携带3xflag基因的重组水痘带状疱疹病毒的构建

Construction of the recombinant varicella zoster virus carrying 3xflag gene

目的利用GalK为基础的同源重组方法,将3xflag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50bp 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(SW102-VZVWTBAC)感受态细胞,通过同源重组、GalK筛选及脱GalK筛选,获得带有3xflag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,3d后,转染SW102-VZVWTBAC及SW102-VZV ORF7-3xflag-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3xflag时ORF7的表达水平有效增强。结论获得了携带3xflag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 3 xflag gene,3 xflag was added to the VZV open reading frame 7（ORF7）using GalK-based homologous recombination.MethodsGalK and 3 xflag 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-VZVWT BAC）.The clones of VZV ORF7 with 3 xflag（SW102-VZV ORF7-3 xflag-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 ORF7-3 xflag on ARPE-19 cells was observed.Results The clones of VZV ORF7 with 3 xflag（SW102-VZV ORF7-3 xflag-BAC）were obtained.The virus patches with green fluorescence were observed three days after SW102-VZVWTBAC and SW102-VZV ORF7-3 xflag-BAC were transfected into ARPE-19 cells. Western blot showed that ORF7 expression was effectively enhanced with3 xflag.Conclusion The recombinant VZV carrying 3 xflag gene was obtained,which suggests that GalK-based homologous recombination is convenient,efficient and accurate in manipulating the gene virus of interest.