核糖体展示嗜热菌精氨酸结合蛋白突变文库筛选小分子农兽药高稳定性模拟抗体的研究

Antibody-based immunoassay is a significant measure for the rapid detection of pesticide and veterinary drug residues and ensuring safety of food. However, on one hand, It proved to be difficult and costly for preparing the monoclonal antibody of pesticide and veterinary drug residues because of its weak immunogenicity. On the other hand, the important demand for antibody is highly stable in low concentration organic solvents when in the detection of pesticide and veterinary drug residues. Aims to achieve rapid screening and to acquire highly stable antibody, arginine-binding protein from Thermotoga maritima(TmArgBP) with a good thermal stability (resistance to 116 ℃) and chemical stability ( resistance to 6M guanidine hydrochloride ) was select as protein skeleton materials and multi-site random mutations were carried out by computational biology technology in its ligand binding region for the construction of ribosome display library. Targeted typical pesticide and veterinary drug residues such as diethylstilbestrol, parathion and paraquat, the mimic antibodies will be acquired based on the biopanning technology and virtual screening and are functionally expressed. The properties of mimic antibodies are overall evaluated and identified. Then the mechanism for the high stability of TmArgBP and for the function of amino acid sites in ligand binding region is elucidated. The rapid screening methods could be established for the preparation of highly stable antibody of pesticide and veterinary drug residues. This project is multidisciplinary research of food safety, computational biology and biology technology. And it has broad future application in the field of inspection and quarantine of the imports and exports, food safe and quality supervision and clinical examination.

基于抗体的免疫分析技术是快速检测农兽药、保障食品安全的重要手段，但农兽药免疫原性弱、单抗制备周期长，要求抗体对有机溶剂具有一定耐受性；因此，本项目拟以稳定性好（耐受116℃和6mol/L盐酸胍）的嗜热菌精氨酸结合蛋白（TmArgBP）为骨架，利用核糖体展示技术实现大容量快速筛选，结合计算生物学技术设计多位点组合随机突变，构建核糖体展示TmArgBP突变模拟抗体库，以典型农兽药己烯雌酚、对硫磷、百草枯等不同小分子为靶标，建立虚拟筛选为辅、生物淘筛为主高稳定性抗体快速筛选技术，阐释TmArgBP高稳定识别靶标机制，进行多轮快速淘筛并高效功能表达，获得农兽药高稳定性模拟抗体，系统评价快速筛选制备TmArgBP模拟抗体的可行性，为高稳定性抗体制备奠定技术基础并提供方法学支撑。本项目是食品安全、计算生物学、生物技术多学科交叉研究，在进出口检验检疫、食品安全、质量监督、临床检测等领域具有广阔应用前景。

该课题利用核糖体展示（ribosome display）方法，用于小分子环境化学污染物模拟抗体(anticalin)筛选，并以典型环境化学污染物雌二醇、百草枯为研究对象，对它们的模拟抗体进行了活性鉴定。.雌二醇是一种小分子甾体类激素。由于雌二醇可在动物组织内形成残留，通过食物链危害人类健康。因此，对肉及其肉制品中雌二醇的监测是确保广大市民食用“放心肉”的必要措施。.百草枯（Paraquat, PQ）又名克芜踪，是一种速效触杀型灭生性季铵盐类除草剂，是我国广泛使用的除草剂之一。百草枯有效成分为 1，1′-二甲基 -4，4′-联吡啶阳离子盐,它在农作物中的残留对人、兽有毒害作用。百草枯可以经胃肠道、呼吸道和皮肤吸收，以导致肺纤维化。利用核糖体展示抗体库库容量大、建库和筛选方法简便的优势，构建Anticalin模拟抗体库。以嗜热菌精氨酸结合蛋白（TmArgBP）为骨架，设计随机突变位点的引物合成TmArgBP模拟抗体库。同时引入核糖体展示必需的全部组件，采用引物重叠PCR技术将TmArgBP库与组件进行拼接，从而构建了核糖体展示的TmArgBP突变模拟抗体库。将构建的文库进行体外转录和体外翻译后，以小分子雌二醇、百草枯为靶标，利用固相和液相亲和方法筛选抗雌二醇、百草枯抗体。通过改变Mg2+浓度将筛选到的模拟抗体-核糖体-mRNA三联复合体解离，获得模拟抗体所对应的mRNA。通过几轮生物淘筛，抗原阳性的模拟抗体得到富集，获得高特异性抗雌二醇、百草枯抗体。将该抗体序列在大肠杆菌中进行高效表达，经WB印迹验证该蛋白为抗雌二醇抗体、百草枯。因为所用模拟抗体库具有通用性，原理上可以以任意抗原为靶标筛选其模拟的亲和抗体，这为利用核糖体展示技术从模拟抗体库中筛选多种抗原尤其是小分子半抗原的特异性模拟抗体奠定了基础。