双启动子双报告基因真核表达质粒的构建及表达

目的构建双启动子双报告基因真核表达质粒,并在293T细胞内进行表达。方法分别以pEGFP-N1和pDsRed-N1为模板,PCR扩增EGFP和DsRed基因,插入表达载体pCI中,分别构建重组表达质粒pCI-EGFP和pCI-DsRed,利用载体上BglⅡ和BamHⅠ为同尾酶的特点,将两个表达质粒酶切后拼接,构建双启动子双报告基因重组真核表达质粒pCI-cmvDsRed-cmvEGFP,转染293T细胞,荧光显微镜观察EGFP和DsRed的表达,Western blot和流式细胞术检测EGFP和DsRed蛋白的表达。结果重组真核表达质粒pCI-cmvDsRed-cmvEGFP经酶切鉴定和测序证实构建正确,两启动子为顺向相连;EGFP和DsRed蛋白在293T细胞中可同时正确表达。结论成功构建了双启动子双报告基因真核表达质粒,并在293T细胞中表达,为实现两种外源基因在真核细胞内的同步表达及示踪提供了有效的分子工具。

Construction and expression of eukaryotic plasmid with dual-promoters and dual-reporter genes

Objective To construct a eukaryotic plasmid with dual-promoters and dual-reporter genes,and express in 293T cells.Methods EGFP and DsRed genes were amplified by PCR using plasmids pEGFP-N1 and pDsRed-N1 as templates respectively and inserted into expression vector pCI.The constructed recombinant plasmids pCI-EGFP and pCI-DsRed were digested with BglⅡand BamHⅠthen spliced to construct recombinant eukaryotic expression vector pCI-cmvDsRed-cmvEGFP containing dual-promoters and dual-reporter genes.293T cells were transfected with plasmid pCI-cmvDsRed-cmvEGFP,in which the expressions of EGFP and DsRed were observed by fluorescent microscopy,Western blot and flow cytometry.Results Recombinant plasmid pCI-cmvDsRed-cmvEGFP was constructed correctly as proved by restriction analysis and sequencing,in which two promoters were linked in the same direction.Both EGFP and DsRed were expressed correctly at the same time.Conclusion A eukaryotic plasmid with dual-promoters and dual-reporter genes was successfully constructed and expressed in 293T cells.