艰难梭状芽孢杆菌谷氨酸脱氢酶的原核表达、纯化及其酶活性  被引量:1

Prokaryotic expression,purification and activity of glutamate dehydrogenase of Clostridium difficile

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作  者:杜茜[1] 吴志鹃[1] 蔡雪飞[1] 袁作为[1] 张君[1] 郑建[1] 

机构地区:[1]重庆医科大学感染性疾病分子生物学教育部重点实验室 重庆医科大学附属二院肝病研究所,重庆400016

出  处:《中国生物制品学杂志》2013年第7期927-930,934,共5页Chinese Journal of Biologicals

摘  要: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 express glutamate dehydrogenase(GDH) of Clostridium difficile in prokaryotic cells,purify the expressed product and determine its activity.Methods GDH gene was amplified from DNA of standard strain ATCC BAA-1805 by PCR and cloned into prokaryotic expression vector pET-28a(+).The constructed recombinant plasmid pET-28-GDH was transformed to E.coli Rosetta(DE3) for expression under induction of IPTG.The expressed product was analyzed by SDS-PAGE and Western blot,purified by Ni-NAT chromatography and determined for activity.Results Restriction analysis and sequencing proved that recombinant plasmid pET-28-GDH was constructed correctly.The expressed recombinant protein,with a relative molecular mass of about 46 000,showed specific binding to monoclonal antibody against His and mainly existed in a soluble form.The purified protein reached a purity of more than 90% and a concentration of 1.7 mg / ml,of which the activities using NADPH and NADP as coenzymes were 42.6 and 63.3 U / L respectively.Conclusion Recombinant GDH of C.difficile was successfully expressed in E.coli,which laid a foundation of preparation of mono-clonal antibody against GDH.

关 键 词:艰难梭状芽孢杆菌 谷氨酸脱氢酶 基因表达 纯化 酶活性 

分 类 号:R378.8[医药卫生—病原生物学] Q786[医药卫生—基础医学]

 

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