巨噬细胞炎性蛋白4的突变和原核表达

Mutation and expression of β-chemokine macro-phage inflammatory protein 4

目的 探索如何抑制嗜酸细胞的趋化作用 ,选择β-趋化因子巨噬细胞炎性蛋白 4 (MIP4 )的突变体 (Met- MIP4 )作为趋化因子受体 3的拮抗剂 ,将 Met- MIP4基因在原核细胞中进行表达 .方法 设计 MIP4基因的 PCR引物并进行氨基酸突变 ,将 MIP4 N末端的丙氨酸突变为蛋氨酸 .以正常人肺 c DNA文库为模板 ,PCR方法获取 Met- MIP4基因 .克隆入载体 p UC19,测序验证序列已得到突变 .将正确的基因插入到 GST融合表达载体 p GEX- 4 T中 ,以 IPTG诱导表达 .结果 PCR产物为 2 2 0 bp左右的片段 ,连接入 p UC19质粒后测序验证获得正确突变 .构建的 p GEX- 4 T融合表达载体在大肠杆菌中表达 ,经 SDS- PAGE凝胶电泳显示有大小约 34k U的新生融合蛋白表达 .结论 成功突变并克隆了 β-趋化因子 MIP4基因 . SDS- PAGE表明 ,与 GST融合的 Met- MIP4突变体已得到表达 。

AIM To screen for antagonist of eosinophils chemotaxis, the gene encoding for β chemokine macrophage inflammatory protein 4 (MIP4), MIP4 was mutated, cloned and expressed in E.coli . METHODS The PCR primers for MIP4 were designed with alanine in place of methionine at N terminal of MIP4 gene. The coding sequence of Met MIP4 was amplified with normal adult human lung cDNA library as the template. The target sequence was cloned into pUC19 cloning vector and was sequenced to confirm the result, and then the sequence was cloned into pGEX 4T expression vector. The target protein was expressed with induction of IPTG. RESULTS A fragment of about 220 bp was amplified from PCR and the sequencing result indicated that Met MIP4 gene was properly mutated. The sequence was cloned into pGEX 4T fusion expression vector. After induction of IPTG, SDS PAGE gel electrophoresis revealed that the fusion protein of GST Met MIP4, an about 34 ku protein, was correctly expressed in E.coli . CONCLUSION The Met MIP4 gene is properly mutated with the alteration of methionine to alanine at N terminal. The GST Met MIP4 is expressed in E.coli with IPTG induction and it may serve as a novel antagonist against chemokine receptor 3.