Construction And Identification Of Eukaryotic Expression Vectors Carrying NPPC Gene

Objective:As a new member in the family of natriuretic peptide, C-type natriuretic peptide have the special functions which regulate the cardiovascular system,central nervous system and many other vasoactive effects.we constructed eukaryotic expression vectors containing the NPPC gene, The plasmid was transfected into human vascular endothelial cells and was transformed into the DH5a cells, The Research contribute to the follow-up study, also can provide a new choice for the treatment of diabetic lower limb arteriosclerosis obliterans.Methods:cDNA library was constructed by RT-PCR method with vascular endothelial tissue from healthy mouse, PCR product and vector were separately digested by appropriate restriction enzymes, and then ligated into pEGFP-C1-NPPC, plasmids were confirmed by restriction enzyme analysis and DNA sequencing.we studied differential expression of C-type natriuretic peptide by Western blotting and realtime fluorescence quantitative PCR,with the cells that was transfected by empty plasmid as control.Results:The recombined plasmid was constructed correctly confirmed by restriction enzymatic analysis and DNA sequencing. Geting transfected strains and cells with resistance. The transfection of pEGFP-Cl-NPPC markledly increased the expression in HUVEC.Conclusion:The eukaryotic expression plasmid pEGFP-C1-NPPC was constructed successfully and high-efficiency expressed in Eukaryotic cells.