携带3xflag基因的重组水痘带状疱疹病毒的构建

摘要

目的 利用 GalK为基础的同源重组方法,将 3 xflag添加到水痘带状疱疹病毒(varicella-zoster virus ,VZV)开放读码框 7(open reading frame7,ORF7),构建携带3xflag与VZVORF7 融合基因的重组水痘带状疱疹病毒.方法 PCR扩增带有 50 bp VZVORF7 左右同源臂的 GalK及 3xflag基因片段,将所得片段纯化后电转到 VZV细菌人工染色体(SW1 02-VZVWTBAC)感受态细胞,通过同源重组、GalK 筛选及脱 GalK 筛选,获得带有 3 xflag 的 VZVORF7 细菌人工染色体(SW102-VZV ORF7-3xflag-BAC)克隆;将所得克隆进行质粒提取后,转染到 ARPE-19 细胞,观察带有3xflag基因的VZV对ARPE-19 细胞的影响.结果 获得了VZV ORF7 带有3xflag的细菌人工染色体克隆(SW102-VZVORF7-3xflag-BAC).将该质粒转染 AARPE-19 细胞,3 d 后,转染 SW102-VZVWTBAC 及 SW102-VZV ORF7-3 xflag-BAC的细胞可见带有绿色荧光的病毒斑块,Western blot检测结果显示 3 xflag时 ORF7 的表达水平有效增强.结论 获得了携带 3 xflag基因的重组 VZV,表明通过 GalK为基础的同源重组,可方便、快捷并准确地对感兴趣的病毒基因进行操作.%Objective To construct the recombinant varicella zoster virus (VZV)carrying 3xflag gene,3xflag was added to the VZV open reading frame 7 (ORF7)using GalK-based homologous recombination.Methods GalK and 3xflag gene fragments with 50 bp VZV ORF7 homologous arms were amplified by PCR.The obtained fragments were purified and transferred to the competent cells of VZV bacterial artificial chromosome (SW102-VZVWTBAC). The clones of VZV ORF7 with 3xflag (SW102-VZV ORF7-3xflag-BAC)were obtained by homologous recombination and selection from medium containing GalK and replaced GalK. The recombinant plasmids were extracted and transfected into ARPE-19 cells.The effect of VZV ORF7-3xflag on ARPE-19 cells was observed.Results The clones of VZV ORF7 with 3xflag (SW102-VZV ORF7-3xflag-BAC)were obtained.The virus patches with green fluorescence were observed three days after SW102-VZVWTBAC and SW102-VZV ORF7-3xflag-BAC were transfected into ARPE-19 cells.Western blot showed that ORF7 expression was effectively enhanced with 3xflag.Conclusion The recombinant VZV carrying 3xflag gene was obtained,which suggests that GalK-based homologous recombination is convenient,efficient and accurate in manipulating the gene virus of interest.