【摘要】 目的在获得乙型脑炎病毒(JEV)全长cDNA分子的基础上,建立感染性转录体,获得恢复病毒,为JEV致病机理、疫苗研制及分子病毒学研究提供方法。方法将全长JEV cDNA分子克隆入改造后的pBluescript KS I(I+)载体上,经T7启动子体外转录,在Lipofectamine2000介导下,将转录产物转染入BHK-21细胞,经RT-PCR、测序、间接免疫荧光试验及噬斑试验鉴定恢复病毒。结果转录产物转染BHK-21细胞后,观察到明显的细胞病变;收获恢复病毒,经RT-PCR、测序、间接免疫荧光试验及噬斑试验证实为JEV。结论已建立了JEV全长cDNA克隆经体外转录获得JEV感染性RNA的方法,为JEV分子生物学及疫苗研究等奠定了基础。更多还原

【Abstract】 Objective To establish an infectious transcript on the basis of full-length cDNA clone of Japanese encephalitis virus(JEV),obtain the recovery virus and provide a method for study on pathogenic mechanism and molecular virology of JEV as well as development of JEV vaccine.Methods The full-length cDNA of JEV was cloned into modified vector pBluescript KS Ⅱ(+),followed by in vitro transcription using T7 promoter and Lipofectamine-mediated transfection to BHK-21 cells.The recovery virus was identified by RT-PCR,sequencing,indirect IFA and plaque formation test.Results Obvious CPE was observed in the BHK-21 cells transfected with transcript.The harvested recovery virus was identified as JEV.Conclusion A method for obtaining infectious JEV RNA by in vitro transcription of full-length cDNA clone was developed,which laid a foundation of study on molecular biology of JEV and vaccine development.更多还原