人p53启动子萤光素酶报告基因的构建及其活性测定

摘要

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.%　　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.