Cloning of human mdr1 gene and preparation of its recombinant adenovirus vector / 重庆医科大学学报

ABSTRACT

Objective:To construct the recombinant adenovirus expressing human mdr1 gene.Methods:The cDNA of mdr1 gene was subcloned into the shuttle plasmid pAdTrack-CMV and then clone the homologous recombinant adenovirus genomic plasmid pAdEasy in bacteria.We tranfected the DNA of identified recombinant plasmid into 293 cells via liposome,and then did the package and amplification of adenovirus.The mononuclear cells of mouse were infected with Ad5-mdr1 and the target gene was determined by fluorescence microscope and flow cytometry.Results:Restriction endonuclease and PCR analysis confirmed that the recombinant adenovirus vector of mdr1 gene was successfully constructed.The titer of the recombinant adenovirus was 8.3?1011pfu/ml,and the infection efficiency to the mononuclear cell achieve 10%~15%.The expression of mdr1 gene was observed at 48h after the transfection of the mononuclearcell with Ad5-mdr1.Conclusion:The recombinant adenovirus expressing mdr1 gene was successfully constructed,which paves the way for the following studies related to mdr1.