乙型脑炎减毒活疫苗(SA14-14-2株)的纯化

Purification of live attenuated Japanese encephalitis vaccine(SA14-14-2 strain)

目的建立纯化乙型脑炎减毒活疫苗(SA14-14-2株)的方法。方法应用超滤浓缩和分子筛凝胶柱层析纯化乙型脑炎病毒(Japanese encephalitis virus,JEV)SA14-14-2病毒液,检测其病毒滴度、原代地鼠肾细胞残留蛋白含量、牛血清白蛋白残留量、庆大霉素残留量,并进行小鼠脑内致病力试验、乳鼠传代返祖试验、异常毒性试验,制备纯化冻干疫苗,进行热稳定性试验。结果纯化后的JEV病毒滴度高于7.0 lg PFU/ml,平均回收率为23%;牛血清白蛋白残留量、庆大霉素残留量及安全性试验均符合《中国药典》三部(2010版)相关规定,去除了约95%的原代地鼠肾细胞残留蛋白;制备的冻干疫苗及热稳定性试验中的病毒滴度均高于5.7 lg PFU/ml,且热稳定性试验中病毒滴度下降未超过1.0 lg,符合《中国药典》三部(2010版)规定。结论建立了乙型脑炎减毒活疫苗(SA14-14-2株)纯化工艺,但病毒回收率较低,不适宜规模化生产,对乙型脑炎病毒减毒活疫苗质量控制具有指导意义。

Objective To develop a method for purification of live attenuated Japanese encephalitis vaccine（JEV）（SA14-14-2 strain）. Methods JEV SA14-14-2 liquid was purified by ultrafiltration concentration and molecular size chromatography, and determined for titer as well as residual host cell protein, bovine serum albumin and gentamicin contents, then subjected to intracerebral virulence test in mice, virulence reversion test in suckling mice and abnormal toxicity test. Purified freeze-dried vaccine was prepared and test for heat stability. Results The titer of purified JEV was more than 7. 0 lg PFU / ml, while the mean recovery rate was 23%, and the residual bovine serum albumin gentamicin contents met the requirements in Chinese Pharmacopoeia（Volume Ⅲ, 2010 edition）, in which about 95% of residual host cell protein was removed. The virus titer of prepared freeze-dried vaccine and the vaccine in heat stability test were more than 5. 7 lg PFU / ml, and the decrease of virus titer in heat stability test was not more than 1. 0 lg, which met the requirements in Chinese Pharmacopoeia（Volume Ⅲ, 2010 edition）. Conclusion The procedure for purification of live attenuated JEV（SA14-14-2 strain） was developed, which was unsuitable for large-scale production because of low virus recovery rate. The study is of guiding significance in the quality control of live attenuated JEV.