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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 the promoter of p53 gene, insert the luciferase reporter gene vector and detect the promoter activity. Methods: The human p53 promoter was amplified by PCR from HepG2 cell line. The luciferase reporter gene vector pGL4.0-empty was inserted into 293T, ZR75-1, HepG2 and A549 cells. The transcriptional activity of the p53 promoter was measured. Results: The luciferase reporter gene of p53 promoter was constructed. The p53 promoter was constructed correctly by sequencing and plasmid restriction enzyme digestion. The activity assay showed that the reporter gene displayed the constructed p53 promoter activity in various cells, And showed a dose-effect; transcription factor USF can increase the transcriptional activity of p53 reporter gene in a dose-response manner. Conclusion: The human p53 promoter was cloned, which laid the foundation for further study on the transcription factor that regulates p53.