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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 Shigella MarA protein and analyze its biological activity. Methods The constructed Escherichia coli BL21 strain was induced by isopropyl-β-D-thiogalactoside (IPTG), and the HisA tagged HisA fusion protein (about 21 ku) was used. The bacteria were disrupted by sonication and extracted Inclusion bodies were lysed with high concentration of urea and purified by nickel affinity chromatography. The purified protein was purified in the presence of small molecule protectant and reduced glutathione (GSH) / oxidized glutathione (GSSG) Fusion protein refolding. Purity by gel thin layer scanning. Results The target protein with the purity of over 90% was obtained from the inclusion body of fusion protein. The results of Western blotting showed that the renaturation of MarA fusion protein could specifically bind to the anti-Shigella flexneri antibody. Conclusion The method of efficiently purifying His-MarA fusion protein was successfully established.