Construction of recombinant replication-defective adenovirus expressing heme oxygenase-1 gene in mouse

Objective:To construct a recombinant adenovirus(Ad) vector expressing heme oxygenase-1(HO-1) gene by the homologous recombination in Escherichia coli strain BJ5183.Methods:HO-1 gene was amplified from embryonic fibroblast cell of mouse by RT-PCR.Digested with BglⅡand XhoⅠ,the HO-1 gene was cloned into shuttle vector pAdTrack-CMV that carried a green fluorescence protein(GFP) gene.PmeⅠlinearized shuttle plasmid was transformed into E.coli BJ5183 containing adenovirus backbone vector pAdEasy-1 to achieve the homologous recombination.The DNA of identified recombinant plasmid was digested with Pac I and transfected to HEK293 cells to package recombinant adenovirus particles Ad-HO-1.The target gene was detected by RT-PCR.Infection efficiency and titre of the Ad-HO-1 in 293 cells was measured through the expression of GFP.Results:It was confirmed by RT-PCR,sequencing and restriction enzyme analysis that the target gene was cloned correctly to the shuttle plasmid.The expression of GFP could be observed by fluorescence microscope.The viral titer purified adenovirus was 1.25×1011 pfu/ml.Conclusion:Recombinant adenovirus vector was constructed successfully and packed in HEK293 cells,which can serve as a good gene transfer vector for the study of gene therapy in chronic liver diseases,such as non-alcoholic steatohepatitis.