STRUCTURE OF HEPATITIS B ANTIGENS

乙型肝炎抗原的结构

• 批准号: 2060317
• 负责人: Darrell L Peterson
• 金额: $ 11.76万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-06-01 至 1996-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2060317
• 关键词: DNA directed DNA polymerase; albumins; aminoacid analyzer; binding proteins; chimeric proteins; chromatography; gel electrophoresis; genetic mapping; guinea pigs; hepatitis B antigens; hepatitis B virus group; immunochemistry; membrane proteins; monoclonal antibody; oligosaccharides; oral administration; peptidases; phosphorylation; posttranscriptional RNA processing; posttranslational modifications; protein biosynthesis; protein sequence; protein structure; radioimmunoassay; receptor binding; surface antigens; viral vaccines; virus DNA; virus protein

The first goal of this project is to determine the structures of the surface proteins of the hepatitis B virus, referred to as the hepatitis B surface antigen (HBsAg), and to determine the relationship between their structures and functions both as essential viral proteins and as the important host targets for prevention of the viral disease. HBsAg is produced in large amounts during the normal course of HBV infection, a fact which has been extensively exploited: HBsAg serves as the basis for the diagnosis of HBV infection and also as the current vaccine against HBV. HBsAg consists of a "nested" set of three proteins, referred to as the S, M, and L proteins, each of which occurs in two forms due to further post-translational modifications. The structure of these will be determined following isolation of the protein from naturally infected human plasma, or following expression of the relevant protein by cloned viral DNA in eukaryotic or prokaryotic expression vectors, using a combination of physical (circular dichroism, FTIR, mass spectrometry, ultracentrifugation), chemical (Edman degradation, carboxypeptidase digestion, reduction and alkylation), and immunological (monoclonal antibody binding studies, studies of immunogenicity) techniques. The function of each of these proteins as binding proteins to cell receptors and polymerized albumin will be examined. The antigenic activity of these proteins will be examined by site specific mutagenesis, chemical modification, use of anti- synthetic peptide and monoclonal antibodies. The second goal of this project will be to utilize the information gained from the above studies to identify specific antigenic domains of the surface proteins (S,M, and L) for incorporation into novel chimeric proteins containing the cholera B toxin subunit for investigation as a potential oral immunogen against HBV. This will be done utilizing synthetic DNA fused to the Cholera B subunit gene, and expression by appropriate prokaryotic expression vectors.

本项目的第一个目标是确定 B型肝炎病毒的表面蛋白，称为B型肝炎 表面抗原（HBsAg），并确定它们之间的关系 结构和功能都是必需的病毒蛋白质， 是预防病毒性疾病的重要宿主靶点。 HBsAg是 在HBV感染的正常过程中大量产生， 已被广泛利用的事实：HBsAg作为 乙肝病毒感染的诊断，也作为目前的疫苗， 乙肝病毒。 HBsAg由一组“嵌套”的三种蛋白质组成，称为 S、M和L蛋白，每种蛋白都有两种形式， 进一步的翻译后修饰。 其结构将是 在从天然感染的病毒中分离蛋白质后测定， 人血浆，或通过克隆表达相关蛋白质后 真核或原核表达载体中的病毒DNA，使用 物理（圆二色性，FTIR，质谱， 超离心）、化学（Edman降解、羧肽酶 消化、还原和烷基化）和免疫学（单克隆 抗体结合研究、免疫原性研究）技术。 的 这些蛋白质中的每一种作为细胞受体的结合蛋白的功能 和聚合白蛋白。 的抗原活性 这些蛋白质将通过位点特异性诱变、化学诱变和免疫荧光法进行检测。 修饰，使用抗合成肽和单克隆抗体。 该项目的第二个目标是利用信息 从上述研究中获得，以鉴定 用于掺入新嵌合体的表面蛋白（S、M和L） 含有霍乱B毒素亚单位的蛋白质， 抗HBV的潜在口服免疫原。 这将通过使用 与霍乱B亚单位基因融合的合成DNA，并通过 合适的原核表达载体。