TfR⁃Ubi融合蛋白稳定表达株的构建及功能初测
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国家自然科学基金(31171289)


Construction and functional analysis of TfR⁃Ubi fusion protein
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    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。

    Abstract:

    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.

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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(自然科学版),2018,(7):909-914

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  • 收稿日期:2017-11-14
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  • 在线发布日期: 2018-07-20
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