人p53启动子萤光素酶报告基因的构建及其活性测定  

Construction of a Luciferase Reporter Gene Vector Directed by p53 Promoter and Characterization of its Promoter Activity

作　　者：刘家宏[1,2] 徐小洁[2] 符静[3] 范忠义[1] 吕朝晖[3] 陆菊明[3] 肖文华[1] 叶棋浓[2] 朱建华[1] 

机构地区：[1]解放军总医院第一附属医院肿瘤科,北京100037 [2]军事医学科学院生物工程研究所,北京100850 [3]解放军总医院内分泌科,北京100853

出　　处：《生物技术通讯》2013年第1期41-44,共4页Letters in Biotechnology

基　　金：国家自然科学基金(30872939;31100604)

摘　　要：目的:克隆p53基因的启动子,插入萤光素酶报告基因载体,并检测启动子活性。方法:采用PCR技术从人肝癌细胞系HepG2基因组中扩增人p53启动子,插入萤光素酶报告基因载体pGL4.0-empty,将重组质粒转染293T、ZR75-1、HepG2、A549细胞,测定p53启动子的转录活性。结果:构建了p53启动子的萤光素酶报告基因;通过测序及质粒酶切鉴定,所构建的p53启动子正确;活性实验表明,报告基因在多种细胞中显示构建的p53启动子活性,并呈现一定的剂量效应;转录因子USF能以剂量效应方式提高p53报告基因的转录活性。结论:克隆了人p53启动子,为进一步研究调控p53的转录因子奠定了基础。Objective： To clone p53 promoter and insert it into a luciferase reporter gene vector, and to charac- terize its promoter activity. Methods： p53 promoter was obtained by PCR from human liver cancer cell line HepG2 genomic DNA and cloned into pGL4.0-empty vector. After transfection of recombinant plasmid into 293T, ZR75-1, HepG2 and A549 cells, the activity of the p53 luciferase reporter was detected by luciferase reporter as- say. Results： The p53 luciferase reporter plasmid was successfully constructed and was proved to be correct by en- zyme digestion and sequencing. Luciferase assay showed that the activity of p53 promoter could be induced in dif- ferent cell lines in a dose dependent manner and this activity could be increased by the transcription factor USF. Conclusion： The p53 promoter was cloned, which will be used in studying p53 transcription factor function.

关 键 词：p53启动子 萤光素酶报告基因 转录活性 

分 类 号：Q78[生物学—分子生物学] Q75