肠道病毒71型灭活疫苗灭活验证超速离心条件的优化

Optimization of condition for ultracentrifugation for verification of inactivation effect of inactivated enterovirus 71 vaccine

目的优化肠道病毒71型(enterovirus 71,EV71)灭活疫苗灭活验证超速离心条件,为EV71灭活疫苗灭活验证样品的处理提供实验依据。方法在不同转速(45 000、40 000、35 000、30 000、25 000 r/min)和时间(120、90、60、30 min)条件下对EV71收获液进行超速离心,检测离心前和离心后病毒液的滴度,计算回收率,比较不同条件下离心前后病毒回收效果。结果在转速相同或离心时间相同的条件下,EV71的沉淀效果与离心时间或离心转速呈正相关(R^2从0.77和0.88分别增大到0.99),其中,当离心转速超过40 000 r/min时,不同离心时间下病毒的回收率均在90%以上,当离心时间为120 min,离心转速为45 000 r/min时,病毒的沉淀效果最好,回收率最高,达98%。综合离心时间因素,选择离心条件为45 000 r/min-60 min较适宜(回收率为97%),与45 000 r/min-120 min相比,差异无统计学意义(P>0.05);如将时间减为45 000 r/min-30 min,与其他3组比较,差异有统计学意义(P<0.05)。结论 EV71的沉淀效果与离心转速和离心时间呈正相关,即随着离心转速增大和离心时间的延长,病毒的沉淀效果越好、回收率越高。

Objective To optimize the condition for uhracentrifugation for verification of inactivation effect of inactivated enterovirus 71 （EV71） vaccine, and provide a basis for treatment of samples of inactivation verification test of the vac- cine. Methods The harvested liquid EV71 was ultraeentrifuged at various rotation rates （45 000, 40 000, 35 000, 30 000 and 25 000 r/rain ） for various （120, 90, 60 and 30） minutes, of which the titers before and after ultracentrifugation were determined, and recovery rates were calculated to compare the reeovery effects under various conditions. Results The precipitation effects of EV71 at the same rotation rate were positively related to the time for ultracentrifugation （R2 increased from 0. 77 to 0. 99 ）, while those at the same time point after ultracentrifugation were positively related to the rotation rate （R2 increased from 0. 88 to 0. 99）. When the rotation rate was more than 40 000 r/min, the recovery rates of EVT1 after ultraeentrifugation for various minutes were more than 90%. When the time for ultracentrifugation was 120 rain, and the rotation rate was 45 000, the precipitation effect of EV71 was satisfactory, while the recovery rate reached a maximum of 98%. The recovery rate of EV71 after ultracentrifugation at 45 000 r/min for 60 rain was 97%, which showed no significant difference with that after ultracentrifugation for 120 min. However, the recovery rate after ul- tracentrifugation for 30 rain showed significant difference with those after uhracentrifugation for 120, 90 and 60 min （P 〈 0. 05）. Conclusion The precipitation effect of EV71 was positively related to rotation rate and time for ultracentrifuga- tion. The effect was improved, while the recovery rate increased, with the increasing rotation rate and time.