【作者】 郭亮；

【导师】 华修国； 崔立； 吴更；

【作者基本信息】 上海交通大学 ， 预防兽医学， 2013， 硕士

【摘要】 戊型肝炎病毒编码三个开放性阅读框: ORF1,ORF2和ORF3。其中ORF1编码病毒的非结构蛋白，对病毒复制起重要作用。ORF2编码病毒的外壳蛋白。而ORF3则编码一个约120个氨基酸的磷酸化且与细胞骨架结合的蛋白，然其确切的生物学功能尚不详。在本研究中，为了研究戊型肝炎病毒ORF3蛋白的功能，我们采用昆虫杆状病毒表达系统和哺乳动物表达系统表达了戊型肝炎病毒ORF3蛋白，并基于本实验室先前酵母双杂交筛选戊型肝炎病毒ORF3在人肝细胞中相互作用蛋白的阳性结果，采用哺乳动物表达系统克隆并表达了RPS11，HPN，ALB，HRG和PIK3CD五个ORF3的候选相互作用蛋白。通过免疫共沉淀验证，我们发现，其中HPN可以和ORF3相互作用。HPN是一种跨膜丝氨酸蛋白酶，其被广泛报道具有抑制肿瘤的功能。因而我们推测，ORF3蛋白可能通过结合HPN蛋白从而具有促进肿瘤发生的作用。同时，我们也利用了本研究克隆和表达的ORF3蛋白结合免疫共沉淀，液相色谱质谱联用的方法筛选并鉴定了七个ORF3的候选相互作用蛋白，这为进一步了解ORF3的功能提供了新的线索以及更丰富的视野。更多还原

【Abstract】 Hepatitis E virus encodes three open reading frames, ORF1, ORF2and ORF3. ORF1encodes viral non-structural proteins, which are very important for viral replication.ORF2encodes viral capsid protein and ORF3encodes an around120amino acids ofphosphorylation protein that associates with cytoskeleton, while its precise biologicalfunction is still unknown.In this study, to understand the function of Hepatitis E virus, we firstly expressed theORF3protein of hepatitis E virus via baculovirus and mammalian expression systems.In order to further understand its functions, mammalian expression system was used toclone and express the RPS11, HPN, ALB, HRG and PIK3CD, basing on our previousyeast two-hybrid screening of hepatitis E virus ORF3interacting proteins in hepatocytes.Five ORF3interacting protein candidates were tested by co-immunoprecipitation. Wefound that, HPN, a transmembrane serine protease that was widely reported to functionas the inhibitor of several tumors, interacts with ORF3. We inferred that the ORF3protein of Hepatitis E virus may promote the tumorigenesis via interacting with hepsin.Also, we made use of the cloned and expressed of ORF3protein to screen and identifyseven ORF3candidate interacting proteins by combining co-immunoprecipitation andliquid chromatography-mass spectrometry methods. Our results provided us a new clueand richer perspective to understand the function of the ORF3of Hepatitis E virus.更多还原