Expression and activity of VEGF165 in mammalian cells

Objective To study VEGF165 gene expression and the activity of the product in mammalian cells transfected by a recombinant plasmid pcDNA3.1(+)-VEGF165.Methods VEGF165 gene was obtained by PCR and was constructed into the expression vector pcDNA3.1(+).The pcDNA3.1(+)-VEGF165 plasmid was transfected into NIH 3T3 cells by Lipofectamine 2000.The positive clones were screened by G418.The expressed product was identified by sandwich ELISA,SDS-PAGE and Western blotting.The biological activities of the product were analyzed by cell ELISA and MTT assay.Results The expression vector pcDNA3.1(+)-VEGF165 was successfully constructed and transfected into NIH 3T3 cells.After screened by G418,the resistant cells were cultured and passaged.The VEGF165 gene expressed in the recombinant NIH 3T3 cells.The product could bind to the receptor cells ECV304 and stimulate the proliferation of ECV304.Conclusion NIH 3T3 cells transfected by the pcDNA3.1(+)-VEGF165 plasmid can express active VEGF165 protein.