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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 study the method for preparing Enterovirus 71 (EV71) on a large scale using microcarrier technology. Methods The microcarriers of Vero cells were cultured with NBSCelliGen 310 5 L bioreactor. The effects of different concentrations of Cytodex-1 (3, 10, 15, 20 g / L) on the growth and metabolism of Vero cells and cell density were investigated. The virus multiplication of Vero cells in Cell factory (CF) was compared. The supernatant, washing solution and elution solution were used to collect the drug, respectively, and the antigen content and virus titer (CCID50) of EV71 were compared among different methods of drug collection. Results The cell density of Vero cells was up to 5.47 × 106 cells / ml after cultured with 10 g / L microcarrier and batch culture for 120 h. When the microcarrier concentration was more than 15 g / L, Culture using perfusion mode. After MOI 0.2, the collection time of microcarriers was 48 h slower than that of CF, but the virus titer reached 8.8 Log10 CCID50 / ml, about 5 times of that of CF. EV71 with the presence of ion exchange adsorption of microcarriers, according to the supernatant plus eluent way to poison, the total amount of antigen up to 1261 U / ml, about three times the CF. Conclusion The method of culturing Vero cell with 5 L biochar by microcarrier and successfully producing EV71 has been established, which lays the foundation for further EV71 large-scale culture and vaccine development.