HEPATITIS B VIRUS GENE EXPRESSION

乙型肝炎病毒基因表达

• 批准号: 3454373
• 负责人: Alan McLachlan
• 金额: $ 10.47万
• 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/3454373
• 关键词: Golgi apparatus; Retroviridae; disease /disorder model; duck hepatitis B virus; gene expression; genetic manipulation; genetic mapping; genetic promoter element; genetic transcription; hepatitis B antigens; laboratory mouse; molecular cloning; operon; protein signal sequence; simian virus 40; surface antigens; transfection; virus antigen; virus cytopathogenic effect; virus genetics; virus infection mechanism; virus protein; 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 传播乙型肝炎病毒的实验动物模型系统 （HBV）限制了对期间发生的事件的分析 乙型肝炎病毒的生命周期。 因此，长期目标是 开发重组表达系统来分析病毒和 调节 HBV 转录、复制和传播的细胞因子 集会。此外，由于推测肝损伤 乙型肝炎病毒感染是由细胞免疫介导的 对表达病毒抗原的肝细胞的反应 将开发双嗜性逆转录病毒表达系统 产生自体人溶细胞性 T 淋巴细胞 (CTL) 表达各种 HBV 抗原的靶细胞/刺激细胞。 这 这些细胞的产生将允许分析这个 肝细胞损伤的假设机制。 四个 HBV 开放阅读框 (ORF) 的表征， 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启动。 分析 表示表面抗原 (HBsAg) 的前 S(1) 区域 具有在高尔基体前或早期隔离 HBsAg 的能力 高尔基体细胞区室，前核心区域具有以下特性 蛋白质分泌信号肽和核心多肽的制备 (HBcAg) 含有蛋白质分泌的信号肽和核心 多肽 (HBcAg) 包含用于定位的信号序列 HBcAg 进入细胞核。 细胞分析 表达的 HBV/标记蛋白融合体的区室化 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结构域。 使用逆转录病毒和 SV40 表达载体，内源性 HBsAg 和 HBcAg 启动子似乎是转录的 积极的。 这些转录单元的详细缺失分析 将允许识别涉及的调控序列 这些抗原的表达。 此外，分析 表达细胞系中内源性 HBV 启动子活性 HBV 抗原将决定这些多肽是否具有反式 代理监管活动。 将表达 HBV 抗原的小鼠细胞系引入 同基因小鼠将被用作模型系统来确定哪些 抗原可以作为 CTL 靶标。 使用两性 表达载体，有效传递HBV抗原的能力 通过逆转录病毒感染原代人类细胞的表达将是 开发使得 HBV 抗原特异性 CTL 的存在 可以在人类身上研究病毒感染期间的情况。