摘要
为建立鸭乙型肝炎病毒ＬＪ－７６的转染细胞系，将ＬＪ－７６病毒ＤＮＡ插入到ｐＵＣ１９的ＥｃｏＲⅠ位点上，分离得到含有双拷贝ＬＪ－７６ＤＮＡ的重组质粒．通过磷酸钙沉淀方法，将经ＣｓＣｌ等密度离心纯化的ＬＪ－７６ＤＮＡ双体导入到人肝癌细胞ＢＥＬ７４０２中．收集转染细胞的培养液进行蔗糖密度梯度离心，所得沉淀经检测发现含有ＬＪ－７６ＤＮＡ并具有特异性ＤＨＢＶ内源性ＤＮＡ多聚酶活性；对上述样品通过ＤｏｔＥＩＡ检测ＤＨＢＶ核心抗原及表面抗原结果为阳性．Ｓｏｕｔｈｅｒｎｂｌｏｔ分析表明转染细胞内存在病毒ＤＮＡ复制中间体ｃｃｃＤＮＡ、ｓｓＤＮＡ和ｒｃＤＮＡ，而ｃｃｃＤＮＡ被认为是复制活动较为活跃的标志．电镜观察转染细胞的上清发现有病毒颗粒的存在．
In order to establish a transfected cell system for production of DHBV in vitro ,DHBV DNA was prepared from pLJ 76 and was inserted into the EcoRⅠ site of pUC18.The recombinant plasmid which consisted of two copies of the head to tail DHBV DNA was transferred into BEL7402 cells by DNA calcium phosphate coprecipitation method.Transfected cell medium was sedimented.DHBV DNA and DHBV DNA polymerase activity were detected in the sediment.Dot EIA analysis of DHBcAg and DHBsAg of DHBV particles from cells transfected for 4,5 days showed that the results of the measurement are positive.Southern blot analysis showed that replicative intermediates ccc DNA,ss DNA and rc DNA existed in the transfected BEL7402 cells.DHBV like particles were detected in the supernatant of transfected cells by electron microscope.
关键词
鸭乙型肝炎病毒
转染
细胞系
乙型肝炎病毒
Duck hepatitis B virus LJ 76(DHBV LJ 76),BEL7402 cells,Transfection
