Construction of eukaryotic expression vector with EGFP and hVEGF121 fusion protein and its expression in mesenchymal stem cells of rats / 中国病理生理杂志

ABSTRACT

AIM: To construct a recombinant plasmid carrying enhanced green fluorescent protein and human vascular endothelial growth factor 121 gene and to detect its expression in rats mesenchymal stem cells (MSCs). METHODS: Human VEGF121 cDNA was amplified with polymerase chain reaction (PCR) from pCD/hVEGF121 and was inserted into the eukaryotic expression vector pEGFP-C1. The recombinant plasmid pEGFP/hVEGF121 was identified with PCR, double enzyme digestion and DNA sequencing. Then this recombinant plasmid was transfected into rat's MSCs with lipofectamine. The expression of EGFP and VEGF121 protein were detected with fluorescence microscope and immunocytochemical staining, respectively. RESULTS: The recombinant plasmid was confirmed with PCR, double enzyme digestion and DNA sequencing. The fluorescence microscope and immunocytochemical staining results showed that the EGFP and VEGF121 protein were expressed in MSCs 48h after transfection. CONCLUSIONS: The recombinant plasmid carrying enhanced green fluorescent protein and human vascular endothelial growth factor was successfully constructed and expressed positively in rat MSCs. It provides a good basis for further research on differentiation of MSC and VEGF gene therapy for ischemial cardiovascular disease.