本文選題：乙型肝炎病毒X蛋白 + 血漿銅藍蛋白��； 參考：《福建醫(yī)科大學》2013年博士論文

【摘要】：乙型肝炎病毒(Hepatitis B virus, HBV)感染是一種呈世界性分布的，危害嚴重的傳染病，是導致慢性肝炎、肝硬化和肝癌（hepatocellular carcinoma, HCC）的主要原因。但其具體致病的機制尚未完全明確。 HBV基因組為全長僅為3.2kb的部分雙鏈環(huán)狀DNA，含四個部分重疊的開放讀碼框（Open reading frame, ORF）：P，S，C，X；其中HBx蛋白是由X讀碼框編碼的，含有154個氨基酸，分子量為16.5kD，是一個多功能蛋白，在HBV感染過程中發(fā)揮著重要的作用，是HBV重要的致病因子之一。本研究在先前CytoTrap酵母雙雜交初步篩選結(jié)果的基礎上，驗證了HBx蛋白可與血漿銅藍蛋白（Ceruloplasmin,Cp）在酵母中發(fā)生相互作用，并進一步確定了二者在體內(nèi)和體外均可發(fā)生相互作用，最后探討二者相互作用后HBx對Cp的功能影響，更有助于深入了解HBV的致病機制。 本研究第一部分在CytoTrap酵母雙雜交初步篩選的基礎上，，抽提出一個推定陽性克隆的獵物文庫質(zhì)粒，經(jīng)酶切鑒定后送測序分析，鑒定為血漿銅藍蛋白（serumceruloplasmin），并將誘餌質(zhì)粒和抽提出的獵物文庫質(zhì)粒重新共轉(zhuǎn)化到酵母中，利用CytoTrap酵母雙雜交系統(tǒng)初步驗證了HBx與Cp可在酵母中發(fā)生相互作用。 本研究第二部分通過GST pull-down和免疫共沉淀（Co-immunoprecipitation, Co-IP）兩個實驗分別進一步證實了HBx蛋白與Cp可以在細胞外和細胞內(nèi)存在相互作用，并且利用CytoTrap酵母雙雜交系統(tǒng)確定了與Cp的發(fā)生相互作用是HBx蛋白的第51-80氨基酸區(qū)段。 本研究的第三部分旨在探討二者相互作用后，HBx對Cp及其相關功能的影響。利用Cp和HBx共轉(zhuǎn)染肝癌細胞HepG2，通過檢測細胞內(nèi)源性活性氧（ROS），氧自由基等氧化應激指標，發(fā)現(xiàn)HBx可通過與Cp的相互作用抑制其抗氧化功能，間接參與了HBV的致病過程，提示Cp可能可作為抗HBV治療的一個新的研究方向。
[Abstract]:Hepatitis B virus (HBV) infection is a worldwide and serious infectious disease, which is the main cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC). However, the mechanism of its specific pathogenesis has not been completely clear. The genome of HBV is a partial double-stranded circular DNA with only a full length of 3.2kb. It contains four overlapping open reading frames (ORF: PX), in which the HBx protein is encoded by X-reading frame and contains 154 amino acids with a molecular weight of 16.5 kD. it is a multifunctional protein. It plays an important role in the process of HBV infection and is one of the important pathogenic factors of HBV. Based on the preliminary screening results of CytoTrap yeast two-hybrid, this study demonstrated that HBx protein could interact with plasma ceruloplasmin protein in yeast, and further confirmed that the two proteins could interact in vivo and in vitro. Finally, the effects of HBx on CP function after the interaction between HBV and HBV were discussed, which was helpful to understand the pathogenicity of HBV. In the first part of this study, based on the screening of CytoTrap yeast two-hybrid, a prey-library plasmid of a presumed positive clone was proposed and sequenced by restriction endonuclease digestion. The plasmids were identified as serumceruloplasminmins, and the bait plasmids and prey library plasmids were retransformed into yeast. The interaction between HBx and CP in yeast was preliminarily verified by CytoTrap yeast two-hybrid system. In the second part of this study, GST pull-down and Co-immunoprecipitation (Co-IP) experiments showed that HBx protein and CP could interact with each other in vitro and in cells, respectively. The interaction with CP was confirmed to be the 51-80 amino acid region of HBx by CytoTrap yeast two-hybrid system. The third part of this study is to investigate the effect of HBX on CP and its related functions. HepG2 cells were co-transfected with CP and HBx. By detecting the oxidative stress indexes such as endogenous reactive oxygen species (Ros) and oxygen free radicals (OFR), it was found that HBx could inhibit the antioxidant function of HepG2 cells through the interaction with CP, and indirectly participate in the pathogenesis of HBV. The results suggest that CP may be a new research direction in anti-HBV therapy.
【學位授予單位】：福建醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R512.62

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