关键词: 亚硝酸盐还原酶, 克隆, 基因表达

Abstract:

Abstract:Objective
To clone and express the nitrite reductase gene  (nir)  from Alcaligenes xylosoxidans and detect the enzyme activity,and to explore the  degration characteristics of the nitrite reductase(NiR) on nitrite.Methods The DNA from Alcaligenes xylosoxidans was purified.The primers were designed according to nir sequences in GenBank.The nir gene was amplified by PCR and cloned into expression vector pET-28a.The recombinant plasmid pET-28a-nir was constructed.After the identification of restriction digestion and sequencing,the positive recombinant plasmid was transferred into E.coli BL21(DE3).The recombinant protein was obtained by induction.The cell fermentation was broken with ultrasonic.The crude enzyme was extracted and the enzyme activity was calculated.Results A 1 083 bp DNA fragment was obtained by PCR.The target gene was induced by IPTG with the final concentration of 0.6 mmol?L-1 at 30℃,and the induction time was 5 h.A kind of recombinant protein with the molecular weight of 37 000 was obtained.Under the condition of pH 6.5 and reaction temperature 35℃,the enzyme activity was 51.74 U•mL^-1.Conclusion Nitrite reductase gene is successfully cloned and expressed in E.coli　BL21(DE3).The expression product has enzyme activity.
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Key words: nitrite reductase, clone, gene expression