纳米多肽竞争物与抗体互作的亲和力调控机制研究

Phage-displayed peptide can be used as heterologous competitor to develop sensitive immunoassay for residual pesticide. Compared with it, nano-peptide has the advantages of small particle size, noninfectious and so on, which is suitable for the development of novel immunoassay methods. However, the affinity regulation mechanism of the interaction between the nano-peptide and antibody is not clear, which has become the bottleneck problem of the development and application of the nano-peptide competitor. This project intends to carry out the following researches on the basis of the preliminary studies for the phage-displayed peptide competitors of organophosphorus pesticides, thiacloprid and benzothiostrobin. The univalent and multivalence (branched peptide) nano-peptide competitors will be prepared through solid phase synthesis, and labeled by different labeling methods, sites and the binding ratio, and the sensitivities of the competitors will be evaluated by immunoassays. Surface plasmon resonance (SPR) technique will be used to determine the affinities of the interactions of labeled and unlabeled competitors and antibodies. The critical factors of affecting the interaction of nano-peptides and antibodies will be sought by the vertical (homologous peptides) and the horizontal (different peptides) comparisons of the affinities. Through the fitting of the curve equation and the construction of the interaction model, we will try to explore the affinity regulation mechanism of the interaction between the nano-peptide competitor and the antibody. Finally, the nano-peptide competitor of imidaclothiz will be designed, prepared, and the affinity will be tested to verify the scientificalness of the regulation mechanism. The results of this project will provide theoretical guidance for the preparation and application of nano-peptide competitor of other pesticides.

噬菌体展示多肽可作为异源竞争物提高农药残留免疫检测的灵敏度。与之相比，纳米多肽具有粒径小、无侵染性等优点，适宜于开发新颖的免疫检测方法，但纳米多肽竞争物与抗体互作的亲和力调控机制不清，成为纳米多肽竞争物研制和应用的瓶颈难题。本项目拟在前期研究有机磷类、噻虫啉、苯噻菌酯等农药噬菌体展示多肽竞争物的基础上，固相合成制备单价和系列多价（分支肽）纳米多肽竞争物，以不同标记方法、标记位点和结合比对其进行标记，并评价其免疫检测灵敏度；利用表面等离子共振技术测定标记和非标记纳米多肽与抗体互作的亲和力，通过亲和力纵向（同种多肽）和横向（不同多肽）比对分析，探寻影响纳米多肽与抗体互作的关键性因素；经曲线方程的拟合和互作模型的构建，解析纳米多肽竞争物与抗体互作的亲和力调控机制；以氯噻啉为对象，经纳米多肽竞争物设计、研制和亲和力测定等，验证调控机制的科学性，为其它农药纳米多肽竞争物的研制与应用提供理论指导。

以赖氨酸为支架，设计合成了1价、2价和4价无噬菌体的苯噻菌酯模拟表位和抗免疫复合物多肽（纳米多肽），并进行异硫氰酸荧光素标记。通过微量等温滴定量热法和免疫法测定了未标记、标记的系列价态纳米多肽与抗体（抗体-分析物免疫复合物）的互作亲和力，明确了价态对亲和力的提升具有指数效应。将未标记的系列模拟表位多肽和抗体分别与上转换荧光纳米材料和金纳米花偶联，建立了均相的荧光免疫分析方法。结果显示高价纳米多肽的灵敏度更高，其原因是高价纳米多肽的亲和力高，可以减少检测体系中游离标记抗体的量，从而降低背景噪音，提高信噪比。利用异硫氰酸荧光素标记的系列价态的模拟表位和抗免疫复合物纳米多肽建立了荧光偏振免疫分析方法，发现偏振信号值随着纳米多肽的价态增加而升高，该趋势与理论计算结果相反，主要原因是理论计算公式未考虑亲和力差异的因素。在灵敏度方面，高价模拟表位和抗免疫复合物纳米多肽的灵敏更好。以上结果表明在纳米多肽研制过程中，多肽的价态是调控多肽与抗体互作亲和力的主要因素，单价和多价纳米多肽均可用于建立免疫分析方法，但多价纳米多肽的灵敏度更高。. 将噬菌体展示多肽的活性多肽区域与标签蛋白（NanoLuc和EmGFP）进行重组表达，率先获得2种重组纳米多肽。该重组多肽一方面可以与抗体发生特异性的结合，另一方面无需化学偶联，可直接提供检测信号，并且响应快、信号强、稳定性好。通过链霉亲和素-生物素的反应研制了多价纳米多肽，该方法经济、简便，为多价纳米多肽的研制提供了方法。使用相关纳米多肽分别建立了多种免疫分析方法，并对检测方法的性能进行了评价。. 相关研究明确了纳米多肽与抗体互作亲和力的主要调控因素，并为单价和多价纳米多肽的研制及应用提供了参考。研究结果发表了SCI论文8篇，其中第1标注4篇；申请发明专利4项，授权1项，完成成果转化1项。.