E2F1基因真核表达载体的构建及其对CD2AP启动子的影响

Construction of an eukaryotic vector expressing E2F1 gene and its effect on CD2AP promoter

目的构建E2F1基因真核表达质粒,并初步探讨E2F1对CD2相关蛋白(CD2AP)启动子的作用。方法 RT-PCR扩增E2F1基因,构建含E2F1基因的重组真核表达质粒,重组质粒转染人胚肾HEK-293细胞。Western blot检测E2F1蛋白表达;双荧光素酶报告基因检测E2F1过表达后人CD2AP启动子的活性。结果核酸序列分析及Western blot结果证实,成功构建含E2F1基因的重组真核表达质粒;过表达E2F1能增强CD2AP启动子的活性。结论 E2F1编码基因在HEK-293细胞中获得正确表达,且E2F1蛋白可促进人CD2AP启动子的转录活性。

Objective To construct an eukaryotic expression vector of E2F1 and investigate the effect of E2F1 on the promoter activity of CD2AP.Methods E2F1 gene was amplified by RT-PCR.Recombinant eukaryotic expression plasmids encoding E2F1 were constructed.HEK-293 cells were transfected with the recombinant plasmids and Western blot was performed to evaluate the protein expression of E2F1.The human CD2AP promoter activity after over-expressing E2F1 was examined by dual luciferase reporter gene analysis.Results DNA sequence analysis and Western blot indicated that an eukaryotic vector expressing E2F1 gene was successfully constructed . Over-expression of E2F1 increased the promoter activity of CD2AP.Conclusion E2F1 is correctly expressed in HEK-293 cells. E2F1 transcription factor can enhance human CD2AP promoter activity.