关键词: 显性负突变, 存活素, 重叠PCR

Abstract: Objective To construct dominant negative mutant plasmid of survivin. Methods Total RNA was extracted from rat cardiac microvascular endothelial cells and reverse transcripted into cDNA. Survivin gene was amplified and the restriction enzyme cutting sites were added using nested PCR, followed by site-specific mutagenesis of survivin by overlap PCR. The product was reconstructed into eukaryotic expression plasmid pEGFP-N3 and sequence analysis was used to examine the recombinant. Cells were transfected with the recombinant and cell apoptosis was then estimated. Results PCR-amplified 496 bp fragment coding for survivin and 452 bp fragment containing restriction enzyme cutting sites were obtained using nested PCR. Site-specific mutagenesis of 452 bp survivin fragment was gained after three PCR reactions by overlap extention. The recombinant was identified by sequence analysis. Transfected cells emitted bright green fluorescence and underwent severe apopotosis compare with control cells. Conclusion The dominant negative plasmid pEGFP-DN-survivin was successfully constructed.

Key words: dominant negative, survivin, overlap PCR