Flag和GFP双标记的BK_(Ca)通道α亚基表达质粒的构建、鉴定和序列分析

Construction and identification of BK_(Ca) α subunit expression plasmid with double labeling of Flag and GFP

目的:采用重叠PCR法构建表达质粒,为下一步研究通道功能奠定基础。方法:在已有编码大电导钙激活钾通道(BKCa)通道α亚单位的表达质粒pcDNA3.1-h Slo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pcDNA3.1-Flag-hSlo-GFP(Flag-hSlo-GFP)。结果:构建的表达质粒Flag标签插入BKCa通道的S1-S2胞外环,GFP标签连接BKCa通道的胞内C末端,测序结果证实质粒构建成功。结论:成功构建BK通道基因表达质粒Flag-hSlo-GFP,重叠PCR能够很好的用于长片段基因扩增和插入片段的实验。

Objective： This study aimed to construct a large conductance calcium activated potassium channel α（BKCa） subunit plasmid with two tags by the overlapping PCR technique to set up a steady base for future ion channel study. Methods： Based on the existing coding BKCa channel α subunit expression plasmid pc DNA3. 1-hSlo,we constructed a double-tag expression plasmid,namely,pc DNA3. 1-Flag-hSlo-GFP（ Flag-hSlo-GFP）. Results： Flag tag was inserted into the S1-S2 extracellular loop of BKCa channel α subunit,and GFP tag was connected to the C-terminus of BKCa channel α subunit. Sequence of the constructed plasmid was confirmed successful. Conclusion： The expression plasmid Flag-hSlo-GFP was constructed successfully with overlapping PCR. Overlapping PCR is a valuable method for amplifying long size genes.