Puma荧光素酶报告基因的构建和鉴定  

Puma luciferase reporter gene construction and identification

作　　者：杨新[1] 邱实[1] 顾寿智 蔡云[1] 高兴[1] 刘泽军[1] 

机构地区：[1]第三军医大学西南医院西南癌症中心,重庆400038 [2]Japan, School of Rehabilitation Sciences, Seirei Christopher University

出　　处：《肿瘤研究与临床》2011年第1期8-10,共3页Cancer Research and Clinic

基　　金：国家自然科学基金（30971140）;重庆市自然科学基金（CSTC,2008BA5003）

摘　　要：目的为研究p53家族调控细胞凋亡的机制，构建并鉴定插入人Puma基因的启动子片段的荧光素酶报告基因载体。方法采用PCR技术从人类HepG2细胞中扩增Puma启动子的含p53反应元件的180bpDNA片段，插入荧光素酶报告基因载体pGL3-basic中。经测序确定所扩增的DNA序列；将其转染人人类肺腺癌141299细胞中，双报告基因实验检测荧光素酶活力。结果测序结果表明扩增的Puma启动子序列正确，活性检测显示构建的报告基因具有启动子活性。结论克隆了含p53反应元件的Puma启动子，成功构建了人类Puma启动子报告基因，为p53家族凋亡通路的功能研究提供了必要的实验材料。Objective To study the mechanism of p53 inducing cell apoptosis, the 180 bp fragment of Puma promoter was cloned into the pGL3-basic luciferase reporter vector. The biological activity of Puma reporter plasmid was verified by cell transfection. Methods The target fragments of Puma were amplified by RT-PCR method and the fragments were inserted into the pGL3-basic luciferase reporter vector. The acquired Puma-Luc plasmid was transfected into H1299 cell line and detected its activity. Results Sequencing indicated that the amplified Puma promoter is correct. Dual-luciferase Reporter Assay showed the Puma-Luc constructs have promoter activity. Conclusion The cloning of human Puma gene promoter and the construction of its reporter vector were successful. This study will lay the foundation for further research on the function of p53 inducing apoptosis through mitochondrial pathway.

关 键 词：PUMA 启动子 荧光素酶 报告基因 凋亡 

分 类 号：R346[医药卫生—基础医学]