泡菜中植物乳杆菌亚硝酸盐还原酶的克隆表达

Cloning and Expression of Nitrite Reductase from Lactobacillus plantarum in Pickles

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pMD19-T-nirS,并转化到大肠杆菌DH5a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2131.5U/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。

Objective:To clone and express the nitrite reductase gene(nirS)of Lactobacillus plantarum,and to detect the expression situation of the recombinant enzyme and its activity.Methods:nirS is amplified by PCR using genomic DNA isolated from Lactobacillus plantarum as template,the TA cloning plasmid pMD 19-T-nirS is reconstructed and transformed into E.coli DH5a for storage,the nirS gene is linked to pET-32a(+)with double enzyme digestion,and the recombinant expression plasmid pET-32a(+)-nirS is obtained,and transformed into E.coli BL21(DE3)to induce the expression of the recombinant enzyme.SDS-PAGE electrophoresis is used to detect the expression of recombinant enzyme after purification,and the activity of recombinant enzyme is detected after renaturation.Results:The nirS gene is amplified and successfully expressed in E.coli BL21(DE3).The recombinant enzyme exists in the form of inclusion body and its activity is 2131.5 U/mg.Conclusion:The nitrite reductase obtained by genetic engineering technology has a certain application prospect.