Prelamin A相互作用蛋白的筛选及验证
Journal of Sun Yat-sen University(Medical Sciences)(2015)
Abstract
[目的]筛选衰老相关蛋白prelaminA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cDNA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RanBPM与绿色荧光蛋白融合表达载体pEGFP-N1-RanBPM,与红色荧光蛋白-prelamin A融合表达质粒pDsRed1-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RanBPM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RanBPM能与带红色荧光蛋白的prelaminA蛋白在核膜共定位.[结论]RanBPM可能通过与Prelamin A发生相互作用参与了细胞衰老的过程.
MoreKey words
prelamin A,RanBPM/RanBP9,yeast two-hybrid,HGPS
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