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: 3748159
• 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/3748159
• 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 in both full-length and truncated forms. Hybridomas have been prepared using the full-length IN clone and the MAbs are reactive with HIV-1 but not HIV-2 by Western blot. IN possesses a specific DNA cleavage activity and a non-specific DNA-binding activity. We have previosly shown that only the C-terminus of IN was capable of binding DNA in a Southwestern blot. Construction of a series of C-terminal deletion clones and analysis of the proteins demonstrated that the DNA binding domain could be localized to within amino acids 180-248. This region contains an array of basic amino acids (lysines and arginines). Site-directed mutagenesis of some of these residues has now been done and the resultant mutant proteins are being purified and will be analysed for their DNA binding ability as well as in a specific oligonucleotide cleavage assay. This newest set of clones was constructed in a gluathione transferase fusion vector, which provides for a simplified purification by affinity chromatography, and additionally, the possibility of producing labelled proteins for use in protein-protein association studies. IN has been postulated to act as a dimer in vivo, but the region of the protein required for this interaction is unknown. We have also purified large quantities of the IN MAb for David Davies to use to generate Fab fragments for X-ray crystallography of IN; attempts to crystallize full- length IN alone have thus far not been successful due to its tendency to aggregate. This may eventually facilitate design of enzyme inhibitors by providing information about IN~s 3-dimensional structure.

HIV整合酶（IN）是病毒整合所必需的病毒酶 DNA进入宿主染色体。 这种整合方式高度 特异性逆转录病毒，因此HIV-1 IN是 抗病毒治疗 整合酶功能的破坏也是可取的 设计减毒活HIV疫苗。 我们已经表达了IN基因 在大肠大肠杆菌中的全长和截短形式。杂交瘤已经被 使用全长IN克隆制备，并且MAb与 通过Western blot检测HIV-1而非HIV-2。IN具有特异性DNA切割 活性和非特异性DNA结合活性。我们之前已经展示了 只有IN的C末端能够结合DNA 西南印迹。一系列C-末端缺失克隆的构建 对蛋白质的分析表明， 可以定位于氨基酸180-248内。该地区包含 一系列碱性氨基酸（赖氨酸和赖氨酸）。定点 现在已经完成了这些残基中的一些的诱变，并且所得到的 突变蛋白质正在被纯化，并将被分析其DNA 结合能力以及在特异性寡核苷酸切割测定中。 这组最新的克隆是在一个谷氨酸转移酶中构建的。 融合载体，其提供了通过亲和纯化的简化纯化 色谱法，以及另外，产生标记的 用于蛋白质-蛋白质缔合研究的蛋白质。在已经 假设在体内作为二聚体，但蛋白质的区域 这种相互作用所需的时间是未知的。我们还净化了大量 大卫戴维斯用于产生Fab的IN MAb的数量 IN的X射线晶体学碎片;试图结晶完整- 到目前为止，单独长度IN由于其倾向于 骨料这可能最终有助于酶抑制剂的设计， 提供IN~s三维结构的信息。