产蛋下降综合征病毒受体及其组织嗜性的研究

Egg drop syndrome is a kind of viral disease characterized by serious decrease in egg production of hen flocks with shell eggs. Its pathogen is egg drop syndrome virus (EDSV).EDSV belongs to Atadenovirus, Adenoviridae. The proliferative titer of EDSV produced in duck embryo was significantly higher than that in chicken embryo. However, the mechanism of pathological changes caused by EDSV in the chicken reproductive system is still unclear. It might be related with the virus receptor. No study on EDSV receptor has been reported up to now. In this program, we will apply yeast two-hybrid, virus overlay protein-binding assay, immunoprecipitation, blocking experiments, receptor reconstruction and confocal lase technology to screen and identify the receptors of the EDSV NE4 isolate that was first isolated in China. Then immunohistochemistry, fluorescence confocal microscope, real-time RT-PCR wil be used to reveal the distribution and expression of the receptor proteins in different tissues and cells. This project will explore the molecular mechanism of reproductive system tropism of EDSV andb lay the foundation to develop a novel adenovirus vector based EDSV.

产蛋下降综合征是一种以产蛋鸡产蛋下降，产薄壳蛋为特征的病毒性疾病，病原是产蛋下降综合征病毒（Egg drop syndrome virus, EDSV）。EDSV为腺病毒科腺胸腺病毒属成员。EDSV在鸭胚增殖滴度显著高于鸡胚，但却能够引起鸡生殖系统病理变化，导致这一现象的原因尚不清楚，推测可能与其病毒受体的差异性有关。目前国内外尚无针对EDSV受体的研究报道，本项目拟选用在国内首次分离到的EDSV NE4分离株，利用酵母双杂交、病毒覆盖蛋白印记、免疫共沉淀、病毒阻断、受体重建及激光共聚焦等技术手段，对EDSV的受体进行筛选和鉴定，再利用免疫组化、荧光显微共聚焦、real-time RT-PCR 分析受体蛋白在不同的组织和细胞中的分布和表达水平，从而为从病毒受体水平上深入探索EDSV生殖系统嗜性的分子机制及为将来构建EDSV新型腺病毒载体奠定基础。

应用酵母双杂交（Y2H）方法筛选产蛋下降综合征病毒 (EDSV) 的受体。筛选出鸭成纤维细胞cDAN文库中鸭GABA（A）受体相关蛋白1（Anas platyrhynchos GABA (A) receptor-associated protein like 1，GABARAPL1)和鸭蛋白质核糖体蛋白SA（Anas platyrhynchos ribosomal protein SA，RPSA) 为阳性互作蛋白。通过体外GST pull-down实验再次证实两种蛋白的互作结果。 GABARAPL1和RPSA蛋白初步鉴定为EDSV受体。通过人工感染EDSV，利用荧光定量PCR技术及免疫组化技术检测EDSV在BALB/c小鼠、Nu小鼠及NSG小鼠不同品系小鼠体内增殖情况以及动态变化规律。结果表明EDSV可刺激三种小鼠机体增殖，并在短期内产生免疫应答，在小鼠体内有肝、脾、子宫等组织嗜性。建立了EDSV 实时荧光环介导等温扩增技术(RealAmp)。无需病毒DNA提取，直接检测来自感染样品（尿囊液和细胞培养物）的EDSV。成功用于稀释样品直接检测EDSV纤维蛋白基因。 在15-30分钟的短时间内扩增EDSV。灵敏度低至（26 fg /μL），该方法特异性强，与其他DNA病毒无交叉反应。