酵母双杂交筛选与乙型肝炎病毒剪接特异性蛋白相互作用的肝细胞蛋白

发布时间：2018-10-23 16:49

【摘要】：乙型肝炎病毒剪接特异性蛋白（hepatitis B spliced protein，HBSP）由2447nt-489nt单剪接型2.2Kb HBV剪接变异体编码产生，与病毒的致病性及持续性感染相关，但其具体机制尚未阐明。本研究首先利用经典的细胞核酵母双杂交筛选与HBSP相互作用的肝细胞蛋白，共获得1166个阳性克隆，，进一步在酵母体内进行验证，去除假阳性和重复克隆后共得到金属硫蛋白（metallothionein-2，MT2），乙醇脱氢酶（alcohol dehydrogenase1A(class I)， alpha polypeptide，ADH1A），tRNA剪接内切酶（tRNA-splicing endonuclease subunit34，TSEN34），乙酰辅酶A酰基转移酶（acetyl-CoAacyltransferase2，ACAA2)等38个与HBSP相互作用的猎物蛋白。选取乙醇脱氢酶1A（ADH1A）验证其与HBSP在哺乳动物细胞体内外的相互作用。为此，构建pGEX-4T-1-HBSP载体，转化大肠杆菌并纯化获得GST-HBSP融合蛋白，以RT-PCR技术从HepG2细胞扩增得到ADH1A全长基因，构建pCMVTNT-ADH1A载体，体外转录翻译出ADH1A蛋白，GST pull-down实验证实ADH1A蛋白可以和HBSP体外相互作用。将HBSP重组腺病毒感染肝癌细胞株，进行内源性免疫共沉淀实验，证实HBSP和哺乳动物细胞内源性ADH1A具有相互作用。本研究为今后进一步探讨HBSP致病性奠定基础。
[Abstract]:Hepatitis B virus splicing specific protein (hepatitis B spliced protein,HBSP) is encoded by 2447nt-489nt single-splicing 2.2Kb HBV splicing variant, which is related to the pathogenicity and persistent infection of the virus, but its specific mechanism has not been elucidated. In this study, 1166 positive clones were obtained by screening hepatocyte proteins interacting with HBSP by classical two-hybrid hybridization of nuclear yeast, which were further verified in yeast. After removing false positive and repeated clones, 38 prey proteins interacting with HBSP were obtained, such as metallothionein (metallothionein-2,MT2), ethanol dehydrogenase (alcohol dehydrogenase1A (class I), alpha polypeptide,ADH1A), tRNA) splicing endonuclease (tRNA-splicing endonuclease subunit34,TSEN34) and acetyl-coenzyme A acyltransferase (acetyl-CoAacyltransferase2,ACAA2). Ethanol dehydrogenase 1A (ADH1A) was selected to determine the interaction between HBSP and ethanol dehydrogenase 1A in mammalian cells in vitro and in vivo. Therefore, we constructed pGEX-4T-1-HBSP vector, transformed Escherichia coli and purified GST-HBSP fusion protein, amplified the full-length ADH1A gene from HepG2 cells by RT-PCR technique, and constructed pCMVTNT-ADH1A vector. ADH1A protein was transcribed and translated in vitro. GST pull-down assay confirmed that ADH1A protein could interact with HBSP in vitro. The recombinant adenovirus (HBSP) was infected with hepatoma cell line, and the result of endogenous co-immunoprecipitation showed that HBSP and mammalian cell endogenous ADH1A interacted with each other. This study will lay a foundation for further study of pathogenicity of HBSP.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R373

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