EXPRESSION OF HIV ANTIGENS IN E COLI

HIV 抗原在大肠杆菌中的表达

• 批准号: 3792517
• 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/3792517
• 关键词: DNA binding protein; DNA directed DNA polymerase; Escherichia coli; X ray crystallography; antiserum; antiviral agents; chimeric proteins; chromosomes; drug design /synthesis /production; enzyme activity; enzyme linked immunosorbent assay; enzyme structure; gene expression; human immunodeficiency virus 1; human immunodeficiency virus 2; hybridomas; immunoglobulin structure; laboratory rabbit; molecular cloning; monoclonal antibody; 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. The cloned IN is reactive with both HIV-1 and HIV-2 positive patient sera in ELISA, while rabbit antisera to the recombinant protein are reactive only with HIV-1 IN but not HIV-2 IN by Western blot. These data have been published in AIDS Res. Hum. Retro. Hybridomas have been prepared using the IN expressing clone and the MAbs are reactive with HIV-1 but not HIV-2 by Western blot. An invention report has been filed and a manuscript is in preparation. Additional clones expressing the N- and C-terminal halves of IN have been constructed and the proteins they express 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 has recently been published in AIDS Res. Hum. Retro. We have purified our full-length recombinant IN and it exhibits activity in a specific in vitro assay for enzyme activity involving cleavage of 2 bp from an oligonucleotide corresponding to the HIV-1 LTR. Since our IN is produced as a fusion protein containing 13 amino acids of lambda cII at its amino terminus, detection of activity with our protein indicates that a free amino-terminus is not required for activity. This may be relevant to the in vivo situation, since IN is initially synthesized as a polyprotein linked to RT at its amino-terminus. To more precisely localize the DNA binding site within the C-terminal half of the molecule, we have constructed a series of additional subclones and we are currently purifying the deleted proteins they produce for analysis of DNA binding. We have recently established a collaboration with David Davies at the NIH to provide him with large quantities of IN MAb for generation of Fab fragments for X-ray crystallography of IN, which may eventually facilitate design of enzyme inhibitors.

HIV整合酶(IN)是病毒整合所必需的一种病毒酶 DNA进入宿主染色体。这种类型的集成是高度 针对逆转录病毒，因此HIV-1IN是 抗病毒治疗。要设计抗病毒药物，至关重要的是 描述一下In。我们已经在大肠杆菌中以融合形式表达了IN基因 蛋白。克隆的IN对HIV-1和HIV-2阳性均有反应 ELISA检测患者血清，兔抗重组蛋白血清 经Western印迹分析，只与HIV-1 IN反应，而不与HIV-2 IN反应。这些 数据已经在AIDS Res.Hum上发表。复古。杂交瘤已经被 用IN表达克隆制备的单抗与 Western印迹结果显示HIV-1不表达HIV-2。已经提交了一份发明报告 一份手稿正在准备中。表达N-的其他克隆 和C-末端的一半已被构建，它们的蛋白质 用西南印迹法检测Express结合DNA的能力 程序。完整的IN分子和C-末端蛋白 结合DNA；N-末端部分没有结合活性， 提示C-末端含有DNA结合位点。一个 手稿最近发表在艾滋病研究中心。复古。我们有 纯化了我们的全长重组IN，它在 含2个碱基对的酶活性的体外特异性测定 从与HIV-1 LTR对应的寡核苷酸中分离出来的。因为我们的IN是 产生的融合蛋白含有13个氨基酸的lambda CII at 它的氨基末端，检测我们的蛋白质的活性表明 活性不需要游离氨基末端。这可能是 与体内情况有关，因为IN最初合成为 一种在其氨基末端与RT相连的多蛋白。 为了更准确地定位C-末端内的DNA结合位点 分子的一半，我们已经构建了一系列额外的 亚克隆，我们目前正在提纯它们缺失的蛋白质 用于DNA结合分析的产品。我们最近建立了一个 与大卫·戴维斯在NIH的合作为他提供了 用于产生X射线FAB碎片的In MAb的量 IN的结晶学，这可能最终促进酶的设计 抑制剂。