基于多残留检测导向的大环内酯类磷酸转移酶与靶标分子识别机理研究

大环内酯类抗生素(Mals)是我国动物养殖和兽医临床上的重要药物。动物源性食品中此类药物的残留是快检领域关注的重点之一。但现有基于传统抗体的快检方法仅能同时识别少数几种Mals，易留下食品安全隐患。本项目以大环内酯类磷酸转移酶(Mphs)为对象，(1)基于等温滴定量热和表面等离子共振等研究不同Mphs与Mals的结合特性；(2)基于同源建模、分子对接等，筛选参与Mphs与Mals互作的关键因素；(3)基于共结晶技术解析表达的Mphs蛋白与配体的晶体结构，分析蛋白与配体作用的活性位点；(4)对蛋白结构进行改造以提高蛋白底物识别特性，验证Mphs体外活性。通过本研究，可揭示Mphs与配体之间的互作机制，阐明Mphs对药物靶标的识别机理。为获得具有体外活性的高亲和力广谱Mphs，实现Mals低成本高效率的多残留快速检测奠定基础，对推动食品安全快检理论和技术发展，确保动物源性食品安全具有重要意义。

Macrolide antibiotics (Mals) play an important role in animal husbandry and veterinary clinics in China. It’s always one of the focuses in the field of rapid detection for the residues of such drugs in animal derived foods. However, the existing rapid detection methods based on the traditional antibodies can only detect a few Mals, which is easy to make risks for food safety. In this project, macrolide phosphotransferase (Mphs), one of Mals receptors, is selected as the object, (1) Based on isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR), the binding properties of different Mphs with Mals will be studied; (2) Based on homologous modeling and molecular docking, the key factors involved in the interaction between Mphs and Mals will be simulated. (3) Based on co-crystallization data, the crystal structure between Mphs and their corresponding ligands will be elucidated and the active sites of protein-ligand interaction will be designed. (4) A series of modifications for the protein structure will be carried out to enhance the ligand binding affinity. The in-vitro activity of Mphs protein will be examined by ITC or/and SPR. Through this study, it will be discovered comprehensively, (1) the interaction mechanism between Mphs and their corresponding ligands and (2) the recognition mechanism between Mphs and their targets. These results will be the base for obtaining a high affinity broad-spectrum Mphs proteins which will offer a new method with a low cost and high competence for the multi-residues detection of macrolides. Moreover, this study delivers a new concept for the multi-residues detection of veterinary drug in animal-derived food. This study accelerates the development of method for the rapid detection of veterinary drug residues and to offer solutions to resolve the problems of food safety and environmental pollution caused by the veterinary drugs residues.