中国病理生理杂志
中國病理生理雜誌
중국병리생리잡지
CHINESE JOURNAL OF PATHOPHYSIOLOGY
2004年
6期
1051-1056
,共6页
付捷%周羽竝%赵迎社%管志文%井柳尧
付捷%週羽竝%趙迎社%管誌文%井柳堯
부첩%주우병%조영사%관지문%정류요
一氧化氮合酶%大肠杆菌%基因表达
一氧化氮閤酶%大腸桿菌%基因錶達
일양화담합매%대장간균%기인표체
Nitric-oxide synthase%Escherichia coli%Gene expression
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.
目的:構建在大腸桿菌中錶達重組人神經型一氧化氮閤酶的高錶達繫統.方法:用PCR方法從cD-NA中擴增目的編碼基因,然後將編碼基因連接入錶達載體pCWori+,將重組的質粒轉染入大腸桿菌BL21進行蛋白高錶達,經Westem blot確認錶達後,進行大量培養,通過層析方法精製此酶,併用吸收光譜法對酶的性質進行測定.結果:從該錶達繫統中可以穫得3 mg/L培養基的高產量的一氧化氮閤酶.結論:從該錶達繫統中可穫得大量有活性的人一氧化氮閤酶.
목적:구건재대장간균중표체중조인신경형일양화담합매적고표체계통.방법:용PCR방법종cD-NA중확증목적편마기인,연후장편마기인련접입표체재체pCWori+,장중조적질립전염입대장간균BL21진행단백고표체,경Westem blot학인표체후,진행대량배양,통과층석방법정제차매,병용흡수광보법대매적성질진행측정.결과:종해표체계통중가이획득3 mg/L배양기적고산량적일양화담합매.결론:종해표체계통중가획득대량유활성적인일양화담합매.
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.