Cloning of rat Smac gene and its expression in cardiocyte H9C2

Objective To clone the rat second mitochondria-derived activator of caspase(rSmac),construct its eukaryotic expression vector,and express it in cardiocytes.Methods The rSmac CDs gene was amplified from the rat kidney tissue by reverse transcriptase-polymerase chain reaction(RT-PCR).It was cloned into pcDNA3.1+ vector to construct the recombinant plasmid pcDNA3.1+-rSmac.Then the recombinant plasmid pcDNA3.1+-rSmac,pcDNA3.1+,and pcDNA3.1+-EGFP were transfected into the rat myocardial cell line,H9C2 mediated by lipofectamine 2000.Among them,pcDNA3.1+-EGFP was used to detect the transfection efficacy indirectly through fluorescence microscoy(FM)and flow cytometry(FCM).After transfection,RT-PCR method was used to detect the expression of exogenous Smac gene.Results The sequencing result indicated that the acquired sequence was in concordance with that published on GenBank.The recombinant plasmid pcDNA3.1+-rSmac was idenfied by PCR and enzyme digesting,and the results were coincident with anticipation.The results detected by FM and FCM showed that the transfection was effective,and the efficacy was up to 38.36%.The mRNA level was increased after transfection with the recombinant plasmid pcDNA3.1+-rSmac for 48 h,and the ratios of Smac/β-actin in control,vacant vector,and recombinant plasmid pcDNA3.1+-rSmac group were 1.19,1.31,1.67,respectively(P0.05,P0.01 vs control group).Conclusion The rSmac gene is cloned and the recombinant plasmid pcDNA3.1+-rSmac is constructed successfully.Moreover,it could be effectively expressed in transfectant H9C2,which establish fundament and benefit the further study on the mechanism of Smac in apoptosis of cardiocytes.