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

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.