| Diphtheria toxin (DT) is an exotoxin produced by C. diphtheriae. Diphtheria toxin is extraordinarily potent. The lethal dose for a cell is about one or two molecules. Interleukin-13 is a cytokine producted by actived T cells. Its receptors include IL-13Rα1 and IL-13Rα2. IL-13α2 is highly expressed in many cancer cells but not in normal human cells.In this study, we expressed, refolded and purified IL-13 and diphtheria toxin fusion protein (DT389-hIL13-13E13K). The fusion protein was purified by diluting and on-column refolding. Then the purified fusion protein was analyzed using SDS-PAGE electrophoresis and Western blot. Further cytotoxicity assay was performed in seven different concentrations in different tumor cell lines in vitro. The protein inhibit proliferation rate was identified at three designed times. In the in vivo study, glioma cells were injected subcutaneous into the nude mouse flank. Five doses of the fusion protein were injected intratumorally into pre-made mouse tumor model on the every other day. The tumor volume and body weight of mouse model were analyzed after injection. The killing effect of this fusion protein was evaluated. At the same time, we introduced siRNA strategy to investigate the role of IL-13Rα2 chain in tumors with the hope that finding the co-relationship between the receptor and cancer develop process as well as the treatment of other cancers by fusion protein DT389-hIL13-13E13K. The sensitivity of interfered cells to fusion protein DT389-hIL13-13E13K was assessed after IL-13Rα2 was interfered with siRNA.The purity of the fusion protein DT389-hIL13-13E13K is over 95%. It significant kills the cancer cells in vitro and the concentration of DT389-hIL13-13E13K at which 50% inhibition(IC50) occurred at 6×10-10M (which is up to the international level) and 1×10-8 M in dilute and column, respectively. In the in vivo experiment, the tumors of the nude mice grew very low when they were injected with the fusion protein but not regressed. Meanwhile, the sensitivity to fusion protein DT389-hIL13-13E13K was decreased in siRNA interfered cells compared with nomal cells. The results suggest that IL-13Rα2 chain is a specific target for IL-13Rα2-directed fusion protein. Some IL-13Rα2-positive tumor cell lines had been detected as well which are unpublished in the world. The Fusion protein shows new hope for the therapy of brain cancer and other cancers overexpress IL-13Rα2.
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