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摘要：亚硝酸盐是潜在的致癌物质，它与蛋白质代谢的中间产物仲胺反应，生成亚硝胺，亚硝胺具有一定的致癌性。亚硝酸盐在各类食品中分布极为广泛，因此控制其浓度对保障食品安全非常重要。乳酸菌具有降解亚硝酸盐的能力。根据已知亚硝酸盐还原酶蛋白序列的保守序列，于蛋白基因序列分析相关网站查找到乳酸菌基因组上的亚硝酸盐还原酶疑似基因。根据大肠杆菌等亚硝酸盐还原酶NIRs的ORF序列设计引物，通过PCR 扩增的方法获得了NIRs基因的完整序列。PCR 产物经双酶切后插入到pET-28a(+)载体中，成功的构建了重组质粒pET-28a(+)-NIRs。构建好的表达载体pET-28a(+)-NIRs在大肠杆菌中经诱导后，高效表达NIRs 蛋白。克隆出基因序列，构建表达载体pET-32a(+)-NiRs。将构建成功的质粒转入TG1表达菌感受态细胞。经诱导表达后的目的蛋白，按照细菌亚硝酸盐还原酶总活性检测试剂盒说明进行酶活测定。实验结果发现，乳酸菌体内的一个hypothetical protein（HP）编码基因表达的蛋白具有显著降解亚硝酸盐的能力。7334
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关键词：乳酸菌；亚硝酸盐还原酶；酶活
Construction of nitrite reductase gene engineering bacteria
Abstract：Nitrite is someting that is potentinal carcinogenic,it reaction on secondary amines with the intermediate of protein metabolism,and generate nitrosamines,nitrosamines is carcinogenic. Nitrite is widely used in various types of food,therfore it's important to controlling the concentration of nitrite in food safety.
Lactobacillus have the ability to degrade nitrite. According to the conserved amino acid sequences of the known four types of nitrite reductase from Lactobacillus plantarum,we have used the protein analysis software and bioinformatics tools to find out the products of genes in Lactobacillus which might be able to degrade nitrite. The genes were cloned from Lactobacillus genome DNA by PCR and we successfully constructed the prokaryotic expression vectors pET-32a(+)-NiRs. Then the recombinant plasmids were transformed into E.coli TG1.
The activity of the proteins were detected in accordance with the total bacterial nitrite reductase activity assay kit instructions. The results showed that hypothetical protein（HP） of Lactobacillus could degrade nitrite. Then we designed the primers for NrfA gene from E.coli. The E.coli NrfA gene was cloned from the E.coli genome DNA by PCR and inserted into the vector pET-28a(+).The prokaryotic expression vector pET-28a(+)-NrfA was constructed successfully. E.coli NrfA protein was expressed by IPTG induction and purified.
Key Words: Lactobacillus; Nitrite Reductase; activity
目 录
毕业设计（论文）1
1绪论 1
1.1 亚硝酸盐还原酶简介.1
1.2 亚硝酸盐的危害与来源.2
1.2.1 亚硝酸盐的危害..2
1.2.2 亚硝酸盐的来源..3
1.3 亚硝酸盐降解.4
1.3.1 一些蔬菜和水果消除亚硝酸盐的研究..4
1.3.2 红曲色素降低亚硝酸盐的研究..4
1.3.3 生物降解法..4
1.3.4茶多酚结合亚硝胺...5
1.3.5酶法处理降解亚硝酸盐 .5
1.4 亚硝酸盐还原酶的性质与研究现状.....5
1.4.1 亚硝酸盐还原酶的性质..5
1.4.2亚硝酸盐还原酶的研究现状...6
1.5实验设计方案..7
1.5.1实验的目的及意义...7
1.5.2 研究内容..7
2实验材料及方法.8
2.1 材料与试剂.....8
2.1.1 材料..8
2.1.2 试剂..8
2.1.3培养基...8
2.1.4 主要试剂盒..8
2.2 实验方法.....8
2.2.1 构建重组表达质粒..8
2.2.2疑似基因的 PCR扩增....9
2.2.3凝胶回收、纯化PCR产物..10
2.2.4 pET-32a(+)载体和PCR产物双酶切..11
2.2.5连接反应.....12
2.2.6 E.coli TG1感受态细胞的制备...12
2.2.7连接产物的转化.....13