水痘-带状疱疹病毒报告细胞系的构建及特性分析  

Establishment and characterization of the reporter cell lines for varicella-zoster virus

作　　者：王官清 井上直树 野沢直树 

机构地区：[1]厦门大学附属中山医院皮肤科,厦门大学医学院临床医学系,361004 [2]日本国立感染症研究所病毒一部

出　　处：《中华微生物学和免疫学杂志》2008年第3期212-216,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目（30671884）;福建省高等学校新世纪优秀人才支持计划资助项目;厦门市卫生局医学科研基金资助项目（WSK0618）

摘　　要：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TgG和T61F，再分别将TgG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-TgG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-TgG、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TgG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-TgG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50PFU VZV 24h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。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, TgG and T61F, which were resulted respectively from the linkage of ORF9G in duplicate and of ORF61F in duplicate, were cloned respectively into an individual reporter plasmid pGL3-basic. In this way, two recombinant promoter-reporter plasmids, pGL-TgG and pGL-T61F were constructed, in which the expression of reporter gene firefly luciferase was under the control of the upstream TgG or T61F. Along with the G418-resistant plasmid pCMV-script, the pGL-TgG and pGL-T61F were respectively transformed into an individual MeWo 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 TgG or T61F was two-fold as that of 9G or 61F. Two reporter cell lines, MV9G containing ORF9 promoter and MV61F 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 sensitivity 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 antiviral mechanisms.

关 键 词：水痘-带状疱疹病毒 报告细胞系 启动子 

分 类 号：R686[医药卫生—骨科学]