人可溶性白介素-4受体(sIL-4R)在甲醇酵母中的表达

白细胞介素-4(IL-4)作为一种多功能的细胞因子在哮喘等变态性炎症反应中具有关键作用。IL-4通过结合细胞表面的白介素-4受体(IL-4R)发挥其生物学效应。sIL-4R缺少跨膜和胞内结构域，结合IL-4后不能产生信号传递来介导IL-4的生物学活性，但sIL-4R与IL-4结合的高度特异性和极高的亲和力使它非常适合作为理想的IL-4拮抗剂应用于哮喘等疾病的治疗。采用RT-PCR方法以人的单核细胞总RNA为模板扩增得到编码sIL-4R的基因片段，经测序证实后插入到甲醇酵母分泌表达质粒pPIC9K中，得到重组质粒pPIC9K／sIL-4R，利用限制性内切酶BalⅡ将其线性化后用电穿孔法导入Pichia pastoris GS115。分别用SDS-PAGE、Western blot和Ligand bioding blot对重组菌发酵上清中的sIL-4R进行鉴定。结果表明，在分子量约为30kD处有sIL-4R特异条带出现，并具有结合其配基IL-4的生物学活性。

Expression of the Human Soluble Interleukin 4 Receptor in Methylotropic Yeast

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 the IgE isotype switch. IL-4 exerts its biological effects through binding to its receptor (IL-4R) complex, with the alpha chain as the high affinity binding subunit. 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. Some companies have embarked the clinical research for asthma treatment with the sIL-4R and the result revealed well therapeutic effect. With RNA extracted from human monocyte as the template, The sIL-4R cDNA encoding the extracellular domain of IL-4R a chain was obtained by RT-PCR. Compared with the sIL-4R encoding sequence in GenBank, the nucleotide sequencing analysis indicated that there was a A-->G mutation at 148bp 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 the only known extracellular variant of human IL-4R. Then the recombinant vector pPIC9K/sIL-4R was constructed, linearized and introduced into Pichia pastoris GS115 by electroporation. The recombinant sIL-4R was identified by SDS-PAGE, Western blot and Ligand binding blot. The SDS-PAGE and Western blot analysis showed that the apparent molecular weight of expressed sIL-4R was about 30kD. And the Ligand binding blot 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).