新烟碱类农药宽谱抗体的识别机制与亲和力调控研究

Neonicotinoid insecticides are highly-concerned because of the ecological safety issues. Thus, development of sensitive, accurate, and rapid detection techniques for multi-residue monitoring is urgently needed. Broad-specific antibodies are core materials of multiple immunoassays for screening different pesticide residues, but they possess different affinity to varies of structural analogues, which hinders the development and application of multi-analyte immunoassays. In this project, based on several cell lines of monoclonal antibodies (mAbs) showing specific or class-selective recognition to 4-6 kinds of neonicotinoid pesticides, we plan to prepare single-chain fragment variable (scFv) with similar performance to the parent mAbs by genetic engineering and phage display technology. Kinetic characteristics of antibody binding to different neonicotinoids would be measured via surface plasmon resonance biosensor. Meanwhile, homology modeling and molecular docking will be used to identify the key amino acids in scFv domain and the mode of interaction with target molecules. Also, recognition mechanism of the broad-spectrum antibody would be studied according to the molecular similarity of related chemical structures. Furthermore, through the site-directed mutation of amino acids, the affinity of scFvs can be adjusted to obtain new recombinant Abs with broader specificity or more similar sensitivity to different neonicotinoids. The purpose of this study is to elucidate the recognition mechanism of broad-specific Abs against neonicotinoids and to regulate the affinity for different analogues. It provides a theoretical basis for the development of multi-residue immunoassays for simultaneous screening and accurate detection of various neonicotinoid pesticides.

新烟碱类杀虫剂因其生态安全问题倍受高度重视，急需研发灵敏、精准的现场多目标速测技术用于残留监控。宽谱抗体是农药多残留免疫筛查方法的核心，但其对不同结构类似物的亲和力差异较大，制约了农药多残留免疫速测技术的发展应用。本项目在前期获得特异或广谱识别4-6种新烟碱农药的多个单抗细胞株基础之上，采用基因工程技术、噬菌体展示筛选制备与亲本单抗性能相似的单链抗体(scFv)；运用表面等离子共振技术解析抗体与不同药物结合的动力学特征；利用同源建模、分子对接，明确scFv识别空腔的关键氨基酸残基及其与目标分子的主要结合方式，并根据相关药物的分子相似度探寻抗体广谱性识别机理；通过氨基酸定向突变，调控scFv对相应新烟碱类农药的亲和力，获得检测谱更广、亲和力差异更小的重组抗体。本研究旨在阐明新烟碱类农药宽谱抗体的识别机制，提出亲和力调控策略，为实现免疫分析法对新烟碱类农药多残留同步筛查、精准速测提供理论依据。

新烟碱类杀虫剂因其对蜜蜂毒性问题倍受全球关注，几大品种陆续在欧盟等地区被禁限用，故急需采用灵敏、高效的多目标免疫快检技术用于该类农药残留的实时现场监测。本研究以吡虫啉等8种常见新烟碱类杀虫剂及其主要代谢物为检测对象，在获得能特异或宽谱识别新烟碱类农药的多株单抗杂交瘤细胞基础之上，运用表面等离子共振（SPR）技术解析了各抗体与不同药物结合的动力学和亲和力特征；采用免疫组库高通量测序结合LC-MS/MS技术准确鉴定了抗体可变区基因和氨基酸序列，并进行了序列功能验证与同源性分析；进而采用同源建模、分子对接和动力学模拟，明确了参与免疫识别的关键氨基酸残基与分子互作方式，并突变验证了主要结合位点，解析了宽谱抗体与多种新烟碱类化合物互作的分子识别机制；获得了一种能有效识别10种新烟碱类化合物的广谱重组抗体，亲和常数 KD范围为0.12~12 nM。.研究表明，新烟碱类农药抗体具有高度多样性；重链可变区中V基因片段序列的同源性，从根本上决定了抗体识别谱的差异性；通过选择不同胚系基因来源的抗体进行亲和力与特异性调控，可实现对不同新烟碱类化合物的免疫检测。项目成果为研究农药等小分子化合物的抗体识别机制、重组抗体的分子设计、多目标识别广谱抗体的研发等提供了参考依据，也为实现免疫分析法对新烟碱类农药多残留同步筛查、精准速测提供了理论基础与技术支撑。在本项目资助下，已发表学术论文7篇；授权国家发明专利4项。