文章摘要
沈婷婷,刘宁生.TfR⁃Ubi融合蛋白稳定表达株的构建及功能初测[J].南京医科大学学报,2018,(7):909~914
TfR⁃Ubi融合蛋白稳定表达株的构建及功能初测
Construction and functional analysis of TfR⁃Ubi fusion protein
投稿时间:2017-11-14  
DOI:10.7655/NYDXBNS20180708
中文关键词: 胞内体分选  泛素  转铁蛋白受体  降解
英文关键词: endosome sorting  ubiquitin  transferrin receptor  degradation
基金项目:国家自然科学基金(31171289)
作者单位
沈婷婷 南京医科大学病理学系江苏 南京 211166 
刘宁生 南京医科大学病理学系江苏 南京 211166 
摘要点击次数: 1053
全文下载次数: 666
中文摘要:
      目的:通过构建转铁蛋白受体?泛素(transferrin receptor?ubiquitin,TfR?Ubi)融合蛋白,研究胞内体蛋白分选机制。方法:首先扩增小鼠泛素和人源转铁蛋白受体基因,构建HA?TfR?Ubi表达质粒,转染A431细胞后,采用Western blot验证融合蛋白的表达。在稳定表达TfR?Ubi的A431和Hrs?KO MEF细胞株中,转铁蛋白刺激,免疫荧光法观察TfR?Ubi融合蛋白在细胞内的位置。结果:成功构建HA?TfR?Ubi表达质粒,免疫荧光结果提示TfR?Ubi融合蛋白被分选到晚期内体(late endosome)/溶酶体,Hrs?KO MEF细胞中TfR?Ubi融合蛋白分选受到影响。结论:TfR?Ubi融合蛋白在胞内体被分选,证明了泛素修饰改变了转铁蛋白受体的降解方式,为胞内体分选转运装置(endosomal sorting complex required for transport,ESCRT)分选机制研究提供了实验工具。
英文摘要:
      Objective:To investigate the mechanism of endosomal sorting by constructing the transferrin receptor?ubiquitin(TfR?Ubi)protein. Methods:Mouse ubiquitin and human transferrin receptor genes were amplified to construct HA?TfR?Ubi plasmid. Then the expression of HA?TfR?Ubi protein was confirmed by Western blot following by transfected into A431 cells. The A431 and Hrs?KO MEF cells stably expressing HA?TfR?Ubi were treated with transferrin,and the location of HA?TfR?Ubi fusion protein was observed in the cells by immunofluorescence. Results:pHA?TfR?Ubi was successfully constructed. Immunofluorescence results showed that HA?TfR?Ubi fusion protein was sorted into late endosome /lysosomal,and its endosomal sorting was affected in Hrs?KO MEF cells. Conclusion:The endosomal sorting of TfR?Ubi fusion protein demonstrated that the ubiquitin label changed the transferrin receptor degradative pathway. TfR?Ubi plasmid provided a useful experimental tool for the study of ESCRT sorting mechanism.
查看全文   查看/发表评论  下载PDF阅读器