earticle

영어

Interleukin-25 (IL–17E) is a novel Th2 pro-inflammatory cytokine belongs to the member of IL–17 cytokine family. In the present study, bioactive recombinant human IL–25 (rhIL–25), the cDNA of mature IL–25 was synthesized using nested PCR and codon bias of prokaryotic host Escherichia coli. The desired template was cloned into the MCS region of expression vector pET28a+. The recombinant vector was transformed into maintenance host Escherichia coli DH5α and the transformants were selected by kanamycin resistance marker. Expression was carried out using IPTG inducible Escherichia coli BL21(DE3) in different media like LB, terrific broth and M9 media. Among all, terrific broth was used for the enhanced production of rhIL–25. SDS–PAGE analysis shows 31 kDa proteins against low molecular weight protein marker. Refolding of inclusion bodies with denaturation buffer (25 mM Tris–HCl [pH 7.2], 5 M Urea, 20 mM β–ME and 200 mM NaCl) yields the rhIL–25 at a concentration of ~ 86 mg/L at 37 0C, where it is high when compared with the expression at 20 0C (~ 16.5 mg/L). Western blot analysis was carried out using anti human IL–17E/IL–25 antibodies. Biological activity of rhIL–25 was determined by the release of IL–6 from PBMC cells. For the first time, under the conditions of current good manufacturing practice (cGMP), bioactive recombinant IL–25 was produced at large scale in soluble form using industrially feasible bacterial host Escherichia coli BL21(DE3).