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.
作者
罗天霞
樊红琨
芮丹丹
孙佳翕
马小芳
章茜
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)
出处
《郑州大学学报(医学版)》
CAS
北大核心
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