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Lactobacillus plantarum nitrite reductase gene, protein coded by gene and applications of protein

A technology of Lactobacillus plantarum and nitrite, applied in the field of high-efficiency affinity chromatography purification, the field of recombinant vectors of nitrite reductase gene of Lactobacillus plantarum, can solve the problem of not being able to well identify whether the expression of nitrite reductase is successful, The target product nitrite reductase cannot be obtained in large quantities, and the separation and purification work is complicated.

Pending Publication Date: 2019-04-12
DALIAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At home and abroad, there are few reports on the extraction of nitrite reductase from lactic acid bacteria and its large-scale expression. In the prior art, simple mechanical crushing and centrifugation methods are used to collect crude enzymes. Since the crude enzyme solution contains a large amount of foreign proteins, the subsequent separation The purification and purification work is complicated, and the target product nitrite reductase cannot be obtained in large quantities
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. Since the microbial engineering bacteria E.coli BL21, E.coli DE 3 and JM105 can all express nitrite reductase before transformation, Through the method of the above invention, it is impossible to well identify whether the expression of nitrite reductase is successful

Method used

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  • Lactobacillus plantarum nitrite reductase gene, protein coded by gene and applications of protein
  • Lactobacillus plantarum nitrite reductase gene, protein coded by gene and applications of protein
  • Lactobacillus plantarum nitrite reductase gene, protein coded by gene and applications of protein

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Experimental program
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Effect test

Embodiment 1

[0034] The preparation of the genomic DNA of plant lactobacillus nitrite reductase comprises the steps:

[0035] 1. PCR primer design

[0036] PCR primers were designed with the DNA of Lactobacillus nitrite reductase in Genbank:

[0037] The upstream primer (SEQ ID NO.3) is 5-CTCGAGAAAAAGACCATGGCCAA-3 (NcoI),

[0038] The downstream primer (SEQ ID NO.4) is 5-CTTAAGAATCCTGTTTGGAC-3 (XhoI), and this pair of upstream and downstream primers is artificially synthesized;

[0039] 2. Extraction of genomic DNA 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. ), wherein the solution is the solution in the kit, and the specific steps are:

[0040] (1) Centrifuge 3-4 mL of Lactobacillus plantarum culture at 12,000 rpm for 1 min, discard the supernatant, place the bacterial cell pellet...

Embodiment 2

[0064] Containing the construction of the recombinant expression vector of the genomic DNA of the Lactobacillus plantarum NiR of the PCR expansion of the gained of embodiment 1, the steps are as follows:

[0065] The amplified fragment DNA of Lactobacillus plantarum nitrite reductase obtained in Example 1 and the plasmid pET-28a(+) were digested with NcoI and XhoI respectively, and the reaction conditions were overnight at 37°C for 14-16h, and then respectively Fragmented DNA and plasmid DNA were recovered.

[0066] Ligate the above two fragments, and refrigerate overnight at 4°C for 14-16 hours to obtain the ligation product. The ligation reaction system is as follows: 19 μL of pET28a(+) was recovered, 2.5 μL of the target gene fragment was recovered, and 1 μL of T4DNA was ligated. T4 DNA ligase buffer 2.5 μL;

[0067] Then transform the above ligation product into E.coli DH5α cells to obtain the recombinant plasmid pET28a(+)-nir;

[0068] The obtained recombinant plasmid p...

Embodiment 3

[0070] The expression vector pET28a(+)-nir plasmid constructed in Example 2 was transferred into E.coli BL21 competent cells to obtain recombinant Escherichia coli for induced expression. The specific steps are as follows:

[0071] Inoculate the constructed expression strains into LB / Kan liquid medium, culture overnight at 37°C with shaking for 14 hours, then inoculate 1% of the bacterial liquid into Erlenmeyer flasks containing 100mL LB / Kan liquid medium, shake at 37°C After culturing for 4 hours (the OD600 of the bacterial solution reached 0.4-0.6), IPTG was added to make the final concentration 1 mmol / L; at the same time, empty vector and no induction were used as controls. Centrifuge the bacteria solution at 8000r / min for 10min, discard the supernatant, wash the precipitated bacteria twice with PBS buffer (pH7.4), and resuspend the washed bacteria in 10mL pre-cooled PBS buffer (pH7.4 ), sonicate for 10 min, each time for 3 s, with an interval of 5 s, and a power of 200 W. ...

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Abstract

The invention discloses a lactobacillus plantarum nitrite reductase gene, a protein coded by the gene and applications of the protein, and belongs to the field of bioengineering. In the present invention, the nitrite reductase gene in lactobacillus plantarum is cloned by a PCR technology and induced to express in escherichia coli, and affinity chromatography purification is utilized to obtain a high-purity recombinant protein. The high-purity protein purified by the method of the invention can effectively degrade nitrite in food, and the structure and properties of the protein can be studied.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant vector of plantar lactobacillus nitrite reductase gene, a host transformed with the vector, and a large amount of expression of nitrite reductase and nitrite reductase using the transformant high-efficiency affinity chromatography purification method. Background technique [0002] Nitrite is a potential carcinogen, which is easy to accumulate in the process of vegetable fermentation, bringing potential product safety problems to the product, and excessive intake of nitrite can induce methemoglobinemia, so strictly control the sub- The nitrate 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 food safety problems of excessive ...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/06C12N15/70C12N1/21C12R1/19
CPCC12N9/0044C12N15/70C12Y107/01004
Inventor 张庆芳李美玉迟乃玉王晓辉于爽胡善松
Owner DALIAN UNIV
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