GST pull-down联合质谱筛选丙型肝炎病毒蛋白NS5A结构域Ⅰ的相互作用蛋白

Screening interacting proteins for HCV NS5A domains Ⅰ protein by GST pull down coupled with LC-MS/MS

目的对1b型丙型肝炎病毒Con1株的NS5A结构域Ⅰ宿主相互作用蛋白进行鉴定分析,为研究NS5A功能及其参与病毒复制机制提供理论基础。方法构建pGEX-4T-1-Con1-NS5A-35-215质粒,诱导表达GST-Con1-NS5A-35-215融合蛋白并纯化。采用GST pull-down联合液相色谱-串联质谱(LC-MS/MS)对其相互作用蛋白进行鉴定,并使用OmicsBean在线软件对Con1-NS5A-35-215相互作用蛋白进行GO功能富集分析和信号通路分析。结果在Huh7.5.1细胞裂解液中鉴定到547个潜在相互作用蛋白。GO功能富集分析发现相互作用蛋白主要参与细胞运输、应激反应、凋亡、蛋白泛素化等生物学过程。信号通路分析发现相互作用蛋白主要参与蛋白酶体、糖代谢、氨基酸合成、RNA转运等通路。通过免疫印迹验证了Con1-NS5A-35-215可与ACBD3和Vps35结合。结论 NS5A结构域Ⅰ通过其相互作用蛋白参与多种生物学过程,如调控细胞内运输、应激反应、蛋白稳态等过程。与ACBD3和Vps35相互作用的NS5A的片段缩短为35-215。

Objective To explore the roles of the hepatitis C virus（HCV）NS5Adomains Ⅰ protein and its involvement in the virus replication mechanism. Methods GEX-4T-1-Con1-NS5A-35-215 plasmid was constructed,and the expression of GST-Con1-NS5A-35-215 fusion protein was induced and purified.GST pull down coupled with LC-MS/MS were performed to screen the Con1-NS5A-35-215 associated proteins in cytosolic extracts from Huh7.5.1cells.The GO（Gene Ontology）annotation and pathway enrichment of Con1-NS5A-35-215 interacting proteins were analyzed by using OmicsBean online software. Results A total of547 potential Con1-NS5A-35-215 interacting proteins were identified.GO（Gene Ontology）annotation analysis shows that these interacting proteins function in regulation of responses to stress,apoptotic process,lipid metabolic process,Golgi vesicle transport,regulation of protein ubiquitination,protein transport.Ⅰn particular,KEGG（Kyoto Enyoolpedia of genes and Genomes）pathway enrichment analysis reveals that these interacting proteins are involved in several important pathways,including Proteasome,Proteasome,Biosynthesis of amino acids,Fatty acid degradation,RNA transport. Conclusions NS5 Adomain Ⅰ of HCV is involved in many biological processes such as the regulation of intracellular transport,stress response,protein homeostasis and so on.By immunoblotting experiments,the interaction of NS5A-Con1-35-215 with ACBD3 and VPS35 were verified,which provided a clue for further study of NS5 Ainvolvement in HCV replication.