CAJ | 학술논문

目的利用水痘-带状疱疹病毒(VZV)ORF9启动子的最短有效序列ORF9G和ORF61启动子的最短有效序列ORF61F构建VZV报告细胞系并分析其特性.方法分别将ORF9G和ORF61F自身首尾连接得到串联启动子T9G和T61F,再分别将T9G和T61F克隆到报告子质粒pGL3-basic中构建串联启动子-报告子重组质粒pGL-T9G和pGL-T61F,质粒中报告基因萤火虫荧光素酶的表达由上游串联启动子控制;将pGL-T9G、pGL-T61F分别同G418抗性质粒pCMV-script恒定转染到人黑色素瘤细胞系Mewo后培养至G418抗性细胞克隆生长;收集G418抗性细胞克隆,测定其感染VZV后萤火虫荧光素酶的表达强度,筛选信噪比高的细胞克隆作为VZV报告细胞系,并分析其灵敏度、特异性等主要特性.结果串联启动子T9G和T61F分别较其单拷贝启动子9G和61F活性增加1倍;分别利用pGL-T9G和pGL-T61F成功构建了VZV报告细胞MV9G和MV61F;两个报告细胞在感染最低50 PFU VZV 24 h时即能检测到萤火虫荧光素酶的强表达,表达强度与感染病毒量呈线性正相关,但感染人巨细胞病毒(HCMV)、人类疱疹病毒6(HHV-6)和HHV-7时无表达;MV9G的灵敏度较MV61F略高.结论 MV9G和MV61F均可作为敏感、特异的VZV报告细胞系供进一步研究用.
목적 이용수두-대상포진병독(VZV)ORF9계동자적최단유효서렬ORF9G화ORF61계동자적최단유효서렬ORF61F구건VZV보고세포계병분석기특성.방법 분별장ORF9G화ORF61F자신수미련접득도천련계동자T9G화T61F,재분별장T9G화T61F극륭도보고자질립pGL3-basic중구건천련계동자-보고자중조질립pGL-T9G화pGL-T61F,질립중보고기인형화충형광소매적표체유상유천련계동자공제;장pGL-T9G、pGL-T61F분별동G418항성질립pCMV-script항정전염도인흑색소류세포계Mewo후배양지G418항성세포극륭생장;수집G418항성세포극륭,측정기감염VZV후형화충형광소매적표체강도,사선신조비고적세포극륭작위VZV보고세포계,병분석기령민도、특이성등주요특성.결과 천련계동자T9G화T61F분별교기단고패계동자9G화61F활성증가1배;분별이용pGL-T9G화pGL-T61F성공구건료VZV보고세포MV9G화MV61F;량개보고세포재감염최저50 PFU VZV 24 h시즉능검측도형화충형광소매적강표체,표체강도여감염병독량정선성정상관,단감염인거세포병독(HCMV)、인류포진병독6(HHV-6)화HHV-7시무표체;MV9G적령민도교MV61F략고.결론 MV9G화MV61F균가작위민감、특이적VZV보고세포계공진일보연구용.
Objective To establish the reporter cell lines for varicella-zoster virus(VZV)with ORF9G,the shortest and efficient sequence of the promoter for VZV ORF9,and ORF61F,the shortest and efficient sequence of the promoter for ORF61,and to characterize the cell lines.Methods The tandem promoters.T9G and T6lF,which were resulted respectively from the linkage of ORF9 in duplicate and of ORF6lF in duplicate.were cloned respectively into an individual reporter plasmid pGL3-basic.In this way,two recombinant promoter-reporter plasmids.pGL-T9G and pGL-T61F were constructed,in which the expression of reporter gene firefly luciferase was under the control of the upstream T9G or T61F.Along with the G418-resistant plasmid pCMV-script.the pGL-T9G and pGL-T6lF were respectively transformed into an in dividual Me Wo cell line.The grown G418-resistant cell clones were collected,and their firefly luciferase expressions post VZV infection was assayed.The best cell clones that have high firefly luciferase activity were chosen as reporter cell lines for VZV,of which the sensitivity and specificity were characterized. Results The activity of T9G or T61 F was two-fold as that of 9G or 61F.Two reporter cell lines,MV9G containing ORF9 ptomoter and MV6lF containing ORF61 promoter,were established successfully.Both cell lines showed fast.sensitive and specific response to VZV infection in a dose-dependent manner although the sensitivitv of MV9G Was somewhat higher than that of MV61F.Conclusion Each of both reporter cell lines for VZV may serve as a sensitive and specific research tool for further study especially on virus entry and antivi ral mechanisms.