融合蛋白TAT-p53-MTS的促细胞凋亡作用
Pro apoptotic effect of the recombinant protein TAT-p53-MTS
作者：吴兰(四川大学生命科学学院生物资源与生态环境教育部重点实验室)；赵梓亦(四川大学生命科学学院生物资源与生态环境教育部重点实验室)；李灵(四川大学生命科学学院生物资源与生态环境教育部重点实验室)
Author：WU Lan(Key laboratory of Bio resources and Eco environment of Ministry of Education,College of Life Sciences, Sichuan University)；ZHAO Zi-Yi(Key laboratory of Bio resources and Eco environment of Ministry of Education,College of Life Sciences, Sichuan University)；LI Ling(Key laboratory of Bio resources and Eco environment of Ministry of Education,College of Life Sciences, Sichuan University)
收稿日期：2013-05-09 年卷（期）页码：2015,52(1):170-174
期刊名称：四川大学学报: 自然科学版
Journal Name：Journal of Sichuan University (Natural Science Edition)
关键字：p53; 细胞凋亡; 线粒体定位; MTS; TAT
Key words：p53; Cell apoptosis; Mitochondrial targeting; MTS; TAT
基金项目：国家自然科学基金（31000579）
中文摘要
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-H1299细胞的凋亡.
英文摘要
The plasmid for prokaryotic production of recombinant p53 fused with TAT and MTS (TAT p53 MTS) was constructed. Purified recombinant protein was incubated with p53 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 membrane ability and the mitochondrial targeting of the fusion protein. What’s more, the fusion protein exhibits ability to interact with Bcl 2, through which apoptosis is significantly induced.
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