摘要
目的:旨在构建稳定表达人大电导钙激活钾通道(BK_(Ca))的稳定细胞系并对重组蛋白分离纯化。方法:利用基因工程技术将人肠系膜动脉平滑肌BK_(Ca)编码基因克隆到表达载体pcDNA5/FRT/TO/FLAG,并转染Flp-InTMT-RExTM-293宿主细胞,用潮霉素B筛选建立稳定表达的单克隆细胞系。BK_(Ca)-pcDNA5-293细胞系经四环素诱导后,用anti-FLAG抗体对表达的BK_(Ca)蛋白质进行纯化。用western blot检测BK_(Ca)通道蛋白质的表达,用单通道膜片钳检测通道的电生理特性。结果:BK_(Ca)-pcDNA5-293细胞系经四环素诱导后表达了高水平的BK_(Ca)-FLAG融合蛋白,BK_(Ca)通道电导值为213.39±9.52pS(n=15,cell-attached)和225.72±10.61pS(n=13,inside-out),BK_(Ca)通道具有典型的大电导特性、电压依赖性、Ca2+依赖性及IbTX敏感性,与在体的人肠系膜动脉平滑肌BK_(Ca)通道具有相同的特征。纯化后的BK_(Ca)蛋白质条带单一,纯度约为89%。结论:成功构建BK_(Ca)-pcDNA5-293细胞系并获得纯化的BK_(Ca)蛋白质,为进一步深入研究通道功能及药物筛选奠定了重要的基础。
Objective：To construct an inducible BK_（Ca）-pcDNA5-293 stable cell line and purify the BK_（Ca）-Flag fusion protein.Methods：The BK_（Ca）ORF amplified from human mesenteric vascular smooth muscle cells was cloned into pcDNA5/FRT/TO/FLAG expression vector,which was transiently transfected intoFlp-InTMT-RExTM-293 cells and positive clones were selected using hygromycin B.The recombinant protein was induced by tetracycline and purified using anti-Flag affinity chromatography.The authenticity of the recombinant BK_（Ca）channel was confirmed by Western blot and its electrophysiological features were characterized by single channel patch clamp.Results：The BK_（Ca）-pcDNA5-293 cell line expressed abundant BK_（Ca）-Flag fusion protein after induction with tetracycline.The conductance values of single BK_（Ca）channel current were 213.39 ± 9.52pS（n=15,cell-attached）and 225.72 ± 10.61pS（n=13,inside-out）,which are typical features of BK_（Ca）channels observed in human mesenteric artery smooth muscle cells,such as large conductance,voltage dependence,calcium sensitivity and IbTX sensitivity.The recombinant BK_（Ca）was also extracted and purified to about 89%.Conclusion：A tetracycline-inducibleBK_（Ca）-pcDNA5-293 stable cell line was successfully established,and the recombinant BK_（Ca）channels were functional.The stable cell line can be used for further characterizing the functions of BK_（Ca）channels and lead drug screening.
作者
毛亮
程俊
黄文俊
谭晓秋
党喜同
曾晓荣
杨艳
Mao Liang Cheng Jun Huang Wen-jun Tan Xiao-qiu Dang Xi-tong Zeng Xiao-rong Yang Yan(Key Laboratory of Medical Electrophysiology, Ministry of Education, Institute of Cardiovascular Research, Southwest Medical University, Sichuan Luzhou 64600)
出处
《四川生理科学杂志》
2017年第2期55-58,共4页
Sichuan Journal of Physiological Sciences
基金
国家自然科学基金项目(编号:81173661)
四川省教育厅项目(编号:11ZB224)
四川省卫生和计划生育委员会(编号:130277)
泸州市科技局项目(编号:2014-S-44(6/8))
泸州医学院院级课题资助
