人SUMO3基因突变体(SUMO3-K11R)真核表达质粒的构建及其鉴定

Construction and characterization of human SUMO3 mutant (SUMO3-K11R) eukaryotic expression vector

构建人SUMO3基因K11R突变体的真核表达质粒并对其进行功能鉴定。方法:PCR扩增人SUMO3基因,将其克隆入真核表达载体pEGFP-C1内,构建含人SUMO基因K11R突变体的真核表达质粒pEGFP-SUMO3-K11R。使用真核转染,Western blot和免疫荧光实验的方法,鉴定SUMO-K11R在真核细胞中的表达和功能。结果:克隆的人SUMO-K11R基因,测序结果显示完全正确。瞬时转染真核细胞后,Western blot在预期的位置检测出目的条带,免疫荧光实验显示SUMO3-K11R突变体蛋白其功能与SUMO1蛋白相似,可作为研究SUMO1蛋白和SUMO3蛋白功能差异的有力工具。结论:成功构建了含人SUMO-K11R基因的真核表达质粒。

Objective:To construct and characterize a eukaryotic system for expressing human SUMO3 gene mutant(SUMO3-K11R).Methods:Human SUMO3 mutant gene(SUMO3-K11R) was amplified by PCR,and subcloned into pEGFP-C1 vector to construct a recombinant plasmid pEGFP-C1-SUMO3-K11R,The expression of human SUMO3 mutant gene(SUMO3-K11R) in eukaryotic cells was verified by Western blot.Immunofluorescence assay was used to detect the localization of SUMO3-K11R with/without MG132 treatment in eukaryotic cells.Results:The sequence of inserted SUMO3 mutant(SUMO3-K11R) gene fragment detected by sequencing was entirely correct.After transfection of eukaryotic cells with pEGFP-C1-SUMO3-K11R,the target band was identified by Western blot.Furthermore,the localization of SUMO3-K11R was similar to SUMO1 under MG132,but not SUMO3.So SUMO3-K11R might be a useful tool for investigating the function difference between SUMO1 and SUMO3.Conclusion:A functional eukaryotic expression plasmid pEGFP-C1-SUMO3-K11R has been established successfully.