响应面法优化鼠细小病毒悬液制备及滴度测定

Establishment of determination of murine minute virus and preparation of the virus by response surface methodology

[目的]研究鼠细小病毒(MMV)感染性滴度测定方法,同时制备高滴度的MMV。[方法]通过Box-Behnken设计-响应面法优化A9细胞制备MMV及MMV感染性滴度测定工艺。[结果]A9细胞的最佳接种浓度、病毒培养时间及病毒吸附时间分别为9×10^(3)个/孔、12 d及0 h,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0.5时,感染后72 h收获,病毒滴度最高。[结论]Box-Behnken设计-响应面法适用于A9细胞制备MMV及MMV感染性滴度测定工艺。制备的病毒液的平均滴度为6.35TCID_(50)/0.1 mL。

[Objective]To develop a method for determination of infectious titer of murine minute virus(MMV)and prepare high titer MMV.[Method]The Box-Behnken based optimization was used to prepare MMV with A9 cell and a median infective dose of cell culture(TCID_(50))method for determination of MMV titer.[Result]The optimal A9 cell concentration for inoculation,virus culture time and virus adsorption time were 9×10^(3) cells/well,12 d and 0 h respectively.The developed TCID_(50) method showed good precision.The MMV titer of A9 cells in precipitate was significantly higher than that in supernatant,and increased gradually with the increasing MOI,which reached the peak value 72 h after infection with a MOI of 0.5.[Conclusion]The Box-Behnken could use in MMV with A9 cell and a median infective dose of cell culture(TCID_(50))method for determination of MMV titer.The mean tite of prepared MMV was 6.35 TCID_(50)/0.1 mL.