鸡传染性支气管炎病毒S蛋白介导的细胞侵染机制研究

项目名称： 鸡传染性支气管炎病毒S蛋白介导的细胞侵染机制研究

项目编号： No.31272569

项目类型： 面上项目

立项/批准年度： 2013

项目学科： 农业科学

项目作者： 李广兴

作者单位： 东北农业大学

项目金额： 85万元

中文摘要： 传染性支气管炎病毒是冠状病毒属成员。IBV通过其囊膜蛋白S与细胞膜受体相互作用进入细胞引起感染。氨肽酶N（APN）已被鉴定是多种冠状病毒的细胞受体，但目前IBV的受体没有得到鉴定，同时其感染机制也不清楚。本研究拟用RT-PCR扩增鸡APN(ChAPN)，表达后制备其抗体。在IBV非易感细胞表达ChAPN后分析IBV对这些细胞的感染。用ChAPN抗体检测ChAPN组织分布及其对IBV感染的阻断。将IBV S蛋白展示在水泡性口炎病毒（VSV）表面制备假病毒V-IBV-S，并感染极性化的IBV靶细胞确定IBV侵染途径；分析IBV感染与ChAPN细胞定位的相关性。用共聚焦免疫荧光和Western blot共定位IBV S蛋白、ChAPN和细胞脂筏Marker，并确定膜胆固醇对IBV感染力、病毒复制和释放的影响，分析IBV感染与细胞脂筏的关系。通过上述实验阐明IBV S蛋白介导的细胞侵染机制。

中文关键词： 冠状病毒；传染性支气管炎病毒；S蛋白；受体；感染

英文摘要： Avian infectious bronchitis virus (IBV) belongs to family of Coronaviruses.Interaction between S protein of IBV and its cellular receptor initiates subsequent infection. Aminopeptidease N （APN） was identifed as functional receptor for several coronaviruses. Nevertheless, The putative receptor of IBV and infection mechanism of this virus are still unknown so far. In this study, the total RNA will be extract from chicken tissues and chicken APN (ChAPN) will be amplified by RT-PCR. The ChAPN was expressed and used to generated both monoclonal and polyclonal antibodies. Unsusceptible cells will be tranfected with ChAPN-expressing plamsmid followed by infection of IBV. Tissue distribution of ChAPN will be analyzed by immunohistochemistry using anti-ChAPN antibody. Blocking effect of anti-ChAPN antibody on IBV infection will be analyzed using plaque and immunofluorescence assays. Cell entry of IBV will be analyzed by VSV-derived pseudotypes bearing IBV S protein. Localization of ChAPN on polarized cells will be analyzed by laser confocal immunofluorescences (LCI) and Western blot. Colocalizaton of IBV S/ChAPN and lipid rafts marker is to be analyzed by LCI and Western blot. Association of cholesterol with IBV entry, replicaiton and release will be investigated by virus plaque, real-time PCR and immunofluorescence assa

英文关键词： Cornavirus；IBV；S protein；Receptor；Infection