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目的利用GST融合基因表达系统表达幽门螺杆菌Catalase/GST融合蛋白,并利用凝血酶柱上切割GST标签。方法将重组表达质粒Catalase/pGEX-4T-1转化大肠杆菌BL21(DE3)感受态中并用IPTG进行诱导表达,菌体经反复冻融、溶菌酶裂解及超声破菌进行裂解。采用谷胱甘肽琼脂糖树脂Glutathione Sepharose4B对Catalase/GST融合蛋白可溶性表达上清进行纯化,并同时利用凝血酶柱上切割GST标签,用鼠抗Catalase抗体对纯化产物进行Western blot鉴定。结果高效表达出相对分子质量约85kDa的Catalase/GST融合蛋白,以部分可溶性的形式表达,凝血酶柱上成功地切割了GST标签,Catalase蛋白能被鼠抗Catalase单克隆抗体识别。结论成功表达了Catalase/GST融合蛋白并柱上切割了GST标签,为深入研究Catalase的功能奠定了基础。
Objective To express Helicobacter pylori Catalase / GST fusion protein by using GST fusion gene expression system, and to cut the GST tag by using thrombin. Methods The recombinant plasmid pGEX-4T-1 was transformed into E. coli BL21 (DE3) competent cells and induced by IPTG. The cells were lysed by repeated freezing and thawing, lysozyme cleavage and sonication. The glutathione sepharose resin Glutathione Sepharose 4B was used to purify the soluble expression supernatant of Catalase / GST fusion protein. Meanwhile, the GST tag was digested with thrombin and the purified product was identified by Western blot using mouse anti-Catalase antibody. As a result, a catalase / GST fusion protein with a relative molecular mass of about 85 kDa was efficiently expressed in a partially soluble form. The GST tag was successfully cleaved on the thrombin column, and the Catalase protein was recognized by the mouse anti-Catalase monoclonal antibody. Conclusion The Catalase / GST fusion protein was successfully expressed and the GST tag was cleaved on the column, which lays the foundation for further study on the function of Catalase.