重组人粒细胞集落刺激因子的摇瓶发酵研究

Studies on the fermentation technology of recombinant E.coli expressing human granulocyte colony-stimulating factor(hG-CSF) in shaking flask

目的 优化重组大肠杆菌生产人粒细胞集落刺激因子摇瓶发酵工艺条件。方法 利用摇瓶系统地考查rhG CSF茵株培养温度、诱导时机、pH值、溶氧、种子菌龄、接种量等工艺条件,选择出最佳的摇瓶培养条件。结果 最佳条件为:培养温度30℃;初始pH值在7 0～7 2;装液量20%;摇床转速180r/min;种子菌龄在A600为1 0～1 5时接种,并在对数生长前期(A600=1 0)时诱导4h。在此优化的培养条件下,在摇瓶中使用优化后的M9培养基时,rhG CSF的表达量占菌体总蛋白的36 5%,光密度达5 85。结论 构建的rhG CSF工程菌发酵的稳定性和重复性良好,可作为rhG CSF的大规模生产提供可靠的放大依据。

AimFermentation technology of recombinant E.coli expressing human granulocyte colony-stimulating factor (rhG-CSF) with shaking flask was optimized.MethodsThe effects of culture temperature,time of inducing,expression,pH,disslved oxygen,inoculum age,inoculum volume and so on were investigated for rhG-CSF.ResultsThe experimental results show that the optimum conditions are: temperature 30℃,initial pH 7.0～7.2, medium volume 20%, rotating speed 180 r/min, and inoculum age 1.0～1.5(A_(600)). It is available for inducing cultivation when the growth of the bacteria is in the initial logarithm growth phase and expressing 4 hours. Under these culture conditions, the expression and optical density(A_(600)) in shaking flask are 36.5% and 5.85,respectively when the optimized M9 medium is used.ConclusionThe results indicate that the rhG-CSF engineered E.Coli strain has better stability and repeatability which provides reliable data for bullk production of rhG-CSF.