重组复合干扰素基因工程菌发酵培养条件的研究

Investigation of fermentation conditions of recombinant consensus interferon in E.coli

目的:研究表达重组复合干扰素(con-IFN)的工程菌E.coliDH5α(pBV220/con-IFN)的发酵条件。方法:在摇瓶实验中,采用正交设计法和均匀设计法确定较佳培养基组分;使用20 L发酵罐进行补料分批发酵实验,研究影响菌体生长和con-IFN表达的因素(如溶氧、补料、温度和诱导时间)。结果:发酵培养基配方为蛋白胨10 g/L,酵母膏7.5 g/L,甘油0.5%,Na2HPO4.12H2O 20.13 g/L,KH2PO410.37 g/L,NaCl 9.31 g/L。确定了高密度发酵工艺,即优化的发酵培养基,初始pH7.2,接种量5%,溶氧15%,先经35℃培养4 h,后降温至30℃培养2 h,再以42℃诱导7 h,诱导的同时补料酵母膏0.5%。在此条件下,发酵液可得15.2 g/L湿菌体,con-IFN的表达量占菌体总蛋白的49.60%,每毫升发酵液的抗病毒活性为9.69×108IU。结论:获得了较满意的优化培养基配方和发酵条件,为下游纯化和进一步大规模生产奠定了基础。

Aim： To assess the key factors influencing the fermentation process of a recombinant E. coli （pBV220 con- IFN DH5a） producing consensus interferon. Methods： Orthogonal design were used to optimize the components of media in shaking-flask, and fed batch fermentation was performed to investigate the factors such as dissolved oxygen, feeding solution, temperature and induction time which would affect the cell growth and the expression of con-IFN. Results：The optimized media was found to be composed of tryptone of 10 g/L, yeast extract of 7.5 g/L , NaHPO4·12H2O of 20.13 g/L, KH2PO4 of 10.37 g/L, and NaCl of 9.31 g/L. The fermentation conditions were confirmed as follows initially pH 7.2, 5 % of inoculation and 15 % of dissolved oxygen at 35℃ for 4 h; then at 30℃ for 2 h; lastly at 42℃ with simultaneous addition of 0.5% yeast extract for 7 h. It was shown that the cell density reached 15.2 g wet cells/L, and the expression of con-IFN taken 49.60% of the total cell proteins. The antiviral activity of con-IFN was estimated to be 9.69×10^8IU/mL broth. Conclusion： The fermentation is efficient for the production of recombinant E. coli （pBV220 con-IFN DH5α） producing consensus interferon, and deserves further exploitation.