Construction of the recombinant varicella zoster virus carrying 3xflag gene / 西安交通大学学报(医学版)

ABSTRACT

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.