鼠CCL5蛋白的原核表达与纯化（英文）

Expression and Purification of Mouse CCL5 Protein in Escherichia coli

趋化因子(CCL5)是由正常T细胞分泌的典型的具有趋化活性的细胞因子,分子量8kD,属于CC型趋化因子的β家族成员之一,并调控T淋巴细胞的活性和分泌能力.本研究旨在克隆小鼠趋化因子CCL5基因并在原核表达系统中实现表达,获得与His标签融合的重组趋化因子mCCL5蛋白,并测定其在体外的趋化活性.将趋化因子CCL5基因扩增并插入pET-28a原核表达载体,转化至Rosetta感受态细胞中.重组mCCL5蛋白以IPTG诱导表达,再以Ni-NTA纯化系统进行纯化.Transwell实验检测重组CCL5蛋白的趋化活性.结果显示pET-28a-mCCL5原核表达载体成功构建,mCCL5蛋白在细菌中大量表达并主要以包涵体的形式存在.表达产物以SDS-PAGE和免疫印迹鉴定,Transwell实验证实趋化因子能够显著促进巨噬细胞RAW264.7细胞的迁移.带有His标签重组鼠趋化因子CCL5的成功表达,为下一步CCL5抑制剂的筛选奠定基础.

Chemokine CCL5(RANTES)is a typical chemotactic cytokine,expressed and secreted by normal T cells,which is a small molecular protein with a molecular weight of 8 kilodaltons.It belongs to theβfamily of CC-chemokines,and regulates the activity and secretion of T-lymphocytes.The aim of this study was to clone the mouse chemokine CCL5 gene(mCCL5)and express it in a prokaryotic expression system,obtain a recombi-nant chemokine CCL5 protein fused with his-tag and test its corresponding chemotactic activity in vitro.The chemokine CCL5 gene was amplified by the polymerase chain reaction(PCR),inserted into the prokaryotic expression vector pET-28a,and were transformed in the competent Rosetta cells by heat shock treatment.Recombinant CCL5 was induced by isopropyl-β-D-thiogalactopyranoside(IPTG),and then purified by the Ni-NTA purification system.Transwell assay was used to detect the chemotaxis of recombinant chemokine CCL5 protein to macrophage RAW264.7 cells.The prokaryotic expression vector of pET-28a-mCCL5 was successfully constructed.The chemokine CCL5 of the mouse was expressed in large amounts in bacterial and mainly in the form of inclusion bodies.The expressed product was identified as recombinant chemokine CCL5 protein by SDS polyacrylamide gel electrophoresis(PAGE)and Western blot.The results of transwell assay showed that chemokine CCL5 significantly improves the mobility of phagocytes in macrophage cells RAW264.7.Recombinant mouse chemokine CCL5 with a his tag could be expressed in in E.coli.and thus provides the basic material for screening of mouse chemokine CCL5 inhibitors.