EXPRESSION OF HIV ANTIGENS IN E COLI

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 为了设计抗病毒药物， 表征IN。 我们在大肠杆菌中表达了IN基因。大肠杆菌作为融合体 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，而不与HIV-2 IN反应。 这些 数据已发表在AIDS Res.2009。复古。杂交瘤已经被 使用IN表达克隆制备，并且MAb与 通过Western blot检测HIV-1而非HIV-2。一份发明报告已经提交 手稿正在准备中表达N- 和C-末端的一半已经构建， 使用Southwestern印迹法检测表达结合DNA的能力 procedure. 完整的IN分子以及C末端蛋白 结合DNA; N-末端部分不显示结合活性， 这表明C-末端区域含有DNA结合位点。一 手稿最近发表在艾滋病研究。复古。我们有 纯化了我们的全长重组IN，它表现出活性， 涉及2bp切割酶活性的特异性体外测定 来自对应于HIV-1 LTR的寡核苷酸。由于我们的IN是 作为含有λ cII的13个氨基酸的融合蛋白产生， 其氨基末端，用我们的蛋白质检测活性表明 活性不需要游离氨基末端。 这可能是 与体内情况相关，因为IN最初合成为 在其氨基末端与RT连接的多聚蛋白。 为了更精确地定位C-末端内的DNA结合位点， 在分子的一半，我们构建了一系列额外的 亚克隆，我们目前正在纯化删除的蛋白质， 用于DNA结合分析。 我们最近成立了一个 与美国国立卫生研究院的大卫戴维斯合作， 用于产生用于X射线的Fab片段的IN MAb的量 IN的晶体学，这可能最终有助于酶的设计 抑制剂的

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.