Objective: To investigate whether human RhoA is modified by SUMO.
Methods: Overlap extension PCR and double digestion technique were used to construct the eukaryotic expression vector pcDNA3-3flag-RhoA, which was identified by sequencing. The plasmid was transfected into HEK293T cells and its expression was detected by Western blotting. Immunofluorescence assay was used to detect whether RhoA is co-localized with SUMO. Co-Immunoprecipitation was used to detect whether RhoA is modified by SUMO.
Results: The recombinant plasmid pcDNA3-3flag-RhoA was successfully constructed and verified. Western blotting showed that the recombinant plasmid pcDNA3-3flag-RhoA expressed abundant fusion protein in HEK293T cells. Immunofluorescence showed that RhoA was co-localized with SUMO2/3 but not with SUMO1. Co-immunoprecipitation verified that RhoA was modified by SUMO2/3 but not SUMO1.
Conclusions: Human RhoA is modified by SUMO2/3 and probably participates in the regulation of axon regrowth after nervous system injury.
目的: 探讨人RhoA是否发生SUMO化修饰。
方法: 运用重叠延伸PCR和双酶切连接方法构建pcDNA3-3flag-RhoA真核表达载体，测序验证。将重组RhoA质粒转染进HEK293T细胞中，免疫印迹检测质粒的表达。将重组RhoA质粒分别和SUMO各型质粒共转染进HEK293T细胞中，细胞免疫荧光检测RhoA与SUMO之间是否存在共定位。免疫共沉淀方法检测RhoA是否发生SUMO化修饰。
结果: 成功构建pcDNA3-3flag-RhoA重组质粒，测序结果提示存在一个同义突变，其余完全正确；免疫印迹检测重组RhoA质粒能高效表达融合蛋白；免疫细胞化学检测到RhoA与SUMO2/3存在共定位，RhoA与SUMO1不存在共定位；免疫共沉淀检测SUMO2/3对RhoA发生修饰作用，SUMO1对RhoA未发生修饰作用。
结论: 人RhoA发生了SUMO2/3参与的SUMO化修饰，可能参与神经系统损伤后轴突再生的调控。
Keywords: RhoA; SUMOylation.