Construction and expression of eukaryotic expression vector of full length human Smac gene

Objective To construct the eukaryotic expression vector of full length human Smac gene(FL hSmac) and express Smac gene in SMMC-7721.Methods The FL hSmac gene was amplified by reverse transcriptase-polymerase chain reaction(RT-PCR),then inserted it into pcDNA3.1+.When the recombinant plasmid pcDNA3.1+-FL hSmac was confirmed correctly through endonucleases digesting and PCR identification,it was used to transfect the SMMC-7721.So was the pcDNA3.1+ as negative control.The pcDNA3.1+-EGFP was transfected into SMMC-7721 to detect the transfection efficacy by flow cytomety(FCM) and fluorescence microscopy(FM).Moreover,the expression of Smac in transfectant SMMC-7721 was detected by RT-PCR and western blot(WB).Results The amplified fragment by PCR was coincident with the anticipated result,and its sequence was in concordance with that published on GenBank.Therefore,the Smac gene was cloned successfully.Furthermore,the recombinant plasmid pcDNA3.1+-FL hSmac was constructed successfully through idenfication.The transfection efficacy was up to 48%,and very bright green fluorescence could be seen under FM,which both indicated the transfection was effective.Both on the mRNA level or the protein level,the expression of Smac gene was increased obviously in the transfected SMMC-7721 detected by RT-PCR and WB respectively.Conclusion The combinant plasmid pcDNA3.1+-FL hSmac was constructed successfully and it could be expressed obviously in SMMC-7721,which is benefit to study the mechanism of Smac gene and explore the gene therapy strategy for tumor.