Construction of recombinant plasmid pcDNA3.1-adrenomedullin and its expression in rat renal epithelial cells

Objective To construct a eukaryotic expression plasmid pcDNA3.1-adrenomedullin(pcDNA3.1-ADM),and to explore its expression in rat renal epithelial cells(NRK-52E).Methods The total RNA was extracted from the adrenal glands in Wistar rats.The full-length cDNA of rat ADM was amplified by RT-PCR and cloned into pMT-18T vector using TA cloning,and then identified with double digestion and sequencing.The pMT-18TADM and plasmid pcDNA3.1 were double digested with BamHⅠ and EcoRⅠ respectively,and the target gene was cloned into pcDNA3.1 directionally.The recombinant plasmid pcDNA3.1-ADM was identified by double digestion,and then transected into NRK-52E by liposome.Expression of ADM in pcDNA3.1-ADM transferred cells were detected by RT-PCR.Results The destination gene,585 bp,was distinctively amplified by PCR,and inserted successfully into the plasmid pcDNA3.1.Expression of ADM in cells transfected with pcDNA3.1-ADM was much higher than in controls.Conclusion The eukaryotic expression plasmid pcDNA3.1-ADM is successfully constructed and expressed in NRK-52E.