Flag和GFP双标记的SK2通道表达质粒的构建、鉴定和序列分析  

Construction of SK2 channel expression plasmid with double labeling of Flag and GFP

作　　者：黄文俊[1] 李涛 范学慧[1] 余奕言 杨艳[1] 曾晓荣[1] 谭晓秋[1] Huang Wenjun Li Tao Fan Xuehui Yu Yiyan Yang Yang Zen Xiaorong Tan Xiaoqiu(Institute of Cardiovascular Research, Southwest Medical University, Luzhou, Sichuan Province,646000, China the Key Laboratory of Medical Electrophysiology of Ministry of Education,Collaborative Innovation Center for Prevention and Treatment of Cardiovascular Disease)

机构地区：[1]西南医科大学心血管医学研究所医学电生理学教育部重点实验室四川省心血管疾病防治协同创新中心,四川泸州646000

出　　处：《泸州医学院学报》2016年第6期527-530,共4页Journal of Luzhou Medical College

基　　金：国家自然科学基金资助项目(NO:31300948;81670310);四川省科技厅支撑计划(2011FZ0106);泸州市-四川医科大学联合资助项目(2015LZCYD-S03)

摘　　要：目的:采用Overlapping PCR(重叠PCR)法进行人源SK2通道Flag和GFP融合表达质粒的构建,为下一步胞外运用Flag抗体进行SK2通道的转运调控研究奠定基础。方法:在既往克隆的人SK2通道基因的表达质粒p IRES-SK2的基础上,采用重叠PCR法构建Flag和GFP双标签标记的表达质粒pEGFP-N3-Flag-SK2(简写为Flag-SK2-GFP)。结果:构建的表达质粒Flag标签插入SK2通道的S1-S2胞外环区域,GFP标签连接SK2通道胞内的C末端,通过酶切、测序等证实质粒构建成功。结论:成功构建人心房肌SK2通道基因表达质粒Flag-SK2-GFP,为下一步研究SK2通道转运过程及调控机制奠定了基础。Objective： This study aimed to construct a SK2 channel plasmid with two tags using overlapping PCR to provide the easiness to track the transportation of the channel protein. Methods： Based on human SK2 channel expression plasmid pIRES-SK2, the full length of SK2 cDNA with a Flag insertion was amplified using overlapping PCR method, which was then subcloned into pEGFP-N3 vector. The double-tagged expression plasmid, pEGFP-N3-Flag-SK2 （Flag-SK2-GFP） was confirmed by DNA sequencing. Results： A Flag tag was inserted in-between the S1-S2 extraeellular loops of SK2 channel whose C-terminus was fused in-frame to GFP. The constructed plasmid was further confirmed by DNA sequencing. Conclusion： The expression plasmid Flag- SK2-GFP was constructed successfully with overlapping PCR.

关 键 词：小电导钙激活钾通道 重叠PCR 基因工程 

分 类 号：R972.4[医药卫生—药品]