M2蛋白的体外进化及其与金刚烷胺类抗病毒药物的互作机制研究

The amantadane-class of antiviral drugs (Amantadine and rimantadine) are banned by China's Ministry of Agriculture in the livestock and poultry breeding in China. So far, no any rapid bioanalysis method specific to these drugs was reported in world, and the absence of recognizing biomaterials is the bottleneck for this situation. These drugs all contain the cage-like structure that is highly symmetrical and hydrophobic, which make the preparation of specific antibody quite challenging. Up to date, no any specific antibody for these drugs is reported. Therefore, in the project proposed here, the M2 receptor protein specific for these drugs is used as the tool to breakthrough the bottleneck. We try to clone and express the binding region of M2 protein, and to embed this region into the double-layer liposome microspheres to improve the stability and solubility of the binding region. And then, the novel M2 protein embedded nanomaterial will be built for bioanalysis. Furthermore, the cocrystallization and molecular stimulation techniques are employed to investigate the interaction mechanism between M2 protein and drugs. And we try to graft the binding region of M2 protein to synthetic antibody-scaffold using the reshaped antibody technique, and the hotspot mutation library will be constructed to achieve the in vitro evolution of the reshaped M2-antibody. The mutant with high affinity and specificity for the target drugs will be finally screened out and used as novel recognizing biomaterial for analysis. The implement of the project should be useful and significant to the advance of key materials and detection techniques of the amantadane-class of antiviral drugs.

金刚烷胺类抗病毒药物（金刚烷胺和金刚乙胺）是我国农业部规定的禁用药物，然而其快速生物分析技术目前尚属空白，其瓶颈在于生物识别材料的获取。该类药物具有高度对称、脂溶性强的笼状结构，其抗体制备极具挑战性，至今未见文献报道。围绕上述问题，本项目拟金刚烷类药物类药物特异性受体M2蛋白为突破口，通过体外基因重组技术表达该蛋白的药物结合域，将其嵌入双分子层脂质体纳米微球表面，改善其体外稳定性和水溶性，构建适用于实际检测的M2蛋白嵌合纳米材料，并利用蛋白质共结晶和分子模拟技术研究其与药物的体外互作机制；通过重构抗体技术（reshaped antibody）将M2蛋白结合域移植至全合成抗体骨架中，结合热点突变技术进行体外进化，筛选出具有高亲和力和良好选择性的M2蛋白重构抗体，为该类药物的生物分析技术提供新型识别材料。本项目的实施对于发展金刚烷胺类的高通量生物分析技术具有重要的理论意义和应用价值。

本项目的研究获得了具有体外结合活性的M2受体蛋白结合域材料及重组抗体，并经体外进化等技术实现了上述识别材料的性能优化和提升，可识别5种金刚烷胺类药物，亲和力达到IC50 0.5 ng/mL水平；基于获得的M2受体蛋白结合域材料及重组抗体，研究建立了ELISA、胶体金免疫层析定性检测技术和时间分辨荧光免疫层析定量检测技术，并建立了鸡鸭肉中的金刚烷胺类药物的快速检测方法，检测限达到LOD可达0.29 μg/kg，线性范围为0.37-19.46 μg/kg，在鸡肉样本中0.5-10 μg/kg添加水平上回收率范围为85.5-116.7%，CVs为6.0-10.6%。并与HPLC-MS/MS确证分析方法的检测结果进行比较，相关性良好（R2 > 0.96），表明本项目建立的快速检测方法可用于鸡肉中实际样本的检测，检测限及灵敏度等性能达到了国家针对该类药物的监控检测要求。本项目的研究成果已经发表SCI收录论文4篇，提交专利申请3项，培养研究生7名。基于本项目研制的重组抗体和M2受体蛋白结合域材料、以及相关的快速检测方法已经形成试剂盒及免疫层析检测卡等产品，已经在全国推广使用。.