【摘要】：乙型肝炎病毒剪接特異性蛋白（hepatitis B spliced protein，HBSP）由2447nt-489nt單剪接型2.2Kb HBV剪接變異體編碼產(chǎn)生，與病毒的致病性及持續(xù)性感染相關(guān)，但其具體機(jī)制尚未闡明。本研究首先利用經(jīng)典的細(xì)胞核酵母雙雜交篩選與HBSP相互作用的肝細(xì)胞蛋白，共獲得1166個(gè)陽(yáng)性克隆，，進(jìn)一步在酵母體內(nèi)進(jìn)行驗(yàn)證，去除假陽(yáng)性和重復(fù)克隆后共得到金屬硫蛋白（metallothionein-2，MT2），乙醇脫氫酶（alcohol dehydrogenase1A(class I)， alpha polypeptide，ADH1A），tRNA剪接內(nèi)切酶（tRNA-splicing endonuclease subunit34，TSEN34），乙酰輔酶A�；D(zhuǎn)移酶（acetyl-CoAacyltransferase2，ACAA2)等38個(gè)與HBSP相互作用的獵物蛋白。選取乙醇脫氫酶1A（ADH1A）驗(yàn)證其與HBSP在哺乳動(dòng)物細(xì)胞體內(nèi)外的相互作用。為此，構(gòu)建pGEX-4T-1-HBSP載體，轉(zhuǎn)化大腸桿菌并純化獲得GST-HBSP融合蛋白，以RT-PCR技術(shù)從HepG2細(xì)胞擴(kuò)增得到ADH1A全長(zhǎng)基因，構(gòu)建pCMVTNT-ADH1A載體，體外轉(zhuǎn)錄翻譯出ADH1A蛋白，GST pull-down實(shí)驗(yàn)證實(shí)ADH1A蛋白可以和HBSP體外相互作用。將HBSP重組腺病毒感染肝癌細(xì)胞株，進(jìn)行內(nèi)源性免疫共沉淀實(shí)驗(yàn)，證實(shí)HBSP和哺乳動(dòng)物細(xì)胞內(nèi)源性ADH1A具有相互作用。本研究為今后進(jìn)一步探討HBSP致病性奠定基礎(chǔ)。
[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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R373

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