养皿中,待细胞汇合至60%左右时,弃去旧培养基,PBS洗一遍,更换含有1.6mM双氧水的生长培养基继续培养,12h后使用碧云天活性氧检测试剂盒(#60063)检测细胞活性氧水平(如图4D),并对检测结果进行量化比较(如图4E)。
结果如图4D和4E所示,BACH1敲除后显著降低了细胞活性氧水平,表明敲除BACH1后可提高细胞抗氧化能力,能抵抗强氧化剂(如双氧水等)诱导的氧化损伤。
实施例5 BACH1敲除后显著抑制其它黄曲霉毒素衍生物诱导的细胞死亡
5.1 BACH1敲除后可抵抗AFM1诱导的细胞死亡
提前一天将PK-15-Cas9细胞与BACH1敲除细胞株铺于96孔板细胞培养皿中(104cells/well);待细胞汇合至60%左右时,弃去旧培养基,PBS洗一遍,更换含不同剂量AFM1的生长培养基(100μL/well)继续培养,AFM1剂量梯度为:0,0.5,1μg/mL;继续培养36h后,弃去旧培养基,更换CCK8溶液(CCK8:10%FBS培养基=1:10),100μL/well,置于培养箱中孵育40min后使用酶标仪测定450nm的吸光度,并进行分析。结果表明,BACH1敲除后可显著抵抗AFM1诱导的细胞死亡(如图5A)。
5.2 BACH1敲除后可抵抗AFG1诱导的细胞死亡
提前一天将PK-15-Cas9细胞与BACH1敲除细胞株铺于96孔板细胞培养皿中(104cells/well);待细胞汇合至60%左右时,弃去旧培养基,PBS洗一遍,更换含不同剂量AFG1的生长培养基(100μL/well)继续培养,AFG1剂量梯度为:0,8,16μg/mL;继续培养36h后,弃去旧培养基,更换CCK8溶液(CCK8:10%FBS培养基=1:10),100μL/well,置于培养箱中孵育40min后使用酶标仪测定450nm的吸光度,并进行分析。结果表明,BACH1敲除后可显著抵抗AFG1诱导的细胞死亡(如图5B)。
实施例6人肝癌细胞上缺失BACH1后显著缓解AFB1诱导的细胞死亡
6.1人肝癌细胞(Huh7)-BACH1敲除细胞系构建
将靶向人BACH1的sgRNA构建到Lenti-CRISPRv2-puro载体上(如表3),包装慢病毒并感染Huh7细胞系。感染3天后,用胰酶消化细胞,将细胞铺于6孔板,同时将等量的野生型Huh7铺于6孔板。待细胞汇合至80%左右时,更换含有2.5μg/mL嘌呤霉素的培养基继续培养,待对照组细胞完全死完时,发现慢病毒感染组大部分细胞都存活,继续扩大培养。
表3.sgRNA序列及sgRNA表达载体构建所用的引物对
接着,收集嘌呤霉素富集后的存活细胞以及Huh7(对照组)细胞,提取基因组DNA,设计PCR扩增引物将sgRNA靶向区域扩增并回收(如表4)。使用T7核酸内切酶对回收产物进行酶切(如表5),结果表明成功突变了Huh7细胞中的BACH1基因(如图6A)。进一步检测BACH1蛋白水平的缺失情况,提取Huh7(对照组)和Huh7-BACH1-KO细胞库的总蛋白,进行Westernblot,实验结果显示,Huh7-BACH1-KO细胞库的BACH1蛋白显著下调(如图6B),表明成功构建Huh7-BACH1-KO细胞库,可用于后续研究。
表4.hBACH1扩增引物对
表5.T7核酸内切酶酶切体系及程序
6.2人肝癌细胞(Huh7)-BACH1敲除细胞库可显著缓解AFB1诱导的细胞死亡
提前一天将Huh7细胞与Huh7-BACH1敲除细胞库细胞铺于96孔板细胞培养皿中(104cells/well);待细胞汇合至60%左右时,弃去旧培养基,PBS洗一遍,更换含不同剂量AFB1的2%FBS的培养基(100μL/well)继续培养,AFB1剂量梯度为:0,4μg/mL;继续培养36h后,弃去旧培养基,更换CCK8溶液(CCK8:10%FBS培养基=1:10),100μL/well,置于培养箱中孵育40min后使用酶标仪测定450nm的吸光度,并进行分析。结果表明,在人肝癌细胞系(Huh7)上缺失BACH1后,可以显著缓解AFB1诱导的细胞死亡(图6C)。
参考文献
Zhao C,Liu H,Xiao T,et al.CRISPR screening of porcine sgRNA libraryidentifies host factors associated with Japanese encephalitis virusreplication[J].Nature Communications,2020,11(1):1234567890.
Claims (5)
1.靶点在制备抗黄曲霉毒素B1毒性的药物中的应用,其特征在于,所述的靶点包括:BACH1,AFM,FOXO1,UCHL1,DEFB124,ENSSSCG00000006349,PKD1,HOXA6,RHPN1,P2RY12,HNF1B,CCDC169,ENSSSCG00000000674,B3GNT6,ENSSSCG00000020901,ENSSSCG00000000264,ERMN,TAF1C,MMP17,FOXN4,ENSSSCG00000026076,ENSSSCG00000009848,ENSSSCG00000023332,SMPD2,CISD2,ENSSSCG00000012735,ENSSSCG00000028077,FBXO32,ENSSSCG00000009706,PDE10A,SMARCA4,ENSSSCG00000007897,ENSSSCG00000026405,XPOT,IZUMO2,GATA5,CCDC66,ENSSSCG00000027964,ENSSSCG00000014233,UBTD1,ENSSSCG00000014579,OSTN,ENSSSCG00000011811,VPS26A,ENSSSCG00000027396,ENSSSCG00000013098,ARL15,EIF4G3,RAB15,ENSSSCG00000023036,OR1I1。
2.BACH1基因作为靶点在制备抗黄曲霉毒素B1或黄曲霉毒素衍生物AFG1、AFB1毒性的药物中的应用。
3.用于防治黄曲霉毒素B1或黄曲霉毒素衍生物AFG1、AFM1毒性的药物,其特征在于,所述的药物为BACH1基因或其编码蛋白的靶向抑制剂。
4.根据权利要求3所述的药物,其特征在于,包括Cas9蛋白和靶向BACH1基因的sgRNA。
5.BACH1基因作为靶点在制备抗黄曲霉毒素B1或黄曲霉毒素衍生物AFG1、AFM1毒性的基因编辑细胞中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110525917.3A CN115341010A (zh) | 2021-05-13 | 2021-05-13 | 参与黄曲霉毒素b1毒性效应的靶点及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110525917.3A CN115341010A (zh) | 2021-05-13 | 2021-05-13 | 参与黄曲霉毒素b1毒性效应的靶点及其应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN115341010A true CN115341010A (zh) | 2022-11-15 |
Family
ID=83946634
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110525917.3A Pending CN115341010A (zh) | 2021-05-13 | 2021-05-13 | 参与黄曲霉毒素b1毒性效应的靶点及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN115341010A (zh) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006017960A1 (fr) * | 2004-08-17 | 2006-02-23 | Guangzhou Co-Win Bioengineering Co., Ltd. | Enzyme de détoxification ayant une activité consistant à transformer l'aflatoxine et gène codant pour celle-ci |
CN103725636A (zh) * | 2014-01-02 | 2014-04-16 | 华中农业大学 | 用于降解黄曲霉毒素b1的芬氏纤维微菌slaq001及其应用 |
CN107012128A (zh) * | 2017-04-10 | 2017-08-04 | 北京勤邦生物技术有限公司 | 一种分泌抗黄曲霉毒素b1单克隆抗体的杂交瘤细胞株及其应用 |
-
2021
- 2021-05-13 CN CN202110525917.3A patent/CN115341010A/zh active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2006017960A1 (fr) * | 2004-08-17 | 2006-02-23 | Guangzhou Co-Win Bioengineering Co., Ltd. | Enzyme de détoxification ayant une activité consistant à transformer l'aflatoxine et gène codant pour celle-ci |
CN103725636A (zh) * | 2014-01-02 | 2014-04-16 | 华中农业大学 | 用于降解黄曲霉毒素b1的芬氏纤维微菌slaq001及其应用 |
CN107012128A (zh) * | 2017-04-10 | 2017-08-04 | 北京勤邦生物技术有限公司 | 一种分泌抗黄曲霉毒素b1单克隆抗体的杂交瘤细胞株及其应用 |
Non-Patent Citations (1)
Title |
---|
JOSELYN PADILLA等: "A Novel Therapeutic Target, BACH1, Regulates Cancer Metabolism", 《CELLS》, 12 March 2021 (2021-03-12), pages 634 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Li et al. | Molecular characterization of a novel hypovirus from the plant pathogenic fungus Fusarium graminearum | |
Pan et al. | A neuron-specific host microRNA targets herpes simplex virus-1 ICP0 expression and promotes latency | |
Brown et al. | Insights into natural products biosynthesis from analysis of 490 polyketide synthases from Fusarium | |
Takahashi-Nakaguchi et al. | Analysis of an intrinsic mycovirus associated with reduced virulence of the human pathogenic fungus Aspergillus fumigatus | |
CN104694533A (zh) | 沙门氏菌的非编码性rna及其鉴定和应用 | |
CN110592172A (zh) | 利用CRISPR/Cas9敲除文库技术筛选JEV抗性基因的方法与靶点 | |
Wang et al. | MFS transporters and GABA metabolism are involved in the self-defense against DON in Fusarium graminearum | |
Zhang et al. | Isolation and identification of a viral haemorrhagic septicaemia virus (VHSV) isolate from wild largemouth bass Micropterus salmoides in China | |
Kong et al. | The basic leucine zipper transcription factor PlBZP32 associated with the oxidative stress response is critical for pathogenicity of the lychee downy blight oomycete Peronophythora litchii | |
Markine-Goriaynoff et al. | The core 2 β-1, 6-N-acetylglucosaminyltransferase-mucin encoded by bovine herpesvirus 4 was acquired from an ancestor of the African buffalo | |
Binh et al. | Establishment of a new and efficient Agrobacterium-mediated transformation system in the nematicidal fungus Purpureocillium lilacinum | |
CN110607280A (zh) | Emc3基因的应用及其定点敲除方法 | |
De Tomás et al. | Genome-wide identification of Reverse Transcriptase domains of recently inserted endogenous plant pararetrovirus (Caulimoviridae) | |
Ray et al. | Mac1-dependent copper sensing promotes Histoplasma adaptation to the phagosome during adaptive immunity | |
CN115341010A (zh) | 参与黄曲霉毒素b1毒性效应的靶点及其应用 | |
CN115779084B (zh) | 激活猪的tusc1基因表达的制剂在制备猪抗伪狂犬病毒感染药物中的应用 | |
Rud et al. | First genetic characterization of sturgeon mimiviruses in Ukraine | |
Soo et al. | Differential STAT gene expressions of Penaeus monodon and Macrobrachium rosenbergii in response to white spot syndrome virus (WSSV) and bacterial infections: Additional insight into genetic variations and transcriptomic highlights | |
CN114470209A (zh) | Trim2在防治猪流行性腹泻病毒感染中的应用 | |
CN110624115B (zh) | 钙网蛋白calr在猪抗病中的应用 | |
CN109652590B (zh) | 一种甲型肝炎病毒的负链rna分子生物学检测方法及应用 | |
KR102081962B1 (ko) | 아밀로이드 전구 단백질 유전자를 절단하기 위한 조성물 | |
Thangadurai et al. | Fundamentals of Molecular Mycology | |
Simbaqueba | Analysis of Fusarium oxysporum effectors shared between strains that infect cape gooseberry and tomato | |
CN114990076B (zh) | 一种蚜虫浓核病毒SmDV-Yuanyang在蚜虫防治中的应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |