發(fā)布時間：2018-06-16 00:44

  本文選題：乙型肝炎病毒 + HbeAg結(jié)合蛋白�。� 參考：《大連醫(yī)科大學(xué)》2007年碩士論文

【摘要】： 乙型肝炎病毒的感染不僅引起急慢性病毒性肝炎,而且與肝纖維化、肝細(xì)胞癌的發(fā)生發(fā)展密切相關(guān),然而對于HBV感染目前尚無特效的治療技術(shù)和方法,這主要是由于HBV感染引起的病毒性肝炎發(fā)病機(jī)制還有諸多方面沒有研究清楚。近年來,隨著對HBV致病機(jī)制研究的深入,人們發(fā)現(xiàn)在HBV致病機(jī)制中存在著其與肝細(xì)胞間復(fù)雜的相互作用關(guān)系。為此,本研究所篩選了肝細(xì)胞內(nèi)能與e抗原相互結(jié)合的蛋白,并將其中一個未知功能新基因命名為乙型肝炎病毒e抗原結(jié)合蛋白2(HBEBP2)。本研究即以此為基礎(chǔ),通過以下研究內(nèi)容,試圖闡釋該基因的生物學(xué)功能: 1.提取HepG2細(xì)胞總RNA,利用RT-PCR方法在HBEBP2基因的兩端分別引入BamHI和EcoRⅠ酶切位點(diǎn),獲得目的基因片段,連接至pGEM-T載體,測序正確后插入至綠色熒光真核表達(dá)載體pEGFP-C1中,轉(zhuǎn)染HepG2細(xì)胞,24hr后在熒光倒置顯微鏡下觀察,發(fā)現(xiàn)細(xì)胞核出現(xiàn)明顯的綠色熒光信號,提示HBENP2主要定位于胞核內(nèi)。 2.利用相同方法將引入EcoRⅠ和SacⅠ酶切位點(diǎn)的HBEBP2基因片段插入至原核表達(dá)載體pET-32a(+)中,轉(zhuǎn)化BL21宿主菌,1mM/ml IPTG、37℃誘導(dǎo)4.5hr獲得了帶有組氨酸標(biāo)簽的重組融合蛋白的優(yōu)化表達(dá)。隨后應(yīng)用Western blot對表達(dá)蛋白的特異性進(jìn)行了驗(yàn)證。 3.將引入EcoRⅠ和BamHⅠ酶切位點(diǎn)的HBEBP2基因片段插入至酵母細(xì)胞表達(dá)載體pGBKT7中,轉(zhuǎn)化AH109酵母菌株并獲得表達(dá)。再與含有肝細(xì)胞文庫質(zhì)粒的Y187酵母菌株配合,進(jìn)行酵母雙雜交篩選獲得與HBEBP2相互結(jié)合的已知功能蛋白4種,包括人線粒體蛋白基因、人α-2-糖蛋白1、人甘露糖-p-長醇利用缺陷1基因和人絲氨酸蛋白酶抑制劑等蛋白基因。結(jié)果提示該蛋白可能與糖基化作用、脂類代謝、細(xì)胞增殖等密切相關(guān)。此外還篩選得到2個新基因,分別命名為HBEBP2結(jié)合蛋白B(HBEBP2BPB)(GenBank Accession:DQ499598)和結(jié)合蛋白C(HBEBP2BPC)(GenBank Accession:DQ499599)。 4.隨后,我們又對HBEBP2BPB進(jìn)行了克隆化研究,并用生物信息學(xué)方法對其作了進(jìn)一步分析。 以上結(jié)果,對深入研究HBEBP2生物學(xué)功能的后續(xù)工作奠定了基礎(chǔ)。
[Abstract]:Hepatitis B virus infection not only causes acute and chronic viral hepatitis, but also is closely related to the occurrence and development of liver fibrosis and hepatocellular carcinoma. However, there is no effective treatment for HBV infection. This is mainly due to the pathogenesis of viral hepatitis caused by HBV infection has not been studied in many aspects. In recent years, with the in-depth study of the pathogenesis of HBV, it has been found that there is a complex interaction between HBV and hepatocytes in the pathogenesis of HBV. In this study, a novel gene was named hepatitis B virus e antigen binding protein 2 (HBEBP2P), which can bind to e antigen in hepatocytes. The new gene was named as hepatitis B virus e antigen binding protein 2 (HBEBP2P), and a novel gene was named as hepatitis B virus e antigen binding protein 2. Based on this, this study attempts to elucidate the biological function of the gene through the following research contents: 1. Total RNAs were extracted from HepG2 cells. BamHI and Ecor 鈪，

本文編號：2024420