表达HPV18晚期蛋白L1重组毕赤酵母在30L Sartorious生物反应器(C_(30)-3)中的培养与诱导工艺摸索及优化

Cultivation and Induction Process Exploration and Optimization of HPV 18 L1 Protein Expressing Pichia Pastorius in 30 L Sartotious Bioreactor

采用改良后的低盐培养基在30 L Sartorious生物反应器(C30-3)对表达18型人乳头瘤病毒(Human papillom avirus,HPV)晚期蛋白L1重组毕赤酵母进行培养,探讨了在重组毕赤酵母细胞积累阶段和HPV18晚期蛋白L1诱导表达阶段不同的培养基配方、p H值、温度、搅拌转速、溶氧(DO)、重组毕赤酵母细胞积累阶段甘油的补加方式、去除阻碍物甘油的去阻碍时间、重组毕赤酵母表达HPV18晚期蛋白L1阶段诱导剂甲醇的补加方式等工艺参数对表达HPV18晚期蛋白L1重组毕赤酵母的影响。优化后的低盐培养基更适合重组毕赤酵母细胞的生长;重组毕赤酵母细胞积累阶段的培养过程中维持培养液p H值5.0,温度31℃,DO 20%~25%,搅拌转速500 r/min,培养到第20 h开始以恒速补加3 000 m L 40%甘油溶液,培养至重组毕赤酵母细胞湿重≥190 g/L,停止甘油补加,继续培养重组毕赤酵母4 h,消耗阻碍HPV18晚期蛋白L1表达的甘油;之后进入重组毕赤酵母表达HPV18晚期蛋白L1诱导阶段,维持培养液p H值5.2,温度29℃,DO含量45%,搅拌转速600 r/min,控制培养液中甲醇浓度为4%在线补加甲醇,诱导60 h,HPV18晚期蛋白L1的表达量达到了311.43 mg/L。利用30 L Sartorious生物反应器(C30-3)对表达HPV18L1重组毕赤酵母进行培养,经过多次试验建立起了稳定的重组毕赤酵母表达HPV18晚期蛋白L1工艺。

Using improved low-salt medium,the recombinant HPV18L1 late protein was expressed by Pichia pastorius at 30 L Sartorious bioreactor( C30-3). Various parameters affecting expression of HPV18L1 protein in accumulation and induction phases,such as medium components,p H value,temperature,stirring speed,dissolved oxygen,addition of glycerol and methanol,were evaluated.The results showed that the optimized low-salt medium is more suitable for the growth of the yeast Pichia cells. During accumulation stage,p H of the culture medium 5. 0,temperature 31℃,DO content of 20% to 25% and stirring speed 500 r / min were maintained,3 000 m L of 40% glycerol solution was added at a constant feed rate for the first 20 h until the Pichia wet weight ≥190 mg / m L and then feeding of glycerol was stopped and Pichia was cultivated for additional 4 h to exhaust glycerol. After entering the recombinant Pichia expression HPV18L1 protein induced late stage,to maintain the culture p H5. 2,temperature 29 ℃,DO content of 45%,stirring speed 600 r / min,methanol concentration control culture medium supplemented with 4% methanol online and induction 60 h.Late HPV18L1 protein expression reached 311. 43 mg / L. 30 L Stable expression of recombinant HPV18L1 protein by Pichia at 30 L Sartorious bioreactor( C30-3) was established.