Abstract:

Abstract:Objective To construct and express a gene engineer bacteria which expresses human  VEGF121 and provide a experimental foundation for targeting therapy of tumor vessel.Methods SUMO-CM-VEGF121 fusion genes were obtained by PCR.The VEGF121 was fused with  SUMO-CM by PCR,and the fused gene was transformed into E.coli Rosetta-gami(DE3).Soluble proteins were obtained at high level by IPTG.The fused protein was purified by DEAE sepharose and Ni-NTA affinity chromatography.Once cleaved from SUMO,the purity of CM-VEGF121 was obtained.Results The acquired gene fragments of SUMO-CM-VEGF121 were identified by digestion and DNA sequencing,and the fusion gene was 774 bp;the best condition for soluble protein was to cultivate the bacteria of  Rosetta-gami (DE3)/pET22b-SUMO-CM-VEGF121 at 20℃ for 24 h,with a soluble expression level at 21%.Western blotting result showed that this protein had the same immunogenicity with human VEGF antibody.Conclusion The prokaryotic expression vector pET22b and host bacterium Roestta-gami are most suitable for CM-VEGF121,which solves the problem of low expression and insolubility.

Key words: cecropin CM； vascular endothelialgrowth factor； fusion protein；purification