亚硝酸盐还原酶基因工程菌的构建
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
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