[1]万滢聪,李春燕,佘佳瑶,等.人RhoA发生了SUMO化修饰[J].南方医科大学学报,2018,(01):75.
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人RhoA发生了SUMO化修饰()
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《南方医科大学学报》[ISSN:1673-4254/CN:44-1627/R]

卷:
期数:
2018年01期
页码:
75
栏目:
出版日期:
2018-01-20

文章信息/Info

Title:
Human RhoA is modified by SUMO2/3
作者:
万滢聪李春燕佘佳瑶王靖雅陈明
关键词:
RhoASUMO化修饰
Keywords:
RhoA SUMOylation
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.
更新日期/Last Update: 1900-01-01