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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 establish a method for the determination of infectious titer of murine parvovirus (MMV) and to prepare a high-titer MMV, which lays a foundation for the verification of biological virus removal / inactivation process. Methods The half cell counting (CCID5) 0 of MMV virus titer assay was established by analyzing the parameters of inoculation amount, culture time and virus adsorption time of NB324K cells, and its precision was analyzed. MMV was prepared from A9 cells and analyzed for infection complex number MOI), harvest time and harvested samples on the virus titer, and test the stability of the virus. Results The optimal inoculation concentration, culture time and virus adsorption time of NB324K cells were 5 × 103 cells / well, 48h / 2h, respectively. The detection precision of the established NB324K cells was better. The virus titer in the A9 cells was higher than that in the supernatant, And gradually increased with the increase of virus MOI. At the MOI of 10, the virus was harvested at 48h after infection, and the highest virus titer was obtained. The average titer of the prepared virus solution was 8.69CCID50 / ml, stored at 4 ° C and 37 ° C for 7 days, and repeatedly frozen and thawed five times. The stability was good. Conclusion The titer of MMV infectivity has been established and high titer virus has been prepared, which lays the foundation for validation of MMV viral clearance / inactivation process.