乙型脑炎减毒株SA14-14-2全长cDNA克隆感染性的研究  

Infectivity of Full-length cDNA Clone of Attenuated Japanese Encephalitis Virus Strain SA14-14-2

作　　者：李静[1] 俞永新[2] 董关木[2] 安祺[2] 杨立宏[2] 孔艳[2] 景川 

机构地区：[1]武汉生物制品研究所,武汉430060 [2]中国药品生物制品检定所疫苗一室,北京100050 [3]华兰生物疫苗有限公司,河南新乡453003

出　　处：《中国生物制品学杂志》2010年第1期13-16,21,共5页Chinese Journal of Biologicals

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。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.

关 键 词：日本乙型脑炎病毒 全长CDNA 感染性RNA 体外转录 转染 

分 类 号：R373.31[医药卫生—病原生物学]