HEPATITIS B VIRUS GENE EXPRESSION

乙型肝炎病毒基因表达

• 批准号: 3454372
• 负责人: Alan McLachlan
• 金额: $ 10.07万
• 依托单位: SCRIPPS CLINIC AND RESEARCH FOUNDATION
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1992-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3454372
• 关键词: Golgi apparatus; Retroviridae; disease /disorder model; duck hepatitis B virus; gene expression; genetic manipulation; genetic mapping; genetic transcription; hepatitis B antigens; molecular cloning; operon; simian virus 40; surface antigens; virus antigen; virus cytopathogenic effect; virus genetics; virus infection mechanism; virus replication

The absence of a tissue culture system and a convenient laboratory animal model system to propagate hepatitis B virus (HBV) has restricted the analysis of the events occurring during the HBV life cycle. The long-term objective, therefore, is to develop recombinant expression systems to analyze the viral and cellular factors which regulate HBV transcription, replication and assembly. In addition, since it is assumed that liver damage resulting from HBV Infection is mediated by a cellular immune response to hepatocytes expressing viral antigens, an amphotropoic retroviral expression system will be developed to generate autologous human cytolytic T-lymphocyte (CTL) target/stimulator cells expressing the various HBV antigens. The production of these cells will permit the analysis of this postulated mechanism of hepatocellular injury. Characterization of the four HBV open reading frames (ORFs), the surface, core, X and polymerase genes, using an amphotropic retroviral expression system, has been initiated. The analysis indicates that the pre-S(1) region of the surface antigen (HBsAg) has the capacity to sequester HBsAg in the pre-golgi or early golgi cellular compartment, the pre-core region has the properties of a signal peptide for protein secretion and the core polypeptide (HBcAg) contains signal peptide for protein secretion and the core polypeptide (HBcAg) contains signal sequences for the localization of HBcAg to the nucleus. Analysis of the cellular compartmentalization of HBV/marker protein fusions expressed using the retroviral expression vector should permit the identification of the various signal domains in these polypeptides. Using retroviral and SV40 expression vectors, the endogenous HBsAg and HBcAg promoters appear to be transcriptionally active. A detailed deletion analysis of these transcription units will permit the identification of regulatory sequences involved in the expression of these antigens. In addition, analysis of the endogenous HBV promoter activities in the cell lines expressing HBV antigens will determine if these polypeptides possess trans- acting regulatory activity. The introduction of mouse cell lines expressing HBV antigens into syngeneic mice will be used as a model system to determine which antigens can act as CTL targets. using the amphotropic expression vectors, the ability to transmit efficiently HBV antigen expression via retroviral infection to primary human cells will be developed so that the presence of HBV antigen specific CTL during viral infection might be investigated in man.

缺乏组织培养系统和方便的 复制乙肝病毒的实验动物模型系统 (乙肝病毒)限制了对期间发生的事件的分析 乙肝病毒的生命周期。因此，长期目标是 建立重组表达系统来分析病毒和 调节乙肝病毒转录、复制和转录的细胞因子 集合。此外，由于假设肝脏损伤 由乙肝病毒感染引起的是由细胞免疫调节的 对表达病毒抗原的肝细胞的反应 两性逆转录病毒表达系统将被开发为 产生自体人溶细胞T淋巴细胞(CTL) 表达各种乙肝病毒抗原的靶/刺激细胞。这个 这些细胞的生产将使分析这一点成为可能 肝细胞损伤的假设机制。 四个乙肝病毒开放阅读框架(ORF)的特征， 表面、核心、X和聚合酶基因，使用两性 逆转录病毒表达系统，已经启动。分析 提示表面抗原的前S(1)区 有能力在高尔基体前或早期隔离乙肝表面抗原 高尔基细胞室，前核心区具有 用于蛋白质分泌的信号肽和核心多肽 (HBcAg)含有蛋白质分泌的信号肽和核心 多肽(HBcAg)含有定位的信号序列 将HBcAg带到细胞核。对细胞的分析 表达的乙肝病毒/标志蛋白融合蛋白的区划 使用逆转录病毒表达载体应该允许 鉴定这些多肽中的各种信号域。 利用逆转录病毒和SV40表达载体，内源性 HBs Ag和HBcAg启动子似乎是转录的 激活。这些转录单位的详细缺失分析 将允许识别涉及的调控序列 这些抗原的表达。此外，还分析了 内源性乙肝病毒启动子在细胞株中的表达 乙肝病毒抗原将决定这些多肽是否具有反式- 代理监管活动。 表达乙肝病毒抗原的小鼠细胞系的导入 同基因小鼠将被用作模型系统来确定 抗原可以作为CTL靶标。使用两性体 高效传递乙肝病毒抗原的表达载体 通过逆转录病毒感染原代人类细胞表达 发展到存在乙肝病毒抗原特异性的CTL 在病毒感染期间，可能会在人类身上进行调查。