猪脑心肌炎病毒2B蛋白与RACK1蛋白相互作用的鉴定

Identification of the interaction between 2B protein of encephalomyocarditis virus and RACK1

为筛选与猪脑心肌炎病毒(EMCV)2B蛋白相互作用的宿主蛋白,本实验利用酵母双杂交方法筛选BHK-21细胞表达文库制备与EMCV 2B蛋白相互作用的宿主蛋白RACK1蛋白。酵母共转化试验和免疫共沉淀试验表明两者可以特异性结合,激光共聚焦试验表明两者共定位于细胞质中。为定位RACK1与2B蛋白相互作用区域,本研究构建了RACK1截短表达重组质粒,并将其与pCAGGS-Flag-2B共转染HEK293细胞,利用免疫共沉淀试验验证其相互作用区域。结果显示2B蛋白与宿主细胞RACK1蛋白存在相互作用,并且证明相互作用区域位于RACK1蛋白的N端。

To screen the host proteins interacted with the 2B protein of encephalomyocarditis virus （EMCV）, we scanned the BHK-21 expression library using EMCV 2B protein as bait protein by yeast-two-hybrid, and found a potential interacting partner protein of RACK1. The interaction of the 2B and RACK1 proteins was further confirmed by co-transformation in the yeast and Co-IP assay in co-transfected HEK293 cells. In addition, they were co-localized in the cytoplasma of co-transfected HEK293 cells examined by laser confocal assays. To map the interacting domain of RACK1, a series of RACK1 truncated eukaryotic expression recombinant plasmids were construct and co-transfected with pCAGGS-Flag-2B into HEK293 cells, respectively, which were subjected to Co-IP and detected by western blot. The results suggested that EMCV 2B interacted with N terminal of RACK1.