鸭黄病毒囊膜E蛋白宿主细胞受体分子的筛选及功能鉴定

Since April 2010, a severe outbreak of duck viral infection, with egg drop, feed uptake decline, ovary-viduct and encephalitis disease, has spread around the major duck-producing provinces of Fujian, zhejiang, jiangsu, henan, hebei, shandong, hubei in China. A new virus, called novel duck flavivirus(DFV), genomic sequencing of the isolated virus revealed that it is closely related to tembusu virus, have made huge economic losses for poultry industries. To date, there is no report overseas, some bionomics, classification, nucleotide sequencing and phylogenetic analysis were studied by the scholars in China, but DFV pathogenesis and the interaction mechanism between virus proteins and host proteins remain elusive. A causative strain named HN1 was isolated from sick shelducks by inoculating filtered spleen homogenates into the allantoic sacs of embryonated duck eggs. The virus was replicated well in embryonated duck and chicken eggs, and also in primary duck embryo fibroblasts . The major envelope glycoprotein(E) is known as the viral attachment protein(VAP) of flavivirus, it contains structural and functional elements that participate in the virus-host cell protein interaction and membrane fusion. In this study, E protein was cloned from HN1 strain and expressed to screening the putative receptor molecule(s) by Phage surface display techniques(PSDT), then the function of the receptor molecule(s) interacting with E was identified and characterized by cell cultured and infected, blocking test of DFV infection, receptor reconstruction, immunofluorescence assay(IFA) and so on. Combined with RNAi, improved stable isotope labeling by amino acids in cell culture(SILAC) technique was used to analyse the proteomic of DFV-infected cultured duck embryo fibroblasts cells quantitatively, all the host cellular proteins were examined globally, which are the actual effector molecules with the receptor molecule(s) of E protein were studied. These results provide important clues to facilitate characterization of receptor molecule(s) interacting with E protein, clarify the invasion method, investigate the tissue tropism, and to further elucidate the pathogenesis of DFV infection.

鸭黄病毒是2010年4月份以来在我国出现的一种新型病毒，主要引起产蛋严重下降和脑炎，给养鸭业造成了重大损失。作为黄病毒属的新成员，鸭黄病毒感染宿主细胞的确切机制尚不清晰。本项目以鸭黄病毒囊膜E蛋白吸附宿主细胞为切入点，采用T7噬菌体展示技术筛选E蛋白宿主细胞受体分子，通过易感细胞病毒结合阻断试验、非易感细胞受体重建及病毒感染试验，对筛选的E蛋白受体分子进行鉴定。运用RNAi技术，抑制宿主细胞E蛋白受体分子的表达，并结合SILAC技术定量解析鸭黄病毒感染宿主细胞重要蛋白差异表达谱，分析重要差异表达蛋白与E蛋白受体的相关性，鉴定E蛋白受体分子在鸭黄病毒感染中的功能。本项目的研究，可从分子水平确证E蛋白受体的基因结构，阐明E蛋白受体分子在病毒感染宿主细胞过程中的作用，初步揭示鸭黄病毒囊膜E蛋白与宿主细胞的相互作用机制。对于深入研究鸭黄病毒的吸附侵入模式、组织嗜性及致病机理具有突破性的意义。

鸭黄病毒为2010年后在我国出现的新病毒，病原命名多样，目前多统一命名为鸭坦布苏病毒（DTMUV），是黄病毒科黄病毒属成员。本项目对鸭坦布苏病毒HN1分离株进行了鉴定，并将其囊膜E蛋白主要抗原区域进行酵母高效表达，利用噬菌体展示技术以E蛋白为靶标，进行随机12肽库三轮筛选，结合ELISA试验和竞争抑制试验，成功筛选了2个能够与E蛋白特异性结合的多肽。测序结果显示，其氨基酸序列分别为：HWSTRQGSTRWN和THRSWQGNSWYM，此两条多肽对鸭坦布苏病毒在鸭胚成纤维细胞增殖方面有抑制作用；构建鸭胚成纤维细胞cDNA T7噬菌体原始文库并进行扩增，用鸭坦布苏病毒E蛋白对鸭胚成纤维细胞T7噬菌体cDNA文库进行三轮生物淘选，将筛选后的噬菌体克隆裂解液进行PCR鉴定和序列测定，结果显示得到1条鸭源目的基因，与层粘连蛋白受体一致；根据E蛋白保守序列设计siRNA，用脂质体法转染鸭胚成纤维细胞，24h后再接种病毒，感染48h后分别进行病毒滴度测定、RT-PCR和Westen blotting分析，表明siRNA能抑制鸭坦布苏病毒HN1株在鸭胚成纤维细胞的增殖及E蛋白的表达；同时，初步制备了鸭坦布苏病毒灭活油乳疫苗，能够有效诱导抗体和细胞免疫反应，以上结果对初步揭示鸭坦布苏病毒囊膜E蛋白与宿主细胞的相互作用提供依据，为鸭坦布苏病毒的防控奠定了一定基础。