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Lactobacillus plantarum nitrite reductase gene, protein encoded by lactobacillus plantarum nitrite reductase gene and preparation method of protein

A technology of Lactobacillus plantarum and nitrite, which is applied in the field of high-efficiency affinity chromatography purification, can solve the problem of not being able to identify whether the expression of nitrite reductase is successful, or obtaining a large amount of target product nitrite reductase, separation and Complicated purification work and other issues

Inactive Publication Date: 2014-11-19
SOUTH CHINA UNIV OF TECH
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  • Claims
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Problems solved by technology

[0003] At home and abroad, there are few reports on the extraction of nitrite reductase from lactic acid bacteria and large-scale expression. The Chinese invention patent application number 201110207974.3 discloses "a production method of lactic acid bacteria nitrite reductase", which uses simple mechanical crushing and The crude enzyme is collected by centrifugation. Since the crude enzyme solution contains a large amount of foreign proteins, the subsequent separation and purification work is complicated, and the target product nitrite reductase cannot be obtained in large quantities. In addition, the medium used for enzyme production in this method The ingredients contain tomato juice. Due to the red color of tomatoes, red pigment is inevitably contained in the final nitrite reductase, which affects the subsequent purification work.
The Chinese invention patent with the application number 201210037774.2 discloses "a nitrite reductase gene of Lactobacillus plantarum and its encoded protein and its application", in which the main basis for verifying the successful expression of nitrite reductase is the whole The results of protein SDS-PAGE electrophoresis did not go further into the separation and purification, enzyme activity and application of the recombinant protein, because the microbial engineering bacteria E.coli BL 21, E.coli DE 3 and JM 105 can all express nitrite reduction before transformation Enzyme, by the method of the present invention, can not well distinguish whether the expression of nitrite reductase is successful

Method used

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  • Lactobacillus plantarum nitrite reductase gene, protein encoded by lactobacillus plantarum nitrite reductase gene and preparation method of protein
  • Lactobacillus plantarum nitrite reductase gene, protein encoded by lactobacillus plantarum nitrite reductase gene and preparation method of protein
  • Lactobacillus plantarum nitrite reductase gene, protein encoded by lactobacillus plantarum nitrite reductase gene and preparation method of protein

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Embodiment 1

[0032] The preparation of the genomic DNA of plant lactobacillus nitrite reductase comprises the steps:

[0033] 1. PCR primer design

[0034] by PCR primers were designed from the DNA in: the upstream primer is 5'-CCGGAATTCATGAGTCAAAGCTTTATGGCA-3' (EcoRI), such as SEQ ID NO.2, and the downstream primer is 5'-CCGCTCGAGTTAATTCCGTACACTGTTTG-3' (Xhol), such as SEQ ID NO.3, And artificially synthesize this pair of upstream and downstream primers;

[0035] 2. Genomic DNA extraction of Lactobacillus plantarum nitrite reductase (bacterial genomic DNA small purification kit TaKaRa MiniBEST Bacterial Genomic DNA Extraction Kit Ver.2.0, a product of Takara Biotechnology Company, purchased from Guangzhou Ruizhen Biotechnology Co., Ltd. company), wherein the solutions are all solutions in the kit, and the specific steps are: (1) 3-4 mL of Lactobacillus plantarum (Lactobacillus plantarum) DMDL 9010 culture was centrifuged at 12000rp for 1 min to discard the supernatant, and the bacteria...

Embodiment 2

[0058] Containing the construction of the recombinant expression vector of the genomic DNA of the PCR-expanded Lactobacillus plantarum Nir of the gained of embodiment 1, the steps are as follows:

[0059] The amplified DNA of the PCR-extended Lactobacillus plantarum nitrite reductase obtained in Example 1 and the plasmid pET-32a (+) (a product of Novagen, USA, purchased from Shanghai Jiran Biotechnology Co., Ltd.) were subjected to EcoRI respectively. , XhoI enzyme digestion, and the reaction conditions were overnight at 37°C for 14-16 hours, and then the fragment DNA and plasmid DNA were recovered respectively.

[0060] Ligate the above two fragments and react overnight in a refrigerator at 4°C for 14-16 hours to obtain the ligation product. The ligation reaction system is: recover 19 μL of pET-32a(+), recover 2.5 μL of the target gene fragment, and T4 DNA ligation Enzyme 1 μL, T4 DNA ligase buffer 2.5 μL;

[0061] Then transform the above ligation product into E.coli DH5α c...

Embodiment 3

[0067] The expression vector pET-32a(+)-Nir plasmid constructed in Example 2 is transferred into E.coli DH5α competent cells to obtain recombinant Escherichia coli, and its specific steps are as follows:

[0068] (1) Add 5 μL of the ligation product to 200 μL of E.coli DH5α competent cell suspension, mix gently, and place on ice for 30 minutes;

[0069] (2) Heat-shock in a water bath at 42°C for 90s, quickly remove and cool on ice for 30min;

[0070] (3) Add 400 μL of LB liquid medium, mix well, and then bathe in water at 37°C for 1 hour to restore the bacteria to normal growth state;

[0071] (4) Centrifuge at 4000rpm for 10 minutes, discard 400 μL of supernatant, take the remaining 200 μL of bacterial solution and evenly spread it on LB solid medium, place it face-up at 37°C for 30 minutes, and culture it upside down for 8-12 hours after the bacterial solution is completely absorbed by the medium;

[0072] (5) Picking a single clone and identifying it by PCR, the positive c...

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Abstract

The invention discloses a lactobacillus plantarum nitrite reductase gene, protein encoded by the lactobacillus plantarum nitrite reductase gene and a preparation method of the protein. A nucleotide sequence of the lactobacillus plantarum nitrite reductase gene is as shown in SEQ ID NO.1. A gene sequence shown in the SEQ ID NO.1 is cloned to a prokaryotic expression vector pET-32a(+); inducible expression is carried out in recombinant escherichia coli; high-purity recombinant protein is obtained by affinity chromatography purification. By using the high-purity protein purified by the method disclosed by the invention, nitrite can be effectively degraded, an enzymic preparation can be produced, the nitrite in food can be effectively degraded, and the protein can also be applied to preparation of a lactobacillus plantarum nitrite reductase antibody as an antigen, and can be applied to research of structure and property of the antibody.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant vector containing the gene of Lactobacillus plantarum nitrite reductase, a host transformed with the vector, and a large amount of expression of nitrite reductase and nitrite reduction by using the transformant High-efficiency affinity chromatography purification method for enzymes. Background technique [0002] Nitrite is a potential carcinogen, which is easy to accumulate in the process of vegetable fermentation, bringing potential food safety problems to the product, and excessive intake of nitrite can induce methemoglobinemia, so strict control of nitrite in food The salt content is very important. Many studies have shown that nitrite is the precursor of nitrosamines, which are strong carcinogens that can induce various cancers in the digestive system, such as gastric cancer, intestinal cancer and liver cancer. In view of the potential ...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/06C12N15/70C12R1/25
Inventor 刘冬梅王盼费永涛陈浩吴晖唐语谦肖性龙
Owner SOUTH CHINA UNIV OF TECH
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