应用硫酸鱼精蛋白处理蔗糖密度梯度超速离心高度纯化流行性乙型脑炎灭活疫苗(非洲绿猴肾细胞)

Purification of Inactivated Japanese Encephalitis Vaccine from Vero Cell by Protamine Sulfate and Sucrose Density Gradient Ultracentrifugation

目的通过离心试验,制备出一种高纯度的流行性乙型脑炎(乙脑)灭活纯化疫苗(非洲绿猴肾细胞)[Purified Inactivated Japanese Encephalitis Vaccine(Vero Cell),JEV]。方法生物反应器培养的Vero细胞乙脑病毒液,超滤浓缩、甲醛灭活后,应用硫酸鱼精蛋白处理,再经过高速离心,蔗糖密度梯度超速离心纯化,超滤除糖,配制成JEV(Vero细胞)。结果经检测,疫苗中Vero细胞脱氧核糖核酸残留量≤10pg(皮克,Picogram)/剂,蛋白含量≤1μg(微克)/剂,宿主蛋白残留量≤30ng(纳克,Nanogram)/ml,并获得较高的抗原回收率。结论经过优化硫酸鱼精蛋白处理和蔗糖密度梯度超速离心纯化,制得了高纯度的JEV(Vero细胞)。

Objective To prepare a purified inactivated Japanese encephalitis vaccine（JEV）by ultracentrifugation.Methods Japanese encephalitis supernatant was collected from bioreactor.After concentrated by ultra-filtration and inactivated by formaldehyde,the inactivated virus suspension was treated with protamine sulfate and purified by sucrose density gradient ultracentrifugation.The sucrose was removed by ultra-filtration.Results The residual of vero cells deaxyribonucleic acid（DNA）was reduced to 10pg/dose and total protein content was reduced to 1μg/dose,the host cell protein was decreased to 15ng/dose,and satisfied recovery was obtained.Conclusion A purified inactivated JEV was prepared by optimizing condition of protamine sulfate treatment and sucrose density gradient ultracentrifugation purification.