Prelamin A相互作用蛋白的筛选及验证（英文）

Screening and Validation of Prelamin A Interacting Proteins

【目的】筛选衰老相关蛋白prelamin A的相互作用蛋白并验证其相互作用情况。【方法】通过酵母双杂交方法从人骨骼肌c DNA文库中筛选prelamin A的相互作用蛋白。一对一回复性杂交,GST pull-down,免疫共沉淀和Western blotting验证其在细胞外的相互作用,构建Ran BPM与绿色荧光蛋白融合表达载体p EGFP-N1-Ran BPM,与红色荧光蛋白-prelamin A融合表达质粒p Ds Red1-N1-prelamin A.共转染HEK293细胞,激光共聚焦显微观察细胞内的共定位情况。【结果】筛选得到包括Ran BPM在内的21个候选相互作用蛋白。一对一回复性杂交,GST pull-down,免疫共沉淀和Western blotting证明两者能在细胞外相互作用。激光共聚焦显微观察发现Ran BPM能与带红色荧光蛋白的prelamin A蛋白在核膜共定位。【结论】Ran BPM可能通过与Prelamin A发生相互作用参与了细胞衰老的过程。

【Objective】 To screen and validate prelamin A interacting proteins. 【Methods】 The yeast two-hybrid system were employed for prolamin A interacting proteins screening using human skeletal muscle c DNA library. One potential partner of Ran BPM was further validated by in vitro GST pull-down and in vivo one to one reverse hybridization and co-immunoprecipitation approaches.Their colocalization was tested by laser confocal microscopy using fusion expression proteins of GFP-Ran BPM and RFP-prelamin A in HEK293 cells from the constructed vectors p EGFP-N1-Ran BPM and p Ds Red1-N1-prelamin A. 【Results】 Totally, 21 potential prelamin A partners, including Ran BPM, were obtained by the yeast two-hybrid system and the interaction of Ran BPM and prelamin A were further confirmed by one to one reverse hybridization, GST pull-down, co-immunoprecipitation approches. The colocalization of the fluoresced proteins GFP-Ran BPM and RFP-prelamin A were also observed by laser confocal microscopy around nuclear envelope regions. 【Conclusions】 The protein of Ran BPM is a novel partner of prelamin A and might function in prelamin A mediated aging process.