Cloning And Expression Of Tumour Vascular Targeted IFN-IFNα2b-NGR, Fusion Protein Purification And Its Preliminary Identification

As a cytokine with broad-wild bioactivity, IFNs can inhibit DNA replication and karyokinesis of cells, especially to tumor cells, which was reported that a 500 to 1000 times inhibition rate higher than to normal cells. IFNs can also enhance LAK activity accompanied with IL-2, induce the expression of major histocompatibility antigen on tumor cells and enhance the sensitivity of killer cells. But numerous clinical experiences demonstrated the anti-tumor activity of IFNs is dose-dependent with increased side-effects, such as fever, cold, even diseases of digestive system and marrow. Targeted therapy has been developed to solve these problems. Other than antibodies, some peptides screened from phage displaying peptide library have been approved to have the ability to bind to some tissues or cells specifically. So there is hope to drop therapeutic molecules to specific organ or cells by peptide targeting.In this experiment, we constructed a fusion protein by recombinant BFN a 2b to CNGRC peptide, which has been approved to bind to aminopeptidase N(CD13) mainly expressed on tumor endothelial cells, and expect this fusion protein can home to tumor specifically by NGR peptide targeting. The recombinant protein IFN a2b-NGR was expressed in Ecoli. Stability of the engineered bacterial was checked. Fermentation process with high cell density was established. The fusion protein has been purified to homogeneity by ion exchange chromatography and affinity chromatography sequentially. The final purity of the fusion protein was morethan 95% and the specific activity was 1 x 109IU/mg. The tissue distribution of IFNa2b-NGR fusion protein in vivo was carried out in tumor bearing mice. ELISA result showed that the concentration of IFNa2b-NGR in tumor tissue was about 75% of that in serum, is about three to four fold more than the concentration of IFNa2b in tumor tissue. Immunohistochemistry result showed a strong positive staining of IFNa2b-NGR in blood vessels of tumor, however IFNa2b was not observed.