猪流行性腹泻病毒S蛋白识别受体pAPN的分子机制研究

Porcine epidemic diarrhea virus (PEDV) is the major causative agent of porcine epidemic diarrhea, which infections have a substantial detrimental effect on the swine industry. Receptor recognition is the first step in viral infection of host cells and one of the most important determinants of viral infectivity and pathogenesis. Determinants of PEDV tropism locate at the S1 region, which mediates attachment of viral particles to cell surface molecules-aminopeptidase (pAPN), initiating virus entry into cells and infection. Most PEDV-neutralizing antibodies cluster at S1 region. To date, there is no detail mechanism available on pAPN recognition by PEDV. In this study, we will express and purify PEDV （Vaccine strain CV777 and virulent strain CHGD-01） truncated spike protein and pAPN ectodomain. The receptor binding domain will be determined by Biacore, Co-immunoprecipitation, and Flow cytometry. Further,We will determine the crystal structures of S1 by itself and S1 in complex with the pAPN ectodomain. Critical contacts at the interface will be confirmed by mutagenesis, providing a structural basis for viral and host specificities of PEDV/APN interactions. The difference of various strains S1 interaction with pAPN will be analysised by Biacore, ELISA and structural comparison. These studies will clarify the receptor recognition mechanism of PEDV, which will provide a molecular and structural basis for tracking future PEDV evolution and guide the design of drug or immunogens that boost CoV-neutralizing immune responses to key motifs for virus cell entry.

猪流行性腹泻病毒（PEDV）是当前严重危害养猪业的一种重要病原体。病毒识别并结合其受体是病毒感染宿主的第一步,也是影响病毒传染性和致病力的最重要因素之一。PEDV S1蛋白在识别受体pAPN和诱导机体产生中和抗体中发挥重要作用。但其与受体pAPN结合的分子机制尚不清楚。本项目拟表达、纯化PEDV弱毒株(CV777)、强毒株（CHGD-01）的S1蛋白及其截短突变体以及受体蛋白pAPN，通过Biacore、流式细胞术、免疫共沉淀等方法确定PEDV S1蛋白的受体结合域；用X-射线晶体衍射来解析S1自身以及S1与pAPN复合物的三维结构，精确定位参与受体识别的关键氨基酸。利用Biacore、ELISA、结构生物学方法，比较强、弱毒株S1结合pAPN的差异。以期阐明PEDV S1蛋白识别受体pAPN的分子机制，为研制PEDV疫苗、抗病毒药物、了解病毒变异规律提供理论依据。

2010年以来猪流行性腹泻病毒(PEDV)变异毒株的出现，对我国乃至世界养猪业造成重大的经济损失，但其识别受体的分子机制以及病毒毒力增强的机理尚未完全阐明。本研究通过昆虫细胞表达PEDV强、弱毒株纤突蛋白S1、受体候选蛋白pAPN，利用Biacore、ELISA、流式细胞术等方法，确定了PEDV受体结合区域位于S1蛋白C端，强、弱毒株结合pAPN没有明显差异；通过结构模拟分析、病毒学试验以及生化试验研究，发现PEDV受体结合机制与其它α冠状病毒(TGEV、NL63)具有不同的模式。进一步通过糖结合试验发现，PEDV结合糖受体或辅助受体的区域位于S1的N端，并且变异毒株结合受体的能力显著高于传统疫苗株；为阐明变异毒株毒力增强的分子机制提供了新思路，相关成果发表1年以来受到国内外学者的高度关注，已被引用15次；在揭示病毒受体识别机制的研究基础上，我们还进一步解析了5个参与冠状病毒复制相关蛋白的晶体结构。目前，已发表项目标注SCI论文6篇(其中2篇发表在Journal of Virology，2篇发表在Virology，1篇发表在Viruses )，培养研究生8名。