EXPRESSION OF HIV ANTIGENS IN E COLI

HIV整合酶(IN)是整合病毒DNA所必需的一种病毒酶 进入宿主染色体。这种类型的集成高度特定于 因此，HIV-1IN是抗病毒治疗的目标。 为了设计抗病毒药物，对IN进行表征是必不可少的。我们有 在大肠杆菌中以含23个氨基酸的融合蛋白的形式表达IN基因 RT和所有IN的酸。克隆的IN与HIV-1和 HIV-2阳性血清。纯化了重组蛋白，并将其用于 给兔子免疫。兔抗血清与HIV-1IN反应，但不与HIV-1IN反应 通过Western印迹检测HIV-2 IN，并与RT反应，表明 抗原性表位位于RT的23个氨基酸末端。这些 数据最近发表在《艾滋病研究和人类逆转录病毒》杂志上 (1990年)。此外，还利用IN制备了21株杂交瘤 表达克隆和亚克隆两次。其中19株单抗具有特异性 IN和RT；都是IgG1，都对HIV-1有反应，但不是 免疫印迹法检测HIV-2。已经提交了一份发明报告和一份 手稿正在准备中。一个仅在IN中表达的额外克隆 用聚合酶链式反应构建了序列，并克隆了表达 IN的N-末端和C-末端的一半。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 在分子中以及C-端蛋白结合DNA；N-端 部分没有结合活性，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。

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.