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目的建立肠道病毒71型(Enterovirus71,EV71)在Cellspin系统中无血清微载体培养工艺,为生物反应器培养EV71工艺的建立奠定基础。方法在Cellspin培养系统中对Vero细胞进行微载体培养,考察不同种类培养基MEM、DMEM/F12、VP-SFM、Opti-SFM、Opti-pro、MD505-199及不同微载体浓度3、5、10 g/L对细胞密度的影响。分别以MOI为0.2及2.0接种EV71至Vero细胞,考察病毒接种量对病毒增殖的影响。分别按培养上清、微载体洗脱液模式收毒,比较不同组分中EV71的抗原含量及TCID50。结果采用5 g/L微载体、VP-SFM培养基时,Vero细胞的密度达4.40×106个/ml;按MOI为2.0染毒,72 h后即可收毒,较MOI为0.2的收毒时间早48 h,按MOI为0.2染毒,收获液上清中的抗原含量达193.5 U/ml,病毒滴度达8.43 Log10TCID50/ml;按上清加洗脱液模式收毒,抗原含量可达573 U/ml。结论成功建立了无血清微载体培养Vero细胞生产EV71的方法,为生物反应器培养EV71工艺的建立奠定了基础。
Objective To establish a serum-free microcarrier culture process of Enterovirus 71 (EV71) in Cellspin system and lay a foundation for the establishment of a bioreactor culture EV71 process. Methods Vero cells were cultured in microcarriers in Cellpin culture system. The effects of different media such as MEM, DMEM / F12, VP-SFM, Opti-SFM, Opti-pro, MD505-199 and different microcarrier concentrations Effect of g / L on cell density. EV71 was inoculated into Vero cells at an MOI of 0.2 and 2.0, respectively. The effect of inoculation amount on virus proliferation was investigated. Respectively according to the culture supernatant, microcarrier elution mode poisoning, compare the different components of the antigen content of EV71 and TCID50. Results With 5 g / L microcarrier and VP-SFM medium, the density of Vero cells reached 4.40 × 106 cells / ml. After exposure to MOI at 2.0, As early as 48 h, the MOI was 0.2, the antigen content in the harvested liquid supernatant reached 193.5 U / ml and the virus titer reached 8.43 Log10 TCID50 / ml. 573 U / ml. Conclusion The method of producing EV71 by culturing Vero cells with serum-free microcarriers was successfully established, which laid the foundation for the establishment of bioreactor culture EV71 technology.