基于分子印迹技术的HIV-1进入抑制剂的合成与性能研究

HIV-1 entry inhibitors is a hotspot in the currently anti-AIDS drug research. The molecularly imprinted nanopartical polymer (MINP) with protein as the template has potential application value in the novel targeted drug delivery system as “artificial antibody”.　Based on the strong accumulation for anti-HIV drugs in our university, combined with our research foundation on molecularly imprinting, in this project, we decide to try the development of MINP used as HIV-1 entry inhibitor..The binding of HIV-1 gp120 with CD4 receptor in the target cell is the first step that the virus enter into the target cell. In this project, the MINP, which has targeting and orientating binding ability with gp120 was synthesized, with gp120 as the template molecular and NBD-556 as anchoring monomer,by molecularly imprinted technique. The synthesized MINP can combine gp120 with high specificity and thus the initial binding site of gp120 with CD4 was occupied, which resulted in that gp120 can not combine with CD4 further.This work will supply a new way for designing HIV-1 entry inhibitor with high inhibition activity; Futhermore, the biocompatibility in vitro , the metabolism in vivo, the Inhibition activity of HIV-1 fusion with target cells and the inhibition mechanism of the MINP were all studied, aimed to supply theoretical foundation for its application as HIV-1 entry inhibitor..

HIV-1进入抑制剂是当前抗艾滋病药物研究的热点之一。蛋白质分子印迹纳米聚合物（MINP）作为“人工抗体”在新型药物靶向传释方面具有潜在应用价值。本项目依托我校在抗艾滋病药物研究方面的积累，结合我们从事分子印迹研究的基础，拟进行HIV-1进入抑制剂的MINP的开发。.HIV-1包膜糖蛋白 gp120与靶细胞上的CD4受体相结合是病毒进入靶细胞的第一步，本项目拟采用分子印迹技术，以gp120为模板分子，NBD-556为靶向定位功能单体，设计并构建一种具有生物相容性和靶向定位结合功能的新型MINP，用于捕获gp120，并将gp120与CD4受体结合的最初位点占据，从而阻止gp120与CD4的结合，为新型HIV-1进入抑制剂的设计提供新思路; 进一步对所合成的MINP的生物相容性、体内代谢等性能以及对HIV-1的抑制活性进行深入研究，并阐明其作用机理，为MINP作为抑制剂类药物应用提供理论依据

本项目采用抗原表位印迹法，通过优化和筛选HIV-1 gp120包膜糖蛋白的抗原表位，以HIV-1 gp120的312-311钛链（GPGRAFVTIG）抗原表位为模板，合成了对gp120具有良好识别性能的分子印迹纳米颗粒MINP。以MINP对模板多肽的吸附识别性能为指标，对合成过程中的影响因素进行了考察，优化了合成工艺；采用SDS-PAGE，对MINP对gp120的吸附识别性能进行了评价，考察了材料对HIV-1 X4以及HIV-1 R5的抗病毒活性，以及对HIV-1靶细胞TZMBl的细胞毒性等性能。采用SEM、DLS等对MINP的表观性能进行了研究。结果表明，采用抗原表位印迹法，以优化的抗原表位GPGRAFVTIG为模板分子，合成的MINP对gp120具有良好吸附识别性能。MINP对gp120的吸附量要大于非印迹聚合物NINP对gp120的吸附量，体现了良好的印迹效果，并且MINP对gp120的吸附优于BSA等其他蛋白，表明所合成MINP对gp120具有良好的选择性识别性能。抗病毒活性实验表明，所合成的MINP 对HIV-1 R5具有一定的抗病毒活性，细胞毒性结果表明MINP细胞毒性低。为将其作为新型HIV-1抑制剂开发与应用提供实验基础。