细胞培养流感病毒H5N1的纯化方法研究

Purification method for cell-cultured influenza virus H5N1

目的降低H5N1甲型流感病毒中宿主细胞残留蛋白质和DNA含量。方法首先使用Core 700分子筛去除宿主残留蛋白质,再使用Capto Q阴离子交换层析柱去除宿主残留DNA,对多批次MDCK细胞培养的H5N1病毒液进行纯化。纯化后的病毒液经荧光定量PCR、血凝试验、单扩试验等结果综合评价纯化效果。除层析外,本实验还设置了广谱核酸酶对照组。结果在不使用广谱核酸酶的条件下,宿主DNA去除率为99.62%,宿主蛋白质残留去除率为98.1%,血凝素(HA)抗原回收率为66.96%。结论本研究提供了一套对于以细胞基质制备的流感疫苗有较好效果的纯化策略,宿主细胞的DNA和蛋白质有较高去除率,且HA回收率较高,纯化结果不劣于添加广谱核酸酶的方法,有可能应用于实际疫苗生产。

Objective To reduce the residual proteins and DNA of host cells in the preparation of H5N1 influenza A virus.Methods Core 700 was firstly used to remove residual host cell proteins,and then Capto Q was used to remove host cell DNA.Several batches of H5N1 influenza A virus cultured in Madin-Darby canine kidney(MDCK)cells were purified using this method.The efficiency of purification was evaluated using many methods including quantitative real-time PCR,hemagglutination(HA)test and single radial immunodiffusion assay.Moreover,Benzonase nuclease was used for comparison.Results Without the use of Benzonase nuclease,the overall removal rates of host cell DNA and residual proteins were 99.62%and 98.1%,and the HA antigen recovery rate was 66.96%.Conclusions This study established a purification strategy with good effect for cell-based influenza vaccines.It can efficiently remove host cell DNA and proteins and achieve a high HA recovery rate.The purification result is no worse than that of adding Benzonase nuclease,suggesting the potential of its application in actual vaccine production.