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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 improve the culture conditions of aG strain rabies virus by improving the buffer system of maintenance solution and prepare high titer virus stock solution. Methods On the basis of the traditional virus maintenance solution, buffer salt and sodium bicarbonate were added. Under the same conditions, the virus was harvested twice with the traditional maintenance solution and the improved maintenance solution. The virus solution was harvested twice and mixed. After purification by ultrafiltration and Sepharose 4FF, the virus stock solution was prepared and the antigen content was detected by ELISA. The virus titers, potency, host DNA and host cell protein residues were determined according to the method of “Chinese Pharmacopoeia” (2010 edition) pH meter for pH measurement. Results The antigen content, virus titer and potency of the virus solution prepared by the modified maintenance solution were higher than those of the control solution. Both the host protein and the host cell DNA residues of the virus solution prepared by the two maintenance solutions were in line with those of the Chinese Pharmacopoeia 2010 version) requirements; improve the maintenance of fluid buffer capacity was significantly enhanced, the pH control of the virus culture was significantly better than the control maintenance solution. Conclusion The titers of virus stock solutions prepared by the improved liquid culture of rabies virus are high, which can improve the low titers of virus stock produced by the traditional aG strain of rabies virus.