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HEPATITIS B VIRUS GENE EXPRESSION

乙型肝炎病毒基因表达

基本信息

批准号：
3454376
负责人：
Alan McLachlan
金额：
$ 11.8万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
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

项目摘要

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一种增殖B型肝炎病毒实验动物模型系统 (HBV)限制了对发生在 HBV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，调节HBV转录、复制和组装件.此外，由于假定肝损伤由HBV感染引起的免疫反应是由细胞免疫介导的，对表达病毒抗原的肝细胞的反应，将开发一种新的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。的这些细胞的产生将允许分析这些细胞。肝细胞损伤的假定机制。四个HBV开放阅读框（ORF）的表征，表面，核心，X和聚合酶基因，使用嗜异性逆转录病毒表达系统已经启动。分析表明表面抗原（HBsAg）的前S（1）区有能力隔离HBsAg在前高尔基体或早期高尔基体细胞隔室，前核心区域具有用于蛋白质分泌的信号肽和核心多肽（HBcAg）含有蛋白分泌的信号肽和核心一种多肽（HBcAg）含有定位信号序列将HBcAg转移到细胞核。细胞分析表达的HBV/标志物蛋白融合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SV 40表达载体， HBsAg和HBcAg启动子似乎在转录水平上与HBsAg和HBcAg启动子相似。活跃这些转录单位的详细缺失分析将允许鉴定涉及以下的调节序列：这些抗原的表达。此外，分析内源性HBV启动子活性在表达 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抗原的小鼠细胞系导入同系小鼠将用作模型系统以确定抗原可以作为CTL靶。利用双温性表达载体，能够有效地传输HBV抗原通过逆转录病毒感染原代人细胞的表达将是因此，HBV抗原特异性CTL的存在在病毒感染过程中，可能会在人类中进行研究。