MIP4的突变体构建及其对嗜酸粒细胞趋化的抑制  

Inhibition of eosinophil chemotaxin by mutant of macrophage inflammatory protein 4

作　　者：李树钧[1] 戚好文[1] 叶江枫[1] 帝新宇[1] 季少平[2] 何鹏[2] 张英起[3] 药立波[2] 

机构地区：[1]第四军医大学西京医院呼吸内科 [2]第四军医大学基础部生物化学与分子生物学教研室 [3]第四军医大学科研部生物技术中心

出　　处：《第四军医大学学报》2003年第6期495-498,共4页Journal of the Fourth Military Medical University

基　　金：国家杰出青年科学基金 (3982 51 1 3) ;国家重点基础研究发展规划 (973)项目 (J1 9990 756)

摘　　要：目的 :为研究MIP4 (巨噬细胞炎性蛋白 4 )的突变体在拮抗嗜酸粒细胞的趋化作用中的活性 ,构建MIP4突变体(Met MIP4 )并进行原核表达和纯化 ,体外观察其拮抗嗜酸粒细胞的趋化作用 .方法 :将Met MIP4基因片段插入到pRSET A融合表达载体中 .诱导表达后 ,表达工程菌经超声裂菌后鉴定可溶性 ,细菌裂解上清经镍螯合亲和层析纯化 .采用葡聚糖沉淀法分离和纯化嗜酸粒细胞 ,利用 2 4孔Transwell双层板鉴定融合表达蛋白的趋化和抗趋化活性 .结果 :构建入融合表达载体pRSETA的基因在大肠杆菌中得到表达 .裂菌后鉴定显示融合蛋白可溶部分占 80 % ,经镍螯合亲和层析纯化后纯度达 87% .分离的嗜酸粒细胞纯度达到 90 % .拮抗嗜酸粒细胞趋化活性实验表明 ,纯化的Met MIP4蛋白具有明显拮抗嗜酸粒细胞的趋化作用 .结论 :Met MIP4突变体基因在pRSETA融合表达载体中获得可溶性表达和纯化 ,生物学活性实验表明融合的pRSET Met MIP4蛋白具有拮抗嗜酸粒细胞的趋化作用的活性 。AIM: To investigate the activity of MIP4 mutant (Met MIP4) in blocking eosinophils chemotaxis in vitro by expressing Met MIP4 in E. coli and purifying it. METHODS: The gene of Met MIP4 was inserted into the expression vector pRSET A and pRSET Met MIP4 fusion protein was expressed by IPTG induction. After E. coli . was lysed by ultrasonic wave, the solubility of the fusion protein was determined by SDS PAGE gel electrophoresis. Protein in supernatant was purified by Ni 2+ NTA agarose beads. The activity of pRSET Met MIP4 protein in inhibiting eosinophils chemotaxis was determined with 24 well chemotaxis plate filter. RESULTS: The pRSET Met MIP4 fusion protein was expressed in E. coli . The solubility of the fusion protein reached 80% and the purity of the fusion protein was 87% by Ni 2+ NTA agarose beads. Eosinophil was purified from asthmatic blood and Eosinophil purity was 90%. Investigation of biological function of the fusion protein showed that the fusion protein inhibited the eosinophils chemotaxis. CONCLUSION: The pRSET Met MIP4 protein has been correctly expressed in E. coli and the fusion protein is soluble. Study of the biological function of the fusion protein indicates that the fusion protein can inhibit eosinophils chemotaxis in vitro .

关 键 词：趋化因子 嗜酸粒细胞 基因表达 遗传载体 

分 类 号：Q78[生物学—分子生物学]