Mpl相互作用蛋白RACK1的筛选与鉴定  

Screening and identification of a new Mpl-interacting protein, RACK1

作　　者：刘红岩[1] 从玉文[1] 陈波[1] 善亚君[1] 杨振[1] 赵振虎[1] 毛秉智[1] 

机构地区：[1]军事医学科学院放射医学研究所,北京100850

出　　处：《军事医学科学院院刊》2005年第3期204-207,共4页Bulletin of the Academy of Military Medical Sciences

摘　　要：目的:筛选新的血小板生成素(TPO)受体胞内区相互作用蛋白并确定相互作用。方法:以TPO受体胞内区为诱饵蛋白,进行酵母双杂交文库的筛选,RT-PCR扩增筛选到的基因并克隆到相应载体上,Western印迹方法检测其在不同组织与细胞内的表达;进行哺乳动物细胞内双杂交、免疫共沉淀、激光共聚焦实验确证相互作用。结果:从酵母双杂交文库筛选到RACK1基因,检测到其在不同组织与细胞内表达都很丰富,哺乳动物细胞内双杂交、免疫共沉淀证明RACK1同TPO受体Mpl的胞内区存在相互作用,而细胞内共定位实验证明两者在哺乳动物细胞内存在共定位现象,即两蛋白的相互作用可能具有生理意义。结论:筛选到的RACK1蛋白同TPO受体Mpl的胞内区存在相互作用。Objective:To find new proteins that interact with Mpl. Methods: Yeast two-hybrid screen was performed to find proteins associated with the cytoplasmic domain of Mpl. One of the identified gene, RACK1, was multiplied by reverse transcription polymerase chain reaction (RT-PCR) and subcloned into corresponding vectors. The interaction of the two proteins was testified by co-immunoprecipitation, mammalian two-hybrid assay and co-localization in cells. The expression of RACK1 in mouse tissues and several cell lines was tested by Western blotting. Results: A protein named RACK1 was screened out in the yeast two-hybrid experiment using cytoplasmic domain of Mpl as a bait. RACK1 is widely expressed in mouse tissues and cell lines. Using laser confocal scanning microscopy, the cytoplasmic localization of RACK1 was found to be related to Mpl, and the interaction was confirmed by Co-IP and mammalian two-hybrid assay. Conclusion: RACK1 is a new Mpl-interacting protein.

关 键 词：MPL RACK1 双杂交系统技术 相互作用 血小板生成素 

分 类 号：Q51[生物学—生物化学]