突变型人TRBP 基因的构建及表达

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。

Construction and Expression of Mutant TRBP Gene

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.