Construction and Expression of the Recombinant Adenovirus Carrying Human β_2-Adrenergic Receptor

Objective: To construct recombinant adenovirus vector bearing the human β2-adrenergic receptor(β2-AR) and amplify the adenovirus in 293 cells.Methods: Human β2-AR cDNA(1242bp) was cloned into adenovirus shuttle plasmid pAdtrackCMV to gener-ate a recombinant plasmid pAdtrackCMV-β2AR,then the linearized plasmid was transformed into E.coli BJ5183 cells which had been electroporated adenovirus backbone plasmid pAdEasy-1.The identified recombinant plasmid was digested with PacⅠand transfected in-to 293 cells to package the adenovirus.Then,the SD rat cardiomyocytes co-cultured with recombinant adenovirus were used to study the expression of β2-adrenergic receptor.Results: The specific fragment(1242bp) of β2-AR gene was detected by agarose gel electrophoresis.Then the fragment was cloned into pAdtrackCMV plasmid and the recombinant plasmid pAdTrackCMV-β2AR was validated by diges-tion with HindIII and XbaI and sequencing.Candidate recombinants was digested by Pac I,which could be released into a 30kb fragment and a smaller fragment of 4.5kb,then the recombinant plasmid was gained.Green fluorescence protein expression in 293 cells was de-tected under fluorescent microscope after transfection.Co-cultured recombinant adenovirus(MOI100) and SD rat cardiomyocytes,ex-pression level of β2-AR was detected by western-blot.Conclusion: The recombinant plasmid Ad-β2AR was established rapidly and sim-ply by homologous recombination.It may be a new tool for gene therapy using β2-AR.