ANALYSIS OF THE HIV INTEGRATION REACTION USING RECOMBINANT INTEGRASE

使用重组整合酶分析 HIV 整合反应

• 批准号: 3770330
• 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/3770330
• 关键词: Escherichia coli; HIV infections; X ray crystallography; chemical binding; chimeric proteins; endodeoxyribonuclease; enzyme activity; enzyme linked immunosorbent assay; esterase inhibitor; gene deletion mutation; human immunodeficiency virus 1; human immunodeficiency virus 2; laboratory rabbit; live vaccine; molecular cloning; molecular site; monoclonal antibody; nucleic acid repetitive sequence; protein sequence; provirus; recombinant DNA; recombinant proteins; transposon /insertion element; virus DNA; 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. Disruption of integrase function is also desirable in design of live attenuated HIV vaccines. 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 positiv 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. 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 file 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. Purified full-length recombinant IN exhibits activity in a specific in vitro assay for enzyme activity involving cleavage of 2 bp from an oligonucleotide corresponding t the HIV-1 LTR. 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 analyzed the deleted proteins for DNA binding. This has enabled us to map the DNA binding site to amino acids 180-248. A manuscript is in press in Nucl. Acids Res. and preparation of additional deletions is in progress. We are also collaborating with David Davies at the NIH and providing 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。 整合酶功能的破坏也是在设计活细胞中期望的。 减毒艾滋病毒疫苗。 我们在大肠杆菌中表达了IN基因。杆菌作为 融合蛋白克隆的IN可与HIV-1和HIV-2阳性反应， 患者血清，而重组蛋白的兔抗血清， Western blot检测仅与HIV-1 IN反应，但不与HIV-2 IN反应。杂交瘤 已使用IN表达克隆制备，并且MAb是反应性的 免疫印迹法检测HIV-1而非HIV-2。发明报告已经归档 手稿正在准备中另外的表达N-和 已经构建了IN的C-末端的一半，并且它们表达的蛋白质 使用Southwestern印迹程序检查结合DNA的能力。 完整的IN分子以及C-末端蛋白结合DNA; N-末端蛋白结合DNA。 末端部分没有表现出结合活性，这表明C- 末端区域含有DNA结合位点。纯化全长 重组IN在酶的特异性体外测定中表现出活性 涉及从对应于T的寡核苷酸切割2bp的活性 HIV-1 LTR为了更精确地定位DNA结合位点在C 在分子的末端，我们构建了一系列额外的 亚克隆并分析缺失蛋白的DNA结合。这 使我们能够将DNA结合位点定位到氨基酸180-248。一份手稿 在Nucl. Acids Res.中出版， 进行中。我们还与美国国立卫生研究院的大卫戴维斯合作， 为他提供了大量的IN MAb，用于产生Fab 用于IN的X射线晶体学的片段，这可能最终有助于 酶抑制剂的设计。