ADENOVIRUS 2 CODED EARLY GLYCOPROTEIN

腺病毒 2 编码的早期糖蛋白

• 批准号: 3166560
• 负责人: WILLIAM SM WOLD
• 金额: $ 18.6万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-05-01 至 1995-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3166560
• 关键词: Adenoviridae; DNA replication; Escherichia coli; X ray crystallography; affinity chromatography; antiserum; binding proteins; biological signal transduction; cell membrane; chimeric proteins; cytotoxic T lymphocyte; epidermal growth factor; gel electrophoresis; gene expression; genetic mapping; genetic transcription; genetic translation; glycoproteins; growth factor receptors; hamsters; human tissue; immunoelectron microscopy; immunofluorescence technique; immunoregulation; laboratory rabbit; major histocompatibility complex; membrane proteins; nucleic acid sequence; oncogenic virus; posttranslational modifications; protein biosynthesis; protein engineering; protein sequence; protein signal sequence; protein structure; radionuclides; radiotracer; site directed mutagenesis; sulfur; transfection; tumor necrosis factor alpha; virus DNA; virus cytopathogenic effect; virus genetics; virus protein

Proteins generally are localized at specific sites within the cell, and therefore regulatory information must exist in the structure of the protein to assure that they arrive at the correct destination. Our major objective is to identify this information, using as a model the 19 kDa plasma membrane glycoprotein (gp19K) coded by early region E3 adenovirus 1 (Ad2) or Ad5, and using a genetic approach. Gp19K is an abundant protein whose structure is known, and viable virus mutants can easily be constructed by in vitro site directed mutagenesis. Antisera are available against purified gp19K as well as synthetic peptides corresponding to gp19K. Gp19K has a 17 amino acid N-terminal "signal" sequence, two high-mannose oligosaccharides, and a 20 amino acid hydrophobic transmembrane domain followed by a 15 amino acid polar domain at the C-terminus. We have isolated virus mutants with sequenced lesions in the signal sequence, the glycosylation sites, and the C-terminal hydrophobic and/or polar domains. We propose to characterize these mutants in terms of processing and transport. Data are now available with some mutants. In a collaborative study, the mutants will be studied as a unique model for viral antigenclass 1 MHC antigen interaction. Other plasmid mutants, some sequenced, are available to construct additional virus mutants. We propose two novel and simple methods that apply standard techniques in bacterial genetics to isolate random missense mutations in gp19K. Region E3 encodes several other membrane proteins whose structures are fundamentally different from gp19K. We propose genetic studies, similar to those with gp19K, to identify the membrane-association and transport signals for these proteins. Virus mutants in some of the these genes, including fusions to gp19K, are currently available and additional mutants are easily constructed. Peptide antisera targeted to each of these proteins are available, and potent antisera directed fusion proteins synthesized by expression vectors will be generated.

蛋白质通常位于细胞内的特定位点，并且 因此调节信息必须存在于蛋白质的结构中 以确保他们到达正确的目的地。 我们的主要目标 是为了识别此信息，使用 19 kDa 等离子体作为模型 由早期区域 E3 腺病毒 1 (Ad2) 编码的膜糖蛋白 (gp19K) 或Ad5，并使用遗传方法。 Gp19K 是一种丰富的蛋白质， 结构已知，并且可以通过以下方式轻松构建可行的病毒突变体 体外定点诱变。 抗血清可用于对抗 纯化的 gp19K 以及与 gp19K 相对应的合成肽。 Gp19K有一个17个氨基酸的N端“信号”序列，两个高甘露糖 寡糖和 20 个氨基酸的疏水性跨膜结构域 随后在 C 末端有一个 15 个氨基酸的极性结构域。 我们有 分离的病毒突变体在信号序列中具有测序损伤， 糖基化位点，以及 C 末端疏水和/或极性结构域。 我们建议在加工和处理方面表征这些突变体 运输。 现在可以获得一些突变体的数据。 在协作中 研究中，突变体将作为病毒抗原类的独特模型进行研究 1 MHC 抗原相互作用。 其他质粒突变体，一些已测序，是 可用于构建额外的病毒突变体。 我们提出了两种新颖且 应用细菌遗传学标准技术的简单方法 分离 gp19K 中的随机错义突变。 E3 区编码其他几种膜蛋白，其结构为 与gp19K根本不同。 我们建议进行遗传学研究，类似于 那些带有 gp19K 的，用于识别膜关联和运输 这些蛋白质的信号。 其中一些基因的病毒突变体， 包括与 gp19K 的融合，目前可用以及其他突变体 很容易建造。 针对其中每一个的肽抗血清 蛋白质是可用的，并且有效的抗血清定向融合蛋白 将生成由表达载体合成的。