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目的 研究乙脑病毒减毒株SA14-14-2在原代地鼠肾细胞(PHKC)传代后E蛋白基因的稳定性。方法 将乙脑病毒(JEV)减毒株SA14-14-2在原代地鼠肾细胞上传至20代,分析第1代(JEVPHKCKC P1)、第5代(JEVPH-KCP5)、第10代(JEVPHKC P10)、第15代(JEVPHKC P15)、第20代(JEVPHK P20)的E蛋白基因的核苷酸序列,并与Genbank的乙脑病毒减毒株(D90195)比较。结果 RT-PCR扩增出的乙脑病毒减毒株E蛋白基因片段大小为1 500bp,与Genbank库中乙脑病毒弱毒株SA14-14-2核苷酸、氨基酸序列比较显示,JEVPHKC P1,P5,P10病毒与GenbankD90195 E蛋白核苷酸和氨基酸完全相同,P15,P20与E425位点核苷酸和E142位点的氨基酸有不同,同源性分别为99.93％和99.8％。结论 乙脑病毒减毒株SA14-14-2的遗传学特性稳定,从分子水平证明了乙脑减毒活疫苗的安全性。
Objective To study the stability of E protein gene after passage of attenuated strain of JE virus in primary hamster kidney cell (PHKC). Methods JEV attenuated strain SA14-14-2 was transfected into primary rat kidney cells for up to 20 generations. The expression of JEVPHKCKC P1, JEVPH-KCP5, JEVPHKC P10), the 15th generation (JEVPHKC P15), the 20th generation (JEVPHK P20), and was compared with the GenBank attenuated strain of JE virus (D90195). Results The size of the E protein gene fragment of JEV attenuated strain amplified by RT-PCR was 1 500 bp. Compared with the nucleotide and amino acid sequence of the attenuated JEV strain SA14-14-2 in Genbank, JEVPHKC P1 and P5 The nucleotide and amino acid identities of the P10 virus and Genbank D90195 E protein were identical. The amino acids at nucleotide positions P15, P20 and E425 and E142 were different, with 99.93% and 99.8% homology respectively. Conclusion The genetic characteristics of attenuated strain J14-14-2 of JE virus are stable, and the safety of attenuated JE vaccine is demonstrated at the molecular level.