Clone, Expression, Purification And Characterization Of Human Interleukin 4 Alternative Splicing Variant(IL-4δ2)

Asthma is a chronic inflammatory disease of the airways, characterized by infiltration of activated eosinophils and activation of resident mast cells and T cells. The specific inflammation in asthma leads to the characteristic physiological abnormality of airway hyperresponsiveness, which makes the airways narrow in response to many environmental triggers and leads to the characteristic symptoms of wheeze, cough and dyspnoea. Asthma and atopy are biologically linked by type-2 cytokine driven inflammatory processes. Interleukin-4 (IL-4), in particular, is central to ther maturation of T helper type-2 (Th2) cells and IgE class switching.Given the importance of IL-4 in the immunopathogenesis of asthma, the relative role of IL-4 and its splice variant (IL-452) in the disease process needs to be understood. The human IL-4 gene contains 4 exons separated by 3 introns. 11-4 messenger RNA contains all 4 exons spliced together, whereas an alternatively spliced mRNA, IL-482, contains the coding sequences of exons 1, 3 and 4 spliced together in an open reading frame with the sequence of exon 2 omitted.One aim of asthma therapy is to reverse the effects of IL-4 and of other type-2 cytokines, so IL-482 may provide a novel treatment strategy. Moreover, an inappropriately low level of expression of IL-482 relative to IL-4 could be an importantbut hitherto unexplored reason for the pathology in patients with severe asthma. Thus, the development of such IL-4 mutant proteins that bind to the IL-4R a-chain and prevent formation of the biologically active IL-4/IL-13R complexes may have better therapeutic benefit in the treatment of allergic airway disease than either antagonists of cytokine or receptor alone.hi our experiment, we sought to distinguish IL-4 and IL-452 mRNA in normal and asthmatics. The results showed that IL-4 and IL-452 were differentially expressed. And significantly more asthmatic subjects than control subjects expressed IL-482. However, expression of IL-452 was unaffected by atopic status. Subsequently we constructed the fusion gen encoding IL-482 successfully. The fusion gene was expressed in prokaryotic expression vector in E.coli efficiently and amounted to 30% of total protein content. Then we established a cheap and easy procedure to purify EL-482 fusion protein from E.coli with refolding and normal pressure ion exchange chromatograph. IL-462 fusion protein was analysis by SDS-PAGE and Western blotting. At last, our results suggested that the purified IL-482 fusion protein was an antagonist of the IL-4-induced synthesis of IgE and expression of CD23 in B cells. We speculate that IL-482 is a natural inhibitor of hIL-4 activities in vivo and that rh IL-452 can be used as a therapeutic agent to block hIL-4 action.