改良维持液培养aG株狂犬病病毒制备病毒原液

Preparation of rabies virus bulk by culture of aG strain in modified maintenance medium

目的通过改良维持液的缓冲体系,改进aG株狂犬病病毒的培养条件,制备高滴度的病毒原液。方法在传统病毒维持液的基础上,补加缓冲盐和碳酸氢钠,在相同的条件下,分别用传统维持液和改良维持液培养病毒,收获2次病毒液,混合,经300 KD膜包超滤、Sepharose 4FF纯化后,制成病毒原液,采用ELISA法检测抗原含量,按《中国药典》三部(2010版)方法检测病毒滴度、效力、宿主DNA和宿主细胞蛋白残留量,用便携式pH计检测pH值。结果改良维持液制备的病毒原液的抗原含量、病毒滴度及效力均高于对照维持液;两种维持液制备的病毒原液的宿主蛋白和宿主细胞DNA残留量均符合《中国药典》三部(2010版)要求;改良维持液的缓冲能力明显增强,对病毒培养过程中pH值的控制明显优于对照维持液。结论改良维持液培养狂犬病病毒制备的病毒原液滴度较高,可改进传统aG株狂犬病病毒生产的病毒原液滴度较低的现状。

Objective To prepare high titer rabies virus（RV） bulk by optimizing the culture condition of aG strain through improving the buffer system of maintenance medium.Methods Conventional virus maintenance medium was supplemented with buffer salt and sodium bicarbonate.RV aG strain was cultured under the same conditions by using conventional and modified maintenance media respectively and harvested for 2 times.The harvested virus liquid was pooled and purified by ultrafiltration with 300 KD membrane and Sepharose 4FF chromatography,and the prepared virus bulk was determined for antigen content by ELISA,for titer,potency,host DNA and residual host cell protein according to the requirements in Chinese Pharmacopoeia（Volume Ⅲ,2010 edition）,and for pH value by portable pH meter.Results The antigen content,virus titer and potency of bulk cultured in modified maintenance medium were higher than those in conventional medium.The host protein and host cell DNA contents in bulks cultured by both the maintenance media met the requirements in Chinese Pharmacopoeia（Volume Ⅲ,2010 edition）.However,the modified maintenance medium was superior to conventional maintenance medium in buffer ability and control of pH value during virus culture.Conclusion As compared with that in conventional maintenance medium,the titer of virus bulk prepared by culture of RV in modified maintenance medium was high.