Construction of mouse PPAR? adenovirus vector and its expression in INS-1 cells / 第三军医大学学报

ABSTRACT

Objective To construct the recombinant adenovirus vector containing mouse PPAR?,and infect the INS-1 cells.Methods The pCDNA3.0-mPPAR? plasmid was digested with KpnⅠ and EcoRⅤ to obtain full-length coding sequence of mPPAR?,then the mPPAR? cDNA was ligated to the pAdtrack-CMV vector.The plasmid of pAdTrack-mPPAR? was linearized with PmeⅠ,and the fragment was reclaimed and transformed into BJ5183 which contains pAdeasy.After being screened,the extracted plasmid of positive bacteria linearized with PacⅠ was transfected into HEK293 cells with lipofectamine~(TM) 2000 and was identified by the green fluorescence protein(GFP).The harvested virus was amplified in HEK293 cells and infected INS-1 cells.After infection,the expression of PPAR? was proved by GFP expression and Western blotting.Results The recombinant adenovirus vector containing mouse PPAR? was constructed successfully and the titer was 1.5?10~(10) pfu/ml.The PPAR? expression in INS-1 cells infected by the virus was much higher than the controls.Conclusion The constructed recombinant adenovirus vector containing mouse PPAR? provides a potent tool to investigate its biological function in pancreatic islets and other tissues.