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目的 探索甲型肝炎减毒活疫苗 (简称甲肝活疫苗 )生产中感染细胞释放病毒的最佳方法。方法 采用超声波细胞粉碎仪、- 70℃及液氮冻融 3种方法粉碎细胞 ,对细胞破碎情况、病毒抗原滴度和感染性滴度进行了比较。结果 超声波连续粉碎 3次 (90秒 /次 )、- 70℃反复冻融粉碎 4次和液氮冻融粉碎 6次 ,细胞破碎率分别为 94%、82 %和 81% ;病毒抗原滴度和感染性滴度均达高峰 ;抗原滴度均为 1∶2 5 6 ,感染性滴度 (logCCID50 /ml)分别为 8 6 7、6 5和 6 0。继续超声和 -70℃冻融粉碎细胞 ,病毒抗原滴度和感染性滴度基本不变 ,而液氮冻融至第 10次时 ,抗原滴度无明显变化 ,感染性滴度下降2 0logCCID50 /ml。结论 超声波破碎率最高 ,细胞碎片最小 ,病毒充分释放 ,且不损伤病毒 ;- 70℃和液氮冻融破碎率较低 ,细胞碎片较大 ,病毒释放效率低 ,病毒损伤以液氮冻融为重。
Objective To explore the best way to release virus from infected cells during the production of live attenuated hepatitis A vaccine (referred to as live hepatitis A vaccine). Methods The cells were pulverized by ultrasonic cell disintegrator, - 70 ℃ and freeze-thaw in liquid nitrogen. The cell disruption, viral antigen titers and infectious titer were compared. Results The results showed that the cell disruption rates were 94%, 82% and 81%, respectively. The titers of virus antigen and The titers of infection reached the peak. The titers of antigens were 1: 256 and the infectivity titers (logCCID50 / ml) were 86.7, 65 and 60, respectively. The cells were further sonicated and frozen-thawed at -70 ° C, and the viral titers and infectivity titers were almost unchanged. However, when the liquid nitrogen was frozen and thawed for 10 times, the titers of the antigens did not change significantly and the infectious titers decreased by 20logCCID50 / ml. Conclusion The highest rate of ultrasonic disruption, minimal cell debris and full release of virus were achieved without any damage to the virus. The freezing and thawing rates at 70 ℃ and liquid nitrogen were lower, the cellular debris was larger, the virus release rate was lower, weight.