Construction of eukaryotic expression vector of human MT1-MMP and its expression in HepG2 cells and significance

Objective To clone human membrane type-1 matrix metalloproteinase(MT1-MMP) gene and construct its eukaryotic expression vector,then detect its expression in human hepatoma cell line HepG2.Methods Full-length human MT1-MMP cDNA was amplified from normal liver by RT-PCR and cloned into pMD18-T simple vector.After sequencing,the fragment was subcloned into the pcDNA3.1 vector and the recombinant eukaryotic expression vector was constructed.MT1-MMP mRNA level of HepG2 cells was evaluated by RT-PCR.Results ① A 704 bp fragment was obtained by PCR,which was the same as the expected fragment.②Two fragments of PCR products(5400 bp and 704 bp) of eukaryotic expression vector pcDNA3.1/MT1-MMP were obtained by EcoRⅠ and BamHⅠ restriction enzyme digestion.The sequencing result of MT1-MMP cDNA was identical with that reported in GenBank.③ MT1-MMP mRNA level of HepG2 cells transfected with the recombinant vector was obviously higher(1.66±0.43) than those of empty vector group(1.21±0.25) and control group(1.19±0.18)(P0.01).Conclusion The recombinant vector pcDNA3.1/MT1-MMP is successfully constructed and expressed stably in HepG2 cells.