Prokaryotic Expression, Purification And Primary Bioactivity Assay For Mouse Soluble Il-13 Receptorα2

Asthma is a chronic allergic airway inflammation mediated by various kinds of cells. IL-13 is a Th2 cytokine that has emerged as a critical regulator of inflammation immune response. It has been conformed as an essential role for this cytokine in driving major correlates of asthma pathology, including airway hyperresponsiveness(AHR), lung eosinophilia, mucus generation, IgE generation and fibrosis. In murine systems, IL-13 blockade by sIL-13Rα2-Fc or by antibody effectively limits asthmatic responses. A number of monoclonal antibodies targeting IL-13 are being developed for the treatment of asthma, with several more in preclinical developments. Results of clinical studies are not yet available, but preclinical data indicates a promising profile. IL-13Rα2 binds IL-13 with high affinity and plays an important role in IL-13 signaling as a decoy receptor. sIL-13Rα2 can inhibit IL-13 mediated bioactivity, has no heteroimmunization and has lower stimulus response to organism. The fusion protein of the extracellular domain of canine IL-13Rα2 and the Fc fragment of canine IgG heavy chain(rcaIL-13Rα2-Fc) and extracellular domain of canine IL-13Rα2(rcaIL-13Rα2) have been expressed in E.coli expression system. In our research, we will express mouse soluble IL-13Rα2(sIL-13Rα2) in an E.coli expression system and it maybe a novel therapeutic reagent.Objective: To express and purify recombinant mouse sIL-13Rα2 in Escherichia coli and analyze its bioactivity in vivo.Methods:The sIL-13Rα2 was inserted into the expression expression vector PProEX HT a and the recombinant plasmid was confirmed by DNA sequencing and restriction endonuclease. Then it was transformed to E.coli BL21(DE3) for expression under IPTG induction. The recombinant protein was purified by Ni2+-NTA Agarose and its biologic activity was identified using the A-549 cell bioassay. SDS-PAGE and Western blot were used to certify whether the purpose protein was correctedly expressed.Results: The recombinant expression vetors pProEX HTa /sIL-13Rα2 was constructed correctly. The sIL-13Rα2 was expressed in Escherichia coli and accounted for 45% of total proteins. After cell breakage, the recombinant protein was recovered in inclusion bodies and could be recongnied by anti-mouse sIL-13Rα2 McAb. The purity was more than 95% after purified with Ni2+-NTA Agarose and it has the right immunology specificity and biologic activity, inhibiting human lung epithelial A-549 cells producing thymus activation-regulated chemokine(TARC) in response to IL-13 stimulation.Conclusion: sIL-13Rα2 was successfully produced in E.coli BL21(DE3) and had biological activity, which laid a foundation of further study on its curative effect on asthma.