青蒿素抗体的分子识别机制及半抗原设计模型研究

The hapten design is a key step for preparing antibodies and developing immunoassay methods for small molecule. In the previous study, we synthesized several haptens of artemisinins, and prepared two broad-spectrum monoclonal antibodies and three high-specific monoclonal antibodies separately. Meanwhile, we proposed that the binding sites of small molecules and carriers should be far away from the position of cluster in the preparation of haptens. In order to explore the hapten design theory, this research will analyze the structural parameters of artemisinins and their haptens by CoMFA and CoMSIA. To clarify the relationship between haptens’ structure and immune activity, we will try to find the intrinsic relationship between the electronic structure, orbital distribution and spatial structure of haptens and antibody specificity. At the same time, this study will also analyze the binding kinetics, main forces and key amino acids between artemisinin-based drugs and antibodies by homologous modeling, molecular docking and surface plasmon resonance. Finally, we will try to quantify the relationship between the antigen binding domain and the modificated site of haptens, and advance the general rule and model of haptens design preliminary, which will provide a new approach for haptens design.

半抗原的设计是制备抗小分子抗体，建立小分子免疫分析方法的关键步骤。申请人在前期研究中合成了多个青蒿素类分子的半抗原，并分别制备了两个广谱的单克隆抗体和三个特异性的单克隆抗体。同时，提出半抗原设计理论，制备半抗原时，小分子与载体结合位点应远离决定簇位置。为进一步探索半抗原设计理论，本研究拟利用CoMFA和CoMSIA分析青蒿素类分子及其半抗原的结构参数，探索半抗原分子的电子结构、轨道分布、空间结构等性质与抗体特异性的内在联系，阐明半抗原结构与免疫活性之间的关系；同时利用同源模建、分子对接分析和表面等离子共振技术，研究青蒿素类药物及抗体间的结合动力学、主要作用力及关键氨基酸。最后，量化半抗原修饰位点与抗体的抗原结合域之间的关系，初步提出半抗原设计的一般规律与模型，为半抗原设计提供新途径。

本项目以前期制备的5个青蒿素类抗体为研究对象，制备单链抗体，并研究其分子识别机制。主要结果包括：（1）测定了5个青蒿素类抗体的可变区基因序列，并分析了抗体序列的差异。（2）构建了双氢青蒿素、蒿甲醚、特异性青蒿素的单链抗体的原核表达载体和真核表达载体，表达了3个带MBP标签的单链抗体，3个带Fc和Nluc标签的单链抗体，并检测了其活性。（3）成功建立了基于双氢青蒿素和蒿甲醚单链抗体原核表达的单链抗体的ELISA方法，IC50分别为2.15 ng mL−1和4.33 ng mL−1，并应用于双氢青蒿素和蒿甲醚药品检测。成功建立了基于双氢青蒿素、蒿甲醚和特异性青蒿素真核表达的单链抗体的ELISA方法，IC50分别为4.95 ng mL−1、3.54 ng mL−1和5.75 ng mL−1。以上抗体ELISA方法的灵敏度、特异性均与其亲本抗体一致。（4）利用AlphaFold2在线网站和SWISS-MODEL在线网站对5个单链抗体进行从头建模和同源建模，选取了合适的模型，并用SAVESv5.0在线服务器验证了模型的合理性。使用autodock 4.2将5个单链抗体三维模型与青蒿素及其衍生物小分子进行分子对接，用Ligplot分析2D相互作用。发现青蒿素及其衍生物的抗体与各小分子的相互作用，主要是重链CDR区（特别是CDR3区）起到作用。而λ型轻链，特别是特异性青蒿素抗体，CDR3区起到重要作用。本研究为抗体对小分子物质的分子识别机制提供了基础数据。

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