人NDRG2启动子的克隆与活性鉴定

目的:克隆人NDRG2的启动子,并进行启动子的活性鉴定。方法:用Advantage-GC Taq酶,采用PCR方法,以BCA克隆R-998D1为模板,克隆人NDRG2(-1455/ 274)的启动子。分别构建NDRG2(-1131/ 274)、NDRG2(-273/ 274)、NDRG2(-135/ 274)、NDRG2(-79/ 274)和NDRG2(-79/ 57)等不同长度的截短体,并分别亚克隆入pGL3-basic报告基因载体,测序鉴定。分别转染HEK293和HeLa细胞后,运用双荧光报告基因系统进行启动子活性分析,从而判断核心启动子的位置。结果:人NDRG2的启动子克隆成功,并构建了不同长度的截短体,DNA测序结果与报道一致。报告基因分析结果将人NDRG2启动子的核心区域定位于NDRG2(-79/ 57)。结论:随着人NDRG2的启动子的长度逐渐被截短,启动子的基础活性也逐渐降低,可将人NDRG2启动子的核心区域定位于NDRG2(-79/ 57)。

Clone and Activity Identification of Human NDRG2 Promoter

Objective: To clone the human NDRG2 promoter and analyze the promoter activity. Methods: The human NDRG2 promoter was amplified from BAC clone R-998D10 with the Advantage-GC genomic polymerase. The resulting amplicon was cloned into the pGL3-basic vector to generate the NDRG2/luciferase reporter plasmid. Various mutants NDRG2(-1131/ 274), NDRG2(-273/ 274), NDRG2(-135/ 274), NDRG2(-79/ 274) and NDRG2(-79/ 57) were generated with PCR using the cloned promoter as template. All of the newly constructed plasmids were confirmed by sequencing. Transfect the plasmids into HEK293 and HeLa cells to analyze the promoter activity and identify the core region of human NDRG2. Results: The promoter of human NDRG2 was amplified successfully and the basic activity was analyzed. The result of sequencing was accordant with the one reported. Identify the core promoter region of human NDRG2. Conclusion: The activity of human NDRG2 promoters weakened with the consecutive truncation. The core region of human NDRG2 located -79 to 57.