Puma luciferase reporter gene construction and identification / 肿瘤研究与临床

ABSTRACT

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.