定点诱变法构建p53基因多态质粒

Construction of p53 polymorphic plasmid by site-directed mutagenesis

目的:为了研究p53codon72多态的功能,采用定点诱变法构建人p53基因的多态质粒。方法:设计带有突变碱基的引物,通过PCR扩增引入突变碱基,再将突变后的p53序列插入载体中,经测序确定所扩增的DNA序列,并用体外转录翻译系统制备p53蛋白和i ASPP蛋白,免疫沉淀法检测蛋白相互作用。结果:通过PCR获得的含突变碱基的p53序列经连接后插入真核表达载体pReceiver-M01中,构建了p53多态位点为72Arg的表达质粒pReceiver-M01-p53(Arg72)。测序证明序列正确,表达的p53蛋白能与i ASPP相互作用,具有生物学功能。结论:成功构建了p53多态(72Arg)真核表达载体,为进一步深入研究p53多态的生物学功能奠定了基础。

OBJECTIVE：To investigate the function of p53 polymorphic variants,we constructed the p53 codon 72 polymorphic plasmid.METHODS：Primers carrying the appropriate mutation were employed in a two-step PCR amplification.The mutated PCR products were subsequently cloned into pReceiver-M01 expression vector.The p53 protein translated in vitro was used to interact with iASPP to certify its biological activity.RESULTS：The sequence of the recombinant eukaryotic expression vector containing CCC to CGC mutation was proved by DNA sequencing.This p53 polymorphic variant could interact with iASPP,implying its biological activitical.CONCLUSION：The recombinant p53 eukaryotic expression vector containing codon 72 polymorphism was constructed successfully,laying a foundation for further studies on the function of p53.