Construction and Identification of pIRES2-NGF-NT-3 Bicistronic Eukaryotic Expression Vector

Construction of double gene co-expression vector pIRES2-NGF-NT-3 and detection of its expression in HEK293 cells were performed. Human nerve growth factor(NGF) and neurotrophin-3(NT-3)genes were obtained from the genomic DNA of human peripheral blood mononuclear cells by direct PCR.The NGF cDNA fragment was inserted into the multiple cloning sites of pIRES2-EGFP to generate the bicistronic eukaryotic expression plasmid pIRES2-NGF-EGFP. Then NT-3 cDNA fragment was cloned into the pIRES2-NGF-EGFP to replace EGFP creating plasmid pIRES2-NGF-NT-3. Then the recombinant plasmid was transfected into HEK293 cells with the cationic liposome. RT-PCR and Western blotting were used to detect NGF and NT-3 mRNA and protein expression in HEK293 cells. Construction of pIRES2-NGF-NT-3 was identified by means of double digestion and sequencing. The expression of mRNA and protein of NGF and NT-3 was significantly up-regulated after transfection. The NGF and NT-3 co-expression plasmid is successfully constructed. It provides a novel expression system, which enable to further study on the functions of NGF and NT-3 genes.