| Interleukin-4 (IL-4) is a pleiotropic cytokine which plays a pivotal role in shaping immune responses. IL-4 mediates important proinflammatory functions in asthma, including of the IgE isotype switch. IL-4 exerts its biological effects through binding to its receptor (IL-4R) complex. IL-4R complex is heterodimer consist of 7 common chain and IL-4Rα chain which is the high affinity binding subunit.A full-length 3.6kb sIL-4R cDNA includes an open reading frame encoding 825 amino acids. IL-4Ra chain contains a predicted 25 amino acid signal peptide, 207 amino acid external domain, 24 amino acid transmembrane region, and 569 amino acid cytoplasmic domain. IL-4Ra chain contains a conserved WSXWS motif which is required for maintaining the receptor in a conformation favorable to cytokine binding. After IL-4 binding to IL-4R, the receptor-associated tyrosine kinases were activated for the initiation of signal transduction. Soluble IL-4R lacks the transmemberane and cytoplasmic domains so it cannot induce cellular activation. By acting as a decoy to circulating IL-4 and neutralize its activity, its high specificity and affinity make it ideal as an IL-4 antagonist. Recombinant sIL-4R has been used for clinical research of asthma therapy and the result revealed well therapeutic effect. With RNA extracted from human umbilical vein endothelial cell as the template, the sIL-4R cDNA encoding the extracellular domain of IL-4R a chain was obtained by RT-PCR. The cDNA was cloned into the plasmid pUC18 and sequenced. Compared with the sIL-4R encoding sequence in GENBANK, the result showed that there was a A→G mutation at 148 bp and the mutation caused Ile→Val at 50th amino acid. According to the references, numerous polymorphisms .have been identified in the IL-4R gene and the Ile50Val was similar to the data published.By inserting sIL-4R cDNA into the shuttle plasmid pPIC9K (Yeast-E.coli) , the recombinant plasmid pPIC9K/sIL-4R was constructed, linearized and transformed into Pichia pastoris GS115 by electroporation. The SDS-PAGE and Western blot alysis showed that the apparent molecular weight of expressed sIL-4R was about 30kD. And the Ligand binding analysis indicated the expressed sIL-4R had the biological activity. The sIL-4R, which had the biological activity, was successfully secretorily expressed in the Pichia pastoris(GS 115) with Mut~s phenotype.The sIL-4R cDNA was inserted into the temperature-regulated expression vector pBV220 with the PrPl promoters. The recombinant plasmid pBV220/sIL-4R was transformed into E.coli DH5a. After induction at 42°C and cell lysis, the recombinant sIL-4R was expressed in the inclusion body form. SDS-PAGE analysis revealed the recombinant sample exhibited a specific band with a molecular weight of 24kDa similar to 23.7kDa calculated from the amino acid sequence. Western blot and the Ligand binding analysis showed that the expressed sIL-4R had the immunological and ligand binding biological activity. Meanwhile, cloning and expression of sIL-4R gene was carried out in S.lividans TK24. The sIL-4R cDNA was inserted into the downstream of gpp signal peptide of the...
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