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【目的】利用聚合酶链反应 (PCR)技术对Wilson病 (WD)基因进行体外定点突变的研究。【方法】采用PCR定点突变技术 ,首先设计两对引物 ,将突变位点设计在引物上 ,通过重叠延伸法两次PCR扩增 ,扩增片段上含有所需要的突变位点 ,最后将扩增片段克隆入pRc/CMV载体中。【结果】DNA测序表明在预期位点已经发生突变 ,WD基因第 778位密码子由精氨酸 (Arg)残基突变为亮氨酸残基 (Leu) ,用PCR定点突变技术成功构建Wilson病基因突变体。【结论】PCR技术诱导定点突变准确、高效。Wilson病基因突变体的构建成功 ,为进一步研究该突变位点导致Wilson病的发病机制和Wilson病蛋白的结构和功能的关系奠定了基础。
【Objective】 Polymerase chain reaction (PCR) was used to study the site-directed mutagenesis of Wilson’s disease (WD) gene in vitro. 【Method】 PCR-based site-directed mutagenesis was used to design two pairs of primers. The mutation sites were designed on the primers and amplified by overlap PCR twice. The amplified fragments contained the required mutation sites and finally amplified The fragment was cloned into the pRc / CMV vector. 【Result】 DNA sequencing showed that mutation had occurred at the expected site. The codon 778 of WD gene was mutated from arginine (Arg) to leu (Leu), and Wilson ’s disease was successfully constructed by PCR site - directed mutagenesis Mutant. 【Conclusion】 PCR-induced site-directed mutagenesis is accurate and efficient. The successful construction of Wilson’s disease gene mutants laid the foundation for further study on the relationship between the pathogenesis of Wilson’s disease and the structure and function of Wilson’s disease protein.