An Aldose Reductase Inhibitor Screening Model Constructed by Transfection of pSNAV-AR into HEK293 Cells / 中国生物工程杂志

ABSTRACT

Objective:Aldose reductase,involved in the pathogenesis of diabetic complications,was recombinated with an adeno associated virus vector pSNAV2.0,and it was transfected into human embryonic kidney 293(HEK 293)cells.The gene engineering produced AR would be used as a target protein to screen aldose reductase inhibitors.Restriction endonuclease digestion and ligation procedures were performed to construct the AR expression plasmid vector pSNAV-hAR.Methods:After confirmation the recombinant plasmid by PCR,restriction endonuclease digestion,and DNA sequencing,pSNAV-hAR was transfected into HEK293 cells.Western blot and immunofluorescence analysis were performed to detect the expression of AR and its enzyme activity.Results:The results of a series of analysis including AR activity assay,Western blot and immunofluorescence analysis shown the expressed protein mediated by the adeno associated virus vector transfecting HEK 293 cells,was functional AR.The traditional aldose reductase inhibitors,Sobinil and Zopolrestat,were used to test and verify the constructed cell model.Conclusion:The established AR expression model can be used in mechanismresearch of activation of polyol pathway on diabetic complications and screening potential aldose reductase inhibitors.