猪札幌病毒感染关键蛋白的筛选、鉴定与分析

Sapovirus (SaV) within the Caliciviridae family are an important cause of gastroenteritis in both humans and animals. SaV are genetically highly heterogeneous and are presently classified in five genogroups that are further subdivided into a number of genotypes. In recent years, a number of novel animal SaV strains, have been partially characterized and proposed to represent novel genogroups or genotypes. Moreover, reports indicated that some porcine strains are genetic and antigenic relative to human strains. The close genetic relationship of SaV found in animals and humans has raised the question whether these viruses have a zoonotic potential. Transmission from animals to humans and vice versa would have far-reaching consequences for epidemiology and food safety. SaV are hard to cell culture except Cowden strain, the only culturable porcine strain, which hinder the unstinting of mechanisms of infection, replication and pathogenesis. In view of this, this proposed study aims to screen proteins relative to infection of SaV from susceptible cells LLC-PK using methods combinating yeast two-hybridization and Co-IP- LC-MS. RNAi, confocal and Infection blocking experiment are used to verify the interaction. Distribution of these proteins in vivo will be further studied in order to reveal the SaV host specificity and tissue tropism. This study is hoped to explain the mechanisms of SaV infection, replication and the potential interspecies transmission from animals to human.

札幌病毒（Sapovirus,SaV）能感染人和猪并导致急性胃肠炎。人源SaV与某些猪SaV毒株基因组高度同源，提示SaV可能存在着跨种间感染与传播能力。目前，SaV的研究主要集中于流行病学，而病毒的感染与致病机制及其跨种间感染与传播能力等尚不清楚。研究易感细胞与病毒相互作用关键蛋白，有助于阐明病毒的致病机制，宿主范围及组织嗜性。因此，本研究拟采用酵母双杂交、免疫共沉淀-LC-MS等技术从其易感细胞或cDNA文库中筛选猪SaV互作蛋白。进而根据上述蛋白性质（包膜蛋白或胞质蛋白），采用RNAi、pull-down以及中和抗体阻断病毒感染等实验技术进一步验证其相互作用，以期揭示参与猪SaV感染及致病的关键蛋白。本研究对阐明猪SaV致病机理，揭示其潜在的跨种间感染与传播机制及研制SaV基因工程疫苗等具有重要意义。

札幌病毒（sapovirus，SaV）属杯状病毒科，与诺如病毒、兔病毒和囊病毒等同属杯状病毒科，是导致人、猪、狗等多种动物罹患急性肠胃炎的重要非细菌性病原体。SaV主要通过粪-口途径传播，被病毒污染的水源、食物和物体表面都可以造成感染，给人和动物的健康均带来一定的威胁。人源SaV与某些猪SaV毒株基因组高度同源，提示SaV可能存在着跨种间感染与传播能力。目前，SaV的研究主要集中于流行病学，而病毒的感染与致病机制及其跨种间感染与传播能力等尚不清楚。本研究（1）：在中国腹泻仔猪中分离到一株SaV毒株，并进行了全基因组测序。遗传进化及重组分析证实该毒株为重组毒株，这是在中国猪群中首次分离到SaV重组毒株，这也是国际上报道的第二株猪SaV重组毒株。SaV中国重组毒株发现为SaV的研究提供了素材，也为SaV在中国可能的跨种间感染与传播敲响了警钟。（2）：利用所分离到的中国SaV毒株建立了小型猪感染模型并对其致病机制进行了研究。（3）分别采用酵母双杂交和Co-IP-LC-MS方法进行SaV受体的筛选，并对筛选到的蛋白进行了验证，证实MHC-I能够与SaV相互作用，这表明CO-IP可能为SaV感染细胞的候选受体。本研究对阐明猪SaV致病机理，揭示其潜在的跨种间感染与传播机制及研制SaV基因工程疫苗等具有重要意义。