目的:构建在大肠杆菌中表达重组人神经型一氧化氮合酶的高表达系统.方法:用PCR方法从cD-NA中扩增目的编码基因,然后将编码基因连接入表达载体pCWori+,将重组的质粒转染入大肠杆菌BL21进行蛋白高表达,经Westem blot确认表达后,进行大量培养,通过层析方法精制此酶,并用吸收光谱法对酶的性质进行测定.结果:从该表达系统中可以获得3 mg/L培养基的高产量的一氧化氮合酶.结论:从该表达系统中可获得大量有活性的人一氧化氮合酶.%AIM:To construct a high-level expression system of recombinant human neuronal nitric oxide synthase(hnNOS)full-length enzyme in Escherichia coli.METHODS:The coding sequence of hnNOS full-length was firstly amplified by PCR,and then ligated into the expression vector pCWori + .The recombinant plasmid was transformed into Escherichia coli BL21 for high-level expression.After having been checked with Western blot,the enzyme was used for large-scale culture and purification.Finally,the property of the enzyme was determined by spectrophotometric method.RESULTS:The constructed expression system could give a yielding of 3 mg/L initial culture.CONCLUSION:The expression system constructed is fully sufficient to express the active human neuronal nitric oxide synthase.
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