人可溶性白介素4受体(sIL4R)在大肠杆菌中的表达和纯化

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 ,应用于哮喘等疾病治疗 .采用RT PCR方法 ,以人单核细胞总RNA为模板扩增得到编码sIL 4R的基因片段 ,经测序确证后插入大肠杆菌高效表达质粒pBV2 2 0 ,得到重组质粒pBV2 2 0 sIL 4R ,重组质粒转化E .coliDH5α .重组菌经温度诱导后超声破碎得到包涵体 ,经SuperdexHR75分子筛柱和DEAE SepharoseFastFlow离子交换柱进行纯化 ,HPLC检测表明纯度达到 90 % .N端测序证明 ,重组sIL 4R与天然sIL 4RN端序列完全一致 .Western印迹、配基结合印迹对重组sIL 4R进行鉴定 .结果表明 ,重组sIL 4R具有结合IL 4的生物学活性

Cloning, Purification and Expression of Human Soluble Interleukin 4 Receptor in E.coli

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 a α chain as the high affinity binding subunit. Soluble IL-4R lacks the transmembrane and cytoplasmic domains, therefore it can not 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. With RNA extracted from human monocyte as the template, the sIL-4R cDNA encoding the extracellular domain of IL-4R α chain was obtained by RT-PCR. Then the recombinant vector pBV220/sIL-4R was constructed and introduced into E.coli DH5α. After induction and cell breakage, the recombinant sIL-4R was recovered in the inclusion body. Through a two-step purification procedure, the recombinant sIL-4R accounted for 90% of total protein. N-terminal assay indicated that recombinant sIL-4R had N-terminal sequence identical to that of native sIL-4R. Western blot and the ligand binding blot analysis also indicated the expressed sIL-4R with biological activity.