运用新型的反向PCR策略高效构建基因的点突变体  

Site-directed Mutagenesis by Novel Inverse PCR Strategy

作　　者：程龙[1] 韩白玉[1,2] 侯莎[1] 韩永健[1] 徐小洁[1] 蒋凯[1] 李法曾[1] 杨智洪[1] 窦京涛[2] 吕朝晖[2] 张浩[1] 叶棋浓[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100850 [2]解放军总医院内分泌科,北京100853

出　　处：《生物化学与生物物理进展》2011年第4期378-382,共5页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金(30800205);军队医药卫生杰出人才基金(06J021);"重大新药创制"科技重大专项(2009ZX09301-002);北京市自然科学基金(7112101)资助项目~~

摘　　要：基因的点突变体在其结构和功能研究中发挥非常关键的作用,如何高效、经济地构建基因点突变体是许多分子生物学研究遇到的棘手问题.以PIAS3点突变体的构建为对象,设计了新型的以反向PCR为基础的点突变构建流程,获得的点突变体质粒经测序后均与预期相符,并在293T细胞内得到了正确表达.以上结果表明,该实验设计方案能够高效、方便地用于基因点突变体的构建,为进一步研究它们的分子功能打下了基础.Site-directed mutagenesis plays important roles to study protein structure-function relationship and the researchers are always confronted with the problem on how to generate a point mutation of a target gene efficiently.A novel strategy to produce a point mutation was depicted based on inverse PCR with PIAS3 as an example.Firstly,the entire PIAS3 coding sequence was amplified from MCF-7 cDNA and then cloned into the expression vector pXJ40-myc.Two sets of PIAS3 primers with specific mutation sequences were synthesized and then phosphorylated at their 5′ terminus.Inverse PCR was performed with the phosphorylated primers as well as with the plasmid pXJ40myc-PIAS3 as the template.Further,the PCR products were subjected to DpnⅠ treatment,agarose gel purification,self-ligation and transformation into DH5α.Three colonies were randomly selected for DNA sequencing.The expression of both the PIAS3 wide type and the point mutants（PIAS3 K110R and K411,412R） was analyzed by transfection of these plasmids into 293T cells.The result showed that the PIAS3 K110R and K411,412R point mutants were successfully constructed and expressed in mammalian cells,which suggested that the novel inverse PCR stratagy can be applied to construct the point mutants of a target gene efficiently and conveniently.

关 键 词：反向PCR PIAS3 点突变 

分 类 号：Q52[生物学—生物化学]