HBV真核表达质粒的构建及其稳定细胞系的建立

发布时间：2018-07-04 10:53

本文选题：乙型肝炎 + HepG2细胞　；参考：《重庆医科大学》2011年硕士论文

【摘要】：乙型病毒性肝炎是全球公共卫生问题,全世界约有3.5亿人因乙型肝炎病毒(Hepatitis B virus; HBV)导致慢性感染,慢性感染可能会发展为肝硬化甚至肝癌( hepatocarcinoma; HCC)。目前对慢性乙肝患者治疗常用的药物为I型干扰素(Interferon,IFNs)与核苷类似物(nucleotide analogs,NAs),它们在临床上得到了广泛应用并且在一定程度上减缓了疾病的恶化。但是由于HBV病毒变异速度快,药物疗效有限,还无法长期有效地遏制HBV对人肝组织细胞的感染及其在胞内DNA的复制。为了寻找新型治疗方法及抗病毒药物,需要深入了解HBV的感染机制,这就有待于筛选理想的HBV感染模型。近二十年来HBV体内感染模型研究进展缓慢,而HBV体外感染模型的研究取得了长足的进展,其中稳定HBV表达细胞系由于其表达水平较稳定且几乎所有细胞都能产生HBV,逐渐成为国内外学者研究的热点。本研究通过构建包含HBV1.1倍体及巨噬细胞病毒(cytomegalovirus;CMV)启动子的重组质粒pneo-CH9/3091,使其表达新霉素(neomycin)抗性并将其稳定转染至人肝癌细胞HepG2,经G418筛选,得到单克隆细胞株。通过多种分子生物学手段,如酶联免疫吸附法(ELISA)检测培养上清表面抗原(HBsAg)及e抗原(HBeAg)的分泌,Western blot检测细胞内HBV核心蛋白(HBcAg)的表达,以及Southern blot检测细胞内核心颗粒HBV DNA的复制情况等,建立了一株HBV稳定整合细胞株HepG2-H7。最后将此细胞株与HepG2.2.15细胞在HBV DNA的复制与蛋白表达水平等方面进行了比较。本研究结果提示HepG2-H7细胞株有望应用于抗病毒药物的筛选和HBV感染机制的研究。
[Abstract]:Hepatitis B is a global public health problem. There are about 350 million people in the world who suffer from chronic infection caused by hepatitis B virus (HBV), which may develop into liver cirrhosis or even hepatocellular carcinoma (HCC). At present, the commonly used drugs in the treatment of chronic hepatitis B patients are interferon I (IFNs) and nucleotide analogs (nucleotide analogs), which have been widely used in clinic and slow down the deterioration of the disease to a certain extent. However, due to the rapid mutation rate of HBV virus and the limited efficacy of drugs, HBV infection of human liver tissue cells and its replication in cellular DNA can not be effectively controlled for a long time. In order to find new treatment methods and antiviral drugs, it is necessary to deeply understand the mechanism of HBV infection, which requires the screening of ideal HBV infection models. In the past two decades, the study of HBV in vivo infection model has been slow, while the study of HBV in vitro infection model has made great progress. Among them, stable HBV expression cell lines have become the focus of domestic and foreign scholars because of their stable expression level and the ability of almost all cells to produce HBV. In this study, a recombinant plasmid pneo-CH9 / 3091 containing HBV 1.1 ploidy and cytomegalovirus virus promoter was constructed and transfected stably into human hepatoma cell line HepG2. Monoclonal cell lines were obtained by G418 screening. The expression of HBV core protein (HBcAg) was detected by enzyme linked immunosorbent assay (Elisa) in culture supernatant surface antigen (HBsAg) and e antigen (HBeAg) by Western blot. A stable integrated HBV cell line HepG2-H7 was established by Southern blot. Finally, the replication and protein expression of HBV DNA were compared between this cell line and HepG2.2.15 cell line. The results suggest that HepG2-H7 cell line may be used in the screening of antiviral drugs and the mechanism of HBV infection.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R346

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