摘要
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 develop a procedure for serum-free culture of enterovirus 71 （EV71） in Vero cells on microcarriers using a Cellspin cell cultivation system. Methods Vero cells were cultured on microcarriers in Cellspin cultivation system, based on which the effects of various media （MEM, DMEM/F12, VP-SFM, Opti-SFM, Opti-pro and MD505-199） and concentrations of mierocarriers （3, 5 and 10 g/L） on cell density were investigated. EV71 was inoculated onto Vero cells at MOIs of O. 2 and 2. 0 respectively, and the effect of MOI on virus proliferation was evaluated. The harvested virus liquid was pooled into three fractions as supernatant, wash and eluate, in which the antigen contents and TCIDs0 were compared. Results By using 5 g/L microcarriers and VP-SFM, the density of Vero cells reached 4. 4 x 106 cells / ml. The harvest time of EV71 inoculated at a MOI of 2. 0 was 72 h, which was 48 h ear/ier than that at a MOI of O. 2. However, the antigen content in harvested supernatant of EV71 inoculated at a MOI of 0. 2 reached 193 U/ml, while the virus titer reached 8. 43 Logl0TCID50/ml. The antigen content in EV71 harvested by combining the supernatant and the eluate factions reached 573 U/ml. Conclusion A method for production of EV71 by serum-free culture of Vero cells on microcarriers was developed, which laid a foundation of development of ero ceils by using a Cellspin cell cultivation system was developed, which laid a foundation of development of a procedure for culture of EV71 by bioreaetor.
出处
《中国生物制品学杂志》
CAS
CSCD
2013年第3期411-415,共5页
Chinese Journal of Biologicals
