Puma荧光素酶报告基因的构建和鉴定

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。

Puma luciferase reporter gene construction and identification

Objective To study the mechanism of p55 inducing cell apoptosis, the 180 bp fragment of Puma promoter was cloned into the pGL3-basic luciferase reporter vector. The biological activity of Pumareporter 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.