CN112481298A - 重组质粒及其应用 - Google Patents
重组质粒及其应用 Download PDFInfo
- Publication number
- CN112481298A CN112481298A CN202011254579.6A CN202011254579A CN112481298A CN 112481298 A CN112481298 A CN 112481298A CN 202011254579 A CN202011254579 A CN 202011254579A CN 112481298 A CN112481298 A CN 112481298A
- Authority
- CN
- China
- Prior art keywords
- protein
- recombinant plasmid
- bad
- plasmid
- ser
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000013612 plasmid Substances 0.000 title claims abstract description 45
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 31
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 30
- 101000936623 Homo sapiens Bcl2-associated agonist of cell death Proteins 0.000 claims abstract description 21
- 102000058044 human BAD Human genes 0.000 claims abstract description 21
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 17
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 17
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 7
- 150000007523 nucleic acids Chemical group 0.000 claims abstract description 7
- 239000013600 plasmid vector Substances 0.000 claims abstract description 5
- 230000035772 mutation Effects 0.000 claims description 15
- 206010028980 Neoplasm Diseases 0.000 claims description 14
- 102220495809 Alkaline ceramidase 3_S99A_mutation Human genes 0.000 claims description 10
- 239000013604 expression vector Substances 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 5
- 239000002773 nucleotide Substances 0.000 claims description 5
- 125000003729 nucleotide group Chemical group 0.000 claims description 5
- 201000011510 cancer Diseases 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 238000010171 animal model Methods 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 3
- 230000002265 prevention Effects 0.000 claims description 3
- 102220468077 Ribonucleases P/MRP protein subunit POP1_S118A_mutation Human genes 0.000 claims description 2
- 102220474385 Solute carrier family 13 member 3_S75A_mutation Human genes 0.000 claims description 2
- 238000012216 screening Methods 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 102000007348 bcl-Associated Death Protein Human genes 0.000 abstract description 30
- 108010007734 bcl-Associated Death Protein Proteins 0.000 abstract description 30
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 64
- 235000018102 proteins Nutrition 0.000 description 25
- 238000002474 experimental method Methods 0.000 description 14
- 210000004881 tumor cell Anatomy 0.000 description 14
- 230000004083 survival effect Effects 0.000 description 12
- 230000006907 apoptotic process Effects 0.000 description 11
- 230000000694 effects Effects 0.000 description 10
- 102000047934 Caspase-3/7 Human genes 0.000 description 9
- 108700037887 Caspase-3/7 Proteins 0.000 description 9
- 230000026731 phosphorylation Effects 0.000 description 9
- 238000006366 phosphorylation reaction Methods 0.000 description 9
- 108020004414 DNA Proteins 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 150000001413 amino acids Chemical class 0.000 description 7
- 102100021569 Apoptosis regulator Bcl-2 Human genes 0.000 description 5
- 101000971171 Homo sapiens Apoptosis regulator Bcl-2 Proteins 0.000 description 5
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 5
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 5
- 201000005202 lung cancer Diseases 0.000 description 5
- 208000020816 lung neoplasm Diseases 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- XQJCEKXQUJQNNK-ZLUOBGJFSA-N Ser-Ser-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O XQJCEKXQUJQNNK-ZLUOBGJFSA-N 0.000 description 4
- 108010047495 alanylglycine Proteins 0.000 description 4
- 230000034994 death Effects 0.000 description 4
- 230000003247 decreasing effect Effects 0.000 description 4
- 230000009462 endogenous apoptosis Effects 0.000 description 4
- 239000013613 expression plasmid Substances 0.000 description 4
- 210000003470 mitochondria Anatomy 0.000 description 4
- 108010031719 prolyl-serine Proteins 0.000 description 4
- 230000001737 promoting effect Effects 0.000 description 4
- 108010048818 seryl-histidine Proteins 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 108010085238 Actins Proteins 0.000 description 3
- 102000007469 Actins Human genes 0.000 description 3
- 206010014733 Endometrial cancer Diseases 0.000 description 3
- 206010014759 Endometrial neoplasm Diseases 0.000 description 3
- 239000000020 Nitrocellulose Substances 0.000 description 3
- 206010033128 Ovarian cancer Diseases 0.000 description 3
- 206010061535 Ovarian neoplasm Diseases 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 238000003782 apoptosis assay Methods 0.000 description 3
- 230000003833 cell viability Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 238000009650 gentamicin protection assay Methods 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 239000010410 layer Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 229920001220 nitrocellulos Polymers 0.000 description 3
- 230000000861 pro-apoptotic effect Effects 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- 102000004899 14-3-3 Proteins Human genes 0.000 description 2
- 101710112812 14-3-3 protein Proteins 0.000 description 2
- NWVVKQZOVSTDBQ-CIUDSAMLSA-N Ala-Glu-Arg Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O NWVVKQZOVSTDBQ-CIUDSAMLSA-N 0.000 description 2
- 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 2
- VENMDXUVHSKEIN-GUBZILKMSA-N Arg-Ser-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O VENMDXUVHSKEIN-GUBZILKMSA-N 0.000 description 2
- NJIKKGUVGUBICV-ZLUOBGJFSA-N Asp-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC(O)=O NJIKKGUVGUBICV-ZLUOBGJFSA-N 0.000 description 2
- QHAJMRDEWNAIBQ-FXQIFTODSA-N Asp-Arg-Asn Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(O)=O QHAJMRDEWNAIBQ-FXQIFTODSA-N 0.000 description 2
- WCFCYFDBMNFSPA-ACZMJKKPSA-N Asp-Asp-Glu Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCC(O)=O WCFCYFDBMNFSPA-ACZMJKKPSA-N 0.000 description 2
- POTCZYQVVNXUIG-BQBZGAKWSA-N Asp-Gly-Pro Chemical compound OC(=O)C[C@H](N)C(=O)NCC(=O)N1CCC[C@H]1C(O)=O POTCZYQVVNXUIG-BQBZGAKWSA-N 0.000 description 2
- LBOVBQONZJRWPV-YUMQZZPRSA-N Asp-Lys-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(O)=O LBOVBQONZJRWPV-YUMQZZPRSA-N 0.000 description 2
- CGVWDTRDPLOMHZ-FXQIFTODSA-N Gln-Glu-Asp Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O CGVWDTRDPLOMHZ-FXQIFTODSA-N 0.000 description 2
- KKCJHBXMYYVWMX-KQXIARHKSA-N Gln-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)N)N KKCJHBXMYYVWMX-KQXIARHKSA-N 0.000 description 2
- LUGUNEGJNDEBLU-DCAQKATOSA-N Gln-Met-Arg Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](CCC(=O)N)N LUGUNEGJNDEBLU-DCAQKATOSA-N 0.000 description 2
- UWMDGPFFTKDUIY-HJGDQZAQSA-N Gln-Pro-Thr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O UWMDGPFFTKDUIY-HJGDQZAQSA-N 0.000 description 2
- JILRMFFFCHUUTJ-ACZMJKKPSA-N Gln-Ser-Ser Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O JILRMFFFCHUUTJ-ACZMJKKPSA-N 0.000 description 2
- JDUKCSSHWNIQQZ-IHRRRGAJSA-N Glu-Phe-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(O)=O)C(O)=O JDUKCSSHWNIQQZ-IHRRRGAJSA-N 0.000 description 2
- BIYNPVYAZOUVFQ-CIUDSAMLSA-N Glu-Pro-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O BIYNPVYAZOUVFQ-CIUDSAMLSA-N 0.000 description 2
- GWCRIHNSVMOBEQ-BQBZGAKWSA-N Gly-Arg-Ser Chemical compound [H]NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O GWCRIHNSVMOBEQ-BQBZGAKWSA-N 0.000 description 2
- IUKIDFVOUHZRAK-QWRGUYRKSA-N Gly-Lys-His Chemical compound NCCCC[C@H](NC(=O)CN)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 IUKIDFVOUHZRAK-QWRGUYRKSA-N 0.000 description 2
- WCORRBXVISTKQL-WHFBIAKZSA-N Gly-Ser-Ser Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O WCORRBXVISTKQL-WHFBIAKZSA-N 0.000 description 2
- DBUNZBWUWCIELX-JHEQGTHGSA-N Gly-Thr-Glu Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O DBUNZBWUWCIELX-JHEQGTHGSA-N 0.000 description 2
- HDXNWVLQSQFJOX-SRVKXCTJSA-N His-Arg-Gln Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N HDXNWVLQSQFJOX-SRVKXCTJSA-N 0.000 description 2
- ZNNNYCXPCKACHX-DCAQKATOSA-N His-Gln-Gln Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O ZNNNYCXPCKACHX-DCAQKATOSA-N 0.000 description 2
- JIUYRPFQJJRSJB-QWRGUYRKSA-N His-His-Gly Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)NCC(O)=O)C1=CN=CN1 JIUYRPFQJJRSJB-QWRGUYRKSA-N 0.000 description 2
- VIJMRAIWYWRXSR-CIUDSAMLSA-N His-Ser-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CN=CN1 VIJMRAIWYWRXSR-CIUDSAMLSA-N 0.000 description 2
- PMGDADKJMCOXHX-UHFFFAOYSA-N L-Arginyl-L-glutamin-acetat Natural products NC(=N)NCCCC(N)C(=O)NC(CCC(N)=O)C(O)=O PMGDADKJMCOXHX-UHFFFAOYSA-N 0.000 description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 2
- UGTHTQWIQKEDEH-BQBZGAKWSA-N L-alanyl-L-prolylglycine zwitterion Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O UGTHTQWIQKEDEH-BQBZGAKWSA-N 0.000 description 2
- BABSVXFGKFLIGW-UWVGGRQHSA-N Leu-Gly-Arg Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCNC(N)=N BABSVXFGKFLIGW-UWVGGRQHSA-N 0.000 description 2
- YFBBUHJJUXXZOF-UWVGGRQHSA-N Leu-Gly-Pro Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N1CCC[C@H]1C(O)=O YFBBUHJJUXXZOF-UWVGGRQHSA-N 0.000 description 2
- FOBUGKUBUJOWAD-IHPCNDPISA-N Leu-Leu-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(C)C)C(O)=O)=CNC2=C1 FOBUGKUBUJOWAD-IHPCNDPISA-N 0.000 description 2
- GQFDWEDHOQRNLC-QWRGUYRKSA-N Lys-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN GQFDWEDHOQRNLC-QWRGUYRKSA-N 0.000 description 2
- PNDCUTDWYVKBHX-IHRRRGAJSA-N Met-Asp-Tyr Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 PNDCUTDWYVKBHX-IHRRRGAJSA-N 0.000 description 2
- YCUSPBPZVJDMII-YUMQZZPRSA-N Met-Gly-Glu Chemical compound CSCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(O)=O YCUSPBPZVJDMII-YUMQZZPRSA-N 0.000 description 2
- XMBSYZWANAQXEV-UHFFFAOYSA-N N-alpha-L-glutamyl-L-phenylalanine Natural products OC(=O)CCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XMBSYZWANAQXEV-UHFFFAOYSA-N 0.000 description 2
- 108010079364 N-glycylalanine Proteins 0.000 description 2
- JSGWNFKWZNPDAV-YDHLFZDLSA-N Phe-Val-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 JSGWNFKWZNPDAV-YDHLFZDLSA-N 0.000 description 2
- ICTZKEXYDDZZFP-SRVKXCTJSA-N Pro-Arg-Pro Chemical compound N([C@@H](CCCN=C(N)N)C(=O)N1[C@@H](CCC1)C(O)=O)C(=O)[C@@H]1CCCN1 ICTZKEXYDDZZFP-SRVKXCTJSA-N 0.000 description 2
- LGMBKOAPPTYKLC-JYJNAYRXSA-N Pro-Phe-Arg Chemical compound C([C@@H](C(=O)N[C@@H](CCCNC(=N)N)C(O)=O)NC(=O)[C@H]1NCCC1)C1=CC=CC=C1 LGMBKOAPPTYKLC-JYJNAYRXSA-N 0.000 description 2
- SEZGGSHLMROBFX-CIUDSAMLSA-N Pro-Ser-Gln Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O SEZGGSHLMROBFX-CIUDSAMLSA-N 0.000 description 2
- FNGOXVQBBCMFKV-CIUDSAMLSA-N Pro-Ser-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(O)=O FNGOXVQBBCMFKV-CIUDSAMLSA-N 0.000 description 2
- BTKUIVBNGBFTTP-WHFBIAKZSA-N Ser-Ala-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)NCC(O)=O BTKUIVBNGBFTTP-WHFBIAKZSA-N 0.000 description 2
- FTVRVZNYIYWJGB-ACZMJKKPSA-N Ser-Asp-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O FTVRVZNYIYWJGB-ACZMJKKPSA-N 0.000 description 2
- UIGMAMGZOJVTDN-WHFBIAKZSA-N Ser-Gly-Ser Chemical compound OC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O UIGMAMGZOJVTDN-WHFBIAKZSA-N 0.000 description 2
- HEYZPTCCEIWHRO-IHRRRGAJSA-N Ser-Met-Phe Chemical compound OC[C@H](N)C(=O)N[C@@H](CCSC)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 HEYZPTCCEIWHRO-IHRRRGAJSA-N 0.000 description 2
- XVWDJUROVRQKAE-KKUMJFAQSA-N Ser-Phe-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CO)CC1=CC=CC=C1 XVWDJUROVRQKAE-KKUMJFAQSA-N 0.000 description 2
- ADJDNJCSPNFFPI-FXQIFTODSA-N Ser-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CO ADJDNJCSPNFFPI-FXQIFTODSA-N 0.000 description 2
- NERYDXBVARJIQS-JYBASQMISA-N Ser-Trp-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](CO)N)O NERYDXBVARJIQS-JYBASQMISA-N 0.000 description 2
- TYIHBQYLIPJSIV-NYVOZVTQSA-N Ser-Trp-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC3=CNC4=CC=CC=C43)C(=O)O)NC(=O)[C@H](CO)N TYIHBQYLIPJSIV-NYVOZVTQSA-N 0.000 description 2
- CAJFZCICSVBOJK-SHGPDSBTSA-N Thr-Ala-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O CAJFZCICSVBOJK-SHGPDSBTSA-N 0.000 description 2
- CDBXVDXSLPLFMD-BPNCWPANSA-N Tyr-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CC1=CC=C(O)C=C1 CDBXVDXSLPLFMD-BPNCWPANSA-N 0.000 description 2
- VCAWFLIWYNMHQP-UKJIMTQDSA-N Val-Glu-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](C(C)C)N VCAWFLIWYNMHQP-UKJIMTQDSA-N 0.000 description 2
- UZFNHAXYMICTBU-DZKIICNBSA-N Val-Phe-Gln Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N UZFNHAXYMICTBU-DZKIICNBSA-N 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 235000004279 alanine Nutrition 0.000 description 2
- 108010069020 alanyl-prolyl-glycine Proteins 0.000 description 2
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000006909 anti-apoptosis Effects 0.000 description 2
- 108010008355 arginyl-glutamine Proteins 0.000 description 2
- 108010069926 arginyl-glycyl-serine Proteins 0.000 description 2
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 108010055341 glutamyl-glutamic acid Proteins 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
- 238000002649 immunization Methods 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 108010003700 lysyl aspartic acid Proteins 0.000 description 2
- 108010054155 lysyllysine Proteins 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-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
- KXOPYFNQLVUOAQ-FXQIFTODSA-N Arg-Ser-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O KXOPYFNQLVUOAQ-FXQIFTODSA-N 0.000 description 1
- 208000003950 B-cell lymphoma Diseases 0.000 description 1
- 108700038897 Bcl-2 family Proteins 0.000 description 1
- 102000051485 Bcl-2 family Human genes 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 206010011985 Decubitus ulcer Diseases 0.000 description 1
- 208000002699 Digestive System Neoplasms Diseases 0.000 description 1
- 208000009849 Female Genital Neoplasms Diseases 0.000 description 1
- 101001056180 Homo sapiens Induced myeloid leukemia cell differentiation protein Mcl-1 Proteins 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 102100026539 Induced myeloid leukemia cell differentiation protein Mcl-1 Human genes 0.000 description 1
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 102000043136 MAP kinase family Human genes 0.000 description 1
- 108091054455 MAP kinase family Proteins 0.000 description 1
- 102000038030 PI3Ks Human genes 0.000 description 1
- 108091007960 PI3Ks Proteins 0.000 description 1
- QGMRQYFBGABWDR-UHFFFAOYSA-M Pentobarbital sodium Chemical compound [Na+].CCCC(C)C1(CC)C(=O)NC(=O)[N-]C1=O QGMRQYFBGABWDR-UHFFFAOYSA-M 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000004520 agglutination Effects 0.000 description 1
- 108010087924 alanylproline Proteins 0.000 description 1
- 230000002424 anti-apoptotic effect Effects 0.000 description 1
- 230000005775 apoptotic pathway Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000001099 axilla Anatomy 0.000 description 1
- 108700000711 bcl-X Proteins 0.000 description 1
- 102000055104 bcl-X Human genes 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000009134 cell regulation Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000030609 dephosphorylation Effects 0.000 description 1
- 238000006209 dephosphorylation reaction Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 208000014829 head and neck neoplasm Diseases 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000003999 initiator Substances 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 230000003387 muscular Effects 0.000 description 1
- 230000012330 negative regulation of anti-apoptosis Effects 0.000 description 1
- 206010061311 nervous system neoplasm Diseases 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 229960002275 pentobarbital sodium Drugs 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 102000034285 signal transducing proteins Human genes 0.000 description 1
- 108091006024 signal transducing proteins Proteins 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000002344 surface layer Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000005748 tumor development Effects 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 230000009750 upstream signaling Effects 0.000 description 1
- 208000029584 urinary system neoplasm Diseases 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
-
- 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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0331—Animal model for proliferative diseases
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
- C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Environmental Sciences (AREA)
- Toxicology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Physics & Mathematics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Animal Husbandry (AREA)
- Animal Behavior & Ethology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Plant Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Peptides Or Proteins (AREA)
- Cell Biology (AREA)
Abstract
本发明公开了重组质粒及其应用。该重组质粒包括质粒载体和外源核酸序列,外源核酸序列编码N‑末端带有蛋白标签的人源BAD蛋白的融合蛋白。根据本发明实施例的重组质粒,至少具有如下有益效果:本发明实施例所构建的重组质粒所表达的人源BAD蛋白在其N‑末端带有蛋白标签,通过该蛋白标签,方便检测外源BAD蛋白在靶细胞中的表达,便于将外源BAD蛋白与内源BAD蛋白进行区分。
Description
技术领域
本发明涉及生物医学技术领域,尤其是涉及重组质粒及其应用。
背景技术
线粒体是细胞凋亡最主要的发起者和执行者,线粒体凋亡通路中,B细胞淋巴瘤2(B-cell lymphoma 2,Bcl-2)家族的各个亚型蛋白广泛参与凋亡信号的转导调控。Bcl-2家族蛋白的一个显著特征是具有Bcl-2同源结构域(Bcl-2 homology domain,BH)。典型的抗凋亡成员如Bcl-2、Bcl-XL、Mcl-1等含有4个短的保守的BH结构域(BH1~BH4)和一个C端疏水尾状结构,而其它一些促凋亡成员则缺少BH4结构域。在这些促凋亡成员中,有一类蛋白质如Bcl-2相关死亡启动子同源基因编码蛋白(Bcl-2 associated agonist of celldeath,BAD),仅含有BH3结构域。相关实验证实,BH3结构域是细胞凋亡的关键性结构域,BAD蛋白在细胞内源性凋亡调节中发挥着重要作用。
BAD蛋白上氨基酸的修饰,如磷酸化,会影响其促凋亡作用的发挥,例如,BAD蛋白丝氨酸位点的磷酸化,会使其功能由促进细胞凋亡转变为促进细胞生存。正常人源野生型BAD蛋白中,在上游信号蛋白激酶(主要为Akt)的作用下,BAD蛋白发生磷酸化。磷酸化后的BAD蛋白会被14-3-3蛋白识别结合并带出线粒体,从而使BAD蛋白失去了对线粒体中抗凋亡蛋白的结合抑制,导致抗凋亡蛋白的活性释放,进而增强了细胞抵抗凋亡的能力。
从肿瘤的发生发展与治疗的角度来看,BAD蛋白的表达和磷酸化能影响到多种肿瘤的形成发展及其对药物治疗的敏感性。研究发现,在绝大多数癌症中,PI3K/AKT和MAPK等重要促癌信号通路都处于高度激活的状态,而处于他们下游的BAD蛋白也处于高度磷酸化激活的状态,BAD蛋白的高度磷酸化激活抑制了肿瘤细胞的内源性凋亡,从而促进肿瘤细胞存活。BAD蛋白作为直接调控细胞内源性凋亡与存活的重要靶点,如果能够对其磷酸化非其上游激酶进行直接抑制,有可能会产生更为优越的抑瘤效果。
为了研究通过靶向抑制BAD的磷酸化来调控肿瘤细胞的内源性凋亡,从而达到肿瘤治疗目的的方案,需要合适的表达载体来表达BAD蛋白。然而,现有质粒产品在后续的蛋白印迹分析中往往难以区分内、外源BAD蛋白。
发明内容
本发明旨在至少解决现有技术中存在的技术问题之一。为此,本发明提出一种容易区分内源和外源蛋白的重组质粒及其应用。
本发明的第一方面,提供重组质粒,该重组质粒包括质粒载体和外源核酸序列,外源核酸序列编码N-末端带有蛋白标签的人源BAD蛋白的融合蛋白。
根据本发明实施例的重组质粒,至少具有如下有益效果:
本发明实施例所构建的重组质粒所表达的人源BAD蛋白在其N-末端带有蛋白标签,通过该蛋白标签,方便检测外源BAD蛋白在靶细胞中的表达,便于将外源BAD蛋白与内源BAD蛋白进行区分。
根据本发明的一些实施例,人源BAD蛋白在S75、S99、S118中至少一个位点具有突变。人源BAD蛋白的三个丝氨酸位点(S75、S99和S118)的磷酸化,会使该蛋白的功能由促进细胞凋亡转变为促进细胞生存。如果这些丝氨酸位点发生突变成其它无法被磷酸化的氨基酸(例如苏氨酸、丝氨酸、酪氨酸以外的不含羟基的氨基酸),该蛋白失去在该位点的磷酸化能力,降低其促进细胞生存功能的转变,增强细胞的内源性死亡。从肿瘤的发生发展与治疗来看,BAD蛋白的磷酸化与多种肿瘤的治疗耐受药相关,这些磷酸化位点具有作为肿瘤治疗靶点的潜力,通过点突变去磷酸化对于肿瘤的预防和治疗具有指导性意义。
根据本发明的一些实施例,人源BAD蛋白具有S75A、S99A、S118A中至少一种突变。S75、S99和S118位点由丝氨酸突变为丙氨酸,该蛋白在上述位点失去其磷酸化能力,细胞的内源性死亡得到增强。
根据本发明的一些实施例,人源BAD蛋白具有S99A突变。S99是野生型人源BAD蛋白发挥其生理功能的重要调节位点。该位点突变为丙氨酸后,BAD蛋白失去在该位点的磷酸化能力,降低了BAD蛋白被14-3-3蛋白带出线粒体的可能性,从而增强了细胞的内源性死亡,对于肿瘤治疗具有重要意义。
根据本发明的一些实施例,外源核酸序列包含如SEQ ID No.1所示的核苷酸序列,该外源核酸序列编码N-末端带有蛋白标签的野生型人源BAD蛋白的融合蛋白。
根据本发明的一些实施例,外源核酸序列包含如SEQ ID No.3所示的核苷酸序列,该外源核酸序列编码N-末端带有蛋白标签的具有S99A突变的人源BAD蛋白的融合蛋白。
根据本发明的一些实施例,融合蛋白包含如SEQ ID No.2所示的氨基酸序列,该融合蛋白为N-末端带有蛋白标签的野生型人源BAD蛋白。
根据本发明的一些实施例,融合蛋白包含如SEQ ID No.4所示的氨基酸序列,该融合蛋白为N-末端带有蛋白标签的具有S99A突变的人源BAD蛋白。
根据本发明的一些实施例,质粒载体为真核表达载体。采用真核表达载体使重组质粒能够在真核细胞中进行表达,从而更方便准确研究靶向于BAD蛋白的肿瘤治疗方案中的分子机制。
根据本发明的一些实施例,蛋白标签为Myc标签、Flag标签。
根据本发明的一些实施例,蛋白标签为Flag标签。Flag标签通常不会与人源BAD蛋白相互作用或影响其功能、性质,有利于对融合蛋白进行下游研究;融合蛋白可以直接通过Flag进行亲和层析,纯化有活性的融合蛋白,提高纯化效率;而且,Flag标签可以被抗Flag的抗体识别,方便对含有Flag标签的融合蛋白进行检测、鉴定,对内源BAD蛋白和外源BAD蛋白进行区分。
本发明的第二方面,提供宿主细胞,该宿主细胞转入由上述的重组质粒。该宿主细胞中,内源BAD蛋白和外源BAD蛋白可以通过蛋白标签进行区分。
根据本发明的一些实施例,宿主细胞可以是任选的原核细胞或真核细胞,根据宿主细胞类型的不同,可以采用本领域熟知的各类能够导入上述质粒的方法。
根据本发明的一些实施例,宿主细胞为肿瘤细胞株。在需要对肿瘤细胞进行体内、体外双重标记时,采用上述重组质粒转染得到的宿主细胞进行实验,方便对肿瘤细胞的定性和定量观察。肿瘤细胞株可以是任选的肿瘤细胞,诸如妇科肿瘤细胞、泌尿系统肿瘤细胞、神经系统肿瘤细胞、头颈部肿瘤细胞、肺癌细胞、消化系统肿瘤细胞等。而且,宿主细胞内重组质粒所编码的BAD蛋白为人源蛋白,避免其它来源的BAD蛋白的引入而造成的分子信号干扰,使肿瘤细胞株所构建的模型更加纯粹。
本发明的第三方面,提供动物模型的构建方法,该构建方法包括以下步骤:向非人动物施用上述的宿主细胞。
本发明的第四方面,提供上述的宿主细胞在制备或筛选用于治疗或预防癌症的药物中的应用。
附图说明
下面结合附图和实施例对本发明做进一步的说明,其中:
图1为真核表达载体pcDNA3.1(+)的质粒图谱;
图2为本发明实施例的真核表达质粒pcDNA3.1(+)-flag-hBAD-WT的质粒图谱;
图3为本发明实施例的真核表达质粒pcDNA3.1(+)-flag-hBAD-S99A的质粒图谱;
图4为本发明实施例的卵巢癌细胞系OVCAR2宿主细胞实验结果;
图5为本发明实施例的子宫内膜癌细胞系AN3宿主细胞实验结果;
图6为本发明实施例的肺癌细胞系PC9宿主细胞实验结果。
具体实施方式
以下将结合实施例对本发明的构思及产生的技术效果进行清楚、完整地描述,以充分地理解本发明的目的、特征和效果。显然,所描述的实施例只是本发明的一部分实施例,而不是全部实施例,基于本发明的实施例,本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例,均属于本发明保护的范围。
下面详细描述本发明的实施例,所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的,仅用于解释本发明,而不能理解为对本发明的限制。
在本发明的描述中,若干的含义是一个以上,多个的含义是两个以上,大于、小于、超过等理解为不包括本数,以上、以下、以内等理解为包括本数。如果有描述到第一、第二只是用于区分技术特征为目的,而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量或者隐含指明所指示的技术特征的先后关系。
本发明的描述中,除非另有明确的限定,设置、安装、连接等词语应做广义理解,所属技术领域技术人员可以结合技术方案的具体内容合理确定上述词语在本发明中的具体含义。
本发明的描述中,参考术语“一个实施例”、“一些实施例”、“示意性实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不一定指的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
实施例1
本实施例提供重组质粒,其制备方法如下:
(1)DNA片段合成
根据人源BAD蛋白的mRNA序列(NM_032989.3)为蓝本,在其5′端添加Flag标签的编码序列后,交由生物公司合成编码融合蛋白Flag-hBAD-WT的DNA片段。DNA片段经琼脂糖凝胶电泳鉴定正确后回收,具有如下的核苷酸序列:
ATGGATTACAAGGACGACGATGACAAGGGCAGCATGTTCCAGATCCCAGAGTTTGAGCCGAGTGAGCAGGAAGACTCCAGCTCTGCAGAGAGGGGCCTGGGCCCCAGCCCCGCAGGGGACGGGCCCTCAGGCTCCGGCAAGCATCATCGCCAGGCCCCAGGCCTCCTGTGGGACGCCAGTCACCAGCAGGAGCAGCCAACCAGCAGCAGCCATCATGGAGGCGCTGGGGCTGTGGAGATCCGGAGTCGCCACAGCTCCTACCCCGCGGGGACGGAGGACGACGAAGGGATGGGGGAGGAGCCCAGCCCCTTTCGGGGCCGCTCGCGCTCGGCGCCCCCCAACCTCTGGGCAGCACAGCGCTATGGCCGCGAGCTCCGGAGGATGAGTGACGAGTTTGTGGACTCCTTTAAGAAGGGACTTCCTCGCCCGAAGAGCGCGGGCACAGCAACGCAGATGCGGCAAAGCTCCAGCTGGACGCGAGTCTTCCAGTCCTGGTGGGATCGGAACTTGGGCAGGGGAAGCTCCGCCCCCTCCCAGTGA(SEQ ID No.1)。
融合蛋白Flag-hBAD-WT的氨基酸序列如下:
MDYKDDDDKGSMFQIPEFEPSEQEDSSSAERGLGPSPAGDGPSGSGKHHRQAPGLLWDASHQQEQPTSSSHHGGAGAVEIRSRHSSYPAGTEDDEGMGEEPSPFRGRSRSAPPNLWAAQRYGRELRRMSDEFVDSFKKGLPRPKSAGTATQMRQSSSWTRVFQSWWDRNLGRGSSAPSQ(SEQ ID No.2)。
(2)重组质粒的构建
图1是真核表达载体pcDNA3.1(+)的质粒图谱,图2是真核表达质粒pcDNA3.1-flag-hBAD-WT的质粒图谱,参考图1和图2,将真核表达载体pcDNA3.1(+)用NheI与EcoR I酶切后回收,然后与步骤1回收产物经酶切后的产物在连接酶作用下连接。连接产物转化感受态细胞后,涂布于含有卡那霉素抗性的LB固体培养基上,过夜培养后挑单克隆扩增培养并抽提质粒。将抽提的质粒送公司进行测序鉴定,阳性质粒即为表达N-末端带有Flag蛋白标签的人源野生型BAD蛋白的融合蛋白的真核表达质粒pcDNA3.1(+)-flag-hBAD-WT(WT表示wild type,野生型)。
(3)点突变PCR引入S99A突变位点
引物设计如下:
S99A-F:CGGAGTCGCCACAGCGCCGCGCCCCCCAACCTC(SEQ ID No.5)
S99A-R:GAGGTTGGGGGGCGCGGCGCTGTGGCGACTCCG(SEQ ID No.6)
以pcDNA3.1-flag-hBAD-WT为模板,按表1配制PCR体系:
表1.PCR体系
体系配制完成后按以下条件进行点突变PCR,在pcDNA3.1-flag-hBAD-WT中的野生型hBAD蛋白中引入S99A点突变:95℃,5min;95℃,30s,55℃,30s,72℃,2min,20个循环;72℃,5min。
琼脂糖凝胶电泳检测PCR产物条带大小正确后,使用DMT酶对PCR产物进行消化,37℃孵育1h。消化产物转化至DMT感受态细胞后涂布于含有卡那霉素抗性的LB固体培养基上,过夜培养后挑单克隆扩增培养并抽提质粒。将抽提的质粒送公司进行测序鉴定。阳性质粒即为pcDNA3.1(+)-flag-hBAD-S99A。图3为pcDNA3.1(+)-flag-hBAD-S99A的质粒图谱。
Flag-hBAD-S99A具有如下核苷酸序列:
ATGGATTACAAGGACGACGATGACAAGGGCAGCATGTTCCAGATCCCAGAGTTTGAGCCGAGTGAGCAGGAAGACTCCAGCTCTGCAGAGAGGGGCCTGGGCCCCAGCCCCGCAGGGGACGGGCCCTCAGGCTCCGGCAAGCATCATCGCCAGGCCCCAGGCCTCCTGTGGGACGCCAGTCACCAGCAGGAGCAGCCAACCAGCAGCAGCCATCATGGAGGCGCTGGGGCTGTGGAGATCCGGAGTCGCCACAGCTCCTACCCCGCGGGGACGGAGGACGACGAAGGGATGGGGGAGGAGCCCAGCCCCTTTCGGGGCCGCTCGCGCGCCGCGCCCCCCAACCTCTGGGCAGCACAGCGCTATGGCCGCGAGCTCCGGAGGATGAGTGACGAGTTTGTGGACTCCTTTAAGAAGGGACTTCCTCGCCCGAAGAGCGCGGGCACAGCAACGCAGATGCGGCAAAGCTCCAGCTGGACGCGAGTCTTCCAGTCCTGGTGGGATCGGAACTTGGGCAGGGGAAGCTCCGCCCCCTCCCAGTGA(SEQ ID No.3)。
融合蛋白Flag-hBAD-S99A的氨基酸序列如下:
MDYKDDDDKGSMFQIPEFEPSEQEDSSSAERGLGPSPAGDGPSGSGKHHRQAPGLLWDASHQQEQPTSSSHHGGAGAVEIRSRHSSYPAGTEDDEGMGEEPSPFRGRSRAAPPNLWAAQRYGRELRRMSDEFVDSFKKGLPRPKSAGTATQMRQSSSWTRVFQSWWDRNLGRGSSAPSQ(SEQ ID No.4)。
实施例2
本实施例提供宿主细胞,其制备方法及验证方法的具体步骤如下:
选择卵巢癌细胞系OVCAR2、子宫内膜癌细胞系AN3及肺癌细胞系PC9作为实验对象接种于6孔板,当细胞密度达70%时,各孔以实施例1中的重组质粒pcDNA3.1(+)-flag-hBAD-WT以及pcDNA3.1(+)-flag-hBAD-S99A分别与脂质体(LipofectamineTM 3000,货号L3000015,购自ThermoFisher公司)按照其产品说明书的方法进行转染,得到对应的宿主细胞。
实施例3
卵巢癌细胞系OVCAR2宿主细胞实验
设置实验组和对照组的OVCAR2宿主细胞,参考实施例2,实验组转染重组质粒pcDNA3.1(+)-flag-hBAD-S99A,对照组转染pcDNA3.1(+)空载体。
1.蛋白免疫印记实验
转染后的细胞培养48h后,收集细胞,提取总蛋白样品。样品电泳分离后转移到硝酸纤维素膜上,实验组和对照组各自分别与S99位点磷酸化BAD抗体(pBad(Ser99))、S75位点磷酸化BAD抗体(pBad(Ser75))、BAD抗体和内参抗体(actin)孵育,二抗免疫作用后,显色反应。结果如图4的A所示。
2.细胞存活及凋亡实验
转染后的细胞培养72h后,检测其细胞存活率。收集细胞,Caspase3/7活性细胞凋亡检测试剂盒检测Caspase 3/7酶活。结果如图4的B所示,从图中可以看到,实验组S99A突变使细胞存活率相比于对照组有显著下降(p<0.01),而Caspase 3/7酶活显著上升(p<0.001),细胞凋亡率更高。
实施例4
子宫内膜癌细胞系AN3宿主细胞实验
设置实验组1、实验组2和对照组的AN3宿主细胞,参考实施例2,实验组1转染重组质粒pcDNA3.1(+)-flag-hBAD-S99A,实验组2转染重组质粒pcDNA3.1(+)-flag-hBAD-WT,对照组转染pcDNA3.1(+)空载体。
1.蛋白免疫印记实验
转染后的细胞培养48h后,收集细胞,提取总蛋白样品。样品电泳分离后转移到硝酸纤维素膜上,实验组1、实验组2和对照组各自分别与S99位点磷酸化BAD抗体(pBad(Ser99))、S75位点磷酸化BAD抗体(pBad(Ser75))、BAD抗体、Flag标签抗体和内参抗体(actin)孵育,二抗免疫作用后,显色反应。结果如图5的A所示。
2.细胞存活及凋亡实验
转染后的细胞培养72h后,检测其细胞存活率。收集细胞,Caspase3/7活性细胞凋亡检测试剂盒检测Caspase 3/7酶活。结果如图5的B所示,从图中可以看到,实验组2中BAD蛋白的高水平表达使细胞存活率相比于对照组有显著下降(p<0.01),同时,实验组1的S99A突变又使得细胞存活率相比于实验组2或对照组均有显著差异。同理,实验组1、实验组2和对照组的Caspase 3/7酶活两两之间均存在显著差异(p<0.001),细胞凋亡率依次递减。
实施例5
肺癌细胞系PC9宿主细胞实验
设置实验组和对照组的PC9宿主细胞,参考实施例2,实验组转染重组质粒pcDNA3.1(+)-flag-hBAD-S99A,对照组转染pcDNA3.1(+)空载体。
1.蛋白免疫印记实验
转染后的细胞培养48h后,收集细胞,提取总蛋白样品。样品电泳分离后转移到硝酸纤维素膜上,实验组和对照组各自分别与S99位点磷酸化BAD抗体(pBad(Ser99))、BAD抗体、Flag标签抗体和内参抗体(actin)孵育,二抗免疫作用后,显色反应。结果如图6的A所示。
2.细胞存活及凋亡实验
转染后的细胞培养72h后,检测其细胞存活率。收集细胞,Caspase3/7活性细胞凋亡检测试剂盒检测Caspase 3/7酶活。结果如图6的B所示,从图中可以看到,实验组S99A突变使细胞存活率相比于对照组有显著下降(p<0.01),而Caspase 3/7酶活显著上升(p<0.001),细胞凋亡率更高。
实施例6
本实施例提供一种动物模型的构建方法,包括以下步骤:
以0.6%戊巴比妥钠对小鼠腹腔注射进行麻醉,取右侧卧位固定于鼠板,以75%乙醇常规消毒,在左腋下行1cm左右切口,钝性分离皮下及肌层至肋骨表面层次,能清晰看见粉红色左肺上下运动,在肋骨上缘以微量进样器垂直刺入3~4mm,缓慢注射含有2×106个实施例5中实验组肺癌细胞系PC9宿主细胞的Matrigel胶悬液100μL于肺内,停止数秒后,缓慢拔出微量进样器,医用胶封闭针孔。逐层缝合关腹,再次乙醇消毒。
上面结合附图对本发明实施例作了详细说明,但是本发明不限于上述实施例,在所属技术领域普通技术人员所具备的知识范围内,还可以在不脱离本发明宗旨的前提下作出各种变化。此外,在不冲突的情况下,本发明的实施例及实施例中的特征可以相互组合。
SEQUENCE LISTING
<110> 清华-伯克利深圳学院筹备办公室
<120> 重组质粒及其应用
<130> 1
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 540
<212> DNA
<213> 人工序列
<400> 1
atggattaca aggacgacga tgacaagggc agcatgttcc agatcccaga gtttgagccg 60
agtgagcagg aagactccag ctctgcagag aggggcctgg gccccagccc cgcaggggac 120
gggccctcag gctccggcaa gcatcatcgc caggccccag gcctcctgtg ggacgccagt 180
caccagcagg agcagccaac cagcagcagc catcatggag gcgctggggc tgtggagatc 240
cggagtcgcc acagctccta ccccgcgggg acggaggacg acgaagggat gggggaggag 300
cccagcccct ttcggggccg ctcgcgctcg gcgcccccca acctctgggc agcacagcgc 360
tatggccgcg agctccggag gatgagtgac gagtttgtgg actcctttaa gaagggactt 420
cctcgcccga agagcgcggg cacagcaacg cagatgcggc aaagctccag ctggacgcga 480
gtcttccagt cctggtggga tcggaacttg ggcaggggaa gctccgcccc ctcccagtga 540
<210> 2
<211> 179
<212> PRT
<213> 人工序列
<400> 2
Met Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser Met Phe Gln Ile Pro
1 5 10 15
Glu Phe Glu Pro Ser Glu Gln Glu Asp Ser Ser Ser Ala Glu Arg Gly
20 25 30
Leu Gly Pro Ser Pro Ala Gly Asp Gly Pro Ser Gly Ser Gly Lys His
35 40 45
His Arg Gln Ala Pro Gly Leu Leu Trp Asp Ala Ser His Gln Gln Glu
50 55 60
Gln Pro Thr Ser Ser Ser His His Gly Gly Ala Gly Ala Val Glu Ile
65 70 75 80
Arg Ser Arg His Ser Ser Tyr Pro Ala Gly Thr Glu Asp Asp Glu Gly
85 90 95
Met Gly Glu Glu Pro Ser Pro Phe Arg Gly Arg Ser Arg Ser Ala Pro
100 105 110
Pro Asn Leu Trp Ala Ala Gln Arg Tyr Gly Arg Glu Leu Arg Arg Met
115 120 125
Ser Asp Glu Phe Val Asp Ser Phe Lys Lys Gly Leu Pro Arg Pro Lys
130 135 140
Ser Ala Gly Thr Ala Thr Gln Met Arg Gln Ser Ser Ser Trp Thr Arg
145 150 155 160
Val Phe Gln Ser Trp Trp Asp Arg Asn Leu Gly Arg Gly Ser Ser Ala
165 170 175
Pro Ser Gln
<210> 3
<211> 540
<212> DNA
<213> 人工序列
<400> 3
atggattaca aggacgacga tgacaagggc agcatgttcc agatcccaga gtttgagccg 60
agtgagcagg aagactccag ctctgcagag aggggcctgg gccccagccc cgcaggggac 120
gggccctcag gctccggcaa gcatcatcgc caggccccag gcctcctgtg ggacgccagt 180
caccagcagg agcagccaac cagcagcagc catcatggag gcgctggggc tgtggagatc 240
cggagtcgcc acagctccta ccccgcgggg acggaggacg acgaagggat gggggaggag 300
cccagcccct ttcggggccg ctcgcgcgcc gcgcccccca acctctgggc agcacagcgc 360
tatggccgcg agctccggag gatgagtgac gagtttgtgg actcctttaa gaagggactt 420
cctcgcccga agagcgcggg cacagcaacg cagatgcggc aaagctccag ctggacgcga 480
gtcttccagt cctggtggga tcggaacttg ggcaggggaa gctccgcccc ctcccagtga 540
<210> 4
<211> 179
<212> PRT
<213> 人工序列
<400> 4
Met Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser Met Phe Gln Ile Pro
1 5 10 15
Glu Phe Glu Pro Ser Glu Gln Glu Asp Ser Ser Ser Ala Glu Arg Gly
20 25 30
Leu Gly Pro Ser Pro Ala Gly Asp Gly Pro Ser Gly Ser Gly Lys His
35 40 45
His Arg Gln Ala Pro Gly Leu Leu Trp Asp Ala Ser His Gln Gln Glu
50 55 60
Gln Pro Thr Ser Ser Ser His His Gly Gly Ala Gly Ala Val Glu Ile
65 70 75 80
Arg Ser Arg His Ser Ser Tyr Pro Ala Gly Thr Glu Asp Asp Glu Gly
85 90 95
Met Gly Glu Glu Pro Ser Pro Phe Arg Gly Arg Ser Arg Ala Ala Pro
100 105 110
Pro Asn Leu Trp Ala Ala Gln Arg Tyr Gly Arg Glu Leu Arg Arg Met
115 120 125
Ser Asp Glu Phe Val Asp Ser Phe Lys Lys Gly Leu Pro Arg Pro Lys
130 135 140
Ser Ala Gly Thr Ala Thr Gln Met Arg Gln Ser Ser Ser Trp Thr Arg
145 150 155 160
Val Phe Gln Ser Trp Trp Asp Arg Asn Leu Gly Arg Gly Ser Ser Ala
165 170 175
Pro Ser Gln
<210> 5
<211> 33
<212> DNA
<213> 人工序列
<400> 5
cggagtcgcc acagcgccgc gccccccaac ctc 33
<210> 6
<211> 33
<212> DNA
<213> 人工序列
<400> 6
gaggttgggg ggcgcggcgc tgtggcgact ccg 33
Claims (10)
1.重组质粒,其特征在于,包括质粒载体和外源核酸序列,所述外源核酸序列编码N-末端带有蛋白标签的人源BAD蛋白的融合蛋白。
2.根据权利要求1所述的重组质粒,其特征在于,所述人源BAD蛋白在S75、S99、S118中至少一个位点具有突变。
3.根据权利要求1所述的重组质粒,其特征在于,所述人源BAD蛋白具有S75A、S99A、S118A中至少一种突变。
4.根据权利要求3所述的重组质粒,其特征在于,所述外源核酸序列包含如SEQ IDNo.3所示的核苷酸序列。
5.根据权利要求3所述的重组质粒,其特征在于,所述融合蛋白包含如SEQ ID No.4所示的氨基酸序列。
6.根据权利要求1~5任一项所述的重组质粒,其特征在于,所述质粒载体为真核表达载体。
7.根据权利要求1~5任一项所述的重组质粒,所述蛋白标签为Flag标签。
8.转入有权利要求1~7任一项所述的重组质粒的宿主细胞。
9.动物模型的构建方法,其特征在于,包括以下步骤:向非人动物施用权利要求8所述的宿主细胞。
10.权利要求8所述的宿主细胞在制备或筛选用于治疗或预防癌症的药物中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011254579.6A CN112481298A (zh) | 2020-11-11 | 2020-11-11 | 重组质粒及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011254579.6A CN112481298A (zh) | 2020-11-11 | 2020-11-11 | 重组质粒及其应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN112481298A true CN112481298A (zh) | 2021-03-12 |
Family
ID=74929572
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202011254579.6A Pending CN112481298A (zh) | 2020-11-11 | 2020-11-11 | 重组质粒及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN112481298A (zh) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5955593A (en) * | 1996-09-09 | 1999-09-21 | Washington University | BH3 interacting domain death agonist |
WO2014008167A2 (en) * | 2012-07-02 | 2014-01-09 | Cell Assay Innovations, Inc. | Cell-based assays for post-translational enzyme activity |
CN110719906A (zh) * | 2017-04-19 | 2020-01-21 | 新加坡国立大学 | Bcl-2相关死亡促进因子(BAD)磷酸化的小分子抑制剂 |
-
2020
- 2020-11-11 CN CN202011254579.6A patent/CN112481298A/zh active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5955593A (en) * | 1996-09-09 | 1999-09-21 | Washington University | BH3 interacting domain death agonist |
WO2014008167A2 (en) * | 2012-07-02 | 2014-01-09 | Cell Assay Innovations, Inc. | Cell-based assays for post-translational enzyme activity |
CN110719906A (zh) * | 2017-04-19 | 2020-01-21 | 新加坡国立大学 | Bcl-2相关死亡促进因子(BAD)磷酸化的小分子抑制剂 |
Non-Patent Citations (4)
Title |
---|
FERNANDO R 等: "Breast cancer cell proliferation is inhibited by BAD: regulation of cyclin D1", 《J BIOL CHEM》 * |
POLZIEN L 等: "Identification of novel in vivo phosphorylation sites of the human proapoptotic protein BAD: pore-forming activity of BAD is regulated by phosphorylation", 《J BIOL CHEM》 * |
周学东: "《中国口腔医学年鉴 2004》", 成都:四川科学技术出版社 * |
无: "Accession number: AAP36710.1,Homo sapiens BCL2-antagonist of cell death, partial [synthetic construct]", 《GENBANK》 * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Wu et al. | Opposing functions of BRD4 isoforms in breast cancer | |
Son et al. | Nucleosome-binding activities within JARID2 and EZH1 regulate the function of PRC2 on chromatin | |
Nicolson et al. | Tumor metastasis-associated human MTA1 gene and its MTA1 protein product: role in epithelial cancer cell invasion, proliferation and nuclear regulation | |
Manojlovic et al. | A novel role of RNA helicase A in regulation of translation of type I collagen mRNAs | |
Franke et al. | The tumor suppressor SASH1 interacts with the signal adaptor CRKL to inhibit epithelial–mesenchymal transition and metastasis in colorectal cancer | |
Vernimmen et al. | Identification of HTF (HER2 transcription factor) as an AP-2 (activator protein-2) transcription factor and contribution of the HTF binding site to ERBB2 gene overexpression | |
Chen et al. | Enhanced expression and phosphorylation of the MET oncoprotein by glioma-specific PTPRZ1–MET fusions | |
KR20100015944A (ko) | Mek 리간드 및 이를 인코딩하는 폴리뉴클레오티드 | |
Saloura et al. | WHSC1 monomethylates histone H1 and induces stem-cell like features in squamous cell carcinoma of the head and neck | |
Peng et al. | Induction of epithelial-mesenchymal transition (EMT) by hypoxia-induced lncRNA RP11-367G18. 1 through regulating the histone 4 lysine 16 acetylation (H4K16Ac) mark | |
Kristjuhan | Studies on transcriptional activator properties of tumor suppressor protein p53 | |
Tsai et al. | Induction of apoptosis by the retinoid inducible growth regulator RIG1 depends on the NC motif in HtTA cervical cancer cells | |
CN112481298A (zh) | 重组质粒及其应用 | |
CN114957399B (zh) | 一种多肽、多肽衍生物及其在制备抗肿瘤药物中的应用 | |
Blundell | The biology of p21 Waf1/Cip1-review paper | |
TW201200151A (en) | Methods and compositions related to reduced MET phosphorylation by leukocyte cell-derived chemotaxin 2 in tumor cells | |
CN109112213A (zh) | 检测黏着斑激酶结构变异体的pcr引物及其检测方法与应用 | |
Zhang et al. | Cloning and functional identification of a novel BCA3 splice | |
Kokura et al. | Identity between rat htf and human xbp-1 genes: determination of gene structure, target sequence, and transcription promotion function for HTF | |
CN110592032B (zh) | 泛素连接酶Smurf1突变体、编码基因及用途 | |
WO2022037666A1 (zh) | Rdr蛋白在肿瘤治疗中的应用 | |
Papa et al. | Biochemistry of cell membranes: a compendium of selected topics | |
KR101385357B1 (ko) | 항암 활성을 갖는 GKN1 brichos 도메인의 용도 | |
Shi et al. | The tRNA Gm18 Methyltransferase TARBP1 Promotes Hepatocellular Carcinoma Progression via Metabolic Reprogramming of Glutamine | |
KR101394828B1 (ko) | 항암활성을 갖는 gkn1의 아미노 말단 조절 모티프의 용도 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
RJ01 | Rejection of invention patent application after publication | ||
RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210312 |