摘要
目的研究利用微载体技术规模化制备肠道病毒71型(Enterovirus 71,EV71)的方法。方法利用NBSCelliGen 310 5 L生物反应器进行Vero细胞微载体培养,考察了不同微载体Cytodex-1浓度(3、10、15、20 g/L)对Vero细胞生长代谢及细胞密度的影响,并且与细胞工厂(Cell factory,CF)中Vero细胞染毒后的病毒繁殖进行比较。分别采用上清液、洗涤液、洗脱液模式收毒,比较不同收毒方式中EV71的抗原含量及病毒滴度(CCID50)。结果采用10 g/L微载体浓度,批次培养方式培养Vero细胞120 h后,细胞密度可达5.47×106个/ml;当微载体浓度大于15 g/L时,由于葡萄糖消耗速度快,需采用灌注模式培养。按MOI 0.2染毒后,微载体培养的收毒时间比CF慢48 h,但其病毒滴度可达8.8 Log10CCID50/ml,约为CF的5倍。EV71与微载体存在离子交换吸附作用,按上清液加洗脱液方式收毒,抗原总量可达12 61 U/ml,约为CF的3倍。结论已成功建立了生物反应器微载体5 L发酵培养Vero细胞生产EV71的方法,为进一步EV71大规模培养以及疫苗研发奠定了基础。
Objective To develop a procedure for production of enterovirus 71 （EV71） in Vero cells grown on microcartiers in a bioreactor. Methods Vero ceils were grown on Cytodex-1 microcarriers in a 5 L NBS CelliGen 310 bioreactor filled with MEM containing 10% new calf serum. The effect of concentrations （3, 10, 15 and 20 g/L） of Cytodex-1 microcarriers on growth, metabolism and density of cells were investigated, while the proliferation profile of virus in bioreactor was compared with that in cell factory （CF）. The harvested virus liquid was pooled into three fractions as supernatant, wash and elution, in which the antigen contents and virus titers （CCID50） were compared. Results The density of Vero ceils reached 5.47 × 10^6 cells/ml 120 h after batch culture on 10 g/L micorocarriers. When the concentration of microcarriers was more than 15 g/L, perfusion culture mode was adopted because of rapid consumption of glucose. The harvest time of EV71 inoculated at a MOI of O. 2 in bioreactor was 48 h later than that in CF. However, the titer of EVT1 in bioreactor reached 8. 8 log10CCID50/ml, which was 5 times higher than that in CF. Since the ion exchange ad- sorption between EV71 and microcarriers, the virus was harvested by combining the supernatant and the eluent fractions, in which the antigen content （12 61 U/ml） was about 3 times higher than that harvested from CF. Conclusion The pro cedure for production of EV71 in Vero cells grown on microcarriers in a bioreactor was successfully developed, which laid a foundation of further large-scale culture of EV71 and development of vaccine.
出处
《中国生物制品学杂志》
CAS
CSCD
2013年第2期256-259,共4页
Chinese Journal of Biologicals
