免疫沉淀联合质谱筛选与纳尔逊湾正呼肠孤病毒σNS互作的宿主RNA结合蛋白

Screening of host RNA binding proteins interacting with Nelson Bay reovirus σ NS by immunoprecipitation combined with mass spectrometry

目的筛选出宿主细胞中与纳尔逊湾呼肠孤病毒(NBV)非结构蛋白σNS互作的RNA结合蛋白并分析其生物信息学功能。方法本研究通过构建NBVσNS真核表达载体pEF-HA-MB-S3,验证正确后转染HEK293T细胞,所得的细胞蛋白裂解液经RNase A处理后,利用免疫沉淀富集σNS结合蛋白,经LC-MS/MS质谱分析技术鉴定分析互作蛋白,并且借助相关生信工具挖掘蛋白性质和具体功能。结果本实验成功筛选出32个与NBVσNS蛋白存在互作的候选RNA结合蛋白;生物分析结果显示,这些蛋白主要定位于细胞质与细胞核中,并且主要参与细胞代谢、生物调控、病毒翻译与转录等生物学过程。结论本研究初步分析与NBVσNS互作的RNA结合蛋白功能,为深入研究σNS蛋白在NBV生命周期中的作用机制奠定基础。

Objective To screen the RNA binding proteins interacting with NBV nonstructural proteinσNS in host cells and to analyze their bioinformatics functions.Methods In this study,the eukaryotic expression vector pEF-HA-MB-S3 of NBVσNS was constructed and transfected into HEK293T cells after verification.After RNase A treatment,the obtained protein lysate was enriched by immunoprecipitation to enrichσNS binding proteins,identified and analyzed by LC-MS/MS mass spectrometry,and the properties and specific functions of proteins were discovered with the help of related Bioinformatics tools.Results In this study,32 candidate RNA binding proteins interacting with NBVσNS proteins were successfully screened,and the results of bioanalysis showed that these proteins were mainly located in cytoplasm and nucleus,and were mainly involved in biological processes such as cell metabolism,biological regulation,virus translation and transcription.Conclusion This study preliminarily analyzes the function of RNA binding proteins interacting with NBVσNS,which lays a foundation for further study on the mechanism ofσNS protein in NBV life cycle.