鼠细小病毒感染性滴度测定方法的建立及病毒制备  

Development of A Method for Determination of Infectious Titer of Murine Minute Virus and Preparation of The Virus

作　　者：孟淑芳[1] 林林[1] 冯建平[1] 李修兰[1] 王佑春[1] 李德富[1] 

机构地区：[1]中国药品生物制品检定所卫生部国家生物技术产品质量标准化重点实验室,北京100050

出　　处：《中国生物制品学杂志》2009年第8期815-818,共4页Chinese Journal of Biologicals

基　　金："国家科技基础条件平台建设-实验细胞资源的整理整合与共享"项目资助(2005DKA21502)

摘　　要：目的建立鼠细小病毒(MMV)感染性滴度测定方法,并制备高滴度的MMV,为生物制品病毒清除/灭活工艺的验证奠定基础。方法通过分析NB324K细胞接种量、培养时间及病毒吸附时间等参数,建立MMV病毒滴度测定的半数细胞感染剂量法(CCID5)0,并分析其精密性;以A9细胞制备MMV,分析感染复数(MOI)、收获时间及收获样本对病毒滴度的影响,并检测病毒的稳定性。结果NB324K细胞的最佳接种浓度、培养时间及病毒吸附时间分别为5×103个/孔、48h及2h,所建立的检测方法精密性较好;A9细胞沉淀中病毒滴度高于上清,且随病毒MOI值的升高而逐渐增加,在MOI为10时,感染后48h收获,病毒滴度最高。制备的病毒液的平均滴度为8.69CCID50/ml,4℃和37℃保存7d及反复冻融5次,稳定性良好。结论已建立了MMV感染性滴度测定方法,并制备了高滴度的病毒,为以MMV为指示病毒进行病毒清除/灭活工艺验证奠定了基础。Objective To develop a method for determination of infectious titer of murine minute virus （MMV） and prepare high titer MMV to lay a foundation of verification of procedure for virus removal / inactivation in biologics. Methods A median infective dose of cell culture （CCID50） method for determination of MMV titer was developed by optimizing the concentration of NB324K cells for inoculation, virus culture time and virus adsorption time. MMV was prepared with A9 cells, and the effects of MOI, time for virus harvest and form of harvested virus on MMV titer as well as stability of virus were analyzed. Results The optimal NB324K cell concentration for inoculation, virus culture time and virus adsorption time were 5 × 10^3 cells/well, 48 h and 2 h respectively. The developed CCID50 method showed good precision. The MMV titer in precipitate of A9 ceils was significantly higher than that in supernatant, and increased gradually with the increasing MOI, which reached a peak value 48 h after infection with a MOI of 10. The prepared MMV was at a mean titer of 8. 69 CCID50/ml, and showed good stability after storage at 4 and 37℃ for 7 d or 5 cycles of freeze-thawing. Conclusion A method for determination of infectious titer of MMV was developed, and high titer MMV was prepared, which laid a foundation of verification of procedure for virus removal / inactivation using MMV as an indicator.

关 键 词：鼠细小病毒 感染性滴度 NB324K细胞 A9细胞 半数细胞感染剂量 感染复数 

分 类 号：R392.1[医药卫生—免疫学]