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目的表达幽门螺杆菌Lpp20-GST融合蛋白,获取切除GST标签的重组蛋白。方法采用异丙基硫代半乳糖苷(IPTG)诱导重组表达质粒Lpp20/pGEX-4T-1在大肠埃希菌BL21(DE3)中表达,收集菌体并采用反复冻融、溶菌酶裂解及超声破菌3种细胞破碎方法,表达产物在谷胱甘肽琼脂糖树脂4B柱上纯化,利用凝血酶切除GST标签,用鼠抗Lpp20单克隆抗体进行纯化产物western blot鉴定。结果高效表达出Lpp20-GST融合蛋白,相对分子质量约为4.5 kDa,产物以部分可溶性形式表达,凝血酶成功切除GST标签,纯化产物能被鼠抗Lpp20单克隆抗体识别。结论凝血酶柱上切除GST标签获得目的蛋白。
Objective To express Helicobacter pylori Lpp20-GST fusion protein and obtain GST-tagged recombinant protein. Methods Recombinant plasmid Lpp20 / pGEX-4T-1 was induced by IPTG in Escherichia coli BL21 (DE3). The cells were harvested and lysed by repeated freeze-thaw, lysozyme and ultrasonography The cells were purified by glutathione Sepharose 4B column, and the GST tag was removed by thrombin. The purified product was identified by western blot with mouse anti-Lpp20 monoclonal antibody. The results showed that the Lpp20-GST fusion protein was highly expressed. The relative molecular mass was about 4.5 kDa. The product was expressed in partially soluble form. The GST tag was successfully excised by thrombin and the purified product was recognized by mouse anti-Lpp20 monoclonal antibody. Conclusion The GST tag was excised from the thrombin column to obtain the target protein.