EXPRESSION OF HIV ANTIGENS IN E COLI

表达HIV的Gag、env和pol3部分基因的DNA克隆。 已经分离并鉴定了大肠杆菌，以便获得关于 人血清不同抗原表位的反应性分析 病毒感染的各个阶段。表达GAG不同区域的8个克隆 基因蛋白pl5、p24和p17已通过重组dna产生。 技巧。用聚合酶链式反应制备了第九个克隆 然后是标准的克隆技术。这9个克隆用于作图 由一组单抗识别的Gag基因上的表位。一个 描述这些研究的手稿已经出版，并发明了 报告已归档。HIV整合酶(IN)是一种整合所需的病毒酶 病毒DNA进入宿主染色体。作为协作项目的一部分 与朱迪思·莱文博士(NICHD/NIH)和唐纳德·考特博士(FCRF)一起克隆Poll PWS4-1已被亚克隆，只表达整合酶(IN)基因 以产生单特异性的抗血清。分离到一个IN克隆，该克隆表达 含27个氨基酸的RT、ALL、IN和 纯化后的蛋白免疫兔。兔抗血清为 免疫印迹显示与HIV-1IN反应，但不与HIV-2IN反应，也有反应 RT表示存在抗原表位。在终端27中 RT的氨基酸组成。另外一个仅按顺序表达的克隆已经被 使用聚合酶链式反应构建，并且该蛋白可以从凝胶中分离 具有酶活性的形式。重组蛋白和我们已有的分析 开发的可能用于艾滋病毒抗病毒药物的大规模筛查和 发明报告已提交。此外，24株杂交瘤 针对IN和RT的表达单抗已经准备好并正在进行 亚克隆和鉴定。

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.