双启动子双报告基因真核表达质粒的构建及鉴定

Construction and identification of eukaryotic expression vector with double promoters and double reporter genes

目的构建双启动子双报告基因真核表达质粒,并在293T细胞中表达,为筛选携带报告基因的肿瘤细胞系提供分子工具。方法以质粒pcDNA3.1、pcDNA6-GFP、pSVA-Luc为模板,分别扩增CMV、GFP和Luc基因序列,通过In-fusion法将3个基因片段与经HindⅢ、XhoⅠ双酶切的pcDNA3.1载体连接,构建双启动子双报告基因重组真核表达质粒pC-T2A-Luc-GFP,转染293T细胞,荧光显微镜直接观察GFP的表达,化学发光仪检测Luc的表达。提取293T细胞总RNA,RT-PCR法扩增p53基因,通过In-fusion法将p53基因连接于质粒pC-T2A-Luc-GFP上,构建质粒pC-p53-T2A-Luc-GFP,转染293T细胞,Western blot法检测p53的表达,化学发光仪检测Luc的表达。结果重组真核表达质粒pC-T2A-Luc-GFP经酶切及测序鉴定构建正确,GFP和Luc双报告基因均可在293T细胞中正确表达,且可用于后续的动物模型建立。pC-p53-T2A-Luc-GFP质粒中p53的插入降低了Luc的表达,但其表达仍为阳性,不影响Luc的正常检测。结论成功构建了双启动子双报告基因真核表达质粒pC-T2A-Luc-GFP,并可在293T细胞中表达,为建立携带报告基因的肿瘤细胞系及肿瘤模型奠定了分子基础。

Objective To construct a eukaryotic expression vector with double promoters and double reporter genes,express in 293 T cells and provide a molecular tool for screening the tumor cell lines carrying reporter gene.Methods CMV,GFP and Luc genes were amplified by PCR using plasmids pcDNA3.1,pcDNA6-GFP and pSVA-Luc as templates,and linked to vector pcDNA3.1 digested with HindⅢand XhoⅠby In-fusion method respectively.The 293 T cells were transfected with the constructed recombinant plasmid pC-T2 A-Luc-GFP with double promoters and double reporter genes,observed for the expression of GFP by fluorescent microscopy,and determined for Luc expression by chemiluminescence analyzer.The total RNA of 293 T cells was extracted,from which p53 gene was amplified by RT-PCR and linked to plasmid pC-T2 A-Luc-GFP by In-fusion method to construct recombinant plasmid pC-p53-T2 A-Luc-GFP,with which 293 T cells were transfected and determined for p53 expression by Western blot,and for Luc expression by chemiluminescence analyzer.Results Restriction analysis and sequencing proved that recombinant plasmid pC-T2 A-Luc-GFP was constructed correctly.Both the reporter genes GFP and Luc were expressed correctly in 293 T cells and used for the subsequent establishment of animal model.The insertion of p53 gene in plasmid pC-p53-T2 A-Luc-GFP decreased the expression level of Luc,while the Luc was still expressed,and the normal determination of Luc was not influenced.Conclusion Recombinant plasmid pC-T2 A-Luc-GFP with double promoters and double reporter genes was constructed successfully and expressed in 293 T cells,which provided a molecular basis for the establishment of tumor cell lines carrying reporter gene and animal model of tumor.