To detect retrospectively the phenotype and stability of the E-protein gene in Japanese Encephalitis （JE） virus strain SA14-14-2 used in the live-attenuated JE vaccine prepears, the viral titer was titrated by plaqu...To detect retrospectively the phenotype and stability of the E-protein gene in Japanese Encephalitis （JE） virus strain SA14-14-2 used in the live-attenuated JE vaccine prepears, the viral titer was titrated by plaque formation in BHK-21 cell cultures, and the neuro-virulence of viruses was assayed in mice with body weight of 12-14 g by intracerebral inoculation. Meanwhile, the total RNA of virus gene was extracted and amplified by RT-PCR with the designed primers, and then it was purified and cloned to the expression vector pGEM-T. The recombinant plasmid was purified and sequenced. It was found that the loss of viral titer of vaccines stored in -20℃ for longer than 10 years was less than 0.5 Lg PFU/ml. No mice inoculated intracerebrally showed signs of illness or even death. The size of plagues of the vaccine virus remained to be small, and the E genes of primary virus seed SA14-14-2 and the vaccines prepared at different years （1987-2001） were unchanged, in- cluding the 8 critical amino acid sites which were different from the parent wild virus strain SA14 and the related neuro-virulence. These results indicate that the genotypic and biological characteristics of the attenuated JE virus strain SA14-14-2 and its vaccines sion noted. prepared are quite stable without any reversion noted.展开更多
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...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。展开更多
文摘To detect retrospectively the phenotype and stability of the E-protein gene in Japanese Encephalitis （JE） virus strain SA14-14-2 used in the live-attenuated JE vaccine prepears, the viral titer was titrated by plaque formation in BHK-21 cell cultures, and the neuro-virulence of viruses was assayed in mice with body weight of 12-14 g by intracerebral inoculation. Meanwhile, the total RNA of virus gene was extracted and amplified by RT-PCR with the designed primers, and then it was purified and cloned to the expression vector pGEM-T. The recombinant plasmid was purified and sequenced. It was found that the loss of viral titer of vaccines stored in -20℃ for longer than 10 years was less than 0.5 Lg PFU/ml. No mice inoculated intracerebrally showed signs of illness or even death. The size of plagues of the vaccine virus remained to be small, and the E genes of primary virus seed SA14-14-2 and the vaccines prepared at different years （1987-2001） were unchanged, in- cluding the 8 critical amino acid sites which were different from the parent wild virus strain SA14 and the related neuro-virulence. These results indicate that the genotypic and biological characteristics of the attenuated JE virus strain SA14-14-2 and its vaccines sion noted. prepared are quite stable without any reversion noted.
文摘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。
