【作者】 林万松；

【导师】 林旭；

【作者基本信息】 福建医科大学 ， 病原生物学， 2011， 博士

【摘要】 持续的乙型肝炎病毒(hepatitis B virus, HBV)感染可引起急慢性乙型肝炎,肝硬化,并与原发性肝细胞癌(HCC)的发生发展关系密切。全球有超过3亿的慢性感染人口,HBV致病的确切分子病理机制仍不清楚。HBV为一种部分双链环状嗜肝DNA病毒,含4个编码病毒蛋白的开放读码框(ORF),其中一个X基因ORF编码HBV X蛋白(HBx),包含了154个氨基酸。HBx被认为是一个多功能蛋白,在病毒的感染、宿主细胞的凋亡、肝癌的发生以及参与病毒与宿主细胞相互作用的过程中都起到十分重要的作用。HBx在参与肿瘤的发生发展过程中所发挥的影响是当前HBV研究的热点。目前的研究表明,HBx的许多生物学功能需通过与其它的蛋白相互作用完成,HBx蛋白可以和许多的细胞因子相互作用,其中包括转录调控因子、细胞信号传导因子以及凋亡相关的蛋白。鉴于已经发现了种类繁多的HBx结合蛋白,我们认为可能还存在一些目前未知的能够和HBx蛋白相互作用的细胞蛋白。为更深入研究HBx的功能,本课题第一部分,我们运用免疫沉淀(Immunoprecipitation)及随后的基质辅助激光解吸电离飞行时间质谱鉴定(MALDI-TOF- MS)的方法筛选获得了五个HBx相互作用的肝细胞蛋白,其中一个为真核延长因子1a1(Eukaryotic Elongation Factor 1 alpha 1,eEF1a1)。eEF1a1又称为真核细胞翻译延长因子1a1,是一个重要的蛋白翻译因子。eEF1a-GTP催化氨酰tRNA结合到核糖体的A位点。eEF1a1不仅是翻译必须的蛋白,而且是一个重要的多功能蛋白,参与许多重要的细胞过程和疾病,包括信号传导、翻译控制、凋亡、细胞骨架组成、病毒复制及癌基因转化等。本课题的第二部分,通过GST pull-down实验和免疫共沉淀(Co-Immunoprecipitation,Co-IP)实验来进一步证实HBx蛋白与eEF1a1在细胞外和细胞内存在相互作用。eEF1a1的功能包括参与蛋白翻译和促进F-肌动蛋白成束,本课题第三部分旨在研究HBx对eEF1a1相关功能的影响。结果提示HBx可以通过与eEF1a1结合,抑制细胞蛋白的总体翻译水平,减少F-肌动蛋白的成束,提示HBx通过与eEF1a1的相互作用,参与了HBV的致病性。更多还原

【Abstract】 Persistent hepatitis B virus (HBV) infection is closely associated with the development of the acute and chronic forms of hepatitis, and with cirrhosis and hepatocellular carcinoma (HCC). But the exact molecular mechanism of how the virus inducing these diseases remains unclear.HBV harbors a circular, partially double-stranded DNA genome consisting of four open reading frames (ORFs) that encode viral proteins. One such ORF, the X gene, encodes the 154-amino-acid HBV X protein (HBx), which has been showed to have multiple functions. HBx protein may play important role during the infection establishment, host cell apoptosis, hepatocarcinogenesis and other virus-host cell interaction processes. However, mechanism considering the association between HBx and hepatocarcinogenesis is far from been fully understood. It was proved that HBx need the help of host cell proteins to exert its function by binding to them. Current studies indicate that the HBx protein interacting with a number of host factors, including transcriptional factors and molecules involved in intracellular signaling and apoptosis.Although many partners of HBx have been discovered, there may be many other partners remain unknown. In the first part of present study, we performed immunoprecipitation and mass spectrometry to identify Eukaryotic Elongation Factor 1 alpha 1( eEF1a1) as a novel HBx-binding protein. eEF1a1 is a major translational factor. eEF1a-GFP catalyzes the binding of aminoacyl-tRNA to the A-site of the ribosome. It is not only necessary for translation but also take part in cellular processes, including signal transduction, translational control, apoptosis, cytoskeletal organization, virus replication and oncogenic transformation.In the second part of this study, the GST pull-down assay and the Co-immunoprecipitation(Co-IP) assay were employed to further confirming the direct interaction between HBx and eEF1a1.In the third part of this study the influence of HBx in vitro and vivo respectively on eEF1a1 was analyzed. The results demonstrated that HBx could inhibit protein translation and reduce F-actin bundles, suggesting that HBx participated in pathogenesis of HBV through interacting with eEF1a1.更多还原