脱氧雪腐镰刀菌烯醇(DON)人工抗原及多克隆抗体的研制

Preparation of Artificial Antigen and Polyclonal Antibody of Deoxynivalenol(DON)

脱氧雪腐镰刀菌烯醇(Deoxyn ivalenol,DON)是一种半抗原,没有免疫原性。为了制备抗DON的抗体,必须对其进行结构修饰,加大DON的分子量。本研究采用保护-去保护策略使DON的C3上的羟基与琥珀酸酐反应,使DON衍生后获得1个羧基。利用薄层色谱法跟踪反应进程,反应产物采用薄层色谱法纯化,高效液相色谱和电喷雾离子化质谱(ESI-MS)分析,得出[M+H]+为397.2,确认为3-半琥珀酰-脱氧雪腐镰刀菌烯醇(3-HS-DON)。3-HS-DON与载体蛋白采用碳化二亚胺法进行偶联,偶联物经SDS-聚丙烯酰胺凝胶电泳鉴定,推断偶联成功,并研制出多克隆抗体。

Deoxynivalenol （DON） is a toxin produced by Fusarium graminearum, which causes ear blight of wheat, barley, corn and other cereal crops worldwide. Consumption of cereal grains containing DON can result in diseases in animals and human beings. It is very necessary to detect DON in food. However, DON is not an antigen, it is essential to modify the structure and design an semi-antigen. The conversion of DON to 3-HS-DON was facilitated by protecting two of the three available hydroxyls with a cyclic boronate ester and making DON react with the C3 hydroxyl. The derived product was purified with TLC and analyzed by HPLC and ESI-MS. The mass spectrum of derived DON, 3-HS-DON, had the expected [ M ＋ H ] ＋ peak at 397. 2 m/z. 3-HS-DON-BSA and 3-HS-DON-OVA were prepared by the carbodiimide reaction, and the conjugates were confirmed by SDS-PAGE .