四川大学学报（自然科学版）
四川大學學報（自然科學版）
사천대학학보（자연과학판）
JOURNAL OF SICHUAN UNIVERSITY（NATURAL SCIENCE EDITION）
2015年
1期
170-174
,共5页
p5 3%细胞凋亡%线粒体定位%MTS%TAT
p5 3%細胞凋亡%線粒體定位%MTS%TAT
p5 3%세포조망%선립체정위%MTS%TAT
p5 3%Cell apoptosis%Mitochondrial targeting%MTS%TAT
本研究首先构建了融合蛋白 TAT-p53-MTS 的原核表达质粒 pET-22b(+)-TAT-p53-MTS,将表达纯化的融合蛋白与p53缺陷型人肺癌细胞NCI-H1299孵育,通过Western blot和免疫共沉淀(co-immunoprecipitation,co-IP)检测融合蛋白的细胞穿膜以及与 Bcl-2蛋白的相互作用,并通过流式细胞术检测融合蛋白对 NCI-H1299细胞的促凋亡作用。结果显示,融合TAT和MTS的p53重组蛋白具有更强的细胞穿膜及线粒体定位能力；并且,融合蛋白能够通过与Bcl-2蛋白的结合显著诱导NCI-H 1299细胞的凋亡。
本研究首先構建瞭融閤蛋白 TAT-p53-MTS 的原覈錶達質粒 pET-22b(+)-TAT-p53-MTS,將錶達純化的融閤蛋白與p53缺陷型人肺癌細胞NCI-H1299孵育,通過Western blot和免疫共沉澱(co-immunoprecipitation,co-IP)檢測融閤蛋白的細胞穿膜以及與 Bcl-2蛋白的相互作用,併通過流式細胞術檢測融閤蛋白對 NCI-H1299細胞的促凋亡作用。結果顯示,融閤TAT和MTS的p53重組蛋白具有更彊的細胞穿膜及線粒體定位能力；併且,融閤蛋白能夠通過與Bcl-2蛋白的結閤顯著誘導NCI-H 1299細胞的凋亡。
본연구수선구건료융합단백 TAT-p53-MTS 적원핵표체질립 pET-22b(+)-TAT-p53-MTS,장표체순화적융합단백여p53결함형인폐암세포NCI-H1299부육,통과Western blot화면역공침정(co-immunoprecipitation,co-IP)검측융합단백적세포천막이급여 Bcl-2단백적상호작용,병통과류식세포술검측융합단백대 NCI-H1299세포적촉조망작용。결과현시,융합TAT화MTS적p53중조단백구유경강적세포천막급선립체정위능력；병차,융합단백능구통과여Bcl-2단백적결합현저유도NCI-H 1299세포적조망。
The plasmid for prokaryotic production of recombinant p5 3 fused with TAT and MTS (TAT-p5 3-MTS)was constructed.Purified recombinant protein was incubated with p5 3-deficient human lung cancer line NCl-H1299,and the cell lysate was subjected to co-immunoprecipitation and Western blot for testing the trans-membrane ability of the fusion protein,as well as the interaction between the fusion protein and Bcl-2.Additionally,flow cytometry was employed to detect fusion protein’s proapoptotic impact on NCl-H1299 cells.The results showed that TAT and MTS can improve both the trans-mem-brane ability and the mitochondrial targeting of the fusion protein.What’s more,the fusion protein ex-hibits ability to interact with Bcl-2,through which apoptosis is significantly induced.
