突变型人TRBP基因的构建及表达

Construction and Expression of Mutant TRBP Gene

目的:探讨RNAi过程中TRBP传递双链RNA的机制,构建RNA结合结构域突变的人TRBP基因真核表达载体。方法:设计突变引物,通过拼接PCR将突变型TRBP基因克隆入pFLAG-CMV4真核表达载体,经双酶切和测序鉴定正确后,命名为pFLAG-CMV4-TRBPm。瞬时转染HEK-293细胞,western-blot检测目的蛋白表达。结果:成功扩增了突变序列,构建了突变型TRBP基因的真核表达载体,转染HEK-293细胞后检测到带标签的目的蛋白表达。结论:突变型TRBP基因真核表达载体的成功构建,为进一步研究TRBP的生物学效应奠定了基础。

Objective： To investigate the mechanism that TRBP deliver dsRNA in the process of RNAi,and to construct the eu-karyotic expression vector carrying mutant TRBP.Methods： Primers for dsRBD domain mutation were designed,and gene splicing by overlapping extension PCR was used to obtain mutant TRBP.Then it was cloned into the Flag-labeled eukaryotic expression vector pFLAG-CMV4,and named pFLAG-CMV4-TRBPm after verified by double-enzyme and sequence detection.After transient transfect into HEK-293 cells,the expression of target gene was detected by Western Blot.Results： The mutant TRBP cDNA was amplified and the Flag-labeled eukaryotic expression vector pFLAG-CMV4-TRBPm which encoding mutant TRBP was constructed.Expression of mutant TRBP was successfully detected by western blot using anti-FLAG monoclonal antibody and anti-TRBP polyclonal antibody respectively.Conclusion： Flag-labeled pFLAG-CMV4-TRBPm vector was successfully constructed,which is of significance for further research on biological function of TRBP.