乙脑病毒减毒株SA14-14-2在原代地鼠肾细胞多次传代后病毒基因稳定性研究

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。

Study of the Genetic Stability of Japanese Encephalitis Attenuated Virus Strain SA14-14-2 After Passaging on Primary Hamster Kidney Cells

Objective To study the genetic stability of Japanese encephalitis(JE) attenuated virus strain SA14-14-2 after passaging on primary hamster kidney cells(PKHC) . Methods The master virus seed of JE live attenuated vaccine was passaged on PHKC for 20 generations, the nucleotide sequences of E protein of various generation viruses were analyzed and compared with those in Genbank( D90195). Results The fragment size of E protein for JEV PI, P5.P10, P15.P20 was a 1 500 bp length each by RT-PCR,and compared the sequences of nucleotide and amino acid for them with weak strain SAM-14-2 in Genbank.It showed the result of sequences of JEVP1, P5, P10 is identical to D90195 in Genbank, At the site of E425 nucleotide and E142 amino acid ,JEVP15,P20 were different from weak strain D90195,the homology were 99.93% ,99. 8% respectively. Conclusion The JEV attenuated strain SA14-14-2 is genetic stability after passaged continuously on PHKC to 20 generations, and proved a good safety on molecular level.