乙型肝炎病毒HBx蛋白与血浆铜蓝蛋白的相互作用及对其功能影响

发布时间：2018-05-06 07:44

本文选题：乙型肝炎病毒X蛋白 + 血浆铜蓝蛋白　；参考：《福建医科大学》2013年博士论文

【摘要】：乙型肝炎病毒(Hepatitis B virus, HBV)感染是一种呈世界性分布的，危害严重的传染病，是导致慢性肝炎、肝硬化和肝癌（hepatocellular carcinoma, HCC）的主要原因。但其具体致病的机制尚未完全明确。 HBV基因组为全长仅为3.2kb的部分双链环状DNA，含四个部分重叠的开放读码框（Open reading frame, ORF）：P，S，C，X；其中HBx蛋白是由X读码框编码的，含有154个氨基酸，分子量为16.5kD，是一个多功能蛋白，在HBV感染过程中发挥着重要的作用，是HBV重要的致病因子之一。本研究在先前CytoTrap酵母双杂交初步筛选结果的基础上，验证了HBx蛋白可与血浆铜蓝蛋白（Ceruloplasmin,Cp）在酵母中发生相互作用，并进一步确定了二者在体内和体外均可发生相互作用，最后探讨二者相互作用后HBx对Cp的功能影响，更有助于深入了解HBV的致病机制。本研究第一部分在CytoTrap酵母双杂交初步筛选的基础上，，抽提出一个推定阳性克隆的猎物文库质粒，经酶切鉴定后送测序分析，鉴定为血浆铜蓝蛋白（serumceruloplasmin），并将诱饵质粒和抽提出的猎物文库质粒重新共转化到酵母中，利用CytoTrap酵母双杂交系统初步验证了HBx与Cp可在酵母中发生相互作用。本研究第二部分通过GST pull-down和免疫共沉淀（Co-immunoprecipitation, Co-IP）两个实验分别进一步证实了HBx蛋白与Cp可以在细胞外和细胞内存在相互作用，并且利用CytoTrap酵母双杂交系统确定了与Cp的发生相互作用是HBx蛋白的第51-80氨基酸区段。本研究的第三部分旨在探讨二者相互作用后，HBx对Cp及其相关功能的影响。利用Cp和HBx共转染肝癌细胞HepG2，通过检测细胞内源性活性氧（ROS），氧自由基等氧化应激指标，发现HBx可通过与Cp的相互作用抑制其抗氧化功能，间接参与了HBV的致病过程，提示Cp可能可作为抗HBV治疗的一个新的研究方向。
[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.
【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R512.62

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