兔肝细胞中与RHDV VP60 P2亚区相互作用蛋白质的筛选和初步鉴定  

Screening and Identification of RHDV VP60 P2 Domain Interacting Proteins in Rabbit Liver Cells

作　　者：李腾[1,2] 姜平[1] 王芳[1,2] 宋艳华[2] 胡波[2] 范志宇[2] 魏后军[2] 仇汝龙[2] 刘星 

机构地区：[1]南京农业大学动物医学院,南京210095 [2]江苏省农业科学院兽医研究所,农业部兽用生物制品工程技术重点实验室,国家兽用生物制品工程技术研究中心,南京210014

出　　处：《畜牧兽医学报》2016年第3期549-558,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：现代农业产业技术体系建设兔体系病毒病预防与控制岗位(CARS-44-C-1);国家自然科学基金(31502073)

摘　　要：兔出血症病毒(RHDV)衣壳蛋白VP60P2亚区在病毒感染宿主细胞过程中可能起重要作用,为找寻RHDV靶细胞——兔肝细胞中与P2亚区相互作用的蛋白质,以原核表达的P2蛋白为诱饵蛋白质,通过免疫共沉淀试验(Co-IP)结合质谱分析筛选与之有相互作用的肝细胞蛋白质。经分析选取层黏连蛋白受体(LamR)蛋白做进一步研究。提取兔肝细胞总RNA,反转录扩增LamR基因,将其插入pGEX-6p-1载体,经IPTG诱导表达,获得GST-LamR融合蛋白,并通过GST pull-down试验验证LamR蛋白与P2蛋白的结合。结果显示,以P2蛋白为诱饵蛋白质,免疫共沉淀试验结合质谱分析共筛选到26个与P2存在相互作用的肝细胞蛋白质,其中LamR蛋白是多种病毒的受体。通过原核表达系统表达了LamR蛋白,经pull-down试验验证了LamR蛋白与P2蛋白有直接的相互作用。本研究证实兔肝细胞LamR蛋白与RHDV VP60P2蛋白的相互作用,为深入研究RHDV的感染过程、揭示RHDV的致病机制提供了线索。According to reports,VP60P2 subdomain of RHDV may play an important role in the viral infection.In this study,we used co-immunoprecipitation（Co-IP）with RHDV VP60P2 expressed in E.coli as bait protein and mass spectrometry analysis to screen the RHDV interacting hepatocellular proteins.Laminin receptor（LamR）,as receptors of many other viruses,was identified and verified as potential interacting partner of P2.LamRgene was amplified by RT-PCR from rabbit liver cells.The recombinant LamR protein was expressed in E.coli and used in GST pulldown to verify the interaction of LamR and P2.Here we screened 26 proteins as potential P2 interacting proteins.After verification,the protein LamR could interact with P2 directly.We foundthat RHDV VP60P2 interacted with LamR which would contributed to further study of the infectious process and pathogenesis of RHDV.

关 键 词：兔出血症病毒(RHDV) VP60P2亚区 免疫共沉淀 层黏连蛋白受体(LamR) 

分 类 号：S855.3[农业科学—临床兽医学] S852.659.6[农业科学—兽医学]