二乙氧基硫代磷酸酯类农药多特异性单链抗体识别机制及其定向进化

At present, the residues of organophosphorus pesticides (OPs) in foods is still one of the most serious food security problems in China. The multi-analyte immunoassays based on a broad-specificity antibody have potential use in the screening of food contaminants due to their simpleness, rapidity high-throughput and low-cost. However, the affinity and specificity of traditional polyclonal and monoclonal antibodies were limited to the structure of haptens, which usually resulted in the unsatisfactory sensitivity or ununiformity of specificity in practice. Previously, we produced a monoclonal antibody which showed broad-specificity against O,O-diethyl phosphorothioate pesticides (DPPs). With the aim to improve the sensitivity and uniformity of specificity of anti-DPPs antibody, this project will focus on: (1) The production of a broad-specificity single-chain variable fragment (scFv) by recombinant technique, the construction of interaction model between scFv and DPPs by homology modeling, molecular docking and molecular dynamics, and the confirmation of amino acid residues which are responsible for antibody recognition. (2) The site-directed mutagenesis of scFv based on the key amino acid residues and the selection of specific scFv by display. (3) The production of bi-functional fusion protein and the development of simpler, faster and more stable immunoassays for the multi-analyte analysis of DPPs. The results of this project will improve the screening ability for OPs residues in foods and also provide a new strategy on the production of antibodies with higher affinity or controllable characteristics.

有机磷农药残留仍然是我国现阶段最突出的食品安全问题之一。基于多特异性抗体的多组分免疫分析技术具有简便、快速、通量高、成本低等特点，应用前景广阔，但传统的多/单克隆抗体亲和力及多特异性受限于半抗原结构，常常难以满足实际应用要求。本项目拟在前期获得抗二乙氧基硫代磷酸酯类有机磷农药多特异性单克隆抗体的基础上，重点开展如下内容研究：① 利用重组抗体技术制备单链抗体，通过同源模建、分子对接及动力学优化构建单链抗体与农药的空间识别模型，明确抗体识别位点；② 基于抗体识别位点，对抗体可变区进行定向进化，表面展示筛选特异性单链抗体；③ 将进化单链抗体与功能性蛋白融合表达，制备双功能重组抗体，建立更简便快速及灵敏度显著提高、多特异性的一步法免疫分析，以期推进有机磷农药多残留快速检测的技术进步，同时为其它小分子有害物抗体的性能改造、建立更高灵敏度或可控特异性的免疫分析方法提供理论依据与指导。

本项目以前期获得的有机磷农药多特异单克隆抗体为基础，开展基因工程抗体制备与鉴定、抗体识别模型与识别机制、抗体定向进化等内容研究，取得的主要成果包括：（1）利用噬菌体表面展示技术筛选制备了与亲本单克隆抗体性能相似的单链抗体（scFv）和抗原结合片段（Fab），并进一步制备了具有抗体活性和酶催化功能的双功能抗体scFv-AP，以及稳定性显著提高的单链抗原识别片段（scFab），建立了相应的免疫检测方法；（2）利用同源模建、分子动力学及分子对接等技术分别构建了scFv、Fab与有机磷农药之间的识别模型，明确了CDR-L3的Trp92、Thr93、Thr94和Arg96，CDR-H2的Gln168、Ala170以及CDR-H3的Leu212、Tyr213是关键氨基酸残基，特别是 Arg96 对抗体识别起着最为主要的作用；（3）利用模拟突变策略，选择重链Tyr107为突变的关键点，分别进行定点突变，发现当突变为Phe107后，突变株对多个有机磷农药的灵敏度提高1-6.5倍左右，特别是对乙基谷硫磷、喹硫磷、三唑磷、伏杀硫磷、甲拌磷、毒死蜱、乙基溴硫磷等药物的灵敏度有比较显著提高，使得抗体的均一性得到提高。项目执行期间，主持人晋升教授，并获得广东省自然科学杰出青年基金、广东省特支计划科技创新青年拔尖人才等称号；发表SCI论文3篇，中国科学•化学1篇，核心期刊1篇，申请发明专利1项，获得中国植物保护学会一等奖1项。本项目研究结果对于指导其他小分子抗体的改造具有重要指导意义。

内容获取失败，请点击重试