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

doi:10.11843/j.issn.0366-6964.2016.03.018

畜牧兽医学报 ›› 2016, Vol. 47 ›› Issue (3): 549-558.doi: 10.11843/j.issn.0366-6964.2016.03.018

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

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²

(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)

收稿日期:2015-08-21 出版日期:2016-03-23 发布日期:2016-03-23
通讯作者: 姜平，教授， E-mail：jiangp@njau.edu.cn；王芳，研究员，E-mail：rwangfang@126.com
作者简介:李腾(1990-)，女，湖南邵东人，硕士生，主要从事家畜传染病学研究，Tel：025-84390337，E-mail:2013107045@njau.edu.cn
基金资助:
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）

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

LI Teng^1，2，JIANG Ping^1* ，WANG Fang^1，2* ，SONG Yan-hua²，HU Bo²，FAN Zhi-yu²，WEI Hou-jun²，QIU Ru-long²，LIU Xing²

(1.Key Laboratory of Animal Diseases Diagnostic and Immunology，College of Veterinary Medicine，Nanjing Agricultural University，Nanjing 210095，China；2.Key Laboratory of Veterinary Biological Engineering and Technology of Ministry of Agriculture，National Center for Engineering Research of Veterinary Bio-products，Institute of Veterinary Medicine，Jiangsu Academy of Agricultural Sciences，Nanjing 210014，China)

Received:2015-08-21 Online:2016-03-23 Published:2016-03-23

摘要/Abstract

摘要：

兔出血症病毒（RHDV）衣壳蛋白VP60 P2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 VP60 P2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。

Abstract:

According to reports，VP60 P2 subdomain of RHDV may play an important role in the viral infection． In this study，we used co-immunoprecipitation(Co-IP) with RHDV VP60 P2 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.LamR gene was amplified by RT-PCR from rabbit liver cells．The recombinant LamR protein was expressed in E.coli and used in GST pull-down 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 found that RHDV VP60 P2 interacted with LamR which would contributed to further study of the infectious process and pathogenesis of RHDV．

中图分类号:

李腾，姜平，王芳，宋艳华，胡波，范志宇，魏后军，仇汝龙，刘星. 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[J]. 畜牧兽医学报, 2016, 47(3): 549-558.

LI Teng，JIANG Ping，WANG Fang，SONG Yan-hua，HU Bo，FAN Zhi-yu，WEI Hou-jun，QIU Ru-long，LIU Xing. Screening and Identification of RHDV VP60 P2 Domain Interacting Proteins in Rabbit Liver Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(3): 549-558.

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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

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