乙型脑炎病毒在Vero细胞上的传代适应性及毒种库的建立  

Adaptation of Japanese encephalitis virus in Vero cells and establishment of virus seed bank

作　　者：刘岩[1] 苑志刚[1] 韩轼奇[1] 谢伟 周长军[1] 李春艳[1] 闫峰[1] 杨屹[1] 

机构地区：[1]长春生物制品研究所有限责任公司,吉林长春130062 [2]长春祈健生物制品有限公司,吉林长春130062

出　　处：《中国生物制品学杂志》2014年第12期1529-1533,共5页Chinese Journal of Biologicals

摘　　要：目的分析乙型脑炎病毒（Japanese encephalitis virus,JEV）在Vero细胞上的传代适应性,进行培养条件及接种方式的优化,并建立毒种库。方法将JEV P3株在Vero细胞上连续适应性传至20代（P3V1-P3V20）,采用小鼠法检测P3V2、P3V3、P3V4的病毒滴度。优化JEV静置培养条件（维持液p H值、培养温度、MOI）及接种方式（单层接种法、混合接种法）,并进行培养规模的放大。采用RT-PCR法扩增P3V0、P3V1、P3V5、P3V10、P3V15及P3V20代病毒的E基因片段,并进行测序分析。建立JEV毒种库,并进行全面检定。结果 JEV在Vero细胞上传代适应性良好,各代次病毒滴度均大于8.5 lg LD50/ml。最适培养条件为：病毒维持液p H值为7.6,培养温度为34℃,病毒MOI为0.1;最佳接种方式为：单层接种法。在不同培养规模时,病毒滴度变化较小,且均维持在较高水平。P3V0、P3V1、P3V5、P3V10、P3V15及P3V20各代次间E基因片段同源性为100%,传代稳定性良好。建立的毒种库的鉴别试验、无菌检查、支原体检查、病毒滴定、病毒外源因子检查及免疫原性检查结果均符合《中国药典》三部（2010版）相关要求。结论已完成JEV在Vero细胞上的适应性传代培养,并成功建立毒种库,为实现以Vero细胞为基质的JEV疫苗的研发奠定了基础。Objective To analyze the adaptation of Japanese encephalitis virus（JEV）in Vero cells,optimize the culture condition and inoculation mode,and establish the virus seed bank. Methods JEV P3 strain was subcultured to passage20（P3V1 - P3V20） in Vero cells,of which P3V2,P3V3 and P3V4 were determined for titers by mouse method. The condition（p H value of maintenance medium,temperature for culture and MOI）for stationary culture of JEV was optimized,based on which the culture was scaled up. The E gene fragments of P3V0,P3V1,P3V5,P3V10,P3V15 and P3V20 were amplified by RT-PCR and sequenced. JEV seed bank was established and subjected to overall control tests. Results JEV showed good adaptability in Vero cells,while the titers of virus of various passages were more than 8. 5 lg LD50/ ml.The optimal p H value of maintenance medium,temperature for culture,MOI and inoculation mode were 7. 6,34 ℃,0. 1and monolayer inoculation respectively. The virus titers cultured in various scales showed no significant change,which were maintained at high levels. The homologies of E gene fragments of P3V0,P3V1,P3V5,P3V10,P3V15 and P3V20 were 100%,indicating a high genetic stability. The results of identity test,sterility test,mycoplasma test,virus titration,adventitious agent test and immunogenicity test met the relevant requirements in Chinese Pharmacopoeia（Volume Ⅲ,2010edition）. Conclusion JEV was adapted in Vero cells,and virus seed bank was established,which laid a foundation of development of Vero cells-based JEV vaccine.

关 键 词：乙型脑炎病毒 VERO细胞 传代 适应性 毒种库 

分 类 号：R373.31[医药卫生—病原生物学] R392-33[医药卫生—基础医学]