抗呕吐毒素驼源单域重链抗体结合活性机理的研究

Nanobodies (Nbs) are the variable region of Heavy Chain antibodies which occur naturally in peripheral blood and milk of camels, dromedaries and llamas. The molecular weight of Nbs is about 1/12 of conventional antibodies. The unique intrinsic properties of Nbs, such as highly soluble, highly stable to extremes of pH, remarkable resistance to high temperatures, make them better options for drug development and biotechnological applications. There are many literatures reporting the isolation, characterization and application of Nbs in recent years. However, few examples of isolation high affinity Nbs against low molecular weight compound. In the field of food safety, a lot of hazard components which should be controlled are low molecular weight compounds, for example mycotoxins, antibiotics, pesticides, etc. The mechanism of interaction between Nbs and its targets needs further investigation to guide designing or modifying the structure Nbs. In previous work, an anti-deoxynivalenol nanobody, designated DONIV2, was isolated from a na?ve phage display library. The three dimensional structure of DONIV2 was generated by homologous modeling, and docked with deoxynivalenol (DON) using AutoDock. The calculation data showed that the probable binding sites were Arg59, Arg103 and Arg105, which were located on the CDR2 and CDR3. Nevertheless, experimental data were required to validate the computational results. This project will combine in vitro molecular evolution and bioinformatics to investigate the binding mechanism of the anti-deoxynivalenol nanobody. Firstly, a random mutation library would be constructed. After several rounds of panning, the mutants with improved affinity would be accumulated and isolated. By comparing the affinity property and amino acid sequence mutation, the critical amino acid residues or regions might be identified. Then couple of rational designed mutation libraries will be constructed to discover how these amino acid residues affect the affinity and specificity of the anti-deoxynivalenol nanobody. Meanwhile, the homologous modeling and docking were conducted with mutants to interpret the improvement of binding activity or changes of specificity. Many affinity improved mutants would be generated and computational results would provide a clew for rational designing or modification nanobodies binding with other low molecular weight compounds. In addition, it will provide a novel approach for food safety and quality control.

驼源单域重链抗体由重链抗体的可变区组成，又称为纳米抗体，分子量小，具有易于制备、水溶性好、耐酸碱、热稳定性好等独特性质。若能应用于食品安全领域，则可有效应对食品成分复杂、基质干扰大等难点，同时避免传统抗体存在的一些缺陷。目前针对小分子抗原的纳米抗体存在亲和力普遍较低的问题，限制了其应用。深入研究纳米抗体与小分子抗原相互作用机制，可为体外分子改造制备高亲和力纳米抗体提供理论基础。 本研究拟采用抗呕吐毒素纳米抗体作为研究模型，将分子体外进化技术与生物信息学方法结合，首先通过比较分析体外进化前后性能变化，探寻可能的关键氨基酸残基或区域，然后再结合计算机分析结果，有针对性地进行设计和改造，并进一步阐释其结构与功能的关系，以期获得高亲和力纳米抗体，为建立相应的新检测方法奠定物质基础。研究成果可为制备、设计以及改造针对其它小分子抗原的纳米抗体提供新思路和线索，为食品安全和质量控制提供一套新的技术途径。

纳米抗体分子量约为普通抗体的1/12，具有易于制备、稳定性好、易溶于水等独特性质。然而，针对小分子化合物的纳米抗体普遍存在亲和力不足，导致建立的免疫学检测方法灵敏度较低，不能满足食品安全检测的要求。. 本研究采用分子体外进化技术、分子模拟及对接技术，对针对脱氧雪腐镰刀菌烯醇（deoxynivalenol，DON）纳米抗体的识别机制进行了研究。首先建立了纳米抗体突变位点均匀分布的随机突变方法，并对随机突变的噬菌体展示文库进行了淘选；通过比较序列的变化，发现在CDR环两侧的氨基酸残基位点存在高频突变位点；对高频突变区域进行了饱和突变实验，获得了与DON人工抗原亲和力提高的突变子；采用分子模拟及分子对接对突变子结构进行了对比分析，结果显示突变后纳米抗体的整体结构没有明显变化，但是对CDR3环的朝向、构型等有明显的影响，提示突变子通过改变CDR环两侧氨基酸对识别表位的结构。研究建立了纳米抗体体外突变方法和纳米抗体建模、优化及评价分析流程，在此基础上，对模拟DON的纳米抗体和针对黄曲霉毒素的纳米抗体进行了分子改造研究。