脱氧雪腐镰刀菌烯醇多功能重组全长抗体识别及定向进化机制研究

Mycotoxin deoxynivalenol (DON) as the most important type B trichothecene, has the highest concentration and frequency in a great variety of foods at global level. Rapid and sensitive detection methods play the key role for the whole monitoring of DON contamination. However, due to the high cross-reactivities against related structures of DON, DON derivatives and masked forms when applying the currently available antibodies, highly false positive results were generally observed. Meanwhile, DON derivatives and masked forms carrying similarly identical toxicities would be considered together with the parental molecule of DON, for the total risk assessment in future as expected. Thus, achieving the ideal antibodies as fundamental materials with the bifunctionalities of either specific or broad spectrum recognitions for DON, ADONs and D3G, are urgently needed for practical uses. Moreover, traditionally-made monoclonal antibody and recombinant antibody fragments have the unavoidable drawbacks of time-consuming preparation, poor stability and inappropriate applicability. In this proposal, we intent to focus on mycotoxin DON, to first construct the desirable hybridomas secreting antibodies with high specificity and broad selectivity against DON-typed mycotoxins. Furthermore, via recombination and cell transient transfection approaches, full-length antibody with utmost specificity to DON will be selected with applicable capabilities in classic immunoassays. And then, subsequent homologous modeling and molecular docking, site-specific mutagenesis of key amino acid residues were performed to elucidate the orthogenesis mechanism of the obtained recombinant full-length antibodies. As concluded, the achieved antibodies with various capabilities of simultaneous recognitions against DON family mycotoxins and merely specific recognition for DON itself, would fulfill the real needs of immunoassays. Also, the revealed orthogenesis mechanisms provide novel venues and theoretical support for acquiring ideal antibodies targeting small molecules concerning the whole frame of food safety.

脱氧雪腐镰刀菌烯醇（DON）是全球范围内食品中污染水平和污染率最高的B型单端孢霉烯族真菌毒素，快速、灵敏的检测方法是DON污染监测的重要前提。而现有的抗体往往对与DON原型结构类似的衍生型、隐蔽型有较高的交叉反应率，非特异性高导致较高的假阳性率；而DON衍生型及隐蔽型的危害性也正逐步被纳入总体危害范畴。因此，相关快速检测方法急需高特异性与广谱性的DON抗体。此外，单克隆抗体存在制备周期长，小分子抗体存在稳定性及适用性差等缺陷。本项目拟聚焦DON及其衍生型、隐蔽型，首先通过多种抗原合成及免疫，结合细胞融合过程高效筛选，同时获得分泌DON高特异性与广谱性抗体的杂交瘤细胞株；然后借助重组及细胞瞬时转染技术，构建重组全长抗体，通过同源建模、分子对接确定抗体参与识别的关键氨基酸残基，继而对其进行定点突变，最终揭示其定向进化机制，为食品中DON毒素污染的特异性与广谱性快速检测提供基础原材料与理论支持。

脱氧雪腐镰刀菌烯醇（DON）是一种由真菌产生的次级代谢物，广泛存在于多种食品和农产品中。DON具有细胞毒性，危害人体及动物健康，同时DON的污染范围广泛，因此我国对DON制定了严格的限量标准。免疫学检测技术作为DON快速检测的常用方法，广泛应用于易受DON污染的粮食及其制品中该毒素的检测。抗DON的特异性抗体的开发是DON免疫学检测的关键。.本项目成功合成了DON完全抗原，用于动物免疫，采取酶联免疫吸附试验检测动物血清的效价，取小鼠脾脏细胞与骨髓瘤细胞混合均匀后，成功融合的杂交瘤细胞，得到了六株分泌特异性抗DON单克隆抗体的细胞株；间接竞争ELISA鉴定单克隆抗体的性能,最终得到了一株分泌抗体灵敏度为50ppb、抗体亚型为Ig2a的细胞株。.通过TRIzol法提取该细胞的总RNA，将其反转录为cDNA；采取RACE法，以cDNA为模板，获取抗体序列的全长信息。IMGT/V-QUEST分析抗体重链和轻链信息，划分序列的可变区和恒定区；SignalP分析序列，找到其信号肽序列和切割位点。发现抗体重链无提前终止密码子，而抗体轻链因缺失4个碱基导致提前终止；通过SOE-PCR改造抗体轻链，使其正常表达。将抗体重链和轻链全长与真核表达载体连接，共转染293T细胞；提取细胞蛋白，经Western Blot鉴定，抗体重链和轻链成功表达。.获得的抗体重链和轻链的可变区通过同源建模方法，得到其三维结构模型；Procheck以及Profile-3D对模型评价打分，结果表明模型可信度高，可用于后续分析。使用AutoDock vina软件，通过分子对接分析抗体与DON的相互作用，PyMol软件分析对接结果，表明抗原-抗体相互作用的关键氨基酸位点为抗体重链可变区109位色氨酸和轻链可变区40位天冬酰胺。根据分子对接结果，采取SOE-PCR法，成功构建了抗体的定点饱和突变体库。.本研究成功制备了一株可分泌高特异性和高灵敏度的抗DON单克隆杂交瘤细胞株，并根据抗体的序列信息得到了抗DON的重组抗体，由分子对接找到抗原-抗体相互作用的关键氨基酸位点，成功构建了定点饱和突变体库，为该抗体的定向进化，提高抗体的灵敏度和特异性奠定了基础。