含p21启动子的荧光素酶报告基因的构建和活性测定

Construction of a Luciferase Reporter Gene Vector Directed by p21 Promoter and Characterization of the Promoter Activity

目的:克隆p21基因的启动子,插入荧光素酶报告基因载体中,并检测其活性。方法:采用PCR技术从人乳腺癌细胞系MCF-7基因组中扩增出p21启动子,插入荧光素酶报告基因载体pGL3-basic中,确定所扩增的DNA序列,在293T细胞中检测其活性。结果:测序结果表明扩增的p21启动子序列正确,活性实验表明构建的报告基因具有启动子活性,雌激素受体(ER)α能以剂量依赖的方式升高p21报告基因的转录。结论:克隆了p21启动子,为ERα共调节子的功能研究提供了重要基础。

Objective： To clone p21 promoter and insert it into a luciferase reporter gene vector, and to characterize the promoter activity. Methods： The promoter of p21 was amplified from human breast cancer cell line MCF-7 genomic DNA by PCR and cloned into pGL3-basic. The resulting plasmid was determined by DNA sequencing. The promoter activity was analyzed in 293T cell line. Results： The result of DNA sequencing showed that the sequence of the cloned promoter was correct. The activity of the p21 luciferase reporter gene was confirmed, and this activity was increased by estrogen receptor（ER） α in a dose-dependent manner. Conclusion： The p21 promoter was cloned successfully, and this will be an important basis for the study of the functions of ERα coregulators.