【摘要】 目的优化表达人肿瘤坏死因子受体Ⅱ-Fc(TNFR-Fc)融合蛋白的重组CHO-DG44细胞的培养温度,并考察该细胞株在最佳温度放大培养下纯化后的产物纯度及活性。方法采用批次培养方式,将重组中国仓鼠卵巢细胞培养于37℃、37℃转31℃、31℃3种不同的温度下,每日取样检测细胞密度、细胞活率、葡萄糖浓度、乳酸浓度、人肿瘤坏死因子受体Ⅱ-Fc融合蛋白浓度,并选择最佳的培养温度对重组中国仓鼠卵巢细胞进行放大(10、50、200 mL、2 L)培养,所得培养上清经Protein A亲和色谱柱纯化,并采用SDS-PAGE,SE-HPLC,WST-8对人肿瘤坏死因子受体Ⅱ-Fc融合蛋白进行相对分子质量,纯度和生物活性检测。结果研究发现,37℃转31℃的转温度培养条件可在保持高密度活细胞的同时维持细胞活率,延长培养周期,显著提高人肿瘤坏死因子受体Ⅱ-Fc融合蛋白产量,并且该转温度培养工艺可成功地应用于重组CHO细胞的规模放大培养中。纯化后的人肿瘤坏死因子受体Ⅱ-Fc融合蛋白相对分子质量约为150×103,纯度在93%以上,生物活性检测显示其可中和重组人肿瘤坏死因子α的细胞毒效应。结论相比单一的37℃培养条件,37℃转31℃的转温度培养工艺可明显提高重组CHO-DG44细胞的人肿瘤坏死因子受体Ⅱ-Fc融合蛋白表达量,且该工艺具有可放大性,这为该工艺的更大规模应用奠定了基础。更多还原

【Abstract】 OBJECTIVE To optimize the culture temperature of recombinant CHO-DG44 cells expressing tumor necrosis factor receptor-Fc fusion protein and to determine the bioactivity of expressed protein at the best culture temperature.METHODS Recombinant CHO cells were batchly cultured at three different temperatures(37 ℃,37 ℃ shifting to 31 ℃ and 31 ℃).Samples were daily tested for cell densities,viabilities,glucose concentration,lactic acid concentration and TNFR-Fc fusion protein concentration,then the best culture temperature was chosen to scale up the fermentation volume(10,50,200 mL,and 2 L).TNFR-Fc fusion protein was purified with Protein A affinity chromatography,determined for relative molecular mass,purity and neutralizing activity by SDS-PAGE,SE-HPLC and WST-8 separately.RESULTS The culture condition of 37 ℃ shifting to 31 ℃ resulted in the maximum viable cell density,high viability and long culture time of the cells as well as high TNFR-Fc protein productivity,and this culture procedure could be successfully applied to the scale-up of recombinant CHO cells.The purified fusion protein,with a relative molecular mass about 150 000,reached the purity of more than 93% and neutralized the cytotoxic effect of TNFα.CONCLUSION Compared to the traditional culture temperature of 37 ℃,the culture temperature of 37 ℃ shifting to 31 ℃ can obviously improve the TNFR-Fc fusion protein output by recombinant CHO-DG44 cells.And this technique can be applied to scale-up,which will lay a foundation of large scale industrial production.更多还原