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目的 原核表达、纯化小鼠CXC亚家族趋化性细胞因子MTCK1 mPBP并测定其生物学活性。方法 构建原核表达载体pQE 30 MTCK1 mPBP ;在大肠杆菌M15菌株中表达重组蛋白 6xHis MTCK1 mPBP ,利用镍 亚硝胺乙酸 组氨酸 (Ni NTA His)亲和层析法对重组蛋白进行纯化。采用Boyden小室对纯化的MTCK1 mPBP蛋白进行趋化活性分析。结果 通过优化表达和纯化条件 ,获得了重组MTCK1 mPBP蛋白 ,并对大肠杆菌包涵体蛋白进行变性和复性处理。趋化试验表明 ,原核表达的MTCK1 mPBP蛋白对转染有CXCR2受体的 2 93细胞系具有明显的趋化能力 ,并呈现浓度依赖性。结论利用原核表达系统 ,表达并纯化了重组趋化性细胞因子 6xHis MTCK1 mPBP蛋白 ,体外功能试验证实有生物学活性。
Objective To prokaryotic express and purify the mouse CXC subfamily chemokine MTCK1 mPBP and determine its biological activity. Methods The prokaryotic expression vector pQE 30 MTCK1 mPBP was constructed. The recombinant protein 6xHis MTCK1 mPBP was expressed in E. coli M15 and the recombinant protein was purified by Ni NTA His affinity chromatography. Chemotactic activity analysis of the purified MTCK1 mPBP protein was performed in a Boyden chamber. Results The recombinant MTCK1 mPBP protein was obtained by optimizing the expression and purification conditions, and denatured and renatured E. coli inclusion protein. The chemotaxis assay showed that the mPBP protein prokaryotic expressed on CXCR2-transfected 293 cell line has obvious chemotactic ability in a concentration-dependent manner. Conclusion The prokaryotic expression system was used to express and purify the recombinant chemotactic cytokine 6xHis MTCK1 mPBP protein. The in vitro functional tests confirmed its biological activity.