轮状病毒NSP1蛋白的泛素连接酶活性研究

Study on the ubiquitin ligase activity of rotavirus NSP1 protein

目的 通过实验确证轮状病毒（Rotavirus,RV）非结构蛋白1（NSP1）的泛素连接酶活性,为阐明其在RV复制和致病机制中的作用提供线索.方法 将NSP1基因及其RING结构域缺失突变体构建到pEGFP-C1质粒上,与表达泛素的质粒pBlue-Script-HA-Ubiquitin共转染人胚肾293FT细胞,用免疫荧光和Western blot方法 确定蛋白的表达,并通过免疫共沉淀方法 分析蛋白的泛素化.结果 NSP1蛋白的表达可增加细胞内的泛素化水平,并且自身也发生泛素化.结论 NSP1蛋白具有E3泛素连接酶活性.可能在泛素化调控机制中发挥作用.

Objective To confirm the activity of non structural protein 1 （ NSP1 ） of Rotavirus （RV） as E3 ubiquitin ligase by experiments and to provide some clues for NSP1 on the pathogenic mechanisms and replication of RV. Methods The whole gene and RING deleted mutation gene of NSP1 were coloned into pEGFPC1expression plasmid, and transfected into human embryonic kidney （HEK） 293 FT cells with pBlue-Script-HA-Ubiquitin. The expression of proteins were proved by using con-focal microscope and western blotting. The ubiquination of proteins were detected by co-immunoprecite. Results The cellular proteins of HEK293FT are ubiquinated by NSP1 protein and NSP1 protein was selfubiquinated also. Conclusions It revealed that RV NSP1 had the activity of E3 ubiquitin ligase and it may play a role on the modulate mechanisms of ubiquination.