小鼠CCL2蛋白原核表达及纯化分析

Prokaryotic expression and purification of mouse CCL2 protein

旨在构建小鼠CCL2原核表达载体并诱导表达纯化CCL2重组蛋白,初步检测CCL2蛋白对白血病细胞生物学功能的影响。通过PCR扩增小鼠CCL2基因,用LIC(ligation-independent cloning)法构建pGEX-4T-CCL2原核表达载体。将重组质粒转化大肠杆菌BL21(DE3),在不同温度及IPTG浓度下诱导表达,采用亲和层析法纯化表达产物,进行SDS-PAGE电泳和Western Blot鉴定,将纯化的重组蛋白处理C1498细胞检测其对白血病细胞黏附及迁移的影响。结果显示,成功构建了pGEX-4T-CCL2原核表达载体,通过优化表达条件,确定20℃、0.1 mmol/L IPTG诱导6 h能够表达目的蛋白。利用谷胱甘肽琼脂糖凝胶纯化出了具有生物学功能的融合蛋白,且CCL2蛋白能够促进白血病细胞黏附及迁移,为CCL2在白血病功能及机制的深入研究奠定基础。

This work aimed to construct a prokaryotic expression for mouse gene CCL2,express and purify recombinant CCL2 protein,and detect the adhesion of leukemia cells.The mouse gene CCL2 was amplified by PCR and cloned into the prokaryotic expression vector pGEX-4T-1 by ligation-independent cloning.The recombinant plasmid pGEX-4T-CCL2 was transformed into Escherichia coli BL21(DE3)and the recombinant protein was purified by affinity chromatography.The purified protein was identified by SDS-PAGE and Western Blot.Finally,the effect of CCL2 on C1498 cell adhesion and migration was detected.Results showed that the recombinant vector pGEX-4T-CCL2 was successfully constructed,and GST-CCL2 was successfully induced by 0.1 mmol/L IPTG at 20℃for 6 h;recombinant protein with biological function was prepared,and CCl2 could promote leukemia cell adhesion and migration.This work would provide a foundation for further study of CCL2 in leukemia.