重组ScFv识别多兽药残留的量子点生物发光共振能量转移技术研究

Residues of veterinary drugs become an increasingly important role affecting food safety. Bioluminescence resonance energy transfer (BRET) has the advantage of high sensitivity and without washing steps, and multi-specific recombinant single chain variable region (ScFv) antibody can simultaneously identify multiple veterinary drugs. The establishment of BRET technology based on multi-specific ScFv is the new strategy for more rapid detection of multi-veterinary drug residues in animal-derived food. In this study, the ScFv gene of amphenicols was obtained through molecular manipulation on anti-amphenicols monoclonal antibody. To improve the affinity and specificity of ScFv antibodies, the ScFv genes of sulfonamides, quinolones and amphenicols were undergone with site-directed mutagenesis, and the Renilla luciferase (Rluc) and the linker gene were incorporated to obtain multi-specific recombinant Rluc-ScFv antibodies recognizing at least more than two types of veterinary drugs simultaneously. The interaction between multi-specific recombinant ScFv antibodies and the veterinary drugs were analyzed with molecular modeling. The three types of veterinary drugs were analyzed and transformed, and then covalently conjugated to quantum dots (QDs) with different emission wavelengths to obtain QD-small molecule veterinary drugs conjugates. Furthermore, the QD-BRET methods were developed for simultaneous detection of multi-veterinary drug residues, to provide adequate technical support for ensuring animal-derived food safety.

多兽药残留已成为影响动物源性食品安全的重要因素。生物发光共振能量转移（BRET）具有灵敏度高以及无需洗涤步骤等优势，多特异性重组单链可变区（ScFv）抗体能够同时识别多类兽药，建立基于重组多特异性ScFv的BRET技术是实现快速检测动物源性食品中多兽药残留的新思路。本项目拟通过对酰胺醇类单克隆抗体的分子操作获得其ScFv基因；对磺胺、喹诺酮（已获得）和酰胺醇类ScFv基因进行定点突变并引入Renilla荧光素酶（Rluc）和连接臂基因，获得同时识别两类以上兽药的多特异性重组Rluc-ScFv抗体；借助分子模拟分析兽药与重组ScFv抗体之间的相互作用关系进而提高多特异性重组Rluc-ScFv的亲和力和广谱性；对三类兽药小分子进行结构分析和改造，与不同发射波长的量子点（QD）定向偶联获得QD-兽药小分子；建立同时检测多种兽药残留的QD-BRET技术，为保证动物源性食品的安全提供充足的技术保障。

采用分子生物学手段，获得能够识别喹诺酮类药物的重组Renilla荧光素酶多特异性重组单链可变区（Rluc-ScFv），建立检测喹诺酮类药物的量子点-生物发光共振能量转移免疫方法（Quantum Dots-Bioluminescence Resonance Energy Transfer, QD-BRET），为其在食品安全中检测兽药小分子的应用提供理论依据；通过分子生物学、计算机辅助设计等手段，获得3种同时识别磺胺类、喹诺酮类和酰胺醇类多种兽药小分子的多特异性重组Rluc-ScFv抗体；初步建立1种可同时检测动物源性食品中磺胺类、喹诺酮类、酰胺醇类等兽药小分子的QD-BRET免疫分析技术，为保证动物源性食品的安全提供充足的技术保障。