脱氧雪腐镰刀菌烯醇的快速分离纯化及其抗体制备

Rapid Separation and Purification of Deoxynivalenol by High-Speed Counter-Current Chromatography Coupled with High Performance Liquid Chromatography and Its Antibody Production

采用高速逆流色谱法结合高效液相色谱法从禾谷镰刀菌固体发酵产物中分离纯化脱氧雪腐镰刀菌烯醇(DON)。先用高速逆流色谱在以乙酸乙酯-水(1∶1,体积比)为两相溶剂系统、温度为25℃、主机转速为890r·min-1、流速为1.0mL·min-1、检测波长为254nm的条件下进行分离,再用高效液相色谱进一步分离纯化,最终从300g大米发酵培养物中得到了8mg DON,纯度为97.7%。将纯化的DON用于DON人工抗原制备,基质辅助激光解吸附电离串联飞行时间质谱分析表明,人工抗原制备成功;免疫动物后,研制出了高效价的多克隆抗体。本研究建立的色谱方法操作简单,分离效果较好,纯化的DON可以用于抗体的制备。

Deoxynivalenol （DON） was separated and purified from rice fermented by Fusariurn grazninea- rum. High-speed counter-current chromatography （HSCCC） with a two-phase solvent system composed of acetic ether-water （1 ： 1, volume ratio） was employed to isolate DON from the extract of fermentation product firstly. The conditions of isolation were as follows： temperature of 25 ℃ ,890 r·min^-1 of rotation speed, 1.0 mL·min^-1 of flow rate and 254 nm of detection wavelength. Under these conditions,the fragments of extract containing DON were eluted and collected. Then,it was further purified by HPLC,and 8 mg of DON with the purity of 97. 7% was finally obtained from 300 g of rice solid fermentation. The purified DON was used to synthesize its completed antigen and MALDI-TOF-MS analyses showed the synt＇hesis was successful. Then,Balb/C mice were immuned and the polyclonal antibody with high titer was got. This methoct is simple and efficient for separation and purification of DON, and the purified DON can be utilized to prepare its complete antigen and an- tibody.