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目的 对乙脑减毒活疫苗SA14 14 2株生产毒种及其疫苗病毒滴度、脑内毒力和E区核苷酸序列进行回顾性测定。方法 应用乳金黄地鼠肾传代细胞 (BHK 2 1)结晶紫蚀斑法进行病毒滴度的测定 ,小鼠脑内法检测疫苗脑内毒力。应用RT PCR方法扩增SA14 14 2生产毒种和疫苗E区的核苷酸 ,经凝胶层析纯化的PCR产物直接用于测序或克隆到pGEM T载体中 ,经酶切和PCR鉴定后进行测序。结果 疫苗在 - 2 0℃保存长达 10多年 ,疫苗病毒滴度下降不超过 0 5lg;脑内毒力未见毒力回升。病毒蚀斑形态仍保持较SA14野毒株形态小的特性 ;以不同批次疫苗和生产毒种提取的病毒RNA作为模板进行RT PCR扩增 ,扩增的基因片段与预期大小一致 ;E区基因序列与野毒株差异的 8个氨基酸没有一个发生回复突变。结论 SA14 14 2生产毒种及其不同年度不同批次疫苗的生物表型和E区核苷酸的特性是十分稳定的
OBJECTIVE To retrospectively determine the virus titer, the virulence of the virus and the nucleotide sequence of E region of SA14 14 2 live attenuated JE vaccine. Methods The titer of virus was determined by the crystal violet violet staining method of kidney passage cells of golden hamster (BHK 2 1). The intracerebral toxicity of the vaccine was tested by intracerebral method. The nucleotides of SA14 14 2 producing strain and vaccine E region were amplified by RT PCR. The PCR products purified by gel chromatography were directly used for sequencing or cloned into pGEM T vector. After digestion and PCR, Sequencing. Results The vaccine was stored at -20 ° C for more than 10 years and the titer of the vaccine virus did not drop down to 0 5 lg. No toxicity was found in the brain. The morphology of virus plaque remained smaller than that of the SA14 wild-type strain. The viral RNA extracted from different batches of vaccines and virulent strains was used as a template for RT-PCR amplification, and the amplified gene fragments were consistent with the expected size. The E-region gene None of the eight amino acid sequences differing from wild-type strains undergoes a back-mutation. Conclusions The biological phenotypes of SA14 14 2 producing strains and the vaccine of different batches in different years and the nucleotide characteristics of E region are very stable