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。
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.
关键词
肿瘤坏死因子 /
人肿瘤坏死因子受体Ⅱ-Fc融合蛋白 /
温度 /
规模放大
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Key words
tumor necrosis factor /
TNFR-Fc fusion protein /
temperature /
scale-up
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雷云，谢奎，洪刚，陈宝琼，杨依丽，谢秋玲，熊盛.
表达人肿瘤坏死因子受体Ⅱ-Fc的CHO-DG44细胞的培养温度优化研究[J]. 中国药学杂志, 2013, 48(2): 90-95
LEI Yun， XIE Kui， HONG Gang， CHEN Bao-qiong， YANG Yi-li， XIE Qiu-ling， XIONG Sheng.
Optimization of the Culture Temperature of Recombinant CHO-DG44 Cells Expressing Tumor Necrosis Factor Receptor-Fc[J]. Chinese Pharmaceutical Journal, 2013, 48(2): 90-95
中图分类号：
Q813
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脚注
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基金
国家重大新药创制资助项目（2012ZX09103301-033，2012ZX09202301）；暨南大学科研培育与创新基金杰出人才培育资助项目（1161-1206）；抗体规模制备技术及肿瘤抗体药物的研究（2010U1-E00541）
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