兔出血症病毒细胞受体的筛选与鉴定

项目名称： 兔出血症病毒细胞受体的筛选与鉴定

项目编号： No.30800045

项目类型： 青年科学基金项目

立项/批准年度： 2009

项目学科： 生物科学

项目作者： 陈柳

作者单位： 浙江省农业科学院

项目金额： 20万元

中文摘要： 本项目拟对RHDV 受体进行克隆与鉴定。所采用的策略是将现代生物分子生物学技术与传统病毒免疫学方法相结合，从宿主细胞的cDNA 文库中筛选RHDV 的细胞受体。研究内容及结果主要体现在以下几个方面：（1）利用昆虫细胞-杆状病毒表达系统成功单独表达了RHDV 主要衣壳蛋白VP60和共表达了VP60与次要结构蛋白VP10，通过纯化获得了表达蛋白组装的RHDV 病毒样颗粒（VLPs）；同时对VP60蛋白在昆虫细胞Sf9细胞中的定位进行了研究，发现VP60定位于细胞膜上；对VLPs的组份分析显示VP10整合入VLPs；VP10蛋白能与VP60互作且VP10蛋白的存在能下调VP60表达；（2）完成RHDV敏感细胞系RK13 cDNA表达型分子文库的构建与鉴定工作；（3）在RHDV全长感染性cDNA克隆的基础上，构建了荧光蛋白标记的RHDV全长cDNA克隆；（4）利用表达获得的VLPs从表达型cDNA文库中进行免疫筛选获得了22个阳性样品，其中4个为膜蛋白，为疑似的RHDV细胞受体，有待于进一步鉴定。本研究对进一步开展RHDV 的分子致病机理、分子免疫学和新型防控技术的研究都将有重大意义。

中文关键词： 兔出血症病毒；病毒样颗粒；cDNA文库；受体

英文摘要： This project has focused on the clone and identification of RHDV receptor.The technology consisting of modern molecular biology and traditional immunology has be applied to screen cellular receptor from cDNA library of RHDV permissive RK13 cell. The study contents and results of this project could be summarized as follow: (1)We have expressed RHDV VP60 alone or co-expressed VP60 with VP10 by insect cells-baculovirus expression system, the proteins were successfully expressed and self-asembled into virus-like particles(VLPs); Localization study of VP60 demonstrated that VP60 protein localized on cellular membrane of Sf9 cells;and the component analysis of VLPs shows VP10 assembled into VLPs; VP10 could interact with VP60 and downregulate the expression of VP60.(2)The full length cDNA library of RK13 cells was constructed to identify RHDV receptors.(3)We have constructed a full-length cDNA infectious clone of RHDV which expression of red fluorescent protein between the RdRp-VP60 region. (4)By immunological screening with purified RHDV VLPs from expressed cDNA library,we have obtained 22 positive clones from cDNA expression library and four of them are membrane proteins, which might be RHDV cellular receptor. This study will be highly beneficial for further revealing the pathogenesis and immunomechanism of RHDV and developing new measures of RHDV prevention and control.

英文关键词： Rabbit hemorrhagic disease virus; virus-like particles; cDNA library; receptor