兽药重组Scfv抗体的分子识别机制及双特异Scfv抗体研究

The core reagent during the development of an immunoassay for multi-determination of different classes of veterinary drugs in animal original foods is the broad specific antibody. In this project application, the generic haptens of phenothiazines drugs and benzodiazepines drugs are synthesized respectively, with which to produce the respective generic monoclonal antibody of the two classes of drugs. The gene fractions that encoded the variable region of antibody heavy-chain and light-chain in different hybridoma cells are extracted and used to produce the respective recombinant Scfv antibody of the two classes of drugs. The sensitivity and specificity of the two Scfv antibodies for respective class of drugs are determined by using of competitive ELISA method. Then, the homology modeling for the two Scfv antibodies are performed by using of the computer simulation and the antibody-hapten interactions are simulated by using of Molecular Docking technique to determine the molecular recognition mechanism of the two Scfv antibodies, including recognition positions, key amino acids and binding model. In order to improve the antibody performances, the gene fractions that encoded the two Scfv antibodies are subjected to directional transformation. Based on the obtained optimal gene fractions, the optimized broad specific Scfv antibodies for phenothiazines drugs and benzodiazepines drugs are produced respectively and the molecular recognition mechanisms of the two Scfv antibodies are studied again. Then the recombinant bi-specific Scfv antibody simultaneous recognizing the two classes of drugs is produced based on the obtained optimal gene fractions and used to determine the residues of the two classes of drugs in animal original foods. The results of this study will provide the basis for production of the bi-specific or multi-specific antibodies of other veterinary drugs and help to study their molecular recognition mechanism.

动物性食品中兽药多残留免疫分析法的核心是广谱特异性抗体。本项目拟分别合成吩噻嗪类和苯二氮卓类药物的通用半抗原，制备两类兽药各自的广谱单克隆抗体；以杂交瘤中编码抗体轻、重链可变区的基因片断为基础，制备两类兽药各自的广谱ScFv抗体，以ELISA法检测Scfv抗体对同类药物的交叉反应性和灵敏度。通过计算机模拟分别对两种Scfv抗体进行蛋白同源模建，获得抗体的三维构象，以分子对接技术建立抗体-抗原互作模型，研究抗体对兽药的分子识别机制（识别位点、关键氨基酸和结合模式）；分别对编码两种ScFv抗体的基因片断进行定向改造，制备两种新的ScFv抗体，以提高抗体性能，并研究抗体的分子识别机制。将两种Scfv抗体的基因片断连接，制备能同时识别吩噻嗪类和苯二氮卓类药物的广谱双特异ScFv抗体用于动物性食品中这两类药物的残留检测。本研究结果将为研究其他兽药的双/多特异性ScFv抗体及其识别机制奠定基础。

吩噻嗪类药物、苯二氮卓类药物、青霉素类药物和喹诺酮类药物在动物性食品中的残留严重危害着消费者的健康。因此，建立简单、快速、可用于兽药多残留检测的免疫分析法至关重要。本项目组针对这些问题，进行了以下六方面的工作：. 一、在国内外首先制备出了能识别5种吩噻嗪类药物的广谱单克隆抗体，并建立了ELISA法用于肉组织中5种药物的残留检测，灵敏度为0.2 -0.4 ng/g。. 二、在国内外首先制备了能识别5种苯二氮卓类药物的广谱单克隆抗体，并建立了ELISA法用于猪组织中5种药物的残留检测，灵敏度为0.2 -1.5 ng/g。. 三、在国内外首先制备了抗阿莫西林的ScFv抗体，采用YASARA和Autodock软件研究了该ScFv抗体对11种青霉素类药物的分子识别机制。然后，基于YASARA软件的虚拟突变分析，设计了2个突变位点，对该抗体的基因进行了定向突变。制备的ScFv突变体对11种药物的灵敏度提高了2-6倍，并可用于牛奶中11种药物的残留检测。. 四、在国内外首先制备了抗沙拉沙星的ScFv抗体，利用YASARA和Autodock研究了其对12种喹诺酮类药物的分子识别机制。然后，基于虚拟突变的分析，设计了1个突变位点，对该抗体的基因进行了定向突变。制备的ScFv突变体对12种药物的灵敏度提高了2-7倍，并可用于牛奶中12种药物的残留检测。. 五、在国内外首先制备了针对吩噻嗪类药物的ScFv抗体和针对苯二氮卓类药物的ScFv抗体，两种抗体的识别性能与其母源单克隆抗体相似。然后，采用YASARA和Autodock研究了它们的分子识别机制，证明氢键和疏水作用是主要分子间力，且抗体-药物的结合能是决定抗体识别性的主要因素。. 六、在国内外首先将吩噻嗪药物ScFv抗体的基因和苯二氮卓类药物ScFv抗体的基因进行拼接、表达，制备出了一种可以同时识别5种吩噻嗪类药物和5种苯二氮卓类药物的双特异性ScFv抗体。. 这些研究工作是将免疫学、分子生物学、基因工程技术和计算化学的技术和方法相结合，形成了一套科学的关于基因重组抗体制备、分子识别机制研究、定向进化的技术路线，具有重要的科学价值，也为研究其他小分子物质的基因重组抗体提供了一条新途径。