基于PAL-ABPP探针的四环素受体的制备及其分子识别机制研究

来自于生物体的受体具有识别特异性底物的特点，可以代替抗体解决制约动物源性食品中兽药多残留快速检测方法发展的问题。本项目以四环素阻遏蛋白（TetR）为研究目标，通过化学合成法制备四环素的光亲和标记活性蛋白谱探针（PAL-ABPP-四环素探针），用探针从耐四环素细菌内直接获取天然TetR作为四环素受体；再利用蛋白质组学、生物信息学、质谱、X-射线衍射、免疫分析等方法对该受体进行分析鉴定，确定其氨基酸组成、三维结构、稳定性、特异性等理化性质；并将同源模拟、分子对接、氨基酸突变技术与竞争ELISA方法相结合，研究TetR结合四环素类药物时的关键氨基酸及它们与亲和力的关系，以此为基础建立四环素类药物多残留快速检测方法。研究结果将获得四环素特异性受体并解析其与四环素类药物相互作用的分子机制，从而为其他小分子物质特异性受体的制备、分子识别机制研究和免疫学残留检测方法的建立提供全新的思路和依据。

The receptors derived from the organism have a feature of recognizing the special substrates and will be able to replace the antibody to resolve the problem which restricts the development of multi-residue immunoassays for veterinary medicine. In this project, the tetracycline repressor protein (TetR) of the tetracycline resistance bacteria is selected as the target. Firstly, the Photoaffinity Labeling-Activity Based Protein Profiling (PAL-ABPP) probe will be linked with tetracycline by chemical synthesis method, and then the probe will be used to isolate and extract natural TetR directly from the tetracycline resistant bacterial strains as the tetracycline specific receptor. Secondly, the physical and chemical properties of the obtained receptor such as amino acid composition, molecular weight, three dimensional structure, stabilization and specificity will be characterized by using proteomics, bioinformatics, mass spectrometry, X-ray diffraction and immunoassay methods. And what’s more, the crucial amino acids of this receptor and their relationship with the affinity when it recognizes and bonds with the tetracyclines will be studied by homology modeling, molecular docking, amino acid mutation technique and competitive ELISA method, and meanwhile a multi-analyte immunoassay for the determination of tetracyclines residues will be established based on these. At the end, the specific receptor of tetracycline will be yielded, and the results of this project will illuminate its interactional molecular mechanism with the tetracyclines and provide a novel strategy for production, molecular mechanism study and establishment of residue immunoassay of the receptors of other compounds.