SK2和JPH2双基因重组真核表达载体的构建及在HEK293细胞中的表达  

Construction of an eukaryotic expression vector carried SK2 and JPH2 genes and expressions of SK2 and JPH2 proteins in HEK293 cells

作　　者：罗天霞[1] 樊红琨[1] 芮丹丹 孙佳翕 马小芳[1] 章茜[1] LUO Tianxia FAN Hongkun RUI Dandan SUN Jiaxi MA Xiaofang ZHANG Qianl(） Department of Physiology, School of Basic Medical Sciences,Zhengzhou University, Zhengzhou 450001 ） School of Basic Medical Sciences ,Xinxiang Medical College, Xinxiang 453000)

机构地区：[1]郑州大学基础医学院生理学教研室,郑州450001 [2]新乡医学院基础医学院,新乡453000

出　　处：《郑州大学学报（医学版）》2016年第5期580-583,共4页Journal of Zhengzhou University(Medical Sciences)

基　　金：国家自然科学基金资助项目 81270248;81570311

摘　　要：目的:构建SK2和JPH2双基因重组真核表达载体p IRES-EGFP/SK2+JPH2,并检测其在转染后HEK293细胞中的表达。方法:以p CMV6-entry/SK2为模板,PCR扩增出SK2片段,通过BgⅢ/HindⅢ酶切位点克隆入真核表达载体p IRES-EGFP,将JPH2序列插入构建的SK2表达载体p IRES-EGFP-SK2,并通过酶切及测序鉴定。脂质体转染法将重组质粒p IRES-EGFP/SK2+JPH2转染HEK293细胞,采用Western blot法检测SK2通道蛋白和JPH2蛋白的表达。结果:构建的重组真核表达载体p IRES-EGFP/SK2+JPH2经酶切和测序鉴定,证实插入的序列与Gen Bank中的SK2和JPH2基因序列完全相同。p IRES-EGFP/SK2+JPH2质粒转染HEK293细胞48 h后,SK2和JPH2蛋白均能成功表达。结论:成功构建了重组真核表达载体p IRES-EGFP/SK2+JPH2。Aim： To construct an eukaryotic expression vector carried SK2 and JPH2 genes and detect the expression of them in the transfected HEK293 cells.Methods： The full-length SK2 gene was obtained from a recombinant vector pCMV6-entry/SK2 by PCR amplification, and cloned it into the eukaryotic expression vector pIRES-EGFP. Then JPH2 gene was inserted into pIRES-EGFP-SK2 vector and was detected by endonuclease digestion and sequencing.The recombinant pIRES-EGFP/SK2＋JPH2 was transfected into HEK293 cells with a liposome infection protocol. The expressions of SK2 and JPH2 protein were detected by Western blot.Results： Both endonuclease digestion and sequence analysis demonstrated that the inserted sequences of pIRES-EGFP/SK2＋JPH2 were identical to those of the full-length of SK2 and JPH2 genes in GenBank. Moreover, both SK2 and JPH2 protein in HEK 293 cells transfected with pIRES-EGFP/SK2＋JPH2 for 48 h were expressed successfully.Conclusion： The eukaryotic expression vector pIRES-EGFP/SK2＋JPH2 as well as the expressions of SK2 and JPH2 cell lines have been successfully constructed.

关 键 词：SK2通道 JPH2蛋白 真核表达载体 HEK293 

分 类 号：R541.7[医药卫生—心血管疾病]