VEGF Receptor Binding Domain-GraB Specifically Inhibits KDR/flt-1 Positive Endothelial Proliferation In Vitro And Angiogenesis In Vivo

Objective This study was to develop an inducible system for expression of vascular endothelial growth factor (VEGF) receptor-binding domain,Granzyme B and VEGF receptor-binding domain-GraB in E. coli on the basis of expressive specifity of VEGF receptor on the vascular endothelial of tumor and of the effect that GraB induces cell apoptosis. In the experiment,the biological function of VEGF receptor-binding domain and VEGF receptor-binding domain-GraB were studied for the purpose of antiangiogenesis research and the feasibility of treating tumor with transfecting VEGF receptor-binding domain-GraB gene was futher investigated.Methods After GraB cDNA and hVEGF receptor-binding domain cDNA were amplified by RT-PCR via extracting lymphocyte total RNA and Lovo cell total RNA respectively,the gene was inserted into E. coli expression vector pTrcHis2A. The prokaryotic expression plasmid pTrcHis2A/VEGFD,pTrcHis2A/GraB and pTrcHis2A/VEGFD-GraB were constructed and transformed into TOPI OF. The VEGFD-GraB cDNA was subcloned to the retroviral vector pLNCXi. After being packaged with PT67 amphotropic packaging cell,pLNCX2-VEGFD-GraB virus was produced,which was transfected to carcinoma LS-174T.Flow cytometer was applied to test the cell apoptosis.Results After 8 h of IPTG induction,the VEGF receptor-binding domain,GraB and the VEGF receptor-binding domain-GraB were expressed to 15% of total proteins by SDS-PAGE assay. They were observed to be of significant antigenicity and specificy with Western blot assay. When the recombinant proteins were purified by affinity chromatography,they couldinhibit and block to a significant extent the proliferation of ECV304 and neovascularization of the chick chorioallantoic membrane. The VEGFD-GraB retrovirus were transfected to LS-174T. In the supernant and cell of LS-174T infected by the VEGFD-GraB virus appeared a protein strip containing 39ku by Western blot assay,while no special strip was found in the target cells transfected with the retro viral. The apoptosis peak in LS-174T infected by the VEGFD-GraB virus was found with flow cytometer.Conclusion The construction of expression system of VEGF,granzyme B and VEGF-GraB in E. coli is of great importance for the purpose of antiangiogenesis investigation. They may be potent inhibitors of tumor angiogenesis and metastasis. In our study,introduction of VEGF receptor-binding domain-GraB gene to retroviral vectors was proved to be an effective way to induce cell apoptosis,which provides theoretical basis for GraB in the tumor gene therapy.