抗黄曲霉毒素B1高亲和力抗体的分子机理研究及改造

项目名称： 抗黄曲霉毒素B1高亲和力抗体的分子机理研究及改造

项目编号： No.31271876

项目类型： 面上项目

立项/批准年度： 2013

项目学科： 农业科学

项目作者： 王小红

作者单位： 华中农业大学

项目金额： 85万元

中文摘要： 在小分子污染物的免疫检测技术中，抗体的获得一直是研究的重点。基因工程抗体的研发，为抗体的制备提供了新途径，但基因工程抗体普遍存在亲和力较低、小分子抗体用于免疫检测的效果不及完整抗体。本项目拟以黄曲霉毒素B1（aflatoxin B1, AFB1）为研究对象，以前期获得的抗AFB1单克隆子为材料，克隆表达抗AFB1单链抗体(ScFv)，综合运用同源模建、分子对接和X-射线衍射技术，分析不同ScFv、McAb与AFB1相互作用时，其基因水平、氨基酸水平和抗原抗体复合物空间结构对抗体亲和力、灵敏度和特异性的影响，揭示其亲和力形成的分子机理。在此基础上采用基因突变技术，对抗AFB1ScFv关键基因位点进行改造，然后将其导入真核细胞表达系统或杂交瘤细胞，筛选高亲和力的抗AFB1完整抗体，探讨建立高亲和力抗体的新方法。为AFB1等小分子抗原高亲和力抗体制备提供关键理论指导，也为抗体分子改造提供新策略。

中文关键词： 黄曲霉毒素B1；单链抗体；同源模建和分子对接；亲和力成熟；抗体表达

英文摘要： The acquirement of antibody is the main emphasis in the research of immune detecting technology for small molecular contaminan. The development of genetic engineering antibody provides new approach for the preparation of antibody. However, the current study have found that genetic engineering antibodies ordinarily exist lower affinity, and testing effect can not catch up with that of intact antibody. In this project, with aflatoxin B1 as the research project, anit AFB1 McAbs of different sensitivity and specificity will be used to clone gene of single chain variable fragment and express anti AFB1 single chain variable fragment(ScFv). The effect of gene level, amino acid composition and antigen antibody complex spatial structure characteristics on antibody affinity, specificity and sensitivity will be analyzed by the methods of homology modeling, molecular docking and single crystal X-ray diffraction to reveal the molecular mechanism of its affinity forming in the process of different single chain variable fragments, monoclonal antibodies and AFB1 interactions. On this basis, the key gene site of anti AFB1 ScFv gene will be transformated, and imported into eukaryotic cell expression system or hybridoma cell by cell transfection or gene transfer techniques, and then obtain high affinity intact antibody agai

英文关键词： Aflatoxin B1；Single chain variable fragment；Homology modeling and molecular docking；Affinity maturation；Antibody expression