促进CHO细胞生长及其产物hNGF表达的培养条件的初步研究

Enhanced Production of Human Nerve Growth Factor by CHO Cells Through the Control of Culture Conditions

以稳定表达人神经生长因子(hNGF)的重组工程CHO细胞株为对象,采用无血清流加悬浮培养(Fed-batch culture)方式,考察使用基础培养基(无特殊添加物),分别添加丁酸钠、DMSO、KH2PO4的培养基及不同培养温度(32℃和37℃)对细胞生长和重组蛋白表达的影响。每日取样检测细胞密度、细胞活率、葡萄糖浓度、重组蛋白浓度。结果表明细胞培养温度由37℃下降至32℃,细胞生长周期明显延长,重组蛋白产量增加。5mmol/L丁酸钠和2%DMSO的加入虽然提高了重组蛋白的表达量,但严重抑制细胞生长。最大的蛋白比生成速率(qNGF)出现在37℃培养且添加2%DMSO的培养条件下,而最高蛋白表达量则出现于32℃培养添加3.65mmol/LKH2PO4的培养条件下。研究表明,将培养温度设为32℃,在基础培养基中添加3.65mmol/LKH2PO4或1%DMSO是提高hNGF表达水平的有效方法。

The enhancement of recombinant protein productivity of a stable cell line is essential for developing an efficient large-scale bioprocess.The effect of some media additives and temperature conditions were studied in an attempt to optimize cell growth and protein productivity from a Chinese hamster ovary （CHO） cell line producing human nerve growth factor.The addition of dimethyl sulfoxide （DMSO）,sodium butyrate （NaB）,KH2PO4 and temperature shift （37℃ and 32℃） were evaluated for the enhancement of hNGF productivity in a fed batch suspension culture.The samples were taken everyday for checking their viable cell density,viability,hNGF productivity and glucose concentration.The culture time was prolonged significantly from 7 days to 14 days when the culture temperature decreased from 37℃ to 32℃.The productivity of hNGF increased in addition of NaB and DMSO though they arrested the cell cycle dramatically.The maximum specific hNGF productivity was achieved in the medium with 2% DMSO cultured at 37℃ and the maximum hNGF productivity was achieved in the medium with 3.65mmol/L KH2PO4 cultured at 32℃.Taken together,the addition of 3.65mmol/L KH2PO4 or 1% DMSO in the culture medium had the greatest effect on hNGF productivity in CHO cells.