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目的原核表达艰难梭状芽孢杆菌(Clostridium difficile,CD)谷氨酸脱氢酶(Glutamate dehydrogenase,GDH),纯化重组蛋白,并检测其酶活性。方法采用PCR法从标准株ATCC BAA-1805中扩增GDH基因,克隆至原核表达载体pET-28a(+)中,构建重组表达质粒pET-28-GDH,转化感受态大肠杆菌Rosetta(DE3),IPTG诱导表达。表达的重组蛋白经SDS-PAGE和Western blot分析,并通过Ni-NAT层析柱分离纯化后,根据脱氢酶酶活测定方法,测定纯化蛋白的酶活性。结果重组表达质粒pET-28-GDH经双酶切及测序,证实构建正确,重组蛋白的相对分子质量约46 000,可与抗His单抗特异性结合,主要以可溶性形式表达。纯化的目的蛋白纯度可达90%,浓度为1.7 mg/ml。纯化蛋白GDH以NADPH和NADP为辅酶,酶活性分别为42.6和63.3 U/L。结论已成功在大肠杆菌中表达了重组CD GDH,纯化的目的蛋白纯度高,具有GDH酶活性,为制备GDH单克隆抗体奠定了基础。
Objective To purify the recombinant protein of Clostridium difficile (CD) glutamate dehydrogenase (GDH) in prokaryotic cells and test its enzymatic activity. Methods The GDH gene was amplified by PCR from the standard strain ATCC BAA-1805 and cloned into prokaryotic expression vector pET-28a (+). The recombinant plasmid pET-28-GDH was transformed into competent E.coli Rosetta (DE3) IPTG induced expression. The expressed recombinant protein was analyzed by SDS-PAGE and Western blot, and purified by Ni-NAT column chromatography. The activity of the purified protein was determined according to the method of determination of dehydrogenase activity. Results The recombinant plasmid pET-28-GDH was confirmed by double enzyme digestion and sequencing. The recombinant plasmid pET-28-GDH was constructed with a molecular weight of about 46,000. It can specifically bind to anti-His monoclonal antibody and mainly expressed in soluble form. Purified target protein purity up to 90%, a concentration of 1.7 mg / ml. The purified protein GDH with NADPH and NADP as coenzyme, enzyme activity were 42.6 and 63.3 U / L. Conclusion The recombinant CD GDH has been successfully expressed in E. coli. The purified protein has high purity and GDH activity, which lays the foundation for the preparation of monoclonal antibody against GDH.