CN110408596A - 基于CRISPR/Cas9基因编辑改造的CHO细胞株及其制备方法 - Google Patents
基于CRISPR/Cas9基因编辑改造的CHO细胞株及其制备方法 Download PDFInfo
- Publication number
- CN110408596A CN110408596A CN201810395069.7A CN201810395069A CN110408596A CN 110408596 A CN110408596 A CN 110408596A CN 201810395069 A CN201810395069 A CN 201810395069A CN 110408596 A CN110408596 A CN 110408596A
- Authority
- CN
- China
- Prior art keywords
- cell
- crispr
- ala
- survivin
- cas9
- 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
- 108091033409 CRISPR Proteins 0.000 title claims abstract description 59
- 238000010362 genome editing Methods 0.000 title claims abstract description 26
- 238000010354 CRISPR gene editing Methods 0.000 title claims abstract description 23
- 101100230376 Acetivibrio thermocellus (strain ATCC 27405 / DSM 1237 / JCM 9322 / NBRC 103400 / NCIMB 10682 / NRRL B-4536 / VPI 7372) celI gene Proteins 0.000 title claims abstract description 21
- 241000699802 Cricetulus griseus Species 0.000 title claims abstract description 21
- 210000001672 ovary Anatomy 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 230000009466 transformation Effects 0.000 title claims abstract description 15
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 39
- 230000014509 gene expression Effects 0.000 claims abstract description 33
- 108010002687 Survivin Proteins 0.000 claims abstract description 28
- 102000000763 Survivin Human genes 0.000 claims abstract description 26
- 238000005516 engineering process Methods 0.000 claims abstract description 17
- 230000006907 apoptotic process Effects 0.000 claims abstract description 9
- 108030003984 Thiol oxidases Proteins 0.000 claims abstract description 7
- 238000000034 method Methods 0.000 claims description 28
- 150000001875 compounds Chemical class 0.000 claims description 2
- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 claims 1
- 102000004169 proteins and genes Human genes 0.000 abstract description 15
- 230000002424 anti-apoptotic effect Effects 0.000 abstract description 5
- 238000006555 catalytic reaction Methods 0.000 abstract description 2
- 238000009776 industrial production Methods 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 173
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 23
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 23
- 239000001963 growth medium Substances 0.000 description 22
- 239000003153 chemical reaction reagent Substances 0.000 description 21
- 108020004414 DNA Proteins 0.000 description 20
- 239000013612 plasmid Substances 0.000 description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 20
- 230000010354 integration Effects 0.000 description 19
- 239000007788 liquid Substances 0.000 description 17
- 238000006243 chemical reaction Methods 0.000 description 13
- 108010053070 Glutathione Disulfide Proteins 0.000 description 12
- YPZRWBKMTBYPTK-BJDJZHNGSA-N glutathione disulfide Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@H](C(=O)NCC(O)=O)CSSC[C@@H](C(=O)NCC(O)=O)NC(=O)CC[C@H](N)C(O)=O YPZRWBKMTBYPTK-BJDJZHNGSA-N 0.000 description 12
- 235000018102 proteins Nutrition 0.000 description 12
- 230000008569 process Effects 0.000 description 11
- 238000012360 testing method Methods 0.000 description 11
- 239000002585 base Substances 0.000 description 10
- 230000000694 effects Effects 0.000 description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 230000029087 digestion Effects 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 239000006285 cell suspension Substances 0.000 description 8
- 230000000295 complement effect Effects 0.000 description 8
- 238000013461 design Methods 0.000 description 8
- 229940088598 enzyme Drugs 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 238000001890 transfection Methods 0.000 description 8
- 239000000872 buffer Substances 0.000 description 7
- 238000004043 dyeing Methods 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 239000000499 gel Substances 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 239000012224 working solution Substances 0.000 description 7
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 6
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 6
- 239000006180 TBST buffer Substances 0.000 description 6
- 238000005138 cryopreservation Methods 0.000 description 6
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 6
- 238000006062 fragmentation reaction Methods 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 6
- 238000004321 preservation Methods 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 238000005303 weighing Methods 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 241000282414 Homo sapiens Species 0.000 description 5
- 101001131204 Homo sapiens Sulfhydryl oxidase 1 Proteins 0.000 description 5
- 239000012097 Lipofectamine 2000 Substances 0.000 description 5
- 102100034371 Sulfhydryl oxidase 1 Human genes 0.000 description 5
- 238000004140 cleaning Methods 0.000 description 5
- 230000002380 cytological effect Effects 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 239000000284 extract Substances 0.000 description 5
- 238000000605 extraction Methods 0.000 description 5
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 238000013467 fragmentation Methods 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 239000006166 lysate Substances 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 238000000386 microscopy Methods 0.000 description 5
- 230000014616 translation Effects 0.000 description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 238000010453 CRISPR/Cas method Methods 0.000 description 4
- 238000007400 DNA extraction Methods 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 108020005004 Guide RNA Proteins 0.000 description 4
- 238000011529 RT qPCR Methods 0.000 description 4
- 108091028113 Trans-activating crRNA Proteins 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000004090 dissolution Methods 0.000 description 4
- 239000000975 dye Substances 0.000 description 4
- 239000003292 glue Substances 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- 238000004659 sterilization and disinfection Methods 0.000 description 4
- 230000009182 swimming Effects 0.000 description 4
- 102000007469 Actins Human genes 0.000 description 3
- 108010085238 Actins Proteins 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 102100031780 Endonuclease Human genes 0.000 description 3
- 108010042407 Endonucleases Proteins 0.000 description 3
- 108700039887 Essential Genes Proteins 0.000 description 3
- 101710163270 Nuclease Proteins 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 229930040373 Paraformaldehyde Natural products 0.000 description 3
- 108091027544 Subgenomic mRNA Proteins 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 102000004142 Trypsin Human genes 0.000 description 3
- 108090000631 Trypsin Proteins 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000029918 bioluminescence Effects 0.000 description 3
- 238000005415 bioluminescence Methods 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 235000019441 ethanol Nutrition 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000012452 mother liquor Substances 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 229920002866 paraformaldehyde Polymers 0.000 description 3
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- 239000012096 transfection reagent Substances 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- 239000012588 trypsin Substances 0.000 description 3
- 239000012137 tryptone Substances 0.000 description 3
- 108010084932 tryptophyl-proline Proteins 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 2
- 102000000412 Annexin Human genes 0.000 description 2
- 108050008874 Annexin Proteins 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- IEGFSKKANYKBDU-QWHCGFSZSA-N Gly-Phe-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)NC(=O)CN)C(=O)O IEGFSKKANYKBDU-QWHCGFSZSA-N 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- KWTVLKBOQATPHJ-SRVKXCTJSA-N Leu-Ala-Lys Chemical compound C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(C)C)N KWTVLKBOQATPHJ-SRVKXCTJSA-N 0.000 description 2
- IUYCGMNKIZDRQI-BQBZGAKWSA-N Met-Gly-Ala Chemical compound CSCC[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O IUYCGMNKIZDRQI-BQBZGAKWSA-N 0.000 description 2
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 description 2
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 2
- XZFYRXDAULDNFX-UHFFFAOYSA-N N-L-cysteinyl-L-phenylalanine Natural products SCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XZFYRXDAULDNFX-UHFFFAOYSA-N 0.000 description 2
- BQVUABVGYYSDCJ-UHFFFAOYSA-N Nalpha-L-Leucyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)CC(C)C)C(O)=O)=CNC2=C1 BQVUABVGYYSDCJ-UHFFFAOYSA-N 0.000 description 2
- 108010019160 Pancreatin Proteins 0.000 description 2
- LRBSWBVUCLLRLU-BZSNNMDCSA-N Phe-Leu-Lys Chemical compound CC(C)C[C@H](NC(=O)[C@@H](N)Cc1ccccc1)C(=O)N[C@@H](CCCCN)C(O)=O LRBSWBVUCLLRLU-BZSNNMDCSA-N 0.000 description 2
- RBRNEFJTEHPDSL-ACRUOGEOSA-N Phe-Phe-Lys Chemical compound C([C@@H](C(=O)N[C@@H](CCCCN)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 RBRNEFJTEHPDSL-ACRUOGEOSA-N 0.000 description 2
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 2
- 238000012181 QIAquick gel extraction kit Methods 0.000 description 2
- QVOGDCQNGLBNCR-FXQIFTODSA-N Ser-Arg-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O QVOGDCQNGLBNCR-FXQIFTODSA-N 0.000 description 2
- 238000010459 TALEN Methods 0.000 description 2
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 description 2
- 229960000583 acetic acid Drugs 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- 108010001271 arginyl-glutamyl-arginine Proteins 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000010009 beating Methods 0.000 description 2
- 239000012148 binding buffer Substances 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 238000006664 bond formation reaction Methods 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000005336 cracking Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000002224 dissection Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 239000012362 glacial acetic acid Substances 0.000 description 2
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 2
- 108010027668 glycyl-alanyl-valine Proteins 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 108010009298 lysylglutamic acid Proteins 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 229940055695 pancreatin Drugs 0.000 description 2
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 2
- 230000023603 positive regulation of transcription initiation, DNA-dependent Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 108010070643 prolylglutamic acid Proteins 0.000 description 2
- 108010029020 prolylglycine Proteins 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 239000012192 staining solution Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000007862 touchdown PCR Methods 0.000 description 2
- 238000012549 training Methods 0.000 description 2
- 108010035534 tyrosyl-leucyl-alanine Proteins 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- BQHFYSWNHZMMDO-WDSKDSINSA-N (2r)-2-[2-[[(1r)-1-carboxy-2-sulfanylethyl]amino]ethylamino]-3-sulfanylpropanoic acid Chemical class OC(=O)[C@H](CS)NCCN[C@@H](CS)C(O)=O BQHFYSWNHZMMDO-WDSKDSINSA-N 0.000 description 1
- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- AXAVXPMQTGXXJZ-UHFFFAOYSA-N 2-aminoacetic acid;2-amino-2-(hydroxymethyl)propane-1,3-diol Chemical compound NCC(O)=O.OCC(N)(CO)CO AXAVXPMQTGXXJZ-UHFFFAOYSA-N 0.000 description 1
- KIUMMUBSPKGMOY-UHFFFAOYSA-N 3,3'-Dithiobis(6-nitrobenzoic acid) Chemical compound C1=C([N+]([O-])=O)C(C(=O)O)=CC(SSC=2C=C(C(=CC=2)[N+]([O-])=O)C(O)=O)=C1 KIUMMUBSPKGMOY-UHFFFAOYSA-N 0.000 description 1
- QEDXSHCYPROEOK-UHFFFAOYSA-N 3-phosphanylpropanoic acid Chemical class OC(=O)CCP QEDXSHCYPROEOK-UHFFFAOYSA-N 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- HHGYNJRJIINWAK-FXQIFTODSA-N Ala-Ala-Arg Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N HHGYNJRJIINWAK-FXQIFTODSA-N 0.000 description 1
- RLMISHABBKUNFO-WHFBIAKZSA-N Ala-Ala-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O RLMISHABBKUNFO-WHFBIAKZSA-N 0.000 description 1
- CXRCVCURMBFFOL-FXQIFTODSA-N Ala-Ala-Pro Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O CXRCVCURMBFFOL-FXQIFTODSA-N 0.000 description 1
- DVWVZSJAYIJZFI-FXQIFTODSA-N Ala-Arg-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(O)=O DVWVZSJAYIJZFI-FXQIFTODSA-N 0.000 description 1
- SVBXIUDNTRTKHE-CIUDSAMLSA-N Ala-Arg-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O SVBXIUDNTRTKHE-CIUDSAMLSA-N 0.000 description 1
- XQGIRPGAVLFKBJ-CIUDSAMLSA-N Ala-Asn-Lys Chemical compound N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)O XQGIRPGAVLFKBJ-CIUDSAMLSA-N 0.000 description 1
- ZIBWKCRKNFYTPT-ZKWXMUAHSA-N Ala-Asn-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O ZIBWKCRKNFYTPT-ZKWXMUAHSA-N 0.000 description 1
- PBAMJJXWDQXOJA-FXQIFTODSA-N Ala-Asp-Arg Chemical compound C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N PBAMJJXWDQXOJA-FXQIFTODSA-N 0.000 description 1
- BTYTYHBSJKQBQA-GCJQMDKQSA-N Ala-Asp-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C)N)O BTYTYHBSJKQBQA-GCJQMDKQSA-N 0.000 description 1
- HWPXGQCMZITGFN-XVYDVKMFSA-N Ala-Cys-His Chemical compound C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N HWPXGQCMZITGFN-XVYDVKMFSA-N 0.000 description 1
- CVHJIWVKTFNGHT-ACZMJKKPSA-N Ala-Gln-Cys Chemical compound C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CS)C(=O)O)N CVHJIWVKTFNGHT-ACZMJKKPSA-N 0.000 description 1
- SFNFGFDRYJKZKN-XQXXSGGOSA-N Ala-Gln-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](C)N)O SFNFGFDRYJKZKN-XQXXSGGOSA-N 0.000 description 1
- FUSPCLTUKXQREV-ACZMJKKPSA-N Ala-Glu-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O FUSPCLTUKXQREV-ACZMJKKPSA-N 0.000 description 1
- ZVFVBBGVOILKPO-WHFBIAKZSA-N Ala-Gly-Ala Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O ZVFVBBGVOILKPO-WHFBIAKZSA-N 0.000 description 1
- FAJIYNONGXEXAI-CQDKDKBSSA-N Ala-His-Phe Chemical compound C([C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CNC=N1 FAJIYNONGXEXAI-CQDKDKBSSA-N 0.000 description 1
- AJBVYEYZVYPFCF-CIUDSAMLSA-N Ala-Lys-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O AJBVYEYZVYPFCF-CIUDSAMLSA-N 0.000 description 1
- PMQXMXAASGFUDX-SRVKXCTJSA-N Ala-Lys-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](C)N)CCCCN PMQXMXAASGFUDX-SRVKXCTJSA-N 0.000 description 1
- VCSABYLVNWQYQE-UHFFFAOYSA-N Ala-Lys-Lys Natural products NCCCCC(NC(=O)C(N)C)C(=O)NC(CCCCN)C(O)=O VCSABYLVNWQYQE-UHFFFAOYSA-N 0.000 description 1
- 108010011667 Ala-Phe-Ala Proteins 0.000 description 1
- XRUJOVRWNMBAAA-NHCYSSNCSA-N Ala-Phe-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=CC=C1 XRUJOVRWNMBAAA-NHCYSSNCSA-N 0.000 description 1
- XWFWAXPOLRTDFZ-FXQIFTODSA-N Ala-Pro-Ser Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O XWFWAXPOLRTDFZ-FXQIFTODSA-N 0.000 description 1
- FFZJHQODAYHGPO-KZVJFYERSA-N Ala-Pro-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](C)N FFZJHQODAYHGPO-KZVJFYERSA-N 0.000 description 1
- NHWYNIZWLJYZAG-XVYDVKMFSA-N Ala-Ser-His Chemical compound C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N NHWYNIZWLJYZAG-XVYDVKMFSA-N 0.000 description 1
- ISCYZXFOCXWUJU-KZVJFYERSA-N Ala-Thr-Met Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCSC)C(O)=O ISCYZXFOCXWUJU-KZVJFYERSA-N 0.000 description 1
- ZVWXMTTZJKBJCI-BHDSKKPTSA-N Ala-Trp-Ala Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](C)C(O)=O)=CNC2=C1 ZVWXMTTZJKBJCI-BHDSKKPTSA-N 0.000 description 1
- AETQNIIFKCMVHP-UVBJJODRSA-N Ala-Trp-Arg Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O AETQNIIFKCMVHP-UVBJJODRSA-N 0.000 description 1
- ZJLORAAXDAJLDC-CQDKDKBSSA-N Ala-Tyr-Leu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(O)=O ZJLORAAXDAJLDC-CQDKDKBSSA-N 0.000 description 1
- IYKVSFNGSWTTNZ-GUBZILKMSA-N Ala-Val-Arg Chemical compound C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N IYKVSFNGSWTTNZ-GUBZILKMSA-N 0.000 description 1
- 102000010565 Apoptosis Regulatory Proteins Human genes 0.000 description 1
- 108010063104 Apoptosis Regulatory Proteins Proteins 0.000 description 1
- BIOCIVSVEDFKDJ-GUBZILKMSA-N Arg-Arg-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O BIOCIVSVEDFKDJ-GUBZILKMSA-N 0.000 description 1
- KJGNDQCYBNBXDA-GUBZILKMSA-N Arg-Arg-Cys Chemical compound C(C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CS)C(=O)O)N)CN=C(N)N KJGNDQCYBNBXDA-GUBZILKMSA-N 0.000 description 1
- MAISCYVJLBBRNU-DCAQKATOSA-N Arg-Asn-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCCN=C(N)N)N MAISCYVJLBBRNU-DCAQKATOSA-N 0.000 description 1
- RCAUJZASOAFTAJ-FXQIFTODSA-N Arg-Asp-Cys Chemical compound C(C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CS)C(=O)O)N)CN=C(N)N RCAUJZASOAFTAJ-FXQIFTODSA-N 0.000 description 1
- TTXYKSADPSNOIF-IHRRRGAJSA-N Arg-Asp-Phe Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O TTXYKSADPSNOIF-IHRRRGAJSA-N 0.000 description 1
- OBFTYSPXDRROQO-SRVKXCTJSA-N Arg-Gln-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CCCN=C(N)N OBFTYSPXDRROQO-SRVKXCTJSA-N 0.000 description 1
- HPKSHFSEXICTLI-CIUDSAMLSA-N Arg-Glu-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O HPKSHFSEXICTLI-CIUDSAMLSA-N 0.000 description 1
- QAODJPUKWNNNRP-DCAQKATOSA-N Arg-Glu-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O QAODJPUKWNNNRP-DCAQKATOSA-N 0.000 description 1
- ZZZWQALDSQQBEW-STQMWFEESA-N Arg-Gly-Tyr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O ZZZWQALDSQQBEW-STQMWFEESA-N 0.000 description 1
- AGVNTAUPLWIQEN-ZPFDUUQYSA-N Arg-Ile-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N AGVNTAUPLWIQEN-ZPFDUUQYSA-N 0.000 description 1
- DNUKXVMPARLPFN-XUXIUFHCSA-N Arg-Leu-Ile Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O DNUKXVMPARLPFN-XUXIUFHCSA-N 0.000 description 1
- VYZBPPBKFCHCIS-WPRPVWTQSA-N Arg-Val-Gly Chemical compound OC(=O)CNC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCN=C(N)N VYZBPPBKFCHCIS-WPRPVWTQSA-N 0.000 description 1
- JWCCFNZJIRZUCL-AVGNSLFASA-N Arg-Val-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCN=C(N)N JWCCFNZJIRZUCL-AVGNSLFASA-N 0.000 description 1
- PFOYSEIHFVKHNF-FXQIFTODSA-N Asn-Ala-Arg Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O PFOYSEIHFVKHNF-FXQIFTODSA-N 0.000 description 1
- JEPNYDRDYNSFIU-QXEWZRGKSA-N Asn-Arg-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CC(N)=O)C(O)=O JEPNYDRDYNSFIU-QXEWZRGKSA-N 0.000 description 1
- PPMTUXJSQDNUDE-CIUDSAMLSA-N Asn-Glu-Arg Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N PPMTUXJSQDNUDE-CIUDSAMLSA-N 0.000 description 1
- BZMWJLLUAKSIMH-FXQIFTODSA-N Asn-Glu-Glu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O BZMWJLLUAKSIMH-FXQIFTODSA-N 0.000 description 1
- FTCGGKNCJZOPNB-WHFBIAKZSA-N Asn-Gly-Ser Chemical compound NC(=O)C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O FTCGGKNCJZOPNB-WHFBIAKZSA-N 0.000 description 1
- YYSYDIYQTUPNQQ-SXTJYALSSA-N Asn-Ile-Ile Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O YYSYDIYQTUPNQQ-SXTJYALSSA-N 0.000 description 1
- LTZIRYMWOJHRCH-GUDRVLHUSA-N Asn-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)N)N LTZIRYMWOJHRCH-GUDRVLHUSA-N 0.000 description 1
- ZYPWIUFLYMQZBS-SRVKXCTJSA-N Asn-Lys-Lys Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)N)N ZYPWIUFLYMQZBS-SRVKXCTJSA-N 0.000 description 1
- HPBNLFLSSQDFQW-WHFBIAKZSA-N Asn-Ser-Gly Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O HPBNLFLSSQDFQW-WHFBIAKZSA-N 0.000 description 1
- HNXWVVHIGTZTBO-LKXGYXEUSA-N Asn-Ser-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O HNXWVVHIGTZTBO-LKXGYXEUSA-N 0.000 description 1
- ZUFPUBYQYWCMDB-NUMRIWBASA-N Asn-Thr-Glu Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O ZUFPUBYQYWCMDB-NUMRIWBASA-N 0.000 description 1
- UXHYOWXTJLBEPG-GSSVUCPTSA-N Asn-Thr-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O UXHYOWXTJLBEPG-GSSVUCPTSA-N 0.000 description 1
- PBVLJOIPOGUQQP-CIUDSAMLSA-N Asp-Ala-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O PBVLJOIPOGUQQP-CIUDSAMLSA-N 0.000 description 1
- JGDBHIVECJGXJA-FXQIFTODSA-N Asp-Asp-Arg Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O JGDBHIVECJGXJA-FXQIFTODSA-N 0.000 description 1
- LKIYSIYBKYLKPU-BIIVOSGPSA-N Asp-Asp-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)O)N)C(=O)O LKIYSIYBKYLKPU-BIIVOSGPSA-N 0.000 description 1
- ICZWAZVKLACMKR-CIUDSAMLSA-N Asp-His-Ser Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CO)C(O)=O)CC1=CN=CN1 ICZWAZVKLACMKR-CIUDSAMLSA-N 0.000 description 1
- DWOGMPWRQQWPPF-GUBZILKMSA-N Asp-Leu-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O DWOGMPWRQQWPPF-GUBZILKMSA-N 0.000 description 1
- PWAIZUBWHRHYKS-MELADBBJSA-N Asp-Phe-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)NC(=O)[C@H](CC(=O)O)N)C(=O)O PWAIZUBWHRHYKS-MELADBBJSA-N 0.000 description 1
- HJZLUGQGJWXJCJ-CIUDSAMLSA-N Asp-Pro-Gln Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(O)=O HJZLUGQGJWXJCJ-CIUDSAMLSA-N 0.000 description 1
- KCOPOPKJRHVGPE-AQZXSJQPSA-N Asp-Thr-Trp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O KCOPOPKJRHVGPE-AQZXSJQPSA-N 0.000 description 1
- XMKXONRMGJXCJV-LAEOZQHASA-N Asp-Val-Glu Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O XMKXONRMGJXCJV-LAEOZQHASA-N 0.000 description 1
- GGBQDSHTXKQSLP-NHCYSSNCSA-N Asp-Val-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)O)N GGBQDSHTXKQSLP-NHCYSSNCSA-N 0.000 description 1
- 238000009020 BCA Protein Assay Kit Methods 0.000 description 1
- 201000004569 Blindness Diseases 0.000 description 1
- 102000047934 Caspase-3/7 Human genes 0.000 description 1
- 108700037887 Caspase-3/7 Proteins 0.000 description 1
- ZOLXQKZHYOHHMD-DLOVCJGASA-N Cys-Ala-Phe Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CS)N ZOLXQKZHYOHHMD-DLOVCJGASA-N 0.000 description 1
- KEBJBKIASQVRJS-WDSKDSINSA-N Cys-Gln-Gly Chemical compound C(CC(=O)N)[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CS)N KEBJBKIASQVRJS-WDSKDSINSA-N 0.000 description 1
- KSMSFCBQBQPFAD-GUBZILKMSA-N Cys-Pro-Pro Chemical compound SC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 KSMSFCBQBQPFAD-GUBZILKMSA-N 0.000 description 1
- GGRDJANMZPGMNS-CIUDSAMLSA-N Cys-Ser-Leu Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O GGRDJANMZPGMNS-CIUDSAMLSA-N 0.000 description 1
- IQXSTXKVEMRMMB-XAVMHZPKSA-N Cys-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CS)N)O IQXSTXKVEMRMMB-XAVMHZPKSA-N 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 102000052510 DNA-Binding Proteins Human genes 0.000 description 1
- 101710096438 DNA-binding protein Proteins 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 101150047030 ERO1 gene Proteins 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 240000001624 Espostoa lanata Species 0.000 description 1
- 235000009161 Espostoa lanata Nutrition 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- 101100246999 Gallus gallus QSOX1 gene Proteins 0.000 description 1
- 241000963438 Gaussia <copepod> Species 0.000 description 1
- -1 Gel Red nucleic acid Chemical class 0.000 description 1
- PRBLYKYHAJEABA-SRVKXCTJSA-N Gln-Arg-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O PRBLYKYHAJEABA-SRVKXCTJSA-N 0.000 description 1
- LMPBBFWHCRURJD-LAEOZQHASA-N Gln-Asn-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCC(=O)N)N LMPBBFWHCRURJD-LAEOZQHASA-N 0.000 description 1
- MCAVASRGVBVPMX-FXQIFTODSA-N Gln-Glu-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O MCAVASRGVBVPMX-FXQIFTODSA-N 0.000 description 1
- KEBACWCLVOXFNC-DCAQKATOSA-N Glu-Arg-Met Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(O)=O KEBACWCLVOXFNC-DCAQKATOSA-N 0.000 description 1
- YYOBUPFZLKQUAX-FXQIFTODSA-N Glu-Asn-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O YYOBUPFZLKQUAX-FXQIFTODSA-N 0.000 description 1
- PHONAZGUEGIOEM-GLLZPBPUSA-N Glu-Glu-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PHONAZGUEGIOEM-GLLZPBPUSA-N 0.000 description 1
- LYCDZGLXQBPNQU-WDSKDSINSA-N Glu-Gly-Cys Chemical compound OC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@@H](CS)C(O)=O LYCDZGLXQBPNQU-WDSKDSINSA-N 0.000 description 1
- GGJOGFJIPPGNRK-JSGCOSHPSA-N Glu-Gly-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)N)C(O)=O)=CNC2=C1 GGJOGFJIPPGNRK-JSGCOSHPSA-N 0.000 description 1
- LGYCLOCORAEQSZ-PEFMBERDSA-N Glu-Ile-Asp Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O LGYCLOCORAEQSZ-PEFMBERDSA-N 0.000 description 1
- HVYWQYLBVXMXSV-GUBZILKMSA-N Glu-Leu-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O HVYWQYLBVXMXSV-GUBZILKMSA-N 0.000 description 1
- LZMQSTPFYJLVJB-GUBZILKMSA-N Glu-Leu-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCC(=O)O)N LZMQSTPFYJLVJB-GUBZILKMSA-N 0.000 description 1
- MCGNJCNXIMQCMN-DCAQKATOSA-N Glu-Met-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@H](CCSC)NC(=O)[C@@H](N)CCC(O)=O MCGNJCNXIMQCMN-DCAQKATOSA-N 0.000 description 1
- QNJNPKSWAHPYGI-JYJNAYRXSA-N Glu-Phe-Leu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C(O)=O)CC1=CC=CC=C1 QNJNPKSWAHPYGI-JYJNAYRXSA-N 0.000 description 1
- UDEPRBFQTWGLCW-CIUDSAMLSA-N Glu-Pro-Asp Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O UDEPRBFQTWGLCW-CIUDSAMLSA-N 0.000 description 1
- WIKMTDVSCUJIPJ-CIUDSAMLSA-N Glu-Ser-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCN=C(N)N WIKMTDVSCUJIPJ-CIUDSAMLSA-N 0.000 description 1
- ZAPFAWQHBOHWLL-GUBZILKMSA-N Glu-Ser-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(=O)O)N ZAPFAWQHBOHWLL-GUBZILKMSA-N 0.000 description 1
- JVYNYWXHZWVJEF-NUMRIWBASA-N Glu-Thr-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCC(=O)O)N)O JVYNYWXHZWVJEF-NUMRIWBASA-N 0.000 description 1
- VIPDPMHGICREIS-GVXVVHGQSA-N Glu-Val-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O VIPDPMHGICREIS-GVXVVHGQSA-N 0.000 description 1
- JRDYDYXZKFNNRQ-XPUUQOCRSA-N Gly-Ala-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN JRDYDYXZKFNNRQ-XPUUQOCRSA-N 0.000 description 1
- VXKCPBPQEKKERH-IUCAKERBSA-N Gly-Arg-Pro Chemical compound NC(N)=NCCC[C@H](NC(=O)CN)C(=O)N1CCC[C@H]1C(O)=O VXKCPBPQEKKERH-IUCAKERBSA-N 0.000 description 1
- KKBWDNZXYLGJEY-UHFFFAOYSA-N Gly-Arg-Pro Natural products NCC(=O)NC(CCNC(=N)N)C(=O)N1CCCC1C(=O)O KKBWDNZXYLGJEY-UHFFFAOYSA-N 0.000 description 1
- WDEHMRNSGHVNOH-VHSXEESVSA-N Gly-Lys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCCN)NC(=O)CN)C(=O)O WDEHMRNSGHVNOH-VHSXEESVSA-N 0.000 description 1
- IBYOLNARKHMLBG-WHOFXGATSA-N Gly-Phe-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 IBYOLNARKHMLBG-WHOFXGATSA-N 0.000 description 1
- FEUPVVCGQLNXNP-IRXDYDNUSA-N Gly-Phe-Phe Chemical compound C([C@H](NC(=O)CN)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 FEUPVVCGQLNXNP-IRXDYDNUSA-N 0.000 description 1
- JJGBXTYGTKWGAT-YUMQZZPRSA-N Gly-Pro-Glu Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O JJGBXTYGTKWGAT-YUMQZZPRSA-N 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- JBJNKUOMNZGQIM-PYJNHQTQSA-N His-Arg-Ile Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JBJNKUOMNZGQIM-PYJNHQTQSA-N 0.000 description 1
- BQYZXYCEKYJKAM-VGDYDELISA-N His-Cys-Ile Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O BQYZXYCEKYJKAM-VGDYDELISA-N 0.000 description 1
- WYWBYSPRCFADBM-GARJFASQSA-N His-Cys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CS)NC(=O)[C@H](CC2=CN=CN2)N)C(=O)O WYWBYSPRCFADBM-GARJFASQSA-N 0.000 description 1
- XKIYNCLILDLGRS-QWRGUYRKSA-N His-Lys-Gly Chemical compound NCCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H](N)CC1=CN=CN1 XKIYNCLILDLGRS-QWRGUYRKSA-N 0.000 description 1
- UMBKDWGQESDCTO-KKUMJFAQSA-N His-Lys-Lys Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(O)=O UMBKDWGQESDCTO-KKUMJFAQSA-N 0.000 description 1
- AYUOWUNWZGTNKB-ULQDDVLXSA-N His-Phe-Arg Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O AYUOWUNWZGTNKB-ULQDDVLXSA-N 0.000 description 1
- UWNUQPZUSRFIIN-JUKXBJQTSA-N His-Tyr-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CC2=CN=CN2)N UWNUQPZUSRFIIN-JUKXBJQTSA-N 0.000 description 1
- 101001131276 Homo sapiens Sulfhydryl oxidase 2 Proteins 0.000 description 1
- 101000611023 Homo sapiens Tumor necrosis factor receptor superfamily member 6 Proteins 0.000 description 1
- 101000666295 Homo sapiens X-box-binding protein 1 Proteins 0.000 description 1
- RWIKBYVJQAJYDP-BJDJZHNGSA-N Ile-Ala-Lys Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCCN RWIKBYVJQAJYDP-BJDJZHNGSA-N 0.000 description 1
- PHIXPNQDGGILMP-YVNDNENWSA-N Ile-Glu-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N PHIXPNQDGGILMP-YVNDNENWSA-N 0.000 description 1
- SAVXZJYTTQQQDD-QEWYBTABSA-N Ile-Phe-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N SAVXZJYTTQQQDD-QEWYBTABSA-N 0.000 description 1
- FADYJNXDPBKVCA-UHFFFAOYSA-N L-Phenylalanyl-L-lysin Natural products NCCCCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FADYJNXDPBKVCA-UHFFFAOYSA-N 0.000 description 1
- SITWEMZOJNKJCH-UHFFFAOYSA-N L-alanine-L-arginine Natural products CC(N)C(=O)NC(C(O)=O)CCCNC(N)=N SITWEMZOJNKJCH-UHFFFAOYSA-N 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- CQQGCWPXDHTTNF-GUBZILKMSA-N Leu-Ala-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O CQQGCWPXDHTTNF-GUBZILKMSA-N 0.000 description 1
- WNGVUZWBXZKQES-YUMQZZPRSA-N Leu-Ala-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O WNGVUZWBXZKQES-YUMQZZPRSA-N 0.000 description 1
- HASRFYOMVPJRPU-SRVKXCTJSA-N Leu-Arg-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(O)=O)C(O)=O HASRFYOMVPJRPU-SRVKXCTJSA-N 0.000 description 1
- MMEDVBWCMGRKKC-GARJFASQSA-N Leu-Asp-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N1CCC[C@@H]1C(=O)O)N MMEDVBWCMGRKKC-GARJFASQSA-N 0.000 description 1
- QCSFMCFHVGTLFF-NHCYSSNCSA-N Leu-Asp-Val Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O QCSFMCFHVGTLFF-NHCYSSNCSA-N 0.000 description 1
- QVFGXCVIXXBFHO-AVGNSLFASA-N Leu-Glu-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O QVFGXCVIXXBFHO-AVGNSLFASA-N 0.000 description 1
- OGUUKPXUTHOIAV-SDDRHHMPSA-N Leu-Glu-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N1CCC[C@@H]1C(=O)O)N OGUUKPXUTHOIAV-SDDRHHMPSA-N 0.000 description 1
- OYQUOLRTJHWVSQ-SRVKXCTJSA-N Leu-His-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(O)=O OYQUOLRTJHWVSQ-SRVKXCTJSA-N 0.000 description 1
- JNDYEOUZBLOVOF-AVGNSLFASA-N Leu-Leu-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O JNDYEOUZBLOVOF-AVGNSLFASA-N 0.000 description 1
- AKVBOOKXVAMKSS-GUBZILKMSA-N Leu-Ser-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O AKVBOOKXVAMKSS-GUBZILKMSA-N 0.000 description 1
- SVBJIZVVYJYGLA-DCAQKATOSA-N Leu-Ser-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O SVBJIZVVYJYGLA-DCAQKATOSA-N 0.000 description 1
- VUBIPAHVHMZHCM-KKUMJFAQSA-N Leu-Tyr-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CO)C(O)=O)CC1=CC=C(O)C=C1 VUBIPAHVHMZHCM-KKUMJFAQSA-N 0.000 description 1
- MVJRBCJCRYGCKV-GVXVVHGQSA-N Leu-Val-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O MVJRBCJCRYGCKV-GVXVVHGQSA-N 0.000 description 1
- WSXTWLJHTLRFLW-SRVKXCTJSA-N Lys-Ala-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(O)=O WSXTWLJHTLRFLW-SRVKXCTJSA-N 0.000 description 1
- JBRWKVANRYPCAF-XIRDDKMYSA-N Lys-Asn-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCCCN)N JBRWKVANRYPCAF-XIRDDKMYSA-N 0.000 description 1
- GCMWRRQAKQXDED-IUCAKERBSA-N Lys-Glu-Gly Chemical compound [NH3+]CCCC[C@H]([NH3+])C(=O)N[C@@H](CCC([O-])=O)C(=O)NCC([O-])=O GCMWRRQAKQXDED-IUCAKERBSA-N 0.000 description 1
- DUTMKEAPLLUGNO-JYJNAYRXSA-N Lys-Glu-Phe Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O DUTMKEAPLLUGNO-JYJNAYRXSA-N 0.000 description 1
- ISHNZELVUVPCHY-ZETCQYMHSA-N Lys-Gly-Gly Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)NCC(O)=O ISHNZELVUVPCHY-ZETCQYMHSA-N 0.000 description 1
- MUXNCRWTWBMNHX-SRVKXCTJSA-N Lys-Leu-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O MUXNCRWTWBMNHX-SRVKXCTJSA-N 0.000 description 1
- PYFNONMJYNJENN-AVGNSLFASA-N Lys-Lys-Gln Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N PYFNONMJYNJENN-AVGNSLFASA-N 0.000 description 1
- WZVSHTFTCYOFPL-GARJFASQSA-N Lys-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CCCCN)N)C(=O)O WZVSHTFTCYOFPL-GARJFASQSA-N 0.000 description 1
- MDDUIRLQCYVRDO-NHCYSSNCSA-N Lys-Val-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCCN MDDUIRLQCYVRDO-NHCYSSNCSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- YRAWWKUTNBILNT-FXQIFTODSA-N Met-Ala-Ala Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O YRAWWKUTNBILNT-FXQIFTODSA-N 0.000 description 1
- 206010054949 Metaplasia Diseases 0.000 description 1
- LSDPWZHWYPCBBB-UHFFFAOYSA-N Methanethiol Chemical compound SC LSDPWZHWYPCBBB-UHFFFAOYSA-N 0.000 description 1
- WUGMRIBZSVSJNP-UHFFFAOYSA-N N-L-alanyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)C)C(O)=O)=CNC2=C1 WUGMRIBZSVSJNP-UHFFFAOYSA-N 0.000 description 1
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 1
- WYBVBIHNJWOLCJ-UHFFFAOYSA-N N-L-arginyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCCN=C(N)N WYBVBIHNJWOLCJ-UHFFFAOYSA-N 0.000 description 1
- 108010079364 N-glycylalanine Proteins 0.000 description 1
- 101100369961 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) erv-1 gene Proteins 0.000 description 1
- 108010027206 Nucleopolyhedrovirus inhibitor of apoptosis Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 239000012124 Opti-MEM Substances 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- UHRNIXJAGGLKHP-DLOVCJGASA-N Phe-Ala-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O UHRNIXJAGGLKHP-DLOVCJGASA-N 0.000 description 1
- CDQCFGOQNYOICK-IHRRRGAJSA-N Phe-Glu-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 CDQCFGOQNYOICK-IHRRRGAJSA-N 0.000 description 1
- KYYMILWEGJYPQZ-IHRRRGAJSA-N Phe-Glu-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 KYYMILWEGJYPQZ-IHRRRGAJSA-N 0.000 description 1
- MMYUOSCXBJFUNV-QWRGUYRKSA-N Phe-Gly-Cys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)NCC(=O)N[C@@H](CS)C(=O)O)N MMYUOSCXBJFUNV-QWRGUYRKSA-N 0.000 description 1
- HNFUGJUZJRYUHN-JSGCOSHPSA-N Phe-Gly-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC1=CC=CC=C1 HNFUGJUZJRYUHN-JSGCOSHPSA-N 0.000 description 1
- FINLZXKJWTYYLC-ACRUOGEOSA-N Phe-His-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 FINLZXKJWTYYLC-ACRUOGEOSA-N 0.000 description 1
- WLYPRKLMRIYGPP-JYJNAYRXSA-N Phe-Lys-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CC1=CC=CC=C1 WLYPRKLMRIYGPP-JYJNAYRXSA-N 0.000 description 1
- PBWNICYZGJQKJV-BZSNNMDCSA-N Phe-Phe-Cys Chemical compound N[C@@H](Cc1ccccc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CS)C(O)=O PBWNICYZGJQKJV-BZSNNMDCSA-N 0.000 description 1
- ZVRJWDUPIDMHDN-ULQDDVLXSA-N Phe-Pro-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CC1=CC=CC=C1 ZVRJWDUPIDMHDN-ULQDDVLXSA-N 0.000 description 1
- ZLAKUZDMKVKFAI-JYJNAYRXSA-N Phe-Pro-Val Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(O)=O ZLAKUZDMKVKFAI-JYJNAYRXSA-N 0.000 description 1
- PTDAGKJHZBGDKD-OEAJRASXSA-N Phe-Thr-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N)O PTDAGKJHZBGDKD-OEAJRASXSA-N 0.000 description 1
- MHNBYYFXWDUGBW-RPTUDFQQSA-N Phe-Tyr-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CC2=CC=CC=C2)N)O MHNBYYFXWDUGBW-RPTUDFQQSA-N 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- IWNOFCGBMSFTBC-CIUDSAMLSA-N Pro-Ala-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O IWNOFCGBMSFTBC-CIUDSAMLSA-N 0.000 description 1
- FZHBZMDRDASUHN-NAKRPEOUSA-N Pro-Ala-Ile Chemical compound CC[C@H](C)[C@H](NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1)C(O)=O FZHBZMDRDASUHN-NAKRPEOUSA-N 0.000 description 1
- IFMDQWDAJUMMJC-DCAQKATOSA-N Pro-Ala-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O IFMDQWDAJUMMJC-DCAQKATOSA-N 0.000 description 1
- CYQQWUPHIZVCNY-GUBZILKMSA-N Pro-Arg-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O CYQQWUPHIZVCNY-GUBZILKMSA-N 0.000 description 1
- KPDRZQUWJKTMBP-DCAQKATOSA-N Pro-Asp-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@@H]1CCCN1 KPDRZQUWJKTMBP-DCAQKATOSA-N 0.000 description 1
- LPGSNRSLPHRNBW-AVGNSLFASA-N Pro-His-Val Chemical compound C([C@@H](C(=O)N[C@@H](C(C)C)C([O-])=O)NC(=O)[C@H]1[NH2+]CCC1)C1=CN=CN1 LPGSNRSLPHRNBW-AVGNSLFASA-N 0.000 description 1
- VTFXTWDFPTWNJY-RHYQMDGZSA-N Pro-Leu-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O VTFXTWDFPTWNJY-RHYQMDGZSA-N 0.000 description 1
- WHNJMTHJGCEKGA-ULQDDVLXSA-N Pro-Phe-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O WHNJMTHJGCEKGA-ULQDDVLXSA-N 0.000 description 1
- KDBHVPXBQADZKY-GUBZILKMSA-N Pro-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 KDBHVPXBQADZKY-GUBZILKMSA-N 0.000 description 1
- JLMZKEQFMVORMA-SRVKXCTJSA-N Pro-Pro-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 JLMZKEQFMVORMA-SRVKXCTJSA-N 0.000 description 1
- FDMKYQQYJKYCLV-GUBZILKMSA-N Pro-Pro-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 FDMKYQQYJKYCLV-GUBZILKMSA-N 0.000 description 1
- GMJDSFYVTAMIBF-FXQIFTODSA-N Pro-Ser-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O GMJDSFYVTAMIBF-FXQIFTODSA-N 0.000 description 1
- CZCCVJUUWBMISW-FXQIFTODSA-N Pro-Ser-Cys Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O CZCCVJUUWBMISW-FXQIFTODSA-N 0.000 description 1
- FDMCIBSQRKFSTJ-RHYQMDGZSA-N Pro-Thr-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O FDMCIBSQRKFSTJ-RHYQMDGZSA-N 0.000 description 1
- GZNYIXWOIUFLGO-ZJDVBMNYSA-N Pro-Thr-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O GZNYIXWOIUFLGO-ZJDVBMNYSA-N 0.000 description 1
- VEUACYMXJKXALX-IHRRRGAJSA-N Pro-Tyr-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(O)=O VEUACYMXJKXALX-IHRRRGAJSA-N 0.000 description 1
- MTMJNKFZDQEVSY-BZSNNMDCSA-N Pro-Val-Trp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O MTMJNKFZDQEVSY-BZSNNMDCSA-N 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 101150026799 QSOX1 gene Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- ZUGXSSFMTXKHJS-ZLUOBGJFSA-N Ser-Ala-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O ZUGXSSFMTXKHJS-ZLUOBGJFSA-N 0.000 description 1
- HRNQLKCLPVKZNE-CIUDSAMLSA-N Ser-Ala-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O HRNQLKCLPVKZNE-CIUDSAMLSA-N 0.000 description 1
- UFKPDBLKLOBMRH-XHNCKOQMSA-N Ser-Glu-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CO)N)C(=O)O UFKPDBLKLOBMRH-XHNCKOQMSA-N 0.000 description 1
- GZFAWAQTEYDKII-YUMQZZPRSA-N Ser-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CO GZFAWAQTEYDKII-YUMQZZPRSA-N 0.000 description 1
- KDGARKCAKHBEDB-NKWVEPMBSA-N Ser-Gly-Pro Chemical compound C1C[C@@H](N(C1)C(=O)CNC(=O)[C@H](CO)N)C(=O)O KDGARKCAKHBEDB-NKWVEPMBSA-N 0.000 description 1
- LRWBCWGEUCKDTN-BJDJZHNGSA-N Ser-Lys-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O LRWBCWGEUCKDTN-BJDJZHNGSA-N 0.000 description 1
- FOOZNBRFRWGBNU-DCAQKATOSA-N Ser-Met-His Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CO)N FOOZNBRFRWGBNU-DCAQKATOSA-N 0.000 description 1
- JLKWJWPDXPKKHI-FXQIFTODSA-N Ser-Pro-Asn Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CO)N)C(=O)N[C@@H](CC(=O)N)C(=O)O JLKWJWPDXPKKHI-FXQIFTODSA-N 0.000 description 1
- AZWNCEBQZXELEZ-FXQIFTODSA-N Ser-Pro-Ser Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O AZWNCEBQZXELEZ-FXQIFTODSA-N 0.000 description 1
- SRSPTFBENMJHMR-WHFBIAKZSA-N Ser-Ser-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O SRSPTFBENMJHMR-WHFBIAKZSA-N 0.000 description 1
- NVNPWELENFJOHH-CIUDSAMLSA-N Ser-Ser-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CO)N NVNPWELENFJOHH-CIUDSAMLSA-N 0.000 description 1
- DYEGLQRVMBWQLD-IXOXFDKPSA-N Ser-Thr-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CO)N)O DYEGLQRVMBWQLD-IXOXFDKPSA-N 0.000 description 1
- PLQWGQUNUPMNOD-KKUMJFAQSA-N Ser-Tyr-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(O)=O PLQWGQUNUPMNOD-KKUMJFAQSA-N 0.000 description 1
- 102100034359 Sulfhydryl oxidase 2 Human genes 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 102100036407 Thioredoxin Human genes 0.000 description 1
- BSNZTJXVDOINSR-JXUBOQSCSA-N Thr-Ala-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O BSNZTJXVDOINSR-JXUBOQSCSA-N 0.000 description 1
- KRPKYGOFYUNIGM-XVSYOHENSA-N Thr-Asp-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N)O KRPKYGOFYUNIGM-XVSYOHENSA-N 0.000 description 1
- IMDMLDSVUSMAEJ-HJGDQZAQSA-N Thr-Leu-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O IMDMLDSVUSMAEJ-HJGDQZAQSA-N 0.000 description 1
- RFKVQLIXNVEOMB-WEDXCCLWSA-N Thr-Leu-Gly Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)O)N)O RFKVQLIXNVEOMB-WEDXCCLWSA-N 0.000 description 1
- MYNYCUXMIIWUNW-IEGACIPQSA-N Thr-Trp-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CCCCN)C(=O)O)N)O MYNYCUXMIIWUNW-IEGACIPQSA-N 0.000 description 1
- OGOYMQWIWHGTGH-KZVJFYERSA-N Thr-Val-Ala Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O OGOYMQWIWHGTGH-KZVJFYERSA-N 0.000 description 1
- XGFYGMKZKFRGAI-RCWTZXSCSA-N Thr-Val-Arg Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N XGFYGMKZKFRGAI-RCWTZXSCSA-N 0.000 description 1
- QGVBFDIREUUSHX-IFFSRLJSSA-N Thr-Val-Gln Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O QGVBFDIREUUSHX-IFFSRLJSSA-N 0.000 description 1
- 102000003978 Tissue Plasminogen Activator Human genes 0.000 description 1
- 108090000373 Tissue Plasminogen Activator Proteins 0.000 description 1
- WQYPAGQDXAJNED-AAEUAGOBSA-N Trp-Cys-Gly Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CS)C(=O)NCC(=O)O)N WQYPAGQDXAJNED-AAEUAGOBSA-N 0.000 description 1
- KIMOCKLJBXHFIN-YLVFBTJISA-N Trp-Ile-Gly Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O)=CNC2=C1 KIMOCKLJBXHFIN-YLVFBTJISA-N 0.000 description 1
- RRVUOLRWIZXBRQ-IHPCNDPISA-N Trp-Leu-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N RRVUOLRWIZXBRQ-IHPCNDPISA-N 0.000 description 1
- NMOIRIIIUVELLY-WDSOQIARSA-N Trp-Val-Leu Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C(O)=O)C(C)C)=CNC2=C1 NMOIRIIIUVELLY-WDSOQIARSA-N 0.000 description 1
- 102100040403 Tumor necrosis factor receptor superfamily member 6 Human genes 0.000 description 1
- GULIUBBXCYPDJU-CQDKDKBSSA-N Tyr-Leu-Ala Chemical compound [O-]C(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]([NH3+])CC1=CC=C(O)C=C1 GULIUBBXCYPDJU-CQDKDKBSSA-N 0.000 description 1
- SBLZVFCEOCWRLS-BPNCWPANSA-N Tyr-Met-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CC1=CC=C(C=C1)O)N SBLZVFCEOCWRLS-BPNCWPANSA-N 0.000 description 1
- WURLIFOWSMBUAR-SLFFLAALSA-N Tyr-Phe-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)NC(=O)[C@H](CC3=CC=C(C=C3)O)N)C(=O)O WURLIFOWSMBUAR-SLFFLAALSA-N 0.000 description 1
- ZLFHAAGHGQBQQN-GUBZILKMSA-N Val-Ala-Pro Natural products CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O ZLFHAAGHGQBQQN-GUBZILKMSA-N 0.000 description 1
- COYSIHFOCOMGCF-WPRPVWTQSA-N Val-Arg-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CCCN=C(N)N COYSIHFOCOMGCF-WPRPVWTQSA-N 0.000 description 1
- COYSIHFOCOMGCF-UHFFFAOYSA-N Val-Arg-Gly Natural products CC(C)C(N)C(=O)NC(C(=O)NCC(O)=O)CCCN=C(N)N COYSIHFOCOMGCF-UHFFFAOYSA-N 0.000 description 1
- PAPWZOJOLKZEFR-AVGNSLFASA-N Val-Arg-Lys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCCN)C(=O)O)N PAPWZOJOLKZEFR-AVGNSLFASA-N 0.000 description 1
- ZMDCGGKHRKNWKD-LAEOZQHASA-N Val-Asn-Glu Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N ZMDCGGKHRKNWKD-LAEOZQHASA-N 0.000 description 1
- OUUBKKIJQIAPRI-LAEOZQHASA-N Val-Gln-Asn Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O OUUBKKIJQIAPRI-LAEOZQHASA-N 0.000 description 1
- NYTKXWLZSNRILS-IFFSRLJSSA-N Val-Gln-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](C(C)C)N)O NYTKXWLZSNRILS-IFFSRLJSSA-N 0.000 description 1
- OXVPMZVGCAPFIG-BQFCYCMXSA-N Val-Gln-Trp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N OXVPMZVGCAPFIG-BQFCYCMXSA-N 0.000 description 1
- FOADDSDHGRFUOC-DZKIICNBSA-N Val-Glu-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N FOADDSDHGRFUOC-DZKIICNBSA-N 0.000 description 1
- JTWIMNMUYLQNPI-WPRPVWTQSA-N Val-Gly-Arg Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCNC(N)=N JTWIMNMUYLQNPI-WPRPVWTQSA-N 0.000 description 1
- JPPXDMBGXJBTIB-ULQDDVLXSA-N Val-His-Tyr Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)N JPPXDMBGXJBTIB-ULQDDVLXSA-N 0.000 description 1
- UMPVMAYCLYMYGA-ONGXEEELSA-N Val-Leu-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O UMPVMAYCLYMYGA-ONGXEEELSA-N 0.000 description 1
- RFKJNTRMXGCKFE-FHWLQOOXSA-N Val-Leu-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)CC(C)C)C(O)=O)=CNC2=C1 RFKJNTRMXGCKFE-FHWLQOOXSA-N 0.000 description 1
- QTXGUIMEHKCPBH-FHWLQOOXSA-N Val-Trp-Lys Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](N)C(C)C)C(=O)N[C@@H](CCCCN)C(O)=O)=CNC2=C1 QTXGUIMEHKCPBH-FHWLQOOXSA-N 0.000 description 1
- 102100038151 X-box-binding protein 1 Human genes 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000000980 acid dye Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 101150063416 add gene Proteins 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 108010008685 alanyl-glutamyl-aspartic acid Proteins 0.000 description 1
- 108010024078 alanyl-glycyl-serine Proteins 0.000 description 1
- 108010078114 alanyl-tryptophyl-alanine Proteins 0.000 description 1
- 108010047495 alanylglycine Proteins 0.000 description 1
- 108010087924 alanylproline Proteins 0.000 description 1
- 108010050025 alpha-glutamyltryptophan Proteins 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 108010008355 arginyl-glutamine Proteins 0.000 description 1
- 108010068380 arginylarginine Proteins 0.000 description 1
- 108010060035 arginylproline Proteins 0.000 description 1
- 210000004507 artificial chromosome Anatomy 0.000 description 1
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 1
- 108010093581 aspartyl-proline Proteins 0.000 description 1
- 108010047857 aspartylglycine Proteins 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000002146 bilateral effect Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 238000005266 casting Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003593 chromogenic compound Substances 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000009194 climbing Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000000571 coke Substances 0.000 description 1
- 238000010960 commercial process Methods 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000007334 copolymerization reaction Methods 0.000 description 1
- 101150070926 ct gene Proteins 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 108010069495 cysteinyltyrosine Proteins 0.000 description 1
- 238000005034 decoration Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 1
- HKSZLNNOFSGOKW-UHFFFAOYSA-N ent-staurosporine Natural products C12=C3N4C5=CC=CC=C5C3=C3CNC(=O)C3=C2C2=CC=CC=C2N1C1CC(NC)C(OC)C4(C)O1 HKSZLNNOFSGOKW-UHFFFAOYSA-N 0.000 description 1
- 230000009088 enzymatic function Effects 0.000 description 1
- 235000013861 fat-free Nutrition 0.000 description 1
- 238000002637 fluid replacement therapy Methods 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000010441 gene drive Methods 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 108010008237 glutamyl-valyl-glycine Proteins 0.000 description 1
- 108010049041 glutamylalanine Proteins 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 108010079413 glycyl-prolyl-glutamic acid Proteins 0.000 description 1
- 230000007438 host cellular process Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 108010044374 isoleucyl-tyrosine Proteins 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 238000003367 kinetic assay Methods 0.000 description 1
- 108010083708 leucyl-aspartyl-valine Proteins 0.000 description 1
- 108010034529 leucyl-lysine Proteins 0.000 description 1
- 108010073472 leucyl-prolyl-proline Proteins 0.000 description 1
- 108010057821 leucylproline Proteins 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 108010017391 lysylvaline Proteins 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 230000015689 metaplastic ossification Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 108010012581 phenylalanylglutamate Proteins 0.000 description 1
- 108010073025 phenylalanylphenylalanine Proteins 0.000 description 1
- 108010051242 phenylalanylserine Proteins 0.000 description 1
- 229940012957 plasmin Drugs 0.000 description 1
- 238000005498 polishing Methods 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 108010090894 prolylleucine Proteins 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 238000009418 renovation Methods 0.000 description 1
- 210000003660 reticulum Anatomy 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 108010026333 seryl-proline Proteins 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 238000010583 slow cooling Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 108010005652 splenotritin Proteins 0.000 description 1
- 210000001324 spliceosome Anatomy 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- HKSZLNNOFSGOKW-FYTWVXJKSA-N staurosporine Chemical compound C12=C3N4C5=CC=CC=C5C3=C3CNC(=O)C3=C2C2=CC=CC=C2N1[C@H]1C[C@@H](NC)[C@@H](OC)[C@]4(C)O1 HKSZLNNOFSGOKW-FYTWVXJKSA-N 0.000 description 1
- CGPUWJWCVCFERF-UHFFFAOYSA-N staurosporine Natural products C12=C3N4C5=CC=CC=C5C3=C3CNC(=O)C3=C2C2=CC=CC=C2N1C1CC(NC)C(OC)C4(OC)O1 CGPUWJWCVCFERF-UHFFFAOYSA-N 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 108010001535 sulfhydryl oxidase Proteins 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 108060008226 thioredoxin Proteins 0.000 description 1
- 229940094937 thioredoxin Drugs 0.000 description 1
- 229960000187 tissue plasminogen activator Drugs 0.000 description 1
- 108010080629 tryptophan-leucine Proteins 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 108010073969 valyllysine Proteins 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- 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
-
- 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
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0051—Oxidoreductases (1.) acting on a sulfur group of donors (1.8)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y108/00—Oxidoreductases acting on sulfur groups as donors (1.8)
- C12Y108/03—Oxidoreductases acting on sulfur groups as donors (1.8) with oxygen as acceptor (1.8.3)
- C12Y108/03002—Thiol oxidase (1.8.3.2)
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Cell Biology (AREA)
- Reproductive Health (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明提供基于CRISPR/Cas9基因编辑改造的CHO细胞株及其制备方法，选取二硫键折叠相关的巯基氧化酶HsQSOX1b和细胞凋亡相关的生存素Survivin作为目的基因，设计成外源表达盒EC#1和EC#2，利用新型的CRISPR/Cas9的基因编辑技术，将其整合到CHO细胞的特定基因位点。本发明利用CRISPR/Cas9基因编辑技术，将人HsQSOX1b和Survivin基因对CHO‑K1细胞进行精准编辑，建立了快速高效的宿主细胞改造技术平台，并获得抗凋亡能力强、催化二硫键效率与蛋白表达质量高的单克隆细胞株，为改造符合工业生产要求的抗体高效细胞株提供理论与技术支撑。
Description
技术领域
本发明涉及基因编辑技术领域，更具体地讲，涉及一种以HsQSOX1b和Survivin作为目的基因的基于CRISPR/Cas9基因编辑技术改造CHO细胞。
背景技术
近年来，以抗体为代表的治疗性重组蛋白已成为生物药物行业的主导产品，临床需求巨大。自1986年首个在CHO细胞中生产的重组蛋白人组织纤溶酶原激活物(humantissue plasminogen activator,tPA)获得批准以来，CHO细胞已成为外源蛋白生产最主要的表达宿主。然而在重组蛋白的工业化生产过程中，目标蛋白表达的质和量较低，应对不利环境的抵抗能力较弱等瓶颈一直影响着重组蛋白生产的经济效益和临床广泛应用。随着CHO及CHO-K1细胞的基因组的测序及组学技术的发展，大批基因的结构和功能将被陆续得到解析，这为基因改造提供了足够的靶位点，即可以选择特定的靶基因，实现针对靶基因的靶位点进行改造，建立稳定和新颖的宿主细胞。
将目的基因整合进CHO细胞通常依据两种整合方式：随机整合和位点特异性整合。传统方式的随机整合是通过逐级增加药物浓度迫使细胞获得外源质粒整合，同时随着药物浓度的增高，标记基因的表达带动目的基因的表达而获得高表达的细胞株。但是随机整合的盲目性大，许多位点是不表达和低表达的，需要经过漫长的药物梯度筛选才能获得高产的单克隆株，而且随机整合往往会因为位置效应导致外源基因的沉默。不同于随机整合，位点特异性整合选择了明确的整合位点实现目的基因的整合。其主要的方式有Cre-LoxP重组酶系统、核基质附着区(MAR或SAR)、泛染色体开放元件(UCOE)和人工染色体。以上这些方法能够使目的基因的整合更加具有目的性，但是往往都依赖于碱基序列特异性的染色体区域，能够在一定程度上拓宽目的基因整合基因组的应用范围。此外，不同于上述几种位点特异性整合方法的是，基于核酸内切酶发展起来的基因编辑技术能够极大的扩宽基因组改造的范围，从而加速宿主细胞改造进程。目前基因编辑技术主要可以分为三代，前两代是依赖Fok I限制性内切酶发展起来的ZFNs和TALENs基因编辑技术；第三代即为目前发展迅速的依赖Cas蛋白的CRISPR/Cas基因编辑技术。ZFNs和TALENs的基因编辑技术是将DNA结合蛋白与Fok I内切酶结合，需要依赖蛋白质工程进行设计，能够对靶基因进行敲除以及研究特定的功能基因。这两种方法均能够进一步扩大了基因编辑的范围，但是设计复杂，价格比较昂贵，一定程度限制其大规模应用。
CRISPR/Cas9是最近利用细菌和古生菌的免疫系统开发而来的，是基于RNA介导的核酸内切酶(RNA-guided engineered nucleases,RGENs)的基因编辑技术。依赖于碱基互补配对原则实现基因组的定点修饰，比蛋白质识别特定碱基序列的作用力更强、更稳定；而且设计简便，快速。此外，进行基因编辑的位点得到了极大的丰富，理论上每隔108个碱基即可出现一个CRISPR/Cas9的潜在切割位点。根据CRISPR位点上游Cas基因保守性差异将CRISPR/Cas系统主要分为三类：I型、II型和III型。目前研究最为透彻的是II型CRISPR/Cas系统，其组成较为简单，通常由单个Cas酶组成，因而可以简便、快速的被开发应用。其中，CRISPR/Cas9系统是应用最为广泛的II型系统，核心部分是Cas9蛋白和向导RNA(gRNA)。Cas9酶含有两个能够发挥切割作用的功能域RuvC和HNH。当CRISPR/Cas簇在引导区的作用转录出pre-crRNA时，与Repeat区互补的反式激活crRNA(Trans-activating crRNA，tracrRNA)也转录出来，然后激活Cas9和RNaseIII核酸酶将pre-crRNA加工形成短的crRNAs，并组装形成crRNA、tracrRNA和Cas9复合体，其中crRNA能够识别与之互补的DNA链并与之结合形成RNA-DNA杂合链，然后Cas9酶的RuvC和HNH结构域分别对DNA两条链进行切割，前者切割与crRNA非互补链，后者切割互补链，使DNA双链产生断裂，形成DSBs。Cas9的剪切位点位于crRNA互补序列下游邻近的PAM区(Protospacer Adjacent Motif)的5’-NGG-3’特征区域中的NGG位点前三个碱基。
巯基氧化酶QSOX1的二硫键转移和形成功能是由具有氧化还原活性的半胱氨酸残基介导的，典型的模式为Cys-X-X-Cys。在QSOX1中这种双半胱氨酸基序在硫氧还蛋白结构域中存在，类似于PDI的氧化还原活性结构域。另一个存在于发挥巯基氧化功能的FAD结合域，类似于线粒体酶Erv1。人体中具有两个QSOX基因，分别为QSOX1和QSOX2。其中QSOX1基因转录的mRNA经不同的剪接方式形成3314bp和2588bp的两种剪接体，分别编码747个氨基酸的QSOX1a(L)(82kDa)和604个氨基酸的QSOX1b(S)(66kDa)。研究表明，HsQSOX1b是分泌型的，体外研究发现其具有二硫键形成功能，在氧化三(2-羧乙基)膦(TCEP)，DTT等含有硫醇结构的物质时，酶活较Ero1和Erv家族的巯基氧化酶高上百倍。因此，选择酶学功能鉴定清楚的HsQSOX1b并将其定位到内质网中，研究其在活细胞内质网中的二硫键形成能力。此外，研究发现Survivin是抗凋亡蛋白家族的最强者，其结构由单个baculovirus IAP repeat(BIR)结构域和一个扩展的α螺旋卷曲组成。Survivin可以直接抑制Caspase-3/7活性来抑制细胞凋亡，也可以抑制由Fas(CD95)，Bax，抗癌药物引起的细胞凋亡过程，显现出广泛的抗凋亡能力。而基于Survivin蛋白的细胞改造方面的研究还很少，因此我们在构建含有HsQSOX1b表达盒时将Survivin一起设计到表达盒中，用于研究内质网中蛋白质量控制，同时探究Survivin在CHO-K1细胞中的抗凋亡性能。
发明内容
本发明的第一个目的在于提供一种基于CRISPR/Cas9基因编辑技术改造CHO细胞的制备方法。
本发明的第二个目的在于提供利用基于CRISPR/Cas9基因编辑技术改造CHO细胞的制备方法制备获得的单克隆细胞株。
为了实现本发明第一个目的，本发明提供了一种基于CRISPR/Cas9基因编辑技术改造CHO细胞的制备方法，其特征在于，选取二硫键折叠相关的巯基氧化酶HsQSOX1b和细胞凋亡相关的生存素Survivin作为目的基因，设计成外源表达盒EC#1和EC#2，利用新型的CRISPR/Cas9的基因编辑技术，将其整合到CHO细胞的特定基因位点。
作为一个优选方案，表达盒EC#1和EC#2的串联组合方式分别为CMV(E+P)+eGFP+HsQSOX1b-KDEL+BGH pA和CMV(E+P)+eGFP+HsQSOX1b-KDEL+T2A+Survivin+BGH pA。
为了实现本发明第二个目的，本发明提供了利用基于CRISPR/Cas9基因编辑技术改造CHO细胞的制备方法制备获得的单克隆细胞株，其特征在于，所述单克隆细胞株HsQSOX1b和Survivin过表达。
巯基氧化酶HsQSOX1b的序列：ATMRRCNSGSGPPPSLLLLLLWLLAVPGANAAPRSALYSPSDPLTLLQADTVRGAVLGSRSAWAVEFFASWCGHCIAFAPTWKALAEDVKAWRPALYLAALDCAEETNSAVCRDFNIPGFPTVRFFKAFTKNGSGAVFPVAGADVQTLRERLIDALESHHDTWPPACPPLEPAKLEEIDGFFARNNEEYLALIFEKGGSYLAREVALDLSQHKGVAVRRVLNTEANVVRKFGVTDFPSCYLLFRNGSVSRVPVLMESRSFYTAYLQRLSGLTREAAQTTVAPTTANKIAPTVWKLADRSKIYMADLESALHYILRIEVGRFPVLEGQRLVALKKFVAVLAKYFPGRPLVQNFLHSVNEWLKRQKRNKIPYSFFKTALDDRKEGAVLAKKVNWIGCQGSEPHFRGFPCSLWVLFHFLTVQAARQNVDHSQEAAKAKEVLPAIRGYVHYFFGCRDCASHFEQMAAASMHRVGSPNAAVLWLWSSHNRVNARLAGAPSEDPQFPKVQWPPRELCSACHNERLDVPVWDVEATLNFLKAHFSPSNIILDFPAAGSAARRDVQNVAAAPELAMGALELESRNSTLDPGKPEMMKSPTNTTPHVPAEGPELI(SEQ ID NO：1)。
生存素Survivin的序列：MGAPTLPPAWQPFLKDHRISTFKNWPFLEGCACTPERMAEAGFIHCPTENEPDLAQCFFCFKELEGWEPDDDPIEEHKKHSSGCAFLSVKKQFEELTLGEFLKLDRERAKNKIAKETNNKKKEFEETAKKVRRAIEQLAAMD(SEQ ID NO：2)。
本发明选取二硫键折叠相关的巯基氧化酶HsQSOX1b和细胞凋亡相关的生存素Survivin作为目的基因设计成外源表达盒EC#1/2。以期证实HsQSOX1b能够在哺乳动物细胞中发挥二硫键折叠功能的同时，达到减少蛋白质在内质网聚集而引起的内质网应激，提高内质网的蛋白质量控制能力。同时，研究Survivin赋予CHO细胞抗凋亡的特性，进一步延长细胞的重组蛋白生产周期。
本发明利用新型的CRISPR/Cas9的基因编辑技术将HsQSOX1b和Survivin整合到CHO细胞的特定基因位点。RISPR/Cas9系统是目前应用最为广泛的亚型，核心部分是Cas9蛋白和向导RNA(gRNA)。Cas9酶含有两个能够发挥切割作用的功能域RuvC和HNH。当CRISPR/Cas簇在引导区的作用转录出pre-crRNA时，与Repeat区互补的反式激活crRNA也转录出来，然后激活Cas9和RNaseIII核酸酶将pre-crRNA加工形成短的crRNAs，并组装形成crRNA、tracrRNA和Cas9复合体，其中crRNA能够识别与之互补的DNA链并与之结合形成RNA-DNA杂合链，然后Cas9酶的RuvC和HNH结构域分别对DNA两条链进行切割，前者切割与crRNA非互补链，后者切割互补链，使DNA双链产生断裂，形成DSBs。Cas9的剪切位点位于crRNA互补序列下游邻近的PAM区的5’-NGG-3’特征区域中的NGG位点前三个碱基。
本发明的优点在于，目前CHO细胞是应用最为广泛的哺乳动物表达宿主，但是工业化生产过程中重组蛋白表达的质量较低，应对不利环境的抵抗能力较弱等瓶颈影响着重组蛋白生产的经济效益和临床广泛应用。针对CHO细胞的改造主要依靠培养基与代谢途径优化，而难于从根上进行多基因精准编辑，强化其整体效能。本发明利用CRISPR/Cas9基因编辑技术，将人HsQSOX1b和Survivin基因对CHO-K1细胞进行精准编辑，建立了快速高效的宿主细胞改造技术平台，并以GLuc作为模式蛋白对单克隆株的蛋白生产性能评价，获得抗凋亡能力强、催化二硫键效率与蛋白表达质量高的单克隆细胞株，为改造符合工业生产要求的抗体高效细胞株提供理论与技术支撑。本发明的HsQSOX1b和Survivin过表达的单克隆细胞株改造具有快速简便、便宜、易于设计和高效的特点。
附图说明
图1为设计和构建的两个外源表达盒示意图。
图2为构建表达盒中各DNA片段电泳图，其中，泳道1为CMV(E+P)+signalpeptideof HsQSOX1b，泳道2为EGFP，泳道3为HsQSOX1b30-604+KDEL，泳道4为BGH polyA。
图3为四个质粒转染CHO-K1细胞的荧光倒置显微镜下和白光下细胞图，其中，左上、左下分别为CHO-K1细胞转染Q1和Q2在荧光倒置显微镜和白光下的细胞图，右上和右下分别是CHO-K1细胞转染B1和B2在荧光倒置显微镜和白光下的细胞图。Scale bar＝100μm。
图4为T7E1酶切试验凝胶电泳结果，其中红色箭头表示经T7E1酶切后形成的两条子带，四个质粒转染CHO-K1细胞得到的indels(％)分别为9.1％、16.7％、13.3％和19.1％。
图5为流式细胞仪分析转共转染整合效率，其中EC#1整合的阳性细胞为5.18％，EC#2整合的阳性细胞为3.85％。
图6为eGFP阳性单克隆细胞株中HsQSOX1b和Survivin表达情况。
图7为激光共聚焦扫描电镜下的细胞染色图，左上为内质网探针ER-Tracker Red染色显示的细胞内质网轮廓，右上为细胞表达绿色荧光蛋白的分布图，左下为染核试剂Hoechst 33342染色获得的核分布图，右下为前三张图片组合下的细胞图。Scale bar＝2μm。
图8为转染EC#1和EC#2的稳定单克隆株EC#1-C1和EC#2-B6，图第一列和第二列分别为单克隆细胞株EC#1-C1在两种不同倍镜下的白光和荧光图，第三列和第四列是单克隆株EC2-B6在两种不同倍镜下的白光和荧光下细胞图。
图9为不同细胞株中GSH/GSSG与GSH/tGSH比值图，其中将对照组CHO-K1细胞的GSH/GSSG和GSH/tGSH比值分别计为100％，单克隆细胞EC#1-C1与单克隆细胞株EC#2-B6的GSH/GSSG的比值分别为40.3％和70.4％(*P＜0.05)，两株单克隆细胞的GSH/tGSH比值分别为91.2％和97.3％(*P＜0.05)。
图10为XBP1剪接试验，其中两株单克隆细胞EC#1-C1和EC#2-B6的XBP1t表达量分别是管家基因β-actin的0.564和0.565倍，XBP1u分别是管家基因β-actin的0.306和0.397倍，而根据XBP1t和XBP1u的Ct值计算得到的XBP1s的表达量分别为管家基因β-actin的2.629和1.343倍。两株单克隆细胞株的XBP1s/XBP1t比值分别为4.665和2.378。
图11为诱导细胞凋亡及结果检测，图中数值表示细胞的凋亡比率，试验组和对照组分别进行三个平行，数值显示mean±SD，且均以SPSS软件进行双侧t检验，*P＜0.05。NC指阴性对照，PC指阳性对照。
图12为无血清培养96h后细胞活性，其中以对照组CHO-K1细胞进行的双侧t检验结果，**P＜0.01，n＝6。
图13为不同细胞株的荧光素酶测定结果图，其中以对照组CHO-K1细胞进行的双侧t检验结果，*P＜0.05，**P＜0.01，n＝3。
具体实施方式
以下，结合具体实施方式对本发明的技术进行详细描述。应当知道的是，以下具体实施方式仅用于帮助本领域技术人员理解本发明，而非对本发明的限制。
实施例1.基于CHO-K1细胞的sgRNA-Cas9与表达盒设计、构建及其功能验证
试剂与试剂盒：Premix Taq DNA polymerase，Pyrobest DNA polymerase，限制内切酶(Hind III、BamH I)，DNA A-Tailing Kit，pMDTM19-T Vector CloningKit，DNAmarker购置于宝生物工程有限公司(大连)。核酸内切酶Bbs I和T4DNA ligase购置于NEB。质粒提取试剂盒、血液/细胞、组织基因组DNA提取试剂盒、EasyGene快速重组克隆试剂盒、DNA凝胶回收试剂盒和UniversalDNA纯化回收试剂盒购置于Tiangen。Lipofectamine 2000购置于Thermo FisherScientific。胰化蛋白胨、酵母提取物购置于Oxoid公司(英国)。Ampicillin购自生工生物工程股份有限公司(上海)。F-12和Opti-MEM培养基、胰蛋白酶购置于Gibco。Penicillin-streptomycin(100×)购置于Solarbio。FBS购置于Hyclone。其他的生化试剂属于国产常规的分析纯试剂。
质粒与菌株：质粒pSpCas9(BB)-2A-GFP(PX458)和pSpCas9(BB)-2A-Puro(PX459)V2.0(Addgene Plasmid#48138和#62988)；质粒pUC19(Tiangen)；质粒pETsumo和pCDNA3.1(Invirogen，USA)；E.coli DH5α(Invirogen，USA)作为质粒扩增菌株；细胞株CHO-K1细胞(中科院细胞库)。
试剂配制：
LB液体培养基：称取胰化蛋白胨10g、酵母提取物5g、氯化钠10g，用去离子水定容到1L，121℃高压灭菌20min，常温保存。
LB固体培养基：称取胰化蛋白胨1g、酵母提取物0.5g、氯化钠1g，琼脂粉1.5g，用去离子水定容到100mL，121℃高压灭菌20min，常温保存。
蓝白斑筛选用LB固体培养基：每100mL的LB固体培养基中依次加入100μL 100mMIPTG、200μL 20mg/mL X-Gal、100μL 100mg/mL Amp左侧对应浓度试剂，混匀制成平板封口膜封口4℃保存备用。
氨苄青霉素(100μg/mL)：称取1g，溶于10mL ddH2O，使用0.22μm的无菌滤膜过滤除菌，分装，-20℃保存。
TAE电泳缓冲液(50×)：称取Tris碱242g，量取冰醋酸57.1mL，0.5MEDTA(pH8.0)100mL，加去离子水定容至1L。
琼脂糖凝胶(1％)：称取0.7g琼脂糖，加入TAE(1×)70mL，加热溶解，待冷却60℃左右，加入Gel Red核酸染液(100×)1mL，插好梳子，倒胶。
DPBS溶液：称取NaCl 8.0g，KCl 0.2g，KH2PO4 0.2g，Na2HPO4 1.42g。将上述试剂充分混匀并定容到1L的超纯水中，高压蒸汽灭菌25min，常温保存备用。
细胞培养基：将100mL F-12培养基，10mL FBS和1mL(100×)青链霉素三种成分混匀，4℃保存。
表达盒EC#1和EC#2的构建
引物设计:
表1.1靶向QSOX1和BIRC5合成的sgRNA寡核苷酸
PCR反应体系：
表1.2表达盒构建中DNA片段PCR扩增反应体系
PCR反应条件：
表1.3表达盒构建中DNA片段PCR扩增反应条件
PCR扩增各部分目的条带后，按表1.4所示将各部分DNA片段加入到PCR管中进行连接反应，将PCR管置于50℃的水浴锅中反应15min(对于3个以上DNA片段重组时，设计引物的重叠序列需要≥20nt，且反应时间延长至1h)。
表1.4各DNA片段重组克隆反应体系
待连接反应结束后，瞬时离心PCR反应管并将离心管置于冰上，进行后续的转化反应。次日，挑取单克隆菌落于5mL的含有Amp的LB液体培养基中培养16h，之后取1mL菌液于无菌的EP管中送上海睿迪生物科技有限公司进行测序，测序以pUC19的通用引物M13F和M13R进行(由公司提供)，检测组装的表达盒是否引入突变。
CHO-K1细胞的培养、转染以及sgRNA-Cas9载体与表达盒EC#1/2的功能验证
1)CHO-K1细胞的复苏：细胞的复苏遵循快速复苏的原则，从液氮中取出的冻存管应当迅速取出，置于37℃水浴中，快速地摇动冻存管使细胞受热均匀，待细胞溶解至还剩一小块冰时，加入少量预温的新鲜培养基于冻存管中上下缓慢吹打细胞混合均匀并将细胞移至T-25的培养瓶中，加入4mL预温的新鲜F-12培养基混合均匀，置于37℃，含有5％CO2的培养箱中培养6h左右，待细胞贴壁后，更换新培养液即可。
2)CHO-K1细胞的换液、传代
细胞的培养需要在无菌的环境进行，因此在操作之前需要紫外灭菌30min以上，同时需要将培养基，胰酶等放入培养箱中预热。进行操作时，时刻注意无菌操作的要求，穿好实验服，带好手套，打开超净台风机，用75％的酒精棉球擦拭操作台面，待风机风速稳定后进行操作。
CHO-K1细胞的换液：CHO-K1细胞的培养通常在T-25培养瓶中进行。换液时将培养瓶中已有的培养基倒入废液缸，以DPBS洗两遍细胞，每次2-3mL，然后加入4mL的预温的新鲜的F-12培养基，然后将细胞重新放回到培养箱中培养。
CHO-K1细胞的传代：在倒置显微镜观察细胞密度，当细胞达到80-90％的汇合度时，倒掉培养瓶中已有的培养基，以DPBS洗涤2次，并用长枪头将培养瓶中残留的DPBS吸除，加入500μL胰蛋白酶消化细胞，边水平前后缓慢小幅度转动培养瓶使胰蛋白酶均匀接触细胞，边在倒置显微镜下观察细胞。(对于不同的细胞消化时间和温度有所不同，CHO-K1细胞通常室温消化1min即可)待细胞变圆后，立即加入2mL已预温的F-12培养基终止胰蛋白酶消化过程，并用无菌的吸管吹打细胞至单细胞悬液，然后将细胞移至1.5mL的无菌EP管中，1000rpm离心10mim。离心结束后，取适量细胞稀释并以血球计数板计数，然后按照比例传代，每个培养瓶接种1.2×105个细胞。对于CHO-K1细胞通常以1:2-4进行传代。
3)CHO-K1细胞的冻存
CHO-K1细胞的冻存：同细胞传代步骤，在1000rpm离心10min结束后，以移液枪轻轻吸去上清，加入1mL的细胞冻存液重悬细胞并将细胞悬液移入冻存管中，拧紧盖子，再以封口膜封口，并标记细胞名称、日期和保存人姓名。细胞冻存遵循缓慢降温的原则，将冻存管先置于4℃冰箱30min，然后移入-20℃冰箱1-2h，再移入-80℃过夜，最后移入液氮中保存。
4)细胞转染
细胞转染主要分为单个sgRNA-pSpCas9载体转染和sgRNA-pSpCas9与EC#1/2共转染。转染试剂以Lipofectamine2000进行，因不同的细胞系，转染试剂与质粒DNA的最佳配比不同，故我们进行了预实验，选取质粒(μg):Lipofectamine2000(μL)为1:2-4的条件进行实验，确定质粒和Lipofectamine2000的最佳转染比例。对于CHO-K1细胞，最终确定质粒(μg):Lipofectamine2000(μL)以1:3的配比进行转染实验。本实验以构建好的sgRNA1-QSOX1-pX458(Q1),sgRNA2-QSOX1-pX458(Q2),sgRNA1-BIRC5-pX458(B1)和sgRNA2-BIRC5-pX458(B2)四个质粒转染CHO-K1细胞，分别靶向QSOX1和BIRC5上两个gDNA靶位点，并以pX458空载作为对照。
5)sgRNA-Cas9载体在CHO-K1细胞发挥酶切活性验证
CHO-K1基因组的提取：基因组的提取包括两种方法：其一：对于培养瓶和6孔板中的细胞以天根血液/细胞、组织基因组DNA提取试剂盒，使用说明和提取流程参见官方说明书进行，最后以微型核酸分析仪定量。细胞处理步骤简述如下：以DPBS系两遍后，加入适量的胰蛋白酶消化1min左右，待细胞变圆后，移去胰酶，加入500μL DPBS吹打细胞形成单细胞悬液，再将细胞移至1.5mL的EP管中，1500g离心5min收集细胞沉淀，以500μL DPBS重悬细胞，洗一遍后，尽量去除DPBS，以试剂盒提取基因组DNA。其二：对于96孔板和24孔板中少量细胞其基因组参照QuickExtract DNA ExtractionSolution进行，该溶液可以直接提取基因组作为PCR的模板，基本操作流程为以96孔板计算，每孔加入10μL的QuickExtractDNAExtraction Solution反复吹打细胞，使细胞悬浮，然后将细胞悬液移入PCR管中，进行65℃,15min，68℃,15min和98℃,10min的程序进行处理。最后取1-2μL的反应产物直接作为PCR的模板。
特异性PCR引物设计：引物设计以Primer-Blast进行设计，网址信息：https://www.ncbi.nlm.nih.gov/tools/primer-blast/。特异性的引物合成后经PCR验证特异性方可用于后续实验验证。引物见表1.5。
表1.5 T7E1酶切试验所用引物表
PCR目的片段以及跑胶纯化回收目的片段：根据表1.5中所列引物，以KOD-FX高效率高保真酶进行sgRNA位点的基因组目的片段扩增。反应所用体系以表1.2做相应调整，反应程序以Touchdown PCR进行，见表1.6。
表1.6降落PCR扩增sgRNA靶位点目的片段反应程序
PCR结束后，取5μL PCR产物进行2％的琼脂糖凝胶电泳，出现目的条带后，将全部产物跑胶纯化，纯化以QIAquick Gel Extraction Kit进行回收，步骤按照试剂盒说明书进行，纯化后以微量核酸分析仪进行定量。纯化的PCR目的片段进行变性，复性处理；T7E1酶切反应。
6)eGFP阳性单克隆细胞株的获得以及表达盒整合情况验证
表达eGFP的阳性单克隆细胞株的获得：主要有两种方法：其一：以流式细胞仪进行，依据FACS获得。共转染的CHO-K1细胞传代两到三代后，以以下流程进行流式分选：①FACS培养基：培养基以无酚红的培养基或者DPBS代替含有酚红的培养基，培养基中必须加入青链霉素以防止污染。②铺板：在96孔板中加入100μL含有青链霉素的上述培养基，根据要收集的细胞数量铺相应的96孔板。(因为进行流式分选时许多细胞会耐受不了机械和电压压力，即使很多存活的细胞状态也会受到很大影响，故需要铺足量的96孔板以获得足够的单克隆阳性细胞。)③细胞准备：以DPBS洗细胞两次，加入适量的胰蛋白酶进行消化，(胰蛋白酶不应过量，能够轻轻铺满细胞表层即可)把握好消化时间，待细胞变圆后立刻加入预温的FACS培养基，轻轻吹打细胞形成单细胞悬液。④以室温200g离心5min，移取培养基，以200μL的FACS培养基重悬细胞，并用400目的细胞滤网过滤细胞，除去聚集成团的细胞，确保形成单细胞悬液，分选前将细胞置于冰上。⑤上机进行分选，过程中时刻注意无菌操作，将单克隆细胞分到②中的96孔板中。分选结束后，将96孔板放回到培养箱中。⑥将孔板置于培养箱中培养2-3周，等待细胞扩增形成细胞团。5d后往每孔中补加100μL的预温的完全培养基。可以根据细胞生长情况没3-5d换液。⑦培养一周后进行倒置显微镜镜检，标记空孔和非单细胞孔。⑧待细胞长到60％的汇合度时，在荧光倒置显微镜下镜检，标记eGFP阳性的单细胞孔。同时进行传代，直接吹打细胞使其分散，将30％的细胞转移到新的复制板中，剩余70％的细胞以QuickExtract DNAExtraction Solution提取其基因组记性junction PCR验证。其二：稀释分离法。在方法一的基础上，步骤⑤的FACS分选结束后，调整程序进行eGFP阳性富集，收集阳性最强的那部分细胞。大约收集5×103-104的细胞，并将细胞置于6孔板中进行培养。48h之后根据方法一中的方法将细胞制成单细胞悬液并以血球计数板对细胞进行计数，然后根据细胞计数结果取适量细胞将其稀释成0.5cell/100μL，最后将细胞悬液加入到96孔板中并放回培养箱培养。一周后，镜检单克隆孔，补加新鲜培养基。按照方法一中所述进行镜检eGFP阳性克隆株、传代及鉴定等后续实验。
获得表达表达eGFP的阳性单克隆细胞株后，将对单克隆细胞株整合情况鉴定。主要采取Junction PCR以及TA克隆和测序等方式验证。
实施例2.过表达HsQSOX1b和Survivin的单克隆稳定细胞株的建立
试剂与试剂盒：PMSF和BCAProtein Assay kit购自上海生工。HRP-conjogatedgoat anti-mouse IgG(H+L)、HRP-conjogated goat anti rabbit、Anti-βactin antibody和Anti-QSOX1antibody购置于Proteintech。Pro-light HRPchemiluminescent kit购置于北京天根。ER-Tracker Red和Hoechst 33342dye购自Thermo Fisher Scientific。其他的生化试剂属于国产常规的分析纯试剂。
细胞、菌株和质粒：重组质粒sgRNA1-QSOX1-pX458(Q1)、sgRNA2-QSOX1-pX458(Q2)、sgRNA1-BIRC5-pX458(B1)和sgRNA2-BIRC5-pX458(B2)为本发明上述阶段所构建。E.coli DH5α(Invirogen，USA)作为质粒扩增菌株；细胞株CHO-K1细胞(中科院细胞库)。
试剂配制：
5×蛋白电泳缓冲液(Tris-甘氨酸)：称取Tris碱15.1g，甘氨酸94g，SDS 5g，加双蒸水定容至1L。
15％蛋白电泳分离胶：量取ddH2O 2.3ml，30％丙烯酰胺5.0ml，Tris-HCI(pH6.8)2.5ml，10％SDS 100μl，10％APS 100μl，TEMED 100μl，混合均匀。
5％蛋白电泳浓缩胶：量取ddH2O 3.15ml，30％丙烯酰胺0.75ml，Tris-HCI(pH6.8)0.57ml，10％SDS 45μl，10％APS 45μl，TEMED 4.5μl，混合均匀。
考马斯亮蓝染色液：称取考马斯亮蓝(R-250)2g，量取乙醇200ml，冰醋酸100ml，去离子水750ml，滤纸过滤，室温保存。
细胞蛋白裂解液：量取1mL NP-40裂解液和100μL 0.1M PMSF，混合上述两种试剂，4℃保存，现配现用。
转移缓冲液：2.9g甘氨酸、5.8g Tris、0.37g SDS、200mL甲醇，以蒸馏水将前三种试剂溶解，再加入甲醇，最后定容到1L，混匀室温保存，可重复使用2-3次。
TBS缓冲液：10mL pH7.5的1M Tris·HCl中加入8.8g NaCl，以蒸馏水溶解并定容至1L，混匀室温保存。
TBST缓冲液：250μL 20％Tween20加入500mL TBS中，将两种成分混匀，现用现配。
封闭液：5g non-fat milk加入100mL TBST中，将两种成分混匀，现用现配。
5×ER-Tracker Red母液：5uL 1mM ER-Tracker Red加入到1mLHBSS/Ca/Mg中，避光保存，现配现用。
Hoechst 33342工作液：将2μL Hoechst 33342母液与4mL的DPBS中混匀，避光保存。
4％的多聚甲醛：100mL pH7.4的0.1M DPBS中加入4g多聚甲醛，混匀两种试剂，60℃以下磁力搅拌器加热搅拌直至溶解。
Western Blot检测阳性单克隆细胞株中HsQSOX1b和Survivin表达情况：①将上述获得的eGFP阳性细胞进行扩大培养，接种5×105的细胞到60mm的培养皿中，加入4mL的培养基并置于培养箱中进行培养。24h后，以DPBS洗细胞两遍，尽量去除多余的DPBS，然后加入适量的含有PMSF的NP-40裂解液，用移液枪吸取裂解液吹打细胞，使裂解液能够接触到细胞，然后将裂解液同裂解的细胞样品全部转移到离心管中，以4℃10000g离心10min，最后将上清转移到新的PCR管置于冰上备用，整个裂解过程均需要在冰上和4℃下进行；②取适量裂解液上清，以BCA蛋白浓度测定试剂盒测定总蛋白浓度，最后取50μg总蛋白进行上样，进行SDS-PAGE电泳；③将凝胶置于转膜缓冲液中，根据预染marker切取目的蛋白区域的凝胶，量取凝胶大小，剪相对应的PVDF膜，并以无水甲醇激活膜5min，然后将膜浸泡在转膜缓冲液中10min；④按照从阴极到阳极的顺序，依次放入经转膜缓冲液浸泡过的海绵-三层滤纸-凝胶-PVDF膜-三层滤纸-海绵，(切记凝胶必须置于阴极)扣好阳极板，置于转膜装置中，75V转膜1.5-2h；⑤将膜以封闭液室温封闭1h，然后以TBST洗膜3×5min；⑥以封闭液稀释一抗，然后与PVDF膜在4℃孵育过夜，再以TBST洗膜3×5min；⑦以封闭液稀释二抗，然后室温与膜孵育1h，再以TBST洗膜3×5min；⑧以TBST洗膜3×10min，然后进行ECL化学发光成像。
活细胞成像：①将2000左右的eGFP阳性细胞均匀铺在20mm的培养皿中，将培养皿置于培养箱中培养24h。②以DPBS洗细胞两次，加入预热的ER-Tracker Red工作液(工作液浓度在1μM左右)，37℃，避光染色15-30min。③缓慢吸去染料，以PBS清洗两次，每次5min，然后加入Hoechst 33342工作液37℃避光染色10-20min。④吸去染料，以DPBS洗两遍，然后加入新鲜的培养基或者DPBS，在荧光倒置显微镜下镜检，拍照记录。(注意：整个操作过程需要避光，防止荧光信号淬灭。)
细胞爬片、固定以及共聚焦激光扫描电镜成像：①细胞的爬片：接种1000个左右的细胞于20mm玻底的共聚焦培养皿中，并将培养皿置于培养箱中培养48h左右。②细胞固定：沿着边缘吸去培养基，缓慢加入DPBS清洗细胞两次，每次5min，加入4％的多聚甲醛室温固定细胞20min。③以DPBS清洗两遍，加入预热的ER-Tracker Red工作液37℃，避光染色15min。④以DPBS清洗细胞两次，加入预热的Hoechst 33342工作液37℃，避光染色10min。⑤以DPBS清洗细胞两次，最后加入新鲜的DPBS，避光保存进行激光共聚焦扫描电镜的拍摄。(注：整个细胞染色过程需要避光进行，且拍照时需要也要在暗处进行，防止荧光信号淬灭。)
实施例3.过表达HsQSOX1b和Survivin的单克隆株的胞内蛋白质量控制初探
试剂与试剂盒：PrimeScriptTM RT Master Mix、Premix Ex Taq和限制内切酶(Hind III、BamH I)购置于TaKaRa。Annexin V-PE apoptosis detection kit和GSHand GSSG Assay Kit购置于Beyotime。Gaussia Luciferase Assay Kit购置于NEB。PI Staining Solution购置于Yeasen。其他的生化试剂属于国产常规的分析纯试剂。
细胞和质粒：细胞：稳定表达HsQSOX1b-KDEL的单克隆株EC#1-C1和稳定表达HsQSOX1b-KDEL和Survivin的单克隆株EC#2-B6；质粒：pGluc-Basic，和pCDNA3.1。
试剂配制：
5mg/mL的MTT母液配制：准确称取250mg MTT溶于50mL的PBS中，然后以0.22μm的无菌滤膜过滤除菌，分装后，-20℃避光保存。
胞内GSH/GSSG水平测定：GSH和GSSG的检测以试剂盒进行，其原理大致如下：谷胱甘肽还原酶能够将GSSG还原成GSH，GSH能够与生色底物DTNB反应生成黄色底物TNB和GSSG，合并两个反应，tGSH(tGSH＝GSH+GSSG)的反应相当于一个颜色产生的限速因素，tGSH的量决定了TNB生成的量，故可以通过检测A412nm的吸收值计算tGSH的量。而GSSG的测定是通过GSH清除试剂将GSH清除，然后以上述原理计算GSSG含量，最后以tGSH减去GSSG的量得到GSH的含量。实验的具体操作按照试剂盒进行。
XBP1splicing assay
1)细胞总RNA的提取以Trizol法提取总RNA：下面以六孔板为例，方法简述如下：①将六孔板中细胞以Rnase-Free水洗两遍，直接加入500μL的Trizol试剂，以移液枪吹打几次，室温静置5min，使核酸蛋白完全水解。②(可选)若样品中含有较多的蛋白、脂质、糖类，可在2-8℃，1000g离心10min，取上清进行下一步实验(沉淀中为外膜，多糖，高分子量的DNA，上清中为RNA)。③加入1/5体积的氯仿，剧烈震荡15s，冰上孵育10min，4℃，12000rpm离心15min。④取上层水相(500μL左右)于另一个1.5mL的EP管中，加入等体积的异丙醇，摇匀，冰上孵育5min，4℃，12000rpm离心15min。⑤弃上清，干燥，加入75％的DEPC水配置的乙醇1mL，颠倒洗涤，4℃，12000rpm离心15min。⑥弃上清，干燥5-10min，加入20μL的DEPC水完全溶解RNA。⑦测定RNA浓度(A260/A280在1.8-2.0之间)，最后以DEPC水稀释到500ng/μL。
2)反转录以PrimeScrip RT Master Mix试剂盒进行：反转录以10μL体系进行，取500ng的总RNA作为模板，再加上2μL的5×PrimeScript RT Master Mix，最后以RNase FreeddH2O补齐到10μL。加样过程需要在冰上操作。样品加好后，以一下程序进行反应：37℃，15min，85℃处理5s，最后置于冰上准备进行荧光定量PCR。
3)荧光定量PCR：qPCR以Premix Ex Taq试剂盒进行。按照表1.7所列加入个组分，加样过程需要在冰上进行。qPCR中所用的引物见表1.8所示。
表1.7 qPCR反应体系
表1.8 qPCR进行XBP1s剪接试验所用引物列表
加样结束后以Bio-Rad CFX96qPCR仪进行，反应程序简述为：预变性：95℃，30s；PCR反应：95℃，5s；60℃，30s；40个循环；获得溶解曲线。PCR结束后，对数据进行分析，基因表达水平以2-ΔΔCt计算获得。公式如下：表达水平＝2-ΔΔCt，其中ΔΔCt＝[(Ct gene intest cell lines-Ct gene in control cell line)-(Ctβ-actin in test cell lines-Ctβ-actin in control cell line)]。
流式细胞仪检测细胞抗凋亡能力：①制备2×105/mL的细胞悬液，并铺在6孔板，每孔2mL，置于培养箱中培养24h。②然后往每个孔中加入终浓度为0.2nM的Staurosporine。③继续培养细胞18h，收集培养基中细胞，并以0.25％的胰蛋白酶消化孔板中的细胞，再以预冷的DPBS洗细胞两次，细胞收集在4℃下以500g离心5min。④弃去上清，以180μL 1×binding buffer重悬细胞，然后加入5μL Annexin V-PE，缓慢混匀，在室温暗处孵育15min，然后加入10μL PI Staining Solution并在暗处室温孵育5min。⑤最后，加入300μL1×binding buffer并及时进行流式细胞仪分析。
MTT法检测细胞活性：①分别制备2×105/mL的单克隆细胞和对照组CHO-K1的细胞悬液，然后铺96孔板，每孔100μL，六个复孔。②将细胞置于培养箱连续培养96h。③将孔板从培养箱中取出，加入20μL终浓度5mg/ml的MTT溶液，继续置于培养箱中培养4h。⑤4h后，将细胞取出，缓慢沿着孔壁将培养基吸去，然后加入100μL的DMSO，并在室温摇床上轻轻避光摇晃10min。⑥最后，在酶标仪上测定490nm处的吸光值。然后根据测得结果计算细胞存活率：细胞存活率(％)＝(实验组/对照组)×100％。
GLuc试验
1)载体和启动子的选择：载体构建和测序引物列于表1.9中。
表1.9 pGLuc-CMV载体构建所用引物和测序序列
2)细胞转染：从宿主菌中提取步骤1)中构建并测序正确的pGLuc-CMV载体，并以微量核酸分析仪测定浓度。细胞转染步骤同上述细胞转染中所述。不同之处如下：铺96孔板的细胞密度为1.5×105cells/mL，每孔100μL；转染试剂为0.2μL/孔；载体量为100ng/孔。将细胞置于培养箱中继续培养24h。实验组和对照组均做三组平行试验。
3)荧光素酶活测定：荧光素酶活性的测定以Gaussia LuciferaseAssay试剂盒进行，实验流程大致如下：①GLuc实验工作溶液：以100个样计算，加入50μL的底物，800μL稳定剂和5mL的GLuc试验测定缓冲液。②轻轻混匀上述试剂(不能震荡)上下颠倒即可。于暗处室温孵育25min。③开启荧光酶标仪，等待仪器冷却，设置程序，将积分时间调成5s。④在不透明的96孔板中加入5-20μL的样品，然后加入孵育好的工作液，每孔50μL，并轻轻混匀，防止气泡产生。⑤室温孵育35s，在生物发光酶标仪的475nm出检测生物发光强度。按照实验进行单点测定法或者动力学测定法。(注：仪器的生物发光线性范围以梯度稀释的转染pGLuc-CMV的96h后的EC#2-B6单克隆细胞培养基上清进行。)
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
SEQUENCE LISTING
<110> 华东理工大学
<120> 基于CRISPR/Cas9基因编辑改造的CHO细胞株及其制备方法
<130> /
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 606
<212> PRT
<213> Homo sapiens
<400> 1
Ala Thr Met Arg Arg Cys Asn Ser Gly Ser Gly Pro Pro Pro Ser Leu
1 5 10 15
Leu Leu Leu Leu Leu Trp Leu Leu Ala Val Pro Gly Ala Asn Ala Ala
20 25 30
Pro Arg Ser Ala Leu Tyr Ser Pro Ser Asp Pro Leu Thr Leu Leu Gln
35 40 45
Ala Asp Thr Val Arg Gly Ala Val Leu Gly Ser Arg Ser Ala Trp Ala
50 55 60
Val Glu Phe Phe Ala Ser Trp Cys Gly His Cys Ile Ala Phe Ala Pro
65 70 75 80
Thr Trp Lys Ala Leu Ala Glu Asp Val Lys Ala Trp Arg Pro Ala Leu
85 90 95
Tyr Leu Ala Ala Leu Asp Cys Ala Glu Glu Thr Asn Ser Ala Val Cys
100 105 110
Arg Asp Phe Asn Ile Pro Gly Phe Pro Thr Val Arg Phe Phe Lys Ala
115 120 125
Phe Thr Lys Asn Gly Ser Gly Ala Val Phe Pro Val Ala Gly Ala Asp
130 135 140
Val Gln Thr Leu Arg Glu Arg Leu Ile Asp Ala Leu Glu Ser His His
145 150 155 160
Asp Thr Trp Pro Pro Ala Cys Pro Pro Leu Glu Pro Ala Lys Leu Glu
165 170 175
Glu Ile Asp Gly Phe Phe Ala Arg Asn Asn Glu Glu Tyr Leu Ala Leu
180 185 190
Ile Phe Glu Lys Gly Gly Ser Tyr Leu Ala Arg Glu Val Ala Leu Asp
195 200 205
Leu Ser Gln His Lys Gly Val Ala Val Arg Arg Val Leu Asn Thr Glu
210 215 220
Ala Asn Val Val Arg Lys Phe Gly Val Thr Asp Phe Pro Ser Cys Tyr
225 230 235 240
Leu Leu Phe Arg Asn Gly Ser Val Ser Arg Val Pro Val Leu Met Glu
245 250 255
Ser Arg Ser Phe Tyr Thr Ala Tyr Leu Gln Arg Leu Ser Gly Leu Thr
260 265 270
Arg Glu Ala Ala Gln Thr Thr Val Ala Pro Thr Thr Ala Asn Lys Ile
275 280 285
Ala Pro Thr Val Trp Lys Leu Ala Asp Arg Ser Lys Ile Tyr Met Ala
290 295 300
Asp Leu Glu Ser Ala Leu His Tyr Ile Leu Arg Ile Glu Val Gly Arg
305 310 315 320
Phe Pro Val Leu Glu Gly Gln Arg Leu Val Ala Leu Lys Lys Phe Val
325 330 335
Ala Val Leu Ala Lys Tyr Phe Pro Gly Arg Pro Leu Val Gln Asn Phe
340 345 350
Leu His Ser Val Asn Glu Trp Leu Lys Arg Gln Lys Arg Asn Lys Ile
355 360 365
Pro Tyr Ser Phe Phe Lys Thr Ala Leu Asp Asp Arg Lys Glu Gly Ala
370 375 380
Val Leu Ala Lys Lys Val Asn Trp Ile Gly Cys Gln Gly Ser Glu Pro
385 390 395 400
His Phe Arg Gly Phe Pro Cys Ser Leu Trp Val Leu Phe His Phe Leu
405 410 415
Thr Val Gln Ala Ala Arg Gln Asn Val Asp His Ser Gln Glu Ala Ala
420 425 430
Lys Ala Lys Glu Val Leu Pro Ala Ile Arg Gly Tyr Val His Tyr Phe
435 440 445
Phe Gly Cys Arg Asp Cys Ala Ser His Phe Glu Gln Met Ala Ala Ala
450 455 460
Ser Met His Arg Val Gly Ser Pro Asn Ala Ala Val Leu Trp Leu Trp
465 470 475 480
Ser Ser His Asn Arg Val Asn Ala Arg Leu Ala Gly Ala Pro Ser Glu
485 490 495
Asp Pro Gln Phe Pro Lys Val Gln Trp Pro Pro Arg Glu Leu Cys Ser
500 505 510
Ala Cys His Asn Glu Arg Leu Asp Val Pro Val Trp Asp Val Glu Ala
515 520 525
Thr Leu Asn Phe Leu Lys Ala His Phe Ser Pro Ser Asn Ile Ile Leu
530 535 540
Asp Phe Pro Ala Ala Gly Ser Ala Ala Arg Arg Asp Val Gln Asn Val
545 550 555 560
Ala Ala Ala Pro Glu Leu Ala Met Gly Ala Leu Glu Leu Glu Ser Arg
565 570 575
Asn Ser Thr Leu Asp Pro Gly Lys Pro Glu Met Met Lys Ser Pro Thr
580 585 590
Asn Thr Thr Pro His Val Pro Ala Glu Gly Pro Glu Leu Ile
595 600 605
<210> 2
<211> 142
<212> PRT
<213> Homo sapiens
<400> 2
Met Gly Ala Pro Thr Leu Pro Pro Ala Trp Gln Pro Phe Leu Lys Asp
1 5 10 15
His Arg Ile Ser Thr Phe Lys Asn Trp Pro Phe Leu Glu Gly Cys Ala
20 25 30
Cys Thr Pro Glu Arg Met Ala Glu Ala Gly Phe Ile His Cys Pro Thr
35 40 45
Glu Asn Glu Pro Asp Leu Ala Gln Cys Phe Phe Cys Phe Lys Glu Leu
50 55 60
Glu Gly Trp Glu Pro Asp Asp Asp Pro Ile Glu Glu His Lys Lys His
65 70 75 80
Ser Ser Gly Cys Ala Phe Leu Ser Val Lys Lys Gln Phe Glu Glu Leu
85 90 95
Thr Leu Gly Glu Phe Leu Lys Leu Asp Arg Glu Arg Ala Lys Asn Lys
100 105 110
Ile Ala Lys Glu Thr Asn Asn Lys Lys Lys Glu Phe Glu Glu Thr Ala
115 120 125
Lys Lys Val Arg Arg Ala Ile Glu Gln Leu Ala Ala Met Asp
130 135 140
Claims (3)
1.一种基于CRISPR/Cas9基因编辑技术改造CHO细胞的制备方法，其特征在于，选取二硫键折叠相关的巯基氧化酶HsQSOX1b和细胞凋亡相关的生存素Survivin作为目的基因，设计成外源表达盒EC#1和EC#2，利用新型的CRISPR/Cas9的基因编辑技术，将其整合到CHO细胞的特定基因位点。
2.根据权利要求1所述的一种基于CRISPR/Cas9基因编辑技术改造CHO细胞的制备方法，其特征在于，表达盒EC#1和EC#2的串联组合方式分别为CMV(E+P)+eGFP+HsQSOX1b-KDEL+BGH pA和CMV(E+P)+eGFP+HsQSOX1b-KDEL+T2A+Survivin+BGH pA。
3.利用权利要求1或2所述基于CRISPR/Cas9基因编辑技术改造CHO细胞的制备方法制备获得的单克隆细胞株，其特征在于，所述单克隆细胞株HsQSOX1b和Survivin过表达。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810395069.7A CN110408596A (zh) | 2018-04-27 | 2018-04-27 | 基于CRISPR/Cas9基因编辑改造的CHO细胞株及其制备方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810395069.7A CN110408596A (zh) | 2018-04-27 | 2018-04-27 | 基于CRISPR/Cas9基因编辑改造的CHO细胞株及其制备方法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110408596A true CN110408596A (zh) | 2019-11-05 |
Family
ID=68346984
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810395069.7A Pending CN110408596A (zh) | 2018-04-27 | 2018-04-27 | 基于CRISPR/Cas9基因编辑改造的CHO细胞株及其制备方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110408596A (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113025578A (zh) * | 2021-03-26 | 2021-06-25 | 江苏中慧元通生物科技有限公司 | 一种抗凋亡单克隆细胞株及其制备方法 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102643847A (zh) * | 2011-02-17 | 2012-08-22 | 华东理工大学 | 多基因共表达体系及含二硫键功能性蛋白的生产方法 |
-
2018
- 2018-04-27 CN CN201810395069.7A patent/CN110408596A/zh active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102643847A (zh) * | 2011-02-17 | 2012-08-22 | 华东理工大学 | 多基因共表达体系及含二硫键功能性蛋白的生产方法 |
Non-Patent Citations (3)
| Title |
|---|
| WANG W.P. 等: "Enhanced biosynthesis performance of heterologous proteins in CHO-K1 cells using CRISPR-Cas9", ACS SYNTH.BIOL., vol. 7, pages 1259 - 1268, XP055787662, DOI: 10.1021/acssynbio.7b00375 * |
| ZHAO X.H.等: "Overexpression of survivin and cyclin D1 in CHO cells confers apoptosis resistance and enhances growth in serum-free suspension culture", BIOTECHNOL LETT, vol. 33, pages 1293 - 1300, XP019916057, DOI: 10.1007/s10529-011-0577-9 * |
| 王文鹏 等: "基于CRISPR-Cas9的动物细胞（CHO-K1）外源蛋白生产质量调控研究", 中国生物工程学会第十一届学术年会暨2017年全国生物技术大会论文集, pages 1 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113025578A (zh) * | 2021-03-26 | 2021-06-25 | 江苏中慧元通生物科技有限公司 | 一种抗凋亡单克隆细胞株及其制备方法 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108504657B (zh) | 利用crispr-cas9技术敲除hek293t细胞kdm2a基因的方法 | |
| CN106868008A (zh) | CRISPR/Cas9靶向敲除人Lin28A基因及其特异性gRNA | |
| CN104592364B (zh) | 定点突变和定点修饰的腺相关病毒、其制备方法及应用 | |
| CN107922944A (zh) | 工程改造的crispr‑cas9组合物和使用方法 | |
| Nakanishi et al. | Association with capsid proteins promotes nuclear targeting of simian virus 40 DNA. | |
| Xue et al. | Genetic modification in human pluripotent stem cells by homologous recombination and CRISPR/Cas9 system | |
| CN109415704A (zh) | 对先已存在的人中和抗体有抗性的新型重组腺相关病毒衣壳 | |
| CN108603174A (zh) | 在适用于临床应用的无血清悬浮细胞培养体系中产生重组腺相关病毒(aav)载体的可扩展方法 | |
| Sasvari et al. | Co-opted cellular Sac1 lipid phosphatase and PI (4) P phosphoinositide are key host factors during the biogenesis of the tombusvirus replication compartment | |
| CN106520772A (zh) | 用于人源肝细胞中Nrf2基因定向敲除的sgRNA、载体对及应用 | |
| Edskes et al. | Mak21p of Saccharomyces cerevisiae, a homolog of human CAATT-binding protein, is essential for 60 S ribosomal subunit biogenesis | |
| CN110408596A (zh) | 基于CRISPR/Cas9基因编辑改造的CHO细胞株及其制备方法 | |
| CN110511903A (zh) | 一种miR-21-5p靶向Smad7调控猪卵巢颗粒细胞的分子机制研究方法及其应用 | |
| CN113969283A (zh) | 一种cho细胞基因nw_003613756.1内稳定表达蛋白质的位点及其应用 | |
| CN106399370B (zh) | 一种建立稳定转基因的牙鲆胚胎细胞株方法 | |
| CN106497973B (zh) | 一种人类和其他哺乳动物细胞附着体表达载体、表达系统、制备方法和应用 | |
| CN109750058A (zh) | 一种白色念珠菌的研究方法 | |
| CN107446949A (zh) | Pls3重组蛋白真核表达质粒及其构建方法和应用 | |
| Nickles et al. | Comprehensive guide to extracting and expressing fungal secondary metabolites with Aspergillus fumigatus as a case study | |
| CN110331186A (zh) | 一种5’-核苷酸酶质控品 | |
| CN102533722B (zh) | 一种通过温度突变提高哺乳动物细胞重组蛋白瞬时表达的方法 | |
| CN109913452A (zh) | 用于靶向HBB RNA的gRNA及基于C2c2的HBB突变检测方法、检测试剂盒 | |
| CN108823205A (zh) | 一种敲除plac8基因的hek293t细胞系构建方法 | |
| CN112941020B (zh) | 一种鸡环状rna在促进成肌细胞的增殖的应用 | |
| Long et al. | RNAe in a transgenic growth hormone mouse model shows potential for use in gene therapy |
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 | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20191105 |