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Method for kluyveromyces lactis eukaryotic expression of nitrite reductase gene in lactobacillus plantarum

A technology of Lactobacillus plantarum and Kluyveromyces cerevisiae, applied in the field of molecular biology, can solve the problems of many impurities, difficult extraction, and low nitrite reductase protein content, and achieve the effect of high enzyme activity and stable enzyme activity

Pending Publication Date: 2019-05-10
DALIAN UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, nitrite reductase in wild bacteria has the disadvantages of less protein content, more miscellaneous proteins, and difficult extraction.

Method used

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  • Method for kluyveromyces lactis eukaryotic expression of nitrite reductase gene in lactobacillus plantarum
  • Method for kluyveromyces lactis eukaryotic expression of nitrite reductase gene in lactobacillus plantarum
  • Method for kluyveromyces lactis eukaryotic expression of nitrite reductase gene in lactobacillus plantarum

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The preparation of the genomic DNA of plant lactobacillus nitrite reductase comprises the steps:

[0029] 1. PCR primer design

[0030] PCR primers were designed using the DNA of Lactobacillus plantarum that can be found in Genbank:

[0031] The upstream primer (SEQ ID NO.2) is 5-CTCGAGAAAAAGACCATGGCCAA-3 (EcoRI),

[0032] The downstream primer (SEQ ID NO.3) is 5-CTTAAGAATCCTGTTTGGAC-3 (XhoI), and this pair of upstream and downstream primers is artificially synthesized;

[0033] 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:

[0034] (1) Centrifuge 3-4 mL of Lactobacillus plantarum culture at 12,000 rpm for 1 min, discard the supernatant, place the bacterial...

Embodiment 2

[0058] 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:

[0059] The amplified fragment DNA of Lactobacillus plantarum nitrite reductase obtained in Example 1 and the plasmid pKLAC2 were respectively digested with EcoRI and XhoI, and the digested products were ligated overnight at 16°C, and the ligated products were transformed into E.coli DH5α , screening positive clone strains, recombinant expression vector pKLAC2-nir enzyme digestion verification electrophoresis as shown in figure 2 shown.

Embodiment 3

[0061] Electrotransformation of linearized plasmid pKLAC2-nir and screening of transformants

[0062] The plasmid of the positive clone strain obtained in Example 2 was linearized by SacII and then electrotransformed into Kluyveromyceslactis, spread on a YCB plate, and picked a single colony for verification after 3 to 5 days, so as to obtain positive clone transformants.

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Abstract

The invention discloses a method for kluyveromyces lactis eukaryotic expression of a nitrite reductase gene in lactobacillus plantarum. The method has the advantages that a lactobacillus plantarum genome is taken as a template, a specific primer is designed to amplify the sequence of the nitrite reductase gene, enzyme cutting sites are introduced into the two ends of the specific primer respectively and subcloned into an expression vector pKLAC2 to construct a recombinant plasmid, after enzyme cutting linearization, kluyveromyces lactis is electrotransformed, positive clone transformants are screened, finally, seed culture and liquid fermentation culture are carried out, a supernatant of a fermentation liquid is taken, and a nitrite reductase expression product is obtained. The method forthe kluyveromyces lactis eukaryotic expression of the nitrite reductase gene in the lactobacillus plantarum has the advantages that prepared recombinant nitrite reductase has higher enzyme activity, the enzyme activity is stable, a foundation is laid for the industrial production of the nitrite reductase, and the method can be used for the fields of food, medicine and the like.

Description

technical field [0001] The invention relates to eukaryotic expression of nitrite reductase gene, belongs to the technical field of molecular biology, and specifically relates to a Kluyveromyces lactis eukaryotic expression method and expression product of a nitrite reductase gene derived from Lactobacillus plantarum application. Background technique [0002] Nitrite is an important substance in the nitrogen cycle in nature. It is widely found in soil, water and plants. It is a potential carcinogen. It is easy to accumulate in people's daily life. Excessive intake of nitrite can induce methemoglobinemia , Excessive intake of nitrite by pregnant women can lead to fetal malformations. 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 aquaculture, nitrite will reduce the oxygen in the fish body, which will aff...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/06C12N15/81
Inventor 张庆芳李美玉迟乃玉王晓辉于爽胡善松
Owner DALIAN UNIVERSITY
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