水痘-带状疱疹病毒基因启动子分析

Analysis on promoters for varicela-zoster virus genes

目的 从水痘-带状疱疹病毒（VZV）主要基因的启动子中找到最容易被VZV感染激活的启动子及其最短有效序列以构建VZV报告细胞系。方法 构建18个VZV开放读码框架（0RF）启动子的重组报告质粒，用基因转染瞬时表达分析法比较各启动子在VZV感染早期启动报告基因产物表达的活性；将最容易被激活的启动子从5′和3′端截短后构建截短启动子的重组报告质粒，比较各截短启动子的活性以找到启动子的最短有效序列；应用点定向突变技术将最短有效序列中细胞转录因子的结合序列进行碱基置换突变后比较突变启动子的启动活性。结果ⅥⅣ感染早期ORF9、ORF61和ORF66的启动子最容易被激活；ORF9启动子的最短有效序列位于-127～-34（93bp），其上游刺激因子（USF）结合序列的碱基置换突变后启动子活性降低50％；ORF61启动子的最短有效序列位于-227～-52（165bp），其Pbx-1结合序列的碱基置换突变后启动子活性增强一倍，但NF-κB结合序列的突变对启动子活性无影响。结论 ORF9启动子的最短有效序列最适合用来构建VZV报告细胞系。

Objective To identify a most suitabl＇e varicela-zester virus（VZV） gene promoter for the establishment of reporter cell line for VZV. Methods Eighteen recombinant promoter-reporter plasmids were constructed, in which firefly luciferase was promoted to express upon VZV infection. The promoter activities upon VZV infection and ORF62 plasmid eotransfection were compared in transient transfection assays. Truncated deletion mutants and site-directed base pair substitution mutants of promoters for ORF9 and ORF61 were further analyzed in transient transfection assays. Results Among 18 promoters, promoters for ORFg, 61 and 66 were most strongly activated by cell-free VZV infection as well as by VZV ORF62 encoding plasmid cotransfection. The minimal but essential region for ORF9 promoter activation was mapped in a -93 bp long sequence. Two base pair substitutions on upstream simulating factor（USF） binding motif in this region resulted in a fifty percent loss of the promoter activities. The minimal but essential region for ORF61 promoter activation was mapped in a -175 bp long sequence. Two base pair substitutions on Pbx- 1 binding motif in this region resulted in a two fold increase of the promoter activities, whereas those on NF -κB binding motif did not show significant change in promoter activities . Conchusion The minimal but essential region for ORF9 promoter activation is mostly suitable for the establishment of reporter cell line for VZV.