一步法定点突变技术快速构建bsh基因突变启动子

Construction of bsh Promoter Mutants by Rapid One-step Site-directed Mutagenesis

目的:建立一种高效便捷的定点突变方法,为基因表达调控以及蛋白质结构和功能的研究提供技术支撑。方法:以构建单核细胞增生李斯特菌(Listeria monocytogenes)中编码胆碱水解酶(bile salt hydrolase,BSH)的bsh基因突变启动子为例,采用一对完全互补并带有突变位点的引物扩增携带bsh基因启动子的重组质粒DNA全序列,通过DpnⅠ消化PCR产物中剩余的甲基化的模板DNA,酶切后的PCR产物直接转化大肠杆菌,从而获得含有突变启动子的重组质粒。结果:通过一步法定点突变技术成功构建了bsh基因的三种突变启动子。结论:该方法简单高效,只要把握好对引物设计,高保真的DNA聚合酶、模板DNA的浓度以及PCR扩增程序的选择,突变成功率可以达到100%。

Objective： A convenient and rapid site-directed mutagenesis method was established for study regulation of gene expression as well as relationship between protein structure and function.Method：The construction of bsh（encoding bile salt hydrolase in Listeria monocytogenes） promoter mutants was used as a sample in this study.A pair of completely complementary primers with mutation sites in the middle was used to amplify the total recombinant plasmid DNA sequences.After digestion of residual methylated template DNA in the PCR products by DpnⅠ,PCR products without purification were directly transformed into E.coli to contain mutations of the recombinant plasmid.Result： Three bsh promoter mutants were successfully constructed by the one-step site-directed mutagenesis technology.Conclusion： The method used in this study is robust and fast.As long as primer design,high-fidelity DNA polymerase,template DNA concentration,and PCR amplification procedure are optimized,the successful rate of mutation could reach 100%.