基于纳米抗体的黄曲霉毒素抗原替代物的定向进化及识别机制研究

Development of non-toxic antigens with low cost and ease of large scale production is the key point to improve the application of immunoassays towards mycotoxins. In our previous study, it was found that the newly developed immunoassay using anti-idiotype nanobody (AId-NAb) as antigen surrogate has a lower sensitivity than traditional immunoassay. Besides, the mechanism of AId-NAb binding with antibody is unclear. In this research, on the basis of three AId-NAbs towards aflatoxins developed in our previous study, the direct evolution of antigen surrogate and its recognition mechanism will be studied. By construction of the recombination library and biopanning, AId-NAbs with high affinities will be isolated. Directed evolution system for AId-NAbs will be constructed. By protein homology modeling and molecular docking, the binding model of AId-NAb with anti-aflatoxin antibody will be developed. By analyzing the key amino acid and main acting force in the binding, the molecular mechanism of anti-idiotype nanobody binding with antibody will be illustrated. Anti-idiotype nanobody with the best activity will be synthesized and applied to developing novel immunoassays towards aflatoxin. This research is to provide theoretical and methodological support to the antigen synthesis of other mycotoxins and small molecular with high toxic, and pave the way for developing novel immunoassays.

研究无毒、低成本的真菌毒素抗原替代物规模化制备技术是推动真菌毒素免疫分析技术广泛应用的关键，也是该领域的创新源头。前期研究将抗独特型纳米抗体（AId-NAb）代替黄曲霉毒素抗原建立免疫分析方法，发现其检测灵敏度比传统分析方法低，且AId-NAb的作用机制尚不明确。因此，本研究拟以AId-NAb为技术核心，以前期制备成功的三种黄曲霉毒素抗原替代物作为研究材料，通过构建随机重组文库及体外淘选，获取具有高特异性的抗原替代物，建立AId-NAb的定向进化体系；采用同源建模、分子对接等手段，构建AId-NAb与抗体互作的空间结构模型，分析影响AId-NAb活性的关键氨基酸与主要作用力，探明其识别抗体的分子机理；生物合成AId-NAb，分析其免疫学特性，初步建立高灵敏度黄曲霉毒素新型免疫分析方法。本研究对真菌毒素及其他有毒小分子抗原替代物的研制具有重要的指导意义，并为免疫分析方法的建立提供新思路。

本项目重点以真菌毒素中毒性最强的黄曲霉毒素为研究对象，在前期研制的抗独特型纳米抗体的基础上，利用噬菌体展示技术构建黄曲霉毒素抗独特型纳米抗体噬菌体展示突变文库，筛选、鉴定特异性抗独特型抗体，通过表达载体构建，表达突变型纳米抗体，并初步建立了基于抗独特型纳米抗体的ELISA免疫分析方法。研究结果表明：1）成功构建噬菌体展示纳米抗体突变基因文库，库容量为1×107 pfu/mL；2）通过富集淘选获得阳性克隆，经表达纳米抗体蛋白分子量约17 kDa；3）淘选获得的抗独特型纳米抗体具有良好的特异性，与黄曲霉毒素M、赭曲霉毒素A和玉米赤霉烯酮均无交叉反应；4）初步建立无毒免疫分析方法，该方法的LOD值为0.015 ng/mL，线性范围是0.018~0.079 ng/mL。研究结果为真菌毒素无毒免疫分析方法的构建提供了新思路，也为纳米抗体在食品污染物的免疫快速检测研究中的应用奠定了良好的基础。