轮状病毒规模化纯化方法的建立

Development of a method for large-scale purification of rotavirus

目的建立一种可规模化纯化轮状病毒(Rotavirus,RV)的方法。方法以氯化铯密度梯度离心法(离心法)纯化RV作为对照,分析采用离心-微滤-超滤-层析法(模块法)纯化RV的可行性。透射电镜观察病毒形态;微量滴定法测定病毒的感染性滴度;Bradford法检测纯化前后病毒液的蛋白含量,计算蛋白回收率;以两种方法纯化的病毒免疫BALB/c小鼠,微量中和试验检测小鼠血清中和抗体滴度。结果模块法纯化的RV结构完整,感染性滴度可达(7.15±0.10)lgCCID50,可清除超过95%的蛋白质,蛋白回收率为(2.58±0.06)%,免疫小鼠可诱导中和抗体。模块法纯化的RV蛋白回收率、感染性滴度及诱导的小鼠中和抗体水平均显著高于离心法(P<0.05或P<0.01)。结论模块法可有效纯化RV,为RV灭活疫苗规模化纯化工艺的开发提供了实验依据。

Objective To develop a method for large-scale purification of rotavirus （RV）. Methods The feasibility of centrifugation-microfiltration-uhrafiltration-chromatography （module method） for purification of RV was evaluated using cesium chloride density gradient centrifugation as control. R~ was observed for morphology by transmission electron microscopy, then determined for infectious titer by microtitration method, and for protein contents before and after purification by Bradford method, based on which the recovery rate of protein was calculated. BALB / c mice were immunized with RV purified by the two methods, and determined for neutralizing antibody titer in sera by micro-neutralization assay. Results RV purified by module method showed intact structure, of which the infectious titer reached （7. 15 ＋- O. 10） lgCCIDs0. More than 95% of protein was removed, and the recovery rate of protein was （2. 58 ＋ 0. 06）%. The purified RV induced neutralizing antibody titer in mice. However, the recovery rate of protein, infectious titer and the induced neutralizing antibody titer of the RV purified by module method were significantly higher than those by cesium chloride density gradient centrifugation （P 〈 0. 05 or P 〈 O. 01 ）. Conclusion The developed module method may be used ibr effective purification of RV, which provides an experimental basis for development of procedure for large- scale purification of inactivated RV vaccine.