EXPRESSION OF HIV ANTIGENS IN E COLI

HIV 抗原在大肠杆菌中的表达

• 批准号: 3804789
• 负责人: C J MARCUS-SEKURA
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3804789
• 关键词: DNA binding protein; Escherichia coli; RNA directed DNA polymerase; antiviral agents; chimeric proteins; chromosomes; epitope mapping; gene expression; human immunodeficiency virus 1; human immunodeficiency virus 2; hybridomas; immunization; immunoglobulin G; laboratory rabbit; molecular cloning; monoclonal antibody; polymerase chain reaction; protein purification; southern blotting; virus DNA; virus antigen; virus protein; western blottings

HIV integrase (IN) is a viral enzyme required for integration of virus DNA into the host chromosome. This type of integration is highly specific for retroviruses, and HIV-1 IN is therefore a target for antiviral therapy. To design antivirals, it is essential to characterize IN. We have expressed the IN gene in E. coli as a fusion protein containing 23 amino acids of RT and all of IN. The cloned IN was reactive with both HIV-1 and HIV-2 positive sera. The recombinant protein was purified and used to immunize rabbits. The rabbit antisera are reactive with HIV-1 IN but not HIV-2 IN by Western blot, and also reactive with RT indicating that an antigenic epitope is present in the terminal 23 amino acids of RT. These data have recently been published in AIDS Research and Human Retroviruses (1990). In addition, 21 hybridomas have been prepared using the IN expressing clone and subcloned twice. 19 of these mAbs have specificity for IN and 2 for RT; all are IgG1, and all are reactive with HIV-1 but not HIV-2 by Western blot. An invention report has been filed and a manuscript is in preparation. An additional clone expressing only IN sequence has been constructed using PCR, as well as clones expressing the N- and C-terminal halves of IN. These clones have been examined for ability to bind DNA using a Southwestern blotting procedure. The complete IN molecule as well as the C-terminal protein bind DNA; the N-terminal portion exhibits no binding activity, suggesting that the C-terminal region contains the DNA binding site. A manuscript describing these findings is in preparation.

HIV整合酶（IN）是病毒DNA整合所必需的病毒酶 插入宿主染色体这种类型的集成非常具体， 逆转录病毒，因此HIV-1 IN是抗病毒治疗的靶点。 为了设计抗病毒药物，必须对IN进行表征。 我们有 在E.大肠杆菌表达的融合蛋白， RT和所有IN的酸。 克隆的IN与HIV-1和 HIV-2阳性血清。纯化重组蛋白并用于 使兔子免疫。 兔抗血清与HIV-1 IN反应，但不与HIV-1 IN反应。 免疫印迹法检测HIV-2 IN，并与RT反应，表明 抗原表位存在于RT的末端23个氨基酸中。 数据最近发表在AIDS Research and Human Retroviruses上 （1990年）。 此外，已经使用IN制备了21个杂交瘤。 表达克隆并亚克隆两次。 其中19种mAb具有特异性 IN和RT各2例;均为IgG 1，均与HIV-1反应，但不与 HIV-2蛋白质印迹法。 已提交发明报告， 手稿正在准备中。仅表达IN的额外克隆 已经使用PCR构建了序列，以及表达该序列的克隆。 IN的N-和C-末端一半。这些克隆人已经被检查过了 使用西南印迹程序结合DNA的能力。 完整的 IN分子以及C-末端蛋白质结合DNA; N-末端蛋白质结合DNA。 部分没有表现出结合活性，这表明C-末端 区域包含DNA结合位点。 一份描述这些的手稿 调查结果正在准备中。