Flag和GFP双标记的SK2通道表达质粒的构建尧鉴定和
序列分析
黄文俊;李涛;范学慧;余奕言;杨艳;曾晓荣;谭晓秋
【期刊名称】《泸州医学院学报》 【年(卷),期】2016(039)006
【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pIRES-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 extracellular loops of SK2 channel whose C-terminus
Flag和GFP双标记的SK2通道表达质粒的构建尧鉴定和序列分析
