人类乙型肝炎病毒e抗原结合蛋白2（HBEBP2）的克隆表达与部分功能研究

发布时间：2018-06-16 00:44

  本文选题：乙型肝炎病毒 + HbeAg结合蛋白　； 参考：《大连医科大学》2007年硕士论文

【摘要】： 乙型肝炎病毒的感染不仅引起急慢性病毒性肝炎,而且与肝纤维化、肝细胞癌的发生发展密切相关,然而对于HBV感染目前尚无特效的治疗技术和方法,这主要是由于HBV感染引起的病毒性肝炎发病机制还有诸多方面没有研究清楚。近年来,随着对HBV致病机制研究的深入,人们发现在HBV致病机制中存在着其与肝细胞间复杂的相互作用关系。为此,本研究所筛选了肝细胞内能与e抗原相互结合的蛋白,并将其中一个未知功能新基因命名为乙型肝炎病毒e抗原结合蛋白2(HBEBP2)。本研究即以此为基础,通过以下研究内容,试图阐释该基因的生物学功能: 1.提取HepG2细胞总RNA,利用RT-PCR方法在HBEBP2基因的两端分别引入BamHI和EcoRⅠ酶切位点,获得目的基因片段,连接至pGEM-T载体,测序正确后插入至绿色荧光真核表达载体pEGFP-C1中,转染HepG2细胞,24hr后在荧光倒置显微镜下观察,发现细胞核出现明显的绿色荧光信号,提示HBENP2主要定位于胞核内。 2.利用相同方法将引入EcoRⅠ和SacⅠ酶切位点的HBEBP2基因片段插入至原核表达载体pET-32a(+)中,转化BL21宿主菌,1mM/ml IPTG、37℃诱导4.5hr获得了带有组氨酸标签的重组融合蛋白的优化表达。随后应用Western blot对表达蛋白的特异性进行了验证。 3.将引入EcoRⅠ和BamHⅠ酶切位点的HBEBP2基因片段插入至酵母细胞表达载体pGBKT7中,转化AH109酵母菌株并获得表达。再与含有肝细胞文库质粒的Y187酵母菌株配合,进行酵母双杂交筛选获得与HBEBP2相互结合的已知功能蛋白4种,包括人线粒体蛋白基因、人α-2-糖蛋白1、人甘露糖-p-长醇利用缺陷1基因和人丝氨酸蛋白酶抑制剂等蛋白基因。结果提示该蛋白可能与糖基化作用、脂类代谢、细胞增殖等密切相关。此外还筛选得到2个新基因,分别命名为HBEBP2结合蛋白B(HBEBP2BPB)(GenBank Accession:DQ499598)和结合蛋白C(HBEBP2BPC)(GenBank Accession:DQ499599)。 4.随后,我们又对HBEBP2BPB进行了克隆化研究,并用生物信息学方法对其作了进一步分析。 以上结果,对深入研究HBEBP2生物学功能的后续工作奠定了基础。
[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，

本文编号：2024420