南方医科大学学报

南方医科大学学报 ›› 2018, Vol. 38 ›› Issue (01): 75-.

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人RhoA发生了SUMO化修饰

万滢聪，李春燕，佘佳瑶，王靖雅，陈明   

  • 出版日期:2018-01-20 发布日期:2018-01-20

Human RhoA is modified by SUMO2/3

  • Online:2018-01-20 Published:2018-01-20

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摘要： 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。

Abstract: 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. Conclusion Human RhoA is modified by SUMO2/3 and probably participates in the regulation of axon regrowth after nervous system injury.