植物乳杆菌DMDL 9010中亚硝酸盐还原酶的基因克隆、表达和纯化

研究了植物乳杆菌DMDL 9010亚硝酸盐还原酶基因克隆、表达及表达产物的纯化。首先以植物乳杆菌DMDL 9010的DNA为模板，利用PCR扩增亚硝酸盐还原酶基因，扩增后nir编码序列大小为1638 bp，再将其编码序列克隆到原核表达载体pET-32a(+)上，转化到感受态细胞DH5α，成功构建重组质粒pET-32a(+)-nir。将重组质粒转化到大肠杆菌表达菌株E. coli BL21中，在IPTG浓度为1 mmol/L和诱导温度为25℃的条件下诱导4 h后诱导表达NIR蛋白，经诱导后的工程菌能将50 ?g/mL的亚硝酸盐降解90 %以上，利用亲和层析柱Ni sepharose 6 Fast flow进行纯化得到重组蛋白，该重组蛋白经SDS-PAGE电泳后的分子量约为60 KDa。总之，经由植物乳杆菌DMDL 9010克隆出亚硝酸还原酶基因，并成功构建了重组质粒pET-32a(+)-nir，利用基因工程方法得到的工程菌能有效降解亚硝酸盐，并能利用亲和层析得到高纯度的NIR。
The nitrate reductase gene (nir), encoding the nitrite reductase enzyme (NiR) in Lactobacillus plantarum DMDL 9010, was cloned, and the expressed product was purified. The nir fragment was amplified by polymerase chain reaction (PCR) using L. plantarum DMDL 9010 DNA as template. Sequencing studies revealed that the nir fragment was 1638 bp in size. The gene was cloned into plasmid pET-32a(+) and was transferred into competent Escherichia coli DH5α cells in order to construct the pET-32a(+)-nir-DH5α recombinant plasmid. pET-32a(+)-nir was transferred into the expression strain E. coli BL21 to construct the denitrification recombinant bacterium, pET-32a(+)-nir-BL21. The recombinant bacterium was induced by 1 mmol/L IPTG to produce NiR protein when the induction temperature was maintained at 30 ℃ for 4 h, and more than 90% of NaNO2 (50 ?g/ml) was degraded. High-purity NiR was obtained by His-Tag nickel affinity chromatography (Ni Sepharose 6 Fast Flow), and the molecular weight of NiR, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was approximately 60 kDa. In summary, nir from L. plantarum DMDL 9010 was successfully cloned, and the recombinant bacterium pET-32a (+)-nir-BL21 effectively degraded nitrite. High-purity NiR can be obtained by affinity chromatography.