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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 eukaryotic expression plasmid of E2F1 gene and to explore the effect of E2F1 on CD2AP promoter. Methods E2F1 gene was amplified by RT-PCR, and a recombinant eukaryotic expression plasmid containing E2F1 gene was constructed. The recombinant plasmid was transfected into human embryonic kidney HEK-293 cells. E2F1 protein was detected by Western blot. Dual luciferase reporter gene was used to detect the activity of human CD2AP promoter after overexpression of E2F1. Results Nucleic acid sequence analysis and Western blot confirmed that the recombinant eukaryotic expression plasmid containing E2F1 gene was constructed successfully. Overexpression of E2F1 enhanced the activity of CD2AP promoter. Conclusion The E2F1 gene is correctly expressed in HEK-293 cells, and E2F1 protein can promote the transcription activity of human CD2AP promoter.