福氏2a志贺菌MarA蛋白纯化复性及活性检测

Renaturation,purification and identification for biological activity of shigella flexneri 2a MarA protein

目的纯化、复性志贺菌MarA蛋白并分析其生物学活性。方法将构建好的BL21大肠埃希菌工程菌经异丙基-β-D-硫代-乳糖苷(IPTG)诱导,His-tag形式表达MarA融合蛋白(约21ku),采用超声波破碎细菌,提取包涵体用高浓度尿素裂解,经镍柱亲和层析纯化,纯化后的蛋白质在小分子保护剂及还原型谷胱甘肽(GSH)/氧化型谷胱甘肽(GSSG)的存在下对融合蛋白进行复性。用凝胶薄层扫描法测纯度。结果从融合蛋白包涵体中获得纯度达90%以上的目的蛋白,免疫蛋白印迹试验结果显示,复性后的MarA融合蛋白能与抗福氏志贺菌抗体发生特异性结合。结论成功建立有效纯化融合蛋白包涵体His-MarA的方法。

Objective To purify and renature the MarA protein of shigella flexneri 2a and study its biological activity. Methods His-tag MarA fusion protein was expressed in Escherichia coli as inclusion body with IPTG induction. Cells were then harvested, sonicated and centrifuged, and the inclusion bodies were isolated and purified by Ni^2＋ -high performance af- finity chromatography,and refolded in the presence of GSH/GSSH. The purity of His-MarA was identified by thin-layer scanning analysis. Results The purity of the MarA protein was more than 90% after Ni^2＋ -high performance affinity chromatography. Conclusion The method of fusion protein His-MarA purification from the inclusion body was developed for further study on MarA. Western blotting showed pecific Ag-Ab binding band between the antiserum and the MarA fusion proteins after the protein was purified and renatured.