Construction and identification of pIRES₂-NGF-VEGF₁₆₅ bicistronic eukaryotic expression vector

We used a simple and efficient method to construct the bicistronic eukaryotic expression vector pIRES2-NGF-VEGF165. The nerve growth factor (NGF) gene was obtained from the genomic DNA of human peripheral blood mononuclear cells by polymerase chain reaction. The NGF cDNA fragment was inserted into the multiple cloning sites of the pIRES2-EGFP vector to generate the bicistronic eukaryotic expression plasmid pIRES2-NGF-EGFP. The vascular endothelial growth factor 165 (VEGF165) gene was obtained from the pIRES2-VEGF165-EGFP plasmid by polymerase chain reaction. Next, the VEGF165 cDNA fragment was cloned into pIRES2-NGF-EGFP in place of enhanced green fluorescent protein creating the plasmid pIRES2-NGF-VEGF165. pIRES2-NGF-VEGF165 was transfected into HEK293 cells and reverse transcription-polymerase chain reaction and Western blot analysis were used to test the co-expression of double genes. The NGF and VEGF165 genes were cloned and the DNA was sequenced, which revealed that NGF and VEGF165 were consistent with the sequence recorded in GenBank. Restriction analysis showed that the NGF and VEGF165 genes were inserted into the expression vector pIRES2-EGFP. Transfection of pIRES2-NGF-VEGF165 into HEK293 cells resulted in expression of the double gene at the mRNA and protein levels. The NGF and VEGF165 coexpression plasmid provides a novel expression system, enabling further study of the functions of the NGF and VEGF165 genes.