HIV-1gp41NHR序列靶点专一性的荧光共振能转移测定

目的确定并利用荧光共振能转移法（FRET）对以HIV-lgp41N端七联重复序列（NHR）为靶点的融合抑制剂进行筛选和作用机制研究。方法FRET采用金属络合物多肽技术设计针对不同结合位点、结合强度可调、涵盖全部NHR序列的靶点和探针，对HIV融合抑制剂进行高通量筛选。由于HIV在进行膜融合时其gp41的N端NHR和C端CHR可形成稳定的六螺旋结构，因此，利用圆二色谱仪对FRET所使用的靶点／探针对的结合强度进行验证，确定对应的靶点／探针对可形成稳定的六螺旋结构；同时，借助细胞活性测试测定抑制荆的活性，验证FRET是否可用于筛选以HIV-1gp41NHR为靶点的抑制剂。结果与结论FRET中使用的靶点／探针均可形成螺旋度较高的六螺旋结构，其中Fe（Env2．0），／CP2及Fe（Env5．0），／CP5形成的01．螺旋度分别高达89．6％和84．7％。FRET所使用的靶点／探针对专一性强、结合作用强，可用于进行HIV一1融合抑制剂的筛选和机制研究。

Determination of binding specificity between receptors and probes covering HIV-1 gp41 NHR in fluorescence resonance energy transfer assay

Objective To identify and optimize a fluorescence resonance energy transfer （ FRET） -based assay to screen fusion inhibitors which target HIV-1 gp41 N-terminal heptad repeats（NHR） and to study their mechanisms. Methods The FRET assay using metallopeptide technology was applied to detect HIV fusion inhibitors in a high-throughput mode, by which receptors and probes were designed to cover all the possible NHR binding sites with flexible binding affinities. The assay biochemically represented a stable fusion-active conformation of the envelope protein gp41 of HIV-1, which was a six- helix bundle formed by NHR and C-terminal heptad repeat （CHR） in the process of virus-cell membrane fusion. The six- helix bundle formed by an NHR-derived receptor and CHR-derived probe was characterized by circular dichroism to verify the effective receptor/probe pair, and the FRET assay was verified by comparing the result of the assay with a well-estab- lished cell-cell fusion assay using known fusion inhibitors. Results and Conclusion The receptors and probes used in FRET assay can form a six-helix bundle with high content of helices. Especially, the complexes of Fe （Env2.0）3/CP2 and Fe（EnvS. 0）3/CP5 could reach 89.6% and 84.7% of a-helical contents, respectively. The receptors and probes in the FRET assay with high specificity and strong interaction can identify fusion inhibitors target HIV-1 gp41 and be used to screen HIV-1 fusion inhibitors and study their mechanisms.