Function Of Fusion Protein IL-1ra-Fcε And Its Therapeutic Effects On Allergenic Asthma

Bronchial asthma normally has some genetic backgrounds, which is a chronic inflammatory disease of respiratory tract caused by multiple factors, and its morbidity and mortality are rising year by year. Interleukin 1(IL-1) is one of stronge pro-inflammatory cytokines, which plays an important role in the generation and progress of asthma. Interleukin 1 receptor antagonist(IL-1ra) is a natural antagonist of IL-1, which displays an antagonist action against IL-1 at receptor level and possesses the therapeutic and preventive actions on asthma, being confirmed by some researches.One of the key factors causing asthma is that the binding of IgE to its high affinity receptor triggers the degranulation of mast cell and basophil. Blocking the binding of IgE with its high affinity receptor is an effective targeting site for the treatment of asthma, that shows a tremendous potentiality in clinic.This study integrated the functions of IL-1ra and Fcεof IgE , and the fusion protein between IL-1ra and the constant region of IgE was constructed to form a new type of protein molecule with bifunctional activity, that antagonizes IL-1 biological effects as well as extend the half-life of IL-1ra. simultaneously it can inhibits the binding of IgE with its high affinity receptor at the other side, thus achieving the goal of prevention and cure of asthma.The human IL-1ra gene was integrated with human Fcεand rat Fcεindividually for the first time.The fusion genes were constructed and highly expressed in a prokaryotic system(E.coli). The expressed products exist mainly in the form of inclusion body and were purified by the molecular sieve and ion-exchange chromatography, then the recombinant protein was renatured by gradually decreasing the concentration of denaturant. Assay on EL-4/CTLL-2 cells showed that this fusion protein can antagonize the activity of IL-1, same as IL-1ra. The analysis of RBL-2H3 cell activity showed that the fusion protein can bind to the FcεRI on the cell surface and also can inhibit IgE- mediated apoptosis.For analyzing the therapeutic effects of the fusion molecule IL-1ra-Fcεon on allergenic asthma through gene administration, the expression level of which in 293T cell and rat lung tissue was investigated. As a result, the fusion gene of IL-1ra-Fcεhad been continuously expressed in rat lung tissue by instilling into trachea.In order to evaluate the therapeutic effects of the fusion molecule IL-1ra-Fcεon the allergenic asthma, we set up a rat model of allergenic asthma, the therapeutic efficiencies of IL-1ra-Fcεon the asthma by intravenous injection of the recombinant protein and by instilling into trachea were analyzed. It had been confirmed that IL-1ra-Fcεpossesses a significant therapeutic effects on the allergenic asthma by means of observation of the rat physiological status, determination of the lung function, the changes of inflammatory cells in BALF, detection of serum IgE, IL-4 and IFN-γin BALF, changes of peripheral blood T cell subpopulation, the penetration of blood vessel and pathophysiological changes of lung tissue. It has been found that the therapeutic effect of IL-1ra-Fcεis superior to that of IL-1ra. Immunohistochemical study demonstrated that STAT6 was correlated with the severity of asthma attack. Although the administration was uncertain caused by dropping the vector of pIRES2-EGFP -IL-1ra-Fcε, it still demonstrated a certain therapeutic effect on asthma.Analyses of the key molecules of IRAK1 and Syk in signaling pathway of IL-1 and IgE receptor, it was found that only the IgE signaling pathway was triggered by the above-mentioned signal molecules on apoptosis of RBL-2H3 cell, but IL-1 signaling pathway was not influenced. At the animal level, only IL-1ra can simultaneously trigger both IL-1 and IgE signaling pathways. Both in vivo and vitro assays displayed that these two signaling pathways can influence the NF-κB activity, therefore, the inflammatory response plays an important role in the therapy of allergenic asthma.The results of pharmacokinetic indicated that the half-life of IL-1ra-Fcεwas significantly extended in comparison with that of IL-1ra.In summary, this study has obtained the following results: 1.the fusion gene of IL-1ra-Fcεwas successfully constructed, which had been highly expressed in E.coli. The recombinant protein with activity was obtained after purification and renaturation; 2. an eukaryotic expression vector pIRES2-EGFP-IL-1ra-Fcεwas constructed, the prevention of asthma with this vector was analyzed; 3.IL-1ra-Fcεdisplayed therapeutic effect superior to IL-1ra on the allergenic asthma; 4. the key role of inflammatory response in the pathogenesis of asthma was confirmed through analysis of IL-1 signaling pathway and the signaling pathway of IgE with its receptor. 5.the half-life of fusion protein IL-1ra-Fcεwas significantly extended in comparison with that of IL-1ra.Generally, our study provided new clues to pathogenesis of asthma and novel therapeutic ways of allergenic diseases.