利用酵母双杂交研究与Ca^(2+)CRAC通道Orail相互作用蛋白

Identification of Orail Interacting Proteins by Yeast Two-hybrid Screen

目的:利用酵母双杂交等技术研究与CRAC通道蛋白Orail发生相互作用的蛋白质,从而揭示CRAC通道分子作用机制。方法:以Orail-C端为诱饵蛋白筛选大鼠海马和皮质cDNA文库,寻找能与之相互作用的蛋白质,运用营养缺陷型培养,X-β-Gal蓝斑筛选等实验去除假阳性克隆,并通过GST-Pulldown体外实验验证其相互作用。结果:以Orai1-C端为诱饵最终筛出了几个阳性克隆,经测序分析,发现了其中一个感兴趣的蛋白。结论:Orail可以和该蛋白发生相互作用,其相互作用可能与CRAC通道功能调节有关。

Objective：To investigate proteins interacting with CRAC channel protein Orail by using Yeast two hybrid.Methods：Yeast two hybrid was applied to screen rat cortex and hippocampus cDNA library,and Orail C domain as the bait.Using auxotroph culture and X-β-Gal assay to removal the false positive colony,and checking the interaction using experiment in vitro of GST-Pulldown.Results：we find a protein which can interact with Orail.Conlusion：Orail can interact with this protein.Their interaction may suggest a new molecular mechanism to regulate the function of CRAC channels.