Cloning,Expression,Purification and Preliminary Identification of Human Vascular Endothelial Growth Factor Mutant II

To obtain the hVEGF121Ⅱprotein for the preparation of anti-tumor vaccines which were stronger to resist angiogenesis,the recombinant prokaryotic plasmid expressing human vascular endothelial growth factor mutant hVEGF121 Ⅱwas constructed.By PCR technique,the gene encoding two tandem repeat sequences of peptide 407-426 inside heat shock protein mHSP70 was introduced to the terminus of the constructed hVEGF121Ⅰmutant gene.PCR product digested with Hind III and NcoI was connected to the prokaryotic expression vector pET-28a(+) digested with the same restriction endonuclease.Then the recombinant plasmid was transformed into E.coli strain BL21(DE3).After the reorganized strain was induced with lactose and lysised by ultrasonic,the resulting inclusion body was washed,dissolved,and diluted to renature and purified by ion-exchange chromatography to get the target protein hVEGF121Ⅱ,and then it was identified by Western-blot.The recombinant plasmid pET-28a(+)-hVEGF121Ⅱ expressing hVEGF121Ⅱ was successfully constructed.After the recombinant strains were induced with galactose,the target protein accounted for 62% of the whole bacteria cell proteins,and its purity reached 95% after separation and purification procedure.Both the monomer and dimmer of the hVEGF121Ⅱprotein could combine with VEGF antibody.The successful expression of hVEGF121Ⅱprotein has laid the foundation for its immunology research.