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

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.