中文摘要：

研究改造了原有的未能高效表达的重组表达载体pRSET-p450nor,通过PCR方法,去除了P450nor起始密码子ATG上游的59个碱基序列。将真菌细胞色素P450nor基因首先克隆至克隆载体pBluescript II上,然后亚克隆到高效表达载体pET-28上,得到了重组表达载体pET-p450nor。该基因在30℃、0.1mM IPTG诱导下在大肠杆菌中获得高效表达融合蛋白His-P450nor。SDS-PAGE蛋白电泳表明在45kD处出现强特异性带。将大量表达的重组蛋白用Ni-NTA亲和层析柱纯化,得到了单一的目的条带。

英文摘要：

P450nor gene was amplified by PCR using previously constucted pRSET-p450nor as template.59 bp of the upstream sequence of P450nor gene was deleted.The recombinant plasmid pBlue-p450nor was constructed by inserting PCR fragment,i.e.P450nor gene,into cloning vector pBluescript II.Then the P450nor gene from pBlue-p450nor was sub-cloned into the prokaryotic expression vector pET-28 to construct recombinant expression plasmids pET-p450nor.The pET-p450nor was transformed into Escherichia coli BL21.The expression level of recombinant protein His-P450nor was high at 30℃ after 0.1mM IPTG inducing.SDS-PAGE analysis showed a specific band（about 45kD）.It indicated that the target protein was expressed successfully.The over-expressed cytochrome P450nor was purified to electrophoretic homogeneity by the facilitation of metal（Ni2＋） chelate affinity chromatography.A single band,about 45kD,was detected.