人轮状病毒非结构蛋白2与宿主互作蛋白的筛选及分子对接研究  

Screening and molecular docking for host proteins interacting with human rotavirus nonstructural protein 2

作　　者：林家锋 胡思漫 蒋卓婧 赵微 李永刚 Lin Jiafeng;Hu Siman;Jiang Zhuojing;Zhao Wei;Li Yonggang(Microbiological Laboratory,Shaoxing Center for Disease Control and Prevention,Shaoxing 312071,China;Department of Pathogenic Biology,School of Basic Medical Sciences,Jinzhou Medical University,Jinzhou 121000,China)

机构地区：[1]绍兴市疾病预防控制中心微生物检验科,绍兴312071 [2]锦州医科大学基础医学院病原生物学教研室,锦州121000

出　　处：《中华微生物学和免疫学杂志》2023年第11期886-892,共7页Chinese Journal of Microbiology and Immunology

基　　金：辽宁省教育厅基本科研项目(LJKMZ20221243)。

摘　　要：目的筛选轮状病毒非结构蛋白2(nonstructural protein 2,NSP2)与宿主细胞的互作蛋白,为抗病毒靶标的发掘提供理论依据。方法NSP2-pGEX-6P-1重组质粒转化E.coli BL21(DE3),IPTG诱导表达,GST亲和层析法获得NSP2-GST纯化蛋白,Western blot进行验证。将NSP2-GST及GST纯化蛋白作为靶蛋白,通过GST pull-down捕获与MA104宿主细胞互作的差异蛋白。经银染后,将差异条带通过蛋白质胶内酶解及质谱法筛选出相应蛋白质,借助Protein Pilot平台过滤肽段,由Paragon算法获得蛋白质名称和相关的生物学功能。通过蛋白质互作网络图和GO功能注释分析,展示互作蛋白间的潜在联系。同时,利用HDOCK软件对排名前三的蛋白质进行分子对接预测,结合对接分数和置信分数加以验证,揭示蛋白质间的可视化对接模型。结果SDS-PAGE和Western blot表明NSP2重组蛋白纯化成功。经GST pull-down联合蛋白质谱鉴定,筛选出PKM2等10种可能与NSP2互作的宿主蛋白质;GO分析及互作图展示出RPS4X、EZR、SUPT16H、EIF2S3介导分子表达,PKM2、LDHA、ATP5A1参与能量代谢,HSP90、ACTB、ANXA2参与生物运动过程,并且彼此之间存在功能联系及互作网络。分子对接显示出PKM2、HSP90、RPS4X与NSP2存在互作关系,相互作用力强,能够形成稳定结构。结论成功发现宿主细胞中PKM2、HSP90、RPS4X等蛋白质与NSP2存在互作关系,为轮状病毒感染过程的探究及相关防控策略的制定提供参考。ObjectiveTo screen host cell proteins interacting with rotavirus nonstructural protein 2(NSP2)and provide a theoretical basis for the discovery of antiviral targets.Methods E.coli BL21(DE3)was transformed with a recombinant plasmid NSP2-pGEX-6P-1 and induced by IPTG to express NSP2-GST.GST affinity chromatography was used to purify NSP2-GST and Western blot was performed for verification.NSP2-GST and GST proteins were used as targets to capture interacting proteins in MA104 cells via GST pull-down.After silver staining,differential proteins were screened by in-gel enzymatic hydrolysis and mass spectrometry.Protein pilot platform was used to filter peptides,and the names and the biological functions of the proteins were obtained by Paragon algorithm.The potential connection between the interacting proteins was demonstrated by protein interaction network diagrams and GO functional annotation analysis.Moreover,the molecular docking of the top three proteins with NPS2 was predicted using HDOCK server and verified by docking and confidence scores,so as to investigate the visual docking model between interacting proteins.ResultsSDS-PAGE and Western blot showed the successful purification of the recombinant protein NSP2.Ten host proteins including PKM2,which might interact with NSP2,were identified by GST pull-down and protein profiling.GO analysis and interaction diagrams revealed that RPS4X,EZR,SUPT16H and EIF2S3 mediated molecular expression;PKM2,LDHA and ATP5A1 participated in energy metabolism;HSP90,ACTB and ANXA2 were involved in biological movement.Besides,there were functional connections and interaction networks among them.Molecular docking further verified PKM2,HSP90 and RPS4X did interact with NSP2,and the interaction force were strong enough to form a stable structure.ConclusionsThis study successfully discovered several host proteins including PKM2,HSP90 and RPS4X that could interact with NSP2,providing reference for investigating the process of rotavirus infection and making related prevention and control

关 键 词：轮状病毒 非结构蛋白2 互作蛋白 分子对接 

分 类 号：R373[医药卫生—病原生物学]