Vero细胞肠道病毒71型灭活疫苗的纯化及鉴定

Purification and Identification of Inactivated Enterovirus 71 Vaccine Derived from Vero Cells

关键词:  肠道病毒71型灭活疫苗  Vero细胞  纯化  鉴定

Key words:  inactivated enterovirus 71 vaccine  Vero cells  purification  identification

摘要:

目的 建立Vero细胞肠道病毒71型(EV71)灭活疫苗的纯化工艺。方法 病毒收获液经离心澄清、福尔马林灭活和超滤浓缩后，采用Sephacryl S-400 HR凝胶过滤层析和Source 30Q离子交换层析2步层析法纯化EV71，进行各项检定及分析后除菌过滤。结果 经纯化的疫苗各项纯化指标均符合中国药典2010年版的要求，杂蛋白去除率>99.9%；病毒纯化液在电镜下观察到典型的EV71病毒颗粒，经SDS-PAGE及Western blot分析，检测到与文献报道大小相符的EV71 VP1、VP2、VP3及特异性免疫反应；制备成疫苗后免疫大鼠能产生特异性中和抗体。结论 本实验建立了Vero细胞的EV71灭活疫苗的纯化工艺，纯化后制备的EV71灭活疫苗具有较好的免疫原性。

Abstract:

OBJECTIVE To develop a purification procedure of inactivated enterovirus 71 (EV71) vaccine derived from Vero cells. METHODS The harvested liquid was pretreated by means of centrifugation, ultrafiltration, inactivated with formmdehyde, and then the inactivated EV71 vaccine was purified by Sephacryl S-400 HR gel filtration chromatography and Source 30Q ion exchange chromatography. The vaccine was subjected to control tests and analysis then sterilized by filtration. RESULTS A1l test results of three lots vaccine after purification accorded with the requirements of Ch.P(2010). All the removal rates of protein in vaccine were >99.9%. Typical morphology of EV71 was observed in purified EV71 samples under transmission electron microscope．SDS-PAGE analysis showed three protein straps with relative molecular weight which was according with the VP1, VP2 and VP3 of EV71 and could be confirmed by Western blot analysis. The purified vaccine induced neutralizing antibody titer in rats. CONCLUSION A purification procedure of EV71 vaccine derived from Vero cells is developed, and the vaccine demonstrates high immunogenicity.