【摘要】 利用PCR技术从大肠杆菌BL21中获取酒石酸脱氢酶β亚基基因(TtdB),并将之克隆到质粒pUC18上,转化大肠杆菌DH5α细胞.经测序证明序列无误后,将之与表达载体pTrcHisC连接,在大肠杆菌BL21中经IPTG诱导表达,通过SDS-PAGE和双波长扫描分析,确定酒石酸脱氢酶β亚基在大肠杆菌中表达时以包涵体形式存在.目的蛋白用TALON金属亲和树脂纯化,通过分步透析逐步去除变性剂的方法复性.复性产率可达90%.更多还原

【Abstract】 By using the Polymerase Chain Reaction(PCR) technique,the 600?bp DNA fragment of Tartrate Dehydratase Beta Subunit gene(TtdB) was amplified from the Escherichia coli BL21 cell.The fragment was inserted into plasmid pUC18 by the sites of two restriction enzyme EcoR Ⅰ and Pst Ⅰ,and the target gene was confirmed by DNA sequencing.Then the target gene was subcloned into the expression plasmid pTrcHisC.The recombinant protein expression was induced by IPTG in Escherichia coli BL21,and the expression was associated with formation of inclusion bodies with SDS-PAGE electrophoresis.The recombinant protein was purified by immobilized metal affinity chromatography(IMAC),and refolded by dialysis which resulted in a recovery rate exceeding 80%.更多还原