【摘要】 为了在病毒的整个基因组中研究基因的功能,分析基因与基因之间的相互作用,含有整个野生型EB病毒(EBV)基因组的BAC-EBV质粒(p2089),首先被转染EBV阴性的HEK293细胞,经潮霉素筛选建立了HEK293/p2089稳定细胞系.再构建pcDNA3.1(+)/BZLF1和pcDNA3.1(+)/BALF4真核表达质粒,共转染至HEK293/p2089细胞内,诱导EBV裂解性复制产生可视化的重组EBV颗粒.重组EBV颗粒感染Raji细胞,在倒置荧光显微镜下和流式细胞仪记数GFP阳性细胞,根据这些"绿色Raji单位"确定病毒的滴度.在国内首次建立这种以细菌人工染色体(BAC)为基础的EBV感染性克隆技术,将允许对EB病毒基因组中任何基因的任何遗传修饰,为在整个基因组中对EB病毒基因功能的研究奠定了基础,也为对EBV与其相关的肿瘤如鼻咽癌发生机理的研究建立了新的技术平台.更多还原

【Abstract】 In order to study single viral gene functions in the context of genome and analysis interactions between one gene with another, the HEK293/p2089 stable cell line was established by transfecting the plasmid of DNA (p2089) into EBV-negative 293 cells and selecting for resistance against hygromycin. The plasmid p2089, which was kindly provided by Prof.Hammerschmidt, contained the whole EBV genome of wild-type B95-8. Two eukaryotic expression vectors (pcDNA3.1(+)/BZLF1 and pcDNA3.1(+)/BALF4) were constructed and then transiently cotransfected into the HEK293/p2089 stable cells, so as to induct EBV lytic replication and product recombinant EBV particles visualized through GFP-expressing. To estimate the EBV production, Raji cells were incubated with supernatants from the induced 293 cells carrying p2089 DNA, as revealed by indirect visualization of the Raji cells. GFP-positive cells were evaluated by inverted fluorescence microscope or FACS analysis. The different virus supernatants were quantified with the help of "green Raji units" per ml as an absolute number of infectious particles. This technique makes it possible for the reconstitution of viral progeny or mutants by transfection of BAC plasmid into eukaryotic cells, and any genetic modification in E. coli, thereby facilitating the analysis of viral gene functions in the context of genome. This new technique has provided a useful tool for the study of pathogenesis mechanism of EBV, especially for that of cancer-associated.更多还原