破伤风梭菌无动物源培养基发酵及纯化工艺的探索

Development of animal-component-free medium for Clostridium tetani and purification process of tetanus toxin

目的:开发优化破伤风梭菌无动物源培养基;摸索层析法纯化破伤风毒素的工艺。方法:分别用蛋白胨和酵母浸出物替代培养基中动物源性成分作为氮源。2 L发酵罐发酵比较菌体生长密度和产毒量;发酵液经离心,切向流过滤回收,通过柱层析方法对发酵回收液进行纯化。柱层析时比较不同洗脱条件下毒素回收率。结果:蛋白胨可更好的供菌体生长,但产毒量较低;酵母浸出物可作为氮源供菌体生长,菌体浓度相对较低,但最高产毒量可达52 Lf·m L-1;阴离子柱层析纯化可使破伤风毒素纯度大幅提高,毒素回收率在80%左右。结论:无动物源培养基发酵破伤风梭菌有高产毒量,阴离子柱层析可作为破伤风毒素纯化方法。

Objective： To develop and optimize animal-component-free medium for culturing Clostridium tetani and explore a tetanus toxin purification process. Methods： Animal component materials in the culuture medium was replaced by soytone or yeast extract as nitrogen source. Cell density and the production of toxin meseaured in Lf·m L^- 1were compared in a 2L fermenter. Toxin was recovered by centrifugation and TFF operation,and was purified by column chromatography. Yields of toxin at different elution conditions were compared. Results： Soytone as nitrogen source supported good growth of tetanus but toxin formation was low; yeast extract as nitrogen source supported less tetanus growth but had higher toxin yield. The maximum concentration of toxin in the fermenter was52 Lf·m L^- 1. Purification of tetanus toxin by DEAE chromatography resulted in a toxin yield of 80% with higher purity. Conclusion： Animal-component-free medium produces a larger amount of toxin; DEAE can be adopted as a method for tetanus toxin purification.