Construction of recombinant adenovirus plasmid containing GST-? gene / 重庆医科大学学报

ABSTRACT

Objective: To constuct recombinant adenovirus vector carrying the human glutathione transferase ?(GST-?)gene and amplify the adenovirus vector in HEK293cells.Methods: GST-? gene with the suitable enzyme site are amplified from the original plasmid through PCR,then subcloned into the shuttle plsmid pAdtrackCMV after digested by the same enzyme.The recombinant plasmid pAdtrack-GST-? 1inearized by PmeI,plasmid pAdtrack-GST-? was electroporated into E.coli BJ5183 cells that had been electroporated adenovirus backbone plasmid pAdEasy-1.Picking up the homologous recombant plasmid AdEasy-GST-?,then it was digested with PacI and transfected into HEK293 cells to package the adenovirus,followed by identification of the recombinant adenovirus by means of observation of green fluorescence protein expression under fluorescent microscope.Recombinant virus supernatant infected HEK293 cells repeatedly,and the green fluorescence protein expression Was detected.Results: Through the restriction enzyme digestion and expression of GFP,recombinant adenoviral vector GST-? gene was constructed successfully.Conclusions: The recombinant adenoviral vector containing GST-? gene was successfuHy constructed,and based for the gene therapy of GST-? transferring the hematopoietic stem cells.