EXPRESSION OF HIV ANTIGENS IN E COLI

HIV 抗原在大肠杆菌中的表达

• 批准号: 3811240
• 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/3811240
• 关键词: Escherichia coli; antiviral agents; drug screening /evaluation; epitope mapping; gene expression; human immunodeficiency virus; hybridomas; laboratory rabbit; molecular cloning; monoclonal antibody; polymerase chain reaction; virus antigen

DNA clones expressing portions of the gag, env, and pol genes of HIV in E. coli have been isolated and characterized in order to derive information on antigenic epitopes and to analyze reactivities of human sera at different stages of virus infection. Eight clones expressing different regions of gag gene proteins pl5, p24, and p17 have been generated by recombinant DNA techniques. A ninth clone was prepared using the polymerase chain reaction followed by standard cloning techniques. These 9 clones were used to map epitopes on the gag gene recognized by a panel of monoclonal antibodies. A manuscript describing these studies has been published and an invention report filed. HIV integrase (IN) is a viral enzyme required for integration of virus DNA into the host chromosome. As part of a collaborative project with Dr. Judith Levin (NICHD/NIH) and Dr. Donald Court (FCRF) the pol clone pWS4-1 has been subcloned to express only the integrase (IN) gene in order to produce monospecific antisera. An IN clone was isolated which expresses a fusion protein containing 27 amino acids of the RT and all of IN and the 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 27 amino acids of RT. An additional clone expressing only IN sequence has been constructed using PCR, and the protein can be isolated from gels in enzymatically active form. The recombinant protein and the assay we have developed may be useful in large-scale screening for HIV antivirals and an invention report has been filed. In addition, twenty-four hybridomas expressing mAbs against IN and RT have been prepared and are being subcloned and characterized.

在E. 大肠杆菌已被分离和表征，以获得信息， 抗原表位，并分析人血清在不同温度下的反应性， 病毒感染的阶段。八个克隆表达不同的gag区域 基因蛋白p15、p24和p17已经通过重组DNA产生 技术.使用聚合酶链反应制备第九克隆 然后是标准的克隆技术这9个克隆用于作图 由一组单克隆抗体识别的gag基因上的表位。一 描述这些研究的手稿已经出版， 报告存档。HIV整合酶（IN）是整合所需的病毒酶 病毒DNA进入宿主染色体。作为合作项目的一部分 与朱迪思·莱文博士（NICHD/NIH）和唐纳德·考特博士（FCRF）一起研究pol克隆 已将pWS 4 -1亚克隆以仅表达整合酶（IN）基因， 以产生单特异性抗血清。分离出IN克隆，其表达 含有RT和所有IN的27个氨基酸的融合蛋白， 纯化蛋白并用于免疫家兔。兔抗血清是 通过Western印迹，与HIV-1 IN反应，但不与HIV-2 IN反应， 其中RT表示抗原表位是present.in，末端27 仅表达IN序列的另外的克隆已经被克隆。 使用PCR构建，并且可以从凝胶中分离蛋白质， 酶活性形式。我们的重组蛋白和检测方法 开发可能是有用的，在大规模筛选艾滋病毒抗病毒药物， 发明报告已提交。此外，24个杂交瘤 已经制备了表达抗IN和RT的mAb， 亚克隆和表征。