四川泡菜中植物乳杆菌亚硝酸盐还原酶基因克隆与表达  

Expression of nitrite reductase gene from Lactobacillus plantarum from Sichuan pao cai

作　　者：董维[1] 陈安均[1] 韩国全[1] 蒲彪[1] 罗惟[1] 侯晓艳[1] 

机构地区：[1]四川农业大学食品学院,四川雅安625014

出　　处：《食品工业科技》2015年第15期143-147,共5页Science and Technology of Food Industry

基　　金：国家自然科学基金项目(31171726);"十二五"国家科技支撑计划项目(2012BAD31B04)

摘　　要：目的:对植物乳杆菌中亚硝酸盐还原酶基因进行克隆并表达,并测定酶的活力。方法:根据Gen Bank中亚硝酸盐还原酶(NIR)基因序列,设计引物。提取植物乳杆菌的DNA,采用PCR技术扩增nir基因,TA克隆构建获得重组质粒p MD19-nir,分析其核苷酸序列同源性。通过双酶切连接到表达载体p ET-32a(+)中,获得重组表达载体p ET-32a-nir,构建成功后转化到E.coli BL(DE3)中进行表达研究。经IPTG诱导表达,得重组蛋白,超声波破碎,提取粗酶液,测定酶活力。结果:克隆的目的基因序列长度为1638 bp。获得重组蛋白分子量为80 ku,酶活力为4.2 U/mg。结论:成功克隆了植物乳杆菌中的亚硝酸盐还原酶基因,成功的导入到E.coli BL(DE3)中,表达产物有酶活。Objective： To clone and the nitrite reductase gene （nir）from Lactobacillus plantarum and detect the enzyme activity. Methods, The primers were designed according to nir sequence in GenBank. The DNA from Lactobacillus plantarum which came from Sichuan pao cai was extracted.The nir gene was amplified by PCR, it was cloned into T vector pMDlg.The recombinant plasmid was constructed for analyzing the sequence homology. The plasmid by double enzyme digestion was connected to the expression plasmid pET-32a （ ＋ ）. It was constructed the recombinant plasmid pET-32a-nir.The plamid was transformed into Ecoli BL （DE3）in order to express the research.The new host bacteria grew with IPTG induction,the cell was broken with ultrasonic. The crude enzyme was extracted and the enzyme activity was measured.Result： The purpose of cloned gene length was 1638 bp.A kind of recombinant protein with the molecular weight of 80 ku was obtained.The enzyme activity was 4.2 U/mg.Conclution. The nitrite reductase gene from Lactobacillus plantarum was successfully cloned and transformed in E coli BL（DE3） ,the expression product had enzyme activity.

关 键 词：亚硝酸盐还原酶 克隆 基因表达 植物乳杆菌 

分 类 号：TS201.3[轻工技术与工程—食品科学]