利用合适的启动子提高外源基因在Jurkat细胞中的表达

目的通过选择合适的启动子来实现外源基因在Jurkat细胞中的高效表达。方法以pGL3-MCS质粒为基础构建不同启动子(SV40、CMV、EF1α和UbC)驱动萤火虫荧光素酶Fluc报告基因表达的重组质粒,并将其与作为内参的表达海肾荧光素酶Rluc报告基因的质粒共转Jurkat细胞,检测Jurkat细胞中这两种荧光素酶与相应底物反应所产生的荧光强度,计算Fluc与Rluc荧光强度的比值即Fluc的相对酶活,通过比较Fluc相对酶活的大小来选取在Jurkat细胞中具有高表达活性的启动子。结果以pGL3-MCS质粒为基础成功构建了不同启动子驱动Fluc报告基因表达的重组质粒pGL3-CMV、pGL3-EF1α和pGL3-UbC。在Jurkat细胞中,不同启动了驱动的Fluc报告基因的相对酶活的强弱关系为pGL3-UbCpGL3-EF1αpGL3-CMVpGL3-MCS。结论可以选择高活性的人源泛素C启动子实现外源基因在Jurkat细胞中的高效表达,以利于Jurkat细胞系在哺乳动物T淋巴细胞以及免疫相关功能研究中的应用。

Enhancement of exogenous gene expression in Jurkat cells by efficient promoter

Objective To select promoters to enhance exogenous gene expression in Jurkat cells. Methods Firefly luciferase(Fluc)-expressing plasmids driven by different promoters(SV40,CMV,EFla,UbC) were constructed on the basis of pGL3-MCS.Flue-expressing plasmids were co-transfected into Jurkat cells with renilla luciferase(Rluc)-expressing plasmid serving as an internal control.The fluorescent signal generated from the reaction of the two luciferases with the respective substrates was measured,and the ratio of fluorescent signal of Fluc and Rluc was calculated,which was defined as the relative activity of the Fluc. Results Using CMV,EFlαand UbC promoters,we constructed pGL3-CMV,pGL3-EF1αand pGL3-UbC recombinant plasmids to express Fluc reporter gene.The relative Flue’s activity driven by different promoters in Jurkat cells is pGL3-UbC>pGL3-EF1α>pGL3-CMV>pGL3-MCS. Conclusion The human ubiquitin C promoter efficiently enhances the expression of exogenous gene in Jurkat cells,which facilitates the application of Jurkat cells in the study of mammalian T lymphocytes and immune function.