【摘要】：持續(xù)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,并與原發(fā)性肝細(xì)胞癌(HCC)的發(fā)生發(fā)展關(guān)系密切。全球有超過3億的慢性感染人口,HBV致病的確切分子病理機(jī)制仍不清楚。 HBV為一種部分雙鏈環(huán)狀嗜肝DNA病毒,含4個(gè)編碼病毒蛋白的開放讀碼框(ORF),其中一個(gè)X基因ORF編碼HBV X蛋白(HBx),包含了154個(gè)氨基酸。HBx被認(rèn)為是一個(gè)多功能蛋白,在病毒的感染、宿主細(xì)胞的凋亡、肝癌的發(fā)生以及參與病毒與宿主細(xì)胞相互作用的過程中都起到十分重要的作用。HBx在參與腫瘤的發(fā)生發(fā)展過程中所發(fā)揮的影響是當(dāng)前HBV研究的熱點(diǎn)。目前的研究表明,HBx的許多生物學(xué)功能需通過與其它的蛋白相互作用完成,HBx蛋白可以和許多的細(xì)胞因子相互作用,其中包括轉(zhuǎn)錄調(diào)控因子、細(xì)胞信號傳導(dǎo)因子以及凋亡相關(guān)的蛋白。 鑒于已經(jīng)發(fā)現(xiàn)了種類繁多的HBx結(jié)合蛋白,我們認(rèn)為可能還存在一些目前未知的能夠和HBx蛋白相互作用的細(xì)胞蛋白。為更深入研究HBx的功能,本課題第一部分,我們運(yùn)用免疫沉淀(Immunoprecipitation)及隨后的基質(zhì)輔助激光解吸電離飛行時(shí)間質(zhì)譜鑒定(MALDI-TOF- MS)的方法篩選獲得了五個(gè)HBx相互作用的肝細(xì)胞蛋白,其中一個(gè)為真核延長因子1a1(Eukaryotic Elongation Factor 1 alpha 1,eEF1a1)。eEF1a1又稱為真核細(xì)胞翻譯延長因子1a1,是一個(gè)重要的蛋白翻譯因子。eEF1a-GTP催化氨酰tRNA結(jié)合到核糖體的A位點(diǎn)。eEF1a1不僅是翻譯必須的蛋白,而且是一個(gè)重要的多功能蛋白,參與許多重要的細(xì)胞過程和疾病,包括信號傳導(dǎo)、翻譯控制、凋亡、細(xì)胞骨架組成、病毒復(fù)制及癌基因轉(zhuǎn)化等。 本課題的第二部分,通過GST pull-down實(shí)驗(yàn)和免疫共沉淀(Co-Immunoprecipitation,Co-IP)實(shí)驗(yàn)來進(jìn)一步證實(shí)HBx蛋白與eEF1a1在細(xì)胞外和細(xì)胞內(nèi)存在相互作用。 eEF1a1的功能包括參與蛋白翻譯和促進(jìn)F-肌動蛋白成束,本課題第三部分旨在研究HBx對eEF1a1相關(guān)功能的影響。結(jié)果提示HBx可以通過與eEF1a1結(jié)合,抑制細(xì)胞蛋白的總體翻譯水平,減少F-肌動蛋白的成束,提示HBx通過與eEF1a1的相互作用,參與了HBV的致病性。
[Abstract]:Continuous hepatitis B virus (hepatitis B virus, HBV) infection can cause acute and chronic hepatitis B, liver cirrhosis, and is closely related to the occurrence and development of (HCC) in hepatocellular carcinoma. With more than 300 million chronic infections worldwide, the exact molecular pathogenetic mechanism of HBV remains unclear. HBV is a partially double-stranded DNA virus with open reading frame containing four viral proteins. One X gene, ORF encoding HBV X protein (HBx), contains 154 amino acids. HBx is considered to be a multifunctional protein. In virus infection, host cell apoptosis, The occurrence of liver cancer and the interaction between virus and host cell play a very important role. The influence of HBx in the process of tumor development is a hot topic in the research of HBV. Current studies have shown that many of the biological functions of HBx are accomplished by interacting with other proteins, and that HBx proteins interact with many cytokines, including transcriptional regulatory factors. Signal transduction factors and apoptosis-related proteins. Given that a wide variety of HBx binding proteins have been identified, we believe that there may still be some unknown cellular proteins that interact with HBx proteins. In order to study the function of HBx more deeply, the first part of this topic, Five hepatocyte proteins interacting with HBx were screened by immunoprecipitation (Immunoprecipitation) and subsequent matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF- MS). One of them is 1a1 (Eukaryotic Elongation Factor 1 alpha 1 eEF1a1). EEF1a1 is also called eukaryotic cell translation extension factor 1a1. EEF1a-GTP catalyzes the binding of aminoacyl tRNA to ribosome A site. EEF1a1 is not only a necessary protein for translation, but also an important multifunctional protein involved in many important cellular processes and diseases. These include signal transduction, translation control, apoptosis, cytoskeleton composition, viral replication and oncogene transformation. In the second part of this study, the interaction between HBx protein and eEF1a1 was further confirmed by GST pull-down and Co-Immunoprecipitation,Co-IP. The functions of eEF1a1 include protein translation and promotion of F- actin bundles. In the third part of this study, the effects of HBx on eEF1a1 related functions are studied. The results suggest that HBx can inhibit the total translation of cellular proteins and reduce the bundles of F- actin by binding to eEF1a1, suggesting that HBx is involved in the pathogenicity of HBV through the interaction with eEF1a1.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R373

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