pIRES-EGFP-GRK2-S670A突变质粒真核表达载体的构建与表达

Construction and expression of the eukaryotic expressing vector of p IRES-EGFP-GRK2-S670A mutants recombinant plasmids

GRK2-S670A突变体导入pI RES-EGFP真核表达载体,为寻找GRK2磷酸化GPCR的位点提供研究基础。利用PCR定点突变试剂盒,获得pG EM-T-GRK2-S670A,用Sal I/Apa I分别对pG EM-T-GRK2-S670A和EGFP-C3双酶切,构建EGFP-C3-GRK2-S670A,Sal I/BamH I双酶切EGFP-C3-GRK2-S670A和pI RES-EGFP,得到pI RES-EGFP-GRK2-S670A。将构建的质粒转染HEK293细胞,荧光显微镜下观察和Western blot法检测融合蛋白的表达。酶切鉴定显示pI RES-EGFP-GRK2-S670A质粒条带大小符合,测序结果正确,成功构建pI RES-EGFP-GRK2-S670A真核表达质粒,转染HEK293细胞后可见融合蛋白表达,为后续研究奠定基础。

The S670A research foundation of mutation of GRK2 was imported to the plRES-EGFP eukaryotic expression vector, providing GRK2 phosphorylation GPCR site （s）. TaKaRa mutanBEST Kit was adopted to obtain pGEM-T-GRK2-S670A, pGEM-T-GRK2-S670A and EGFP-C3 plasmids were double-digested by Sal I/Apa I, to construct EGFP-C3-GRK2-S670A plasmid. Then EGFP-C3-GRK2-S670A and pIRES-EGFP were double-digested by Sal I/BamH I, in order to build a complete eukaryotic expression plasmid plRES-EGFP-GRK2-S670A. The plasmid was transfected in HEK293 ceils and recombinant protein was detected with fluorescence microscope and Western blot assays. The results of digestion and DNA sequencing of the plasmid were correct, eukaryotic ex- pressed vector pIRES-EGFP-GRK2-S670A mutant recombinant plasmid was successfully constructed, and the re- combinant protein was expressed in HEK293 cell lines after cell transfection, which may be the foundation of the following research.