Construction of recombinant eukaryotic expression plasmid containing human ACE2 gene and its transfection in human endothelia cells

Objective:To clone and construct recombinant eukaryotic expression plasmid containing human angiotensin converting enzyme 2(ACE2)and transfected it to human endothelial cells.Methods:The full-length cDNA of human ACE2 was amplified from a Human Fetal Heart cDNA Library by PCR,and cloned into pcDNA3.1-D/V5-His expression vector(phACE2).After the identification of enzyme digestion and sequencing,phACE2 was transfected to human endothelial cells by liposome,and the mRNA and protein expression of ACE2 was determined by real-time PCR and Western blot in endothelial cells respectively.Results:The inserted fragment of recombinant vector(2419 bp)was the sequence of target gene by PCR,enzyme digestion and sequencing determination,and ACE2 mRNA,and protein expression were markedly enhanced(P0.01)in the transfected endothelial cells compared with controls.Conclusion:The recombinant eukaryotic expression plasmid pcDNA 3.1-hACE2 is successfully constructed and transfected in cells,which provided the basis for the study of ACE2 functions in the prevention and treatment of human essential hypertension.