Cloning,Expression and Activity Detection of Vascular Endothelial Growth Factor / 中山大学学报(医学科学版)

ABSTRACT

【Ojective】To clone,express and detect activity of VEGF165.【Methods】Using human heart cDNA library as template,amplified the VEGF gene by PCR.The PCR product was ligated to PUC19 plasmid and sequenced.A eukaryotic expression plasmid AdtrackCMV harbouring VEGF165 was constructed.,then transformed into 293 cells.RNA dot blot and Western blotting were performed to demonstrated whether the tranfermants expressed VEGF165 at mRNA and protein level respectively.Furthermore the bio-activity of VEGF165 was preliminarily detected with Miles test.【Results】Sequence of 582bp VEGF165 cDNA was proved correct by sequencer analysis.The expression of VEGF165 mRNA was identified by RNA dot blot,Western blotting indicated that the molecular weight of VEGF165 protein was 22ku.VEGF165 also is of vascular permeability.【Conclusion】VEGF165 gene has been successfully cloned and expressed,which makes a basis for the further study in vivo.