Experimental Study On Biodistribution And Immunogenesis Of A New Fusion Protein

Hematopoietic function obstacle,especially serious thrombocytopenia was dangerous and difficult to cure at present. One of problem in hematopoietic rebuild was how to promote thrombocytopoiesis. Thrombocytopoiesis was regulated by thrombopoietin(TPO), interleukin-3 (IL-3)and interleukin-11 (IL-11)etc.The TPO receptor path was an important proliferation signal passage of megakaryocyte. It was reported that TPO genetic engineering product could promote thrombocytopoiesis markedly but it also exist strong side effect and immunogenesis. In recent researchers has discovered a kind of heterogeneous TPO imitate peptides which was constituted by 14 amino acid. This imitate peptides could selectively bind to the TPO receptor and activate the proliferation signal. But the half- life in the body of the imitate peptides is very short because of molecular weight, it's application was limited thus.Human growth hormone is a kind of growth regulate factor that has extensive physiology function. Recently some researchers including our laboratory had found that human growth hormone can also stimulate proliferation and differentiation of megakaryocyte. But because of its extensive physiology function, the long-term application of human growth hormone will produce some other side effects.Therefore if we can make the hormone selectively bind to megakaryocyte, we can not only raise its effect in thrombocytopoiesis but also reduce its dosage and side effects.Therefore we creative expressed the TPO imitate peptides with human growth hormone on fusion and obtain a kind of new efficient fusion protein which could promote thrombocytopoiesis markedly. Currently, our laboratory has already achieved the small-scale product, which purity was above 98%. It's activity had proved to match with recombinant human IL-11 by animal experiment.Based on the reports mentioned above we study the distribution and metabolism of this new fusion protein. It's regulation to marrow megakaryocyte and the immunoreaction after it's application had also been studied. Through our study the biology characteristic and the action mechanism of the fusion protein would be further understood. Thus it would provide useful experimental data for further research and application of the fusion protein in the future.The new fusion protein was labeled with ¹²⁵I and injected to mice by intravenously in the experiment. The radioactivity of blood,marrow and other 16 kinds of tissue were dynamic examinated to clarify metabolizable characteristic of the fusion protein in the body.The regulation to marrow megakaryocyte of combine-injure rat reorganization has also been analyzed contrast to recombinant human IL-11 and human growth hormone.Then the fusion protein was well emulsify with Freund's adjuvant and injected according to the regular method. After continuously inject for 15 days(similar treatment project) the antibody was examinated by ELISA. Whether the antibody appear and in serum can represent the immunogenesis of the new fusion protein.Main results and conclusions are as follows:1. After injected the ¹²⁵I labeled fusion protein, radioactivity mainly distribute in the blood and marrow in 6 hours. Then radioactivity of marrow was higher than other organs. The distribution of fusion protein have obvious marrow deflection.2. Application of the new fusion protein can increase quantity of megakaryocyte in rat marrow. The effect even better than recombinant human IL-11. 7 and 14 days after injected the new fusion protein, the rat megakaryocyte increase obviously compare to controlled group.It suggested the new fusion protein could promote proliferation and differentiation of megakaryocyte.3. No corresponding antibody in mice was detected under our experiment condition. It suggested the new fusion protein has no obvious immunogenesis.4. Combine the previous results we concluded the new fusion protein could promote proliferation and differentiation of megakaryocyte selectively and obviously. It would not induce the produce of antibody so that new fusion protein deserve to develop further.