Construction of heme oxygenase-1 gene recombinant adenovirus

Objective To construct the recombinant adenovirus of heme oxygenase-1 by using the method of homologous recombination in bacteria.Methods Heme oxygenase-1 gene was liberated from the vector of PRHO-1 via XhoⅠ+HindⅢ digestion,and subcloned into clone plasmid of Puc18,forming transfer vector of Puc18-PRHO1.Then it was digested by KpnⅠ+HindⅢ and subcloned into shuttle vector of pAdTrack-CMV,forming transfer vector of pAdTrack-Puc18-PRHO1.Then it was linearized with PmeⅠ and cotransformed into BJ5183 cells with adenovirus genomic plasmid of pAdEasy-1.The DNA of identified recombinant plasmid was digested with PacⅠand transfected to 293 cells to package recombinant adenovirus particles.The PCR technique was used to detect target gene.The titre and its infection rate of the Ad-H01 was measured with the aid of GFP expression.Results Positive recombinant bacterial clones were obtained after cotransformation of BJ5183 bacterial cells with pAdTrack-Puc18-PRHO1 and pAdEasy-1 by method of CaCl_2.PCR test indicated the recombinant adenovirus contained the insert of HO1.The titre of purified recombinant adenovirus was 1.4×10~(10)pfu/ml.Conclusion The method of homologous recombination in bacteria is a convenient and efficient method to prepare recombinant adenovirus,the prepared Ad-H01 can effectively madiate target gene expression in cultured cells and paves a sound foundation for further study.