利用定点突变技术构建突变nm23-H1和EGFP融合基因

背景与目的以前的研究已经证明nm23-H1基因是肿瘤转移抑制基因，但其抑制肿瘤转移的分子机制目前还不完全清楚。基因定点突变技术可以精确地改变基因的特定碱基序列，获得突变蛋白质。本研究的目的是应用基因定点突变技术构建突变型nm23-H1-增强型绿色荧光蛋白（EGFP）融合基因，为进一步研究肿瘤抑制基因nm23-H1的功能、生化作用机制提供理论基础和实验依据。方法以逆转录病毒PLXSN-野生型nm23-H1-EGFP质粒为突变模板，应用QuikChange^TM定点突变方法，简单、快速、高效地引入nm23-H1^S44A、nm23-H1^P96S、nm23-H1^H118F、nm23-H1^S120G四个点突变和一个联合位点突变nm23-H1^P96S-S120G，构建了突变型nm23-H1-EGFP融合基因。结果成功构建了nm23-H1^S44A-EGFP、nm23-H1^P96S-EGFP、nm23-H1^H118F-EGFP、rim23-H1^S120G-EGFP、nm23-H1^P96S-S120G-EGFP五个突变型nm23-H1-EGFP融合基因，经DNA序列分析突变的碱基序列与实验设计完全一致。结论成功构建了五个具有不同突变位点的突变型nm23-H1-EGFP融合基因。QuikChange^TM定点突变技术是一种简单、快速、高效的基因定点突变方法。

Construction of human nm23-H1 mutant and EGFP fusion genes using site-directed mutagenesis

Background and objective Previous researches have proven that nm23-H1 gene is a tumor metastatic suppressor gene, however, it is still unknown about its exact molecular mechanisms. Site-directed mutagenesis can correctly change the base sequence and get mutant proteins. The aim of this study is to construct nm23-H1 mutant and EGFP fusion genes by site-directed mutagenesis, and to provide basement for studying the functional and biochemical mechanisms of nm23-H1 gene. Methods Site-directed mutagenesis of nm23-H1 gene was performed by modified QuikChange TM Site-directed Mutagenesis Kit method. Pure plasmid containing fusion gene of nm23-H1 and EGFP (PLXSN-nm23-H1-EGFP) was mini-prepared. Four pairs of mutagenic primers were synthesized in vitro and the desired five mutations, S44A, P96S, H118F, S120G and P96S-S120G were introduced into nm23-H1-EGFP fusion gene by PCR. Results Five nm23-H1 mutant and EGFP fusion genes, nm23-H1 S44A-EGFP, nm23-H1 P96S-EGFP, nm23-H1 H118F-EGFP, nm23-H1 S120G-EGFP and nm23-H1 P96S-S120G-EGFP, were successfully constructed. The results of DNA sequencing confirmed that the base sequences of the mutant fusion genes were completely concordant with experiment design. Conclusion Five nm23-H1 mutant and EGFP fusion genes are successfully constructed, which can be used in further studies. QuikChange TM site-directed mutagenesis is a simple, rapid and efficient method.