基于可视化技术的磺胺类和甲氧苄啶类双特异分子识别元件与其靶标分子识别机理研究

The veterinary drug residues in animal derived food are a major safety problem for human health, therefore, it’s a crucial desire to develop and validate a method with higher sensitivity, rapidity, and high effectiveness. However, it’s difficult to solve this problem by used the method based on the traditional antibodies such as polyclonal antibodies and monoclonal antibodies. This problem will be solved by using the method based on the bispecific single chain Fvs (BisFvs). In the current study, two kinds of antibacterial agent, sulfonamides (SAs) and its synergists trimethoprim’s (TMPs), were selected as a model drugs. After immunization, from numerous cell fusion and culture, the obtained SAs and TMPs hybridoma cells that produced specific antibody have been chosen to paradigm the phage antibody libraries of SAs and TMPs. The obtained scFv genes of SAs and TMPs were linked each other by an optimized linker to attain the BisFvs gene. Whereas, the study on the molecular recognition mechanism amongst the BisFvs of SAs, TMPs and their target drugs were performed by visual technology such as DataBase of Web Antibody Modelling and the Software of Autodock 4.2. While, the action mechanism amongst the structure of hapten, immunization methodology, screening strategy, design of linker and the performance of the BisFvs including sensitivity, specificity, affinity and stability has been revealed. As well, the molecular recognition mechanism amongst the BisFvs of SAs, TMPs and its target drugs were also clarified. This entire result delivers a new method with low cost and high efficiency for the multi-residue detection of SAs and its antibacterial synergists TMPs. The present study will offer a new approach to the rapid and simultaneous detection of veterinary drugs in animal derived food. In a word, this study would be of great theoretical and practical significance to promote the development of the rapid detection theory and technology of veterinary drug residues, as well, to solve the problems of food safety and environmental pollution caused by the veterinary drug residues.

动物源性食品中的兽药残留严重威胁人类健康，急需灵敏快速高效的残留检测方法。基于常规抗体的检测方法难以满足该要求。基于双特异单链抗体的免疫分析方法有望破解这一难题。本项目以磺胺类与甲氧苄啶类为模式化合物，采用噬菌体表面展示技术，构建和表达其双特异单链抗体基因；通过Web Antibody Modelling数据库和Autodock 4.2等可视化技术，对双特异单链抗体与药物靶标的识别机理进行研究。本项目研究将揭示双特异单链抗体的灵敏度、特异性、亲和力以及稳定性与抗原结构、免疫程序、筛选策略、linker设计等因素之间的相互作用机制，阐明双特异单链抗体对药物靶标的识别机理，为磺胺类和甲氧苄啶类低成本高效率的多残留快速检测提供新方法，为动物源性食品中多类兽药的同时快速检测提供新思路，对推动兽药多残留快速检测理论和技术的快速发展，解决因兽药残留引发的食品安全和环境污染问题具有重要的理论和实践意义。

动物源性食品中的兽药残留严重威胁人类健康，急需灵敏快速高效的残留检测方法，然而基于常规抗体的检测方法难以满足该需求。本项目以磺胺类与甲氧苄啶类为模式化合物，通过半抗原设计，制备出磺胺类和甲氧苄啶类单克隆抗体，采用噬菌体表面展示技术，成功构建了高库容的磺胺类和甲氧苄啶类噬菌体免疫文库，并筛选得到了能够分别识别磺胺类和甲氧苄啶类的scFv，获取的scFv对磺胺二甲嘧啶和甲氧苄啶的IC50分别为52 ng/mL和24 ng/mL，能够满足市场上的残留检测限量需求。解析了磺胺类和甲氧苄啶类scFv识别药物靶标的分子机制，发现磺胺类scFv与其靶标的结合能为-6.3 kcal/mol，关键氨基酸为HIS-39，ASP-227，ASPP-229，分别与药物靶标形成氢键，π-H键和离子键。甲氧苄啶类scFv与其靶标的结合能为-6.4 kcal/mol，共有六个关键氨基酸与甲氧苄啶之间的相互作用力维持着复合物的形成，其中在scFv识别甲氧苄啶过程中起着至关重要的作用。采用DS进行虚拟饱和突变，结合定点突变技术对甲氧苄啶类scFv实现了体外进化，其识别甲氧苄啶的IC50达到了3.4 ng/mL。同时，构建了磺胺类和甲氧苄啶类双特异单链抗体并阐明了磺胺类和甲氧苄啶类双特异单链抗体对药物靶标的识别机理，发现双特异单链抗体与甲氧苄啶类药物的结合能为-5.7 kcal/mol，关键氨基酸为ASP-159，SER-200和ASP-203，其中ASP-159贡献了65%的结合能，表明ASP-159与靶标小分子之间形成的分子间作用力对维持复合物的形成起着关键作用。本项目研究为进一步通过体外进化增加磺胺类和甲氧苄啶类双特异单链抗体的灵敏度奠定了基础，为动物源性食品中多类兽药的同时快速检测提供新思路，对推动兽药多残留快速检测理论和技术的快速发展，解决因兽药残留引发的食品安全和环境污染问题具有重要的理论和实践意义。

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