泡菜中植物乳杆菌亚硝酸盐还原酶的克隆表达-《中国调味品》

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Title:: Cloning and Expression of Nitrite Reductase Gene from Lactobacillus plantarum in Pickle

摘要:: 目的：对植物乳杆菌亚硝酸盐还原酶基因(nirS)进行克隆及表达，检测重组酶表达情况及其酶活力。方法：以分离自传统泡菜植物乳杆菌的基因组DNA为模版进行PCR扩增nirS；重组构建TA 克隆质粒pMD 19-T-nirS，并转化到大肠杆菌DH5α中保存；通过双酶切消化将nirS基因连接到pET-32a(+)上，获得重组表达质粒pET-32a(+)-nirS，并将其转入大肠杆菌BL21(DE3)中诱导表达；重组酶经纯化后进行SDS-PAGE电泳检测其表达情况；重组酶经复性后检测其酶活力。结果：扩增得到nirS基因并成功在大肠杆菌BL21(DE3)中表达，所得重组酶以包涵体形式存在，酶活力为2131.5 U/mg。结论：采用基因工程技术获得亚硝酸盐还原酶具有一定的应用前景。

Abstract:: Objective:To clone and express the nitrite reductase gene (nirS) of Lactobacillus plantaum,detect the expression of recombinant enzyme and its enzymeacitivity.Method:Amplicon of the nitrite reductase gene nirS was obtain by PCR with the target of Lactobacillus plantaum genomic DNA isolated from traditional pickle.TA cloning plasmid pMD?19-T-nirS?and expression plasmid pET-32a(+)-nirS were constructed and transformed into E.?coli?DH5α and E.coli BL21(DE3)respectively.NirS was induced to expression,which were then extracted and purified.Recombinase were determined by SDS-PAGE and enzyme activity was detected by spectrophotometry.Result:Amplification of the nirS gene was successfully expressed in E.coli BL21(DE3),the recombinant enzyme exists as an iclusion body,and the enzyme activity is 2131.5 U/mg.Conclusion:Producing of nitrite reductase NiRs used genetic engineering has broad development prospect