【作者】 张勇；

【导师】 李元；

【作者基本信息】 中国协和医科大学 ， 微生物与生化药学， 2004， 博士

【摘要】 白细胞介素-4(Interleukin 4，IL-4)作为一种多功能细胞因子在免疫反应中起重要作用。由于IL-4能够调控IgE合成，因此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细胞介素-4受体(IL-4 Receptor，IL-4R)发挥生物学效应。IL-4R是异二聚体复合物，通常由IL-4Rα链与γc(Common γ)链组成。IL-4R复合物中IL-4Rα链对结合IL-4起主导作用。 IL-4Rα链cDNA全长3.6kb，含有一个编码825个氨基酸的开放阅读框架，根据推测其中包括25个信号肽氨基酸，207个细胞因子结合域氨基酸，24个跨膜氨基酸，569个细胞内氨基酸。IL-4Rα链胞外结构域包含有WSXWS模块，是受体结合细胞因子必需的最佳构象。在结合配基后，与IL-4R受体偶联的酪氨酸激酶被激活，从而产生信号传导。由于可溶性IL-4Rα链(Soluble interleukin 4 receptor，sIL-4R)缺少跨膜和胞质内结构域所以它不能诱导细胞内的活化过程，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做为理想的IL-4拮抗剂。重组sIL-4R已进入临床研究，结果显示对哮喘有较好疗效。 本研究以人脐静脉内皮细胞总RNA为模板，通过RT-PCR得到编码IL-4Rα链胞外结构域的基因片段sIL-4R cDNA。测序结果同已知sIL-4R基因编码序列进行比较，发现第148位碱基发生A→G的突变，造成第50位氨基酸Ile突变成为Val，根据文献报道，IL-4R基因序列具有多种单核苷酸多态性(SNP)，其中在IL-4Rα链的胞外区表现为Ile50Val，本实验测序结果同文献报道的sIL-4R基因序列的单核苷酸多态性一致。 将sIL-4R基因片段插入甲醇酵母—大肠杆菌穿梭质粒pPIC9K，构建了重组质粒pPIC9K／sIL-4R，将重组质粒转化至甲醇酵母Pichia pastoris GS115，获得了重组菌株P．pastoris[pPIC9K／sIL-4R]。经SDS-PAGE、Western blot分析显示分子量约为30kD，配基结合(Ligand binding)实验结果表明：Mut~s分泌表达的重组sIL-4R具有配基结合的生物活性。 将sIL-4R基因片段插至含有P_RP_L启动子的温敏型表达载体pBV220，获得重组质粒pBV220／sIL-4R，转化至E．coli DH5α作为宿主进行蛋白表达。经42℃诱导和细胞裂解，重组sIL-4R以包涵体形式表达。 S-PAGE分析表明，表达产物在24kDa处有特异蛋白条带出现，与已知sIL-4R分子量23717.75 Da基本一致，Western bloting及受体配基结合实验显示，重组sIL-4R具有免疫学及与配基结合的生物活性。 利用本实验室构建的链霉菌—大肠杆菌穿梭质粒pSGLgpp作为sIL-4R基因的表达载体。将sIL-4R基因片段插入到质粒pSGLgpp中gpp信号肽编码序列下游，利用gpp信号肽介导sIL-4R分泌表达。重组质粒转化至转化至S．lividans TK24原生质体，获得了重组菌S．lividans[pSGLgpp／sIL-4R]。SDS-PAGE、Western bloting及受体配基结合实验结果表明，重组菌株能分泌表达分子量为23.7kD的重组sIL-4R，表达的sIL-4R具有更多还原

【Abstract】 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更多还原