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目的利用Vero细胞生物反应器微载体培养技术规模化培养轮状病毒重配株LH9(G4型)。方法用3 L生物反应器,以4 g/L载体浓度培养Vero细胞,待细胞浓度达1.8×106个/ml时,分别用含2、1、0.75、0μg/ml胰酶的DMEM培养液,35℃连续培养21 d,观察细胞生长状态。分别按0.5、0.1、0.05、0.01 MOI接种轮状病毒重配株LH9(G4),培养过程中每日取样,检测病毒滴度以及葡萄糖、乳酸含量。根据培养过程中葡萄糖、乳酸含量的变化,确定病毒培养过程中的收获时间,优化培养工艺。结果在反应器中用含不同浓度胰酶的DMEM培养Vero细胞,细胞形态良好,与不含胰酶的DMEM培养液培养的细胞无明显差异;按0.05 MOI接种后的病毒收获液滴度较高,可达7.0 lg CCID50/ml,且持续时间较长;通过对病毒培养液中葡萄糖、乳酸含量的监测,调整了培养过程中的收获时间,收获次数增加至4次,可收获病毒液4个工作体积,病毒高峰滴度可达8.5 lg CCID50/ml。结论采用生物反应器微载体系统培养轮状病毒重配株LH9(G4),可显著提高病毒滴度,通过对培养过程的优化,增加了病毒收获量,为进一步规模化生产轮状病毒疫苗奠定了基础。
OBJECTIVE: To develop a recombinant rotavirus strain LH9 (G4 type) using Vero cell bioreactor microcarrier culture technology. Methods Vero cells were cultured in a 3 L bioreactor at a concentration of 4 g / L. When the cell concentration reached 1.8 × 106 cells / ml, DMEM medium containing 2, 1, 0.75, 0 μg / ml trypsin, 35 ℃ continuous culture 21 d, observe the cell growth status. Rotavirus rearrangement strain LH9 (G4) was inoculated by 0.5, 0.1, 0.05 and 0.01 MOI respectively. Daily samples were taken during the cultivation to detect the virus titer and the content of glucose and lactate. According to the changes of glucose and lactic acid content in the process of cultivation, the harvesting time in the process of virus cultivation was determined and the culture process was optimized. Results Vero cells were cultured in DMEM with different concentrations of trypsin in the reactor. The morphology of the cells was good, and there was no significant difference between the cells cultured in DMEM medium without trypsin. The titer of virus harvested after inoculation with 0.05 MOI was higher , Up to 7.0 lg CCID50 / ml, and longer duration; by monitoring the concentration of glucose and lactate in the virus culture medium, the harvesting time in the culture process was adjusted, the number of harvests increased to 4 times, and 4 virus harvests Working volume, the virus titer up to 8.5 lg CCID50 / ml. Conclusion The culture of rotavirus reassortant LH9 (G4) with the bioreactor microcarrier system can significantly increase the virus titer. Through the optimization of the culture process, the amount of virus harvested is increased, which will lay the foundation for further large-scale production of rotavirus vaccine The foundation.