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目的对高拷贝重组毕赤酵母表达X-HBsAg的诱导策略进行优化。方法在摇瓶中分别诱导1、4、8拷贝数的重组毕赤酵母,ELISA检测X-HBsAg的表达量;在15L发酵罐中对8拷贝的重组毕赤酵母进行诱导表达,分别考察甘油浓度对菌体比生长速率的影响、过渡阶段混合补料对菌体生长及细胞相对活性的影响、诱导阶段比生长速率对目的蛋白表达的影响以及诱导阶段混合补料对目的蛋白表达的影响。结果 8拷贝的重组毕赤酵母在摇瓶中诱导表达的X-HBsAg量明显高于1和4拷贝重组子。优化的发酵诱导策略为:初始补加甘油阶段控制甘油浓度在15g/L;过渡阶段采用甲醇与甘油混合补料,控制甘油浓度在限制性浓度以下;诱导阶段采用甲醇与甘油混合诱导X-HBsAg表达,诱导初始阶段控制比生长速率在0.015/h以上,中后期在0.002/h左右;发酵92h,X-HBsAg的表达量比单纯甲醇诱导提高约67%。结论混合补料诱导较单纯甲醇诱导策略更有利于高拷贝重组毕赤酵母表达X-HBsAg。
Objective To optimize the strategy of induction of X-HBsAg expression in high-copy recombinant Pichia pastoris. Methods Recombinant Pichia pastoris were induced in 1, 4 and 8 copies in shake flasks respectively. The expression of X-HBsAg was detected by ELISA. Eight copies of recombinant Pichia pastoris were induced in a 15 L fermenter. The concentrations of glycerol The effect of mixed feed on the growth of cells and the relative activity of cells at the transitional stage, the effect of the specific growth rate on the expression of the target protein and the effect of the mixed feed on the expression of the target protein in the induction phase. Results The amount of X-HBsAg induced by 8 copies of recombinant Pichia pastoris in shake flask was significantly higher than that of 1 and 4 copies of recombinant. Optimized fermentation induction strategy was as follows: initial glycerin glycerol control phase at 15g / L; transitional phase using methanol and glycerol mixed feed, control glycerol concentration below the limit concentration; induction phase mixed with methanol and glycerol to induce X-HBsAg The specific growth rate was higher than 0.015 / h in the initial stage of induction and 0.002 / h in the middle and late stage. The expression of X-HBsAg was increased by about 67% at 92h after fermentation. Conclusion Compared with simple methanol induction strategy, mixed fed induction is more beneficial to high-copy recombinant Pichia pastoris expression of X-HBsAg.