流行性乙型脑炎减毒活疫苗生产毒种(SA14-14-2株)及其疫苗的表型和E基因稳定性研究

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

Stability of Phenotype and E Protein Gene of Primary Virus Seed and Final Product of Live Attenuated Japanese Encephalitis vaccine

Objective Retrospective determination of virus titer,neurovirulence and nucleotide sequence of E protein of SA14 14 2 strain and live attenuated Japanese encephalitis vaccine prepared with the strain.Methods Determine the virus titer by plaque formation test in BHK21 cells and neurovirulence by i.c. injection in mice.Amplify the nucleotide sequences of E proteins of SA14 14 2 strain and Japanese encephalitis vaccine by RT PCR.The PCR products were directly sequcenced after purification by gel chromatography,or cloned into pGEM T vector and identified by digestion with restriction enzyme and PCR then sequenced.Results The virus titer of vaccine decreased by not more than 0.5 lg after storage at -20℃ for more than 10 years,and no reversion of neurovirulence was observed.The sizes of plaques formed by SA14 14 2 strain were still smaller than those by wild SA14 strain.Gene fragments were amplified by RT PCR using the RNA extracted from JE vaccines and SA14 14 2 strains of different batches as templets,and the lengths of the amplified fragments were consistent with those respected.Gene sequencing of E protein showed no reverse mutation in the 8 amino acids different from those of wild strain.Conclusion The phenotype and E protein gene of SA14 14 2 strain and the vaccine prepared with the strain were quite stable.