Flag和GFP双标记的SK2通道表达质粒的构建、鉴定和序列分析

发布时间：2018-04-04 23:07

本文选题：小电导钙激活钾通道　切入点：重叠PCR　出处：《泸州医学院学报》2016年06期

【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。
[Abstract]:Aim: to construct the fusion expression plasmid of human SK2 channel Flag and GFP by Overlapping PCR, and to lay a foundation for the further study on the regulation of SK2 channel transport by extracellular Flag antibody.Methods: based on the previously cloned expression plasmid p IRES-SK2 of human SK2 channel gene, the expression plasmid pEGFP-N3-Flag-SK2 (Flag-SK2-GFPN) was constructed by overlapping PCR method.Results: the constructed expression plasmid Flag tag was inserted into the S1-S2 outer loop region of the SK2 channel and connected to the C-terminal of the SK2 channel. The plasmid was successfully constructed by restriction endonuclease digestion and sequencing.Conclusion: the gene expression plasmid Flag-SK2-GFPin of human atrial muscle SK2 channel was successfully constructed, which laid a foundation for the further study of SK2 channel transport process and regulation mechanism.
【作者单位】：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;
【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)
【分类号】：R91

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