巨噬细胞炎性蛋白4的非融合表达载体的构建和表达

Expression of mutated β-chemokine macrophage inflammatory protein 4 in pBV220 vector

目的探索如何抑制嗜酸性粒细胞的趋化作用,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非融合表达。方法设计MIP4基因的PCR引物并进行氨基酸突变,将MIP4N末端的丙氨酸突变为蛋氨酸。以正常人肺cDNA文库为模板,PCR方法获取Met-MIP4基因。克隆入载体pUC19,测序验证序列已得到突变。将正确的基因插入到非融合表达载体pBV220中进行表达。结果PCR产物为220bp左右的片段,连接入pUC19质粒后测序验证获得正确突变。构建的pBV220非融合表达载体在大肠杆菌DH5-α中表达,经Tricine-SDS-PAGE凝胶电泳显示有大小约8×103的非融合蛋白表达。结论成功突变并克隆了β-趋化因子MIP4基因。Tricine-SDS-PAGE表明,非融合的Met-MIP4突变体已得到表达,为进一步研究其对哮喘的生物治疗奠定了基础。

Objective To screen for antagonist of eosinophils chemotaxis, me gene encoding for β-chemo- kine macrophage inflammatory protein 4 （MIP4） , a potential antagonist of chemokine receptor 3, was mutated, cloned and expressed in E. coli. Methods The PCR primers for MIP4 were designed with the alteration of methionine to alanine 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 interested sequence was cloned into pUC19 cloning vector and sequenced, and then cloned into pBV220 expression vector. The target protein was expressed. 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 pBV220 expression vector. SDS-PAGE gel electrophoresis revealed that the protein of MIP4 about 8×10^3 was correctly expressed in E. coli. Conclusion The Met-MIP4 gene was properly mutated with the alteration of methionine to alanine at N-terminal. The MIP4 was expressed in E. coli and it may serve as a novel antagonist against chemokine receptor 3.