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Lactococcus lactis food-level double-screening marker secretory expression vector, engineering strain containing vector and construction method and application of vector

A technology of Lactococcus lactis, secretion expression, applied in the field of engineering strains, to achieve the effect of wide selection and increased reliability

Active Publication Date: 2017-08-18
INNER MONGOLIA AUTONOMOUS REGION ACAD OF AGRI & ANIMAL HUSBANDRY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Apply genetic engineering technology to transform the pMG36e vector, construct a new expression vector with the function of secreting and expressing foreign proteins with double selection markers in Lactococcus lactis, and use it to express the endoglucanase of Filamentosa succinate, establishing a safer Effectively secreting endoglucanase strains, which are of great significance for the fermentation of silage and the production of enzyme preparations, are expected to have considerable social and economic value, and have not been reported yet

Method used

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  • Lactococcus lactis food-level double-screening marker secretory expression vector, engineering strain containing vector and construction method and application of vector
  • Lactococcus lactis food-level double-screening marker secretory expression vector, engineering strain containing vector and construction method and application of vector
  • Lactococcus lactis food-level double-screening marker secretory expression vector, engineering strain containing vector and construction method and application of vector

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Example 1, Construction of L.lactis secretion expression vector pMG36e+p32+SPusp45

[0078] 1 Extraction of total DNA of Lactococcus MG1363

[0079] (1) Add 3ml of sterilized normal saline to a 50ml centrifuge tube containing the collected specimen to elute, squeeze gently along the tube wall, draw 2ml into a 2ml centrifuge tube at 12000rpm, and centrifuge for 10min.

[0080] (2) Add 20 μl Proteinase K solution to the tube and mix well.

[0081] (3) Add 220 μl buffer GB, shake for 15 sec, place at 70°C for 10 min, the solution should become clear, and briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0082] (4) Add 220 μl of absolute ethanol, shake and mix well for 15 seconds, at this time flocculent precipitation may appear, centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0083] (5) Put the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (the adsorption col...

Embodiment 2

[0115] Embodiment 2, melAL (alpha-galactosidase) is the expression vector SL1-1 of selectable marker

[0116] Construction of (pMG36e+p32+SPusp45+melA)

[0117] 1 Extraction of Lactobacillus (preserved in laboratory) total DNA

[0118] The method is the same as the extraction of total DNA of Lactococcus MG1363.

[0119] 2 PCR amplification of melAL (α-galactosidase) gene

[0120] 2.1 Primer design and synthesis

[0121] A pair of primers were designed according to the coding sequence (codingsequence, CDS) of the melA gene sequence (accession number: AF189765) known in GenBank, as shown in Table 2. Primers were synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0122] Table 2

[0123]

[0124] 2.2 PCR amplification of melA (α-galactosidase) gene

[0125] PCR reaction system of melA gene (25 μL): 12.5 μL of 2X XUltra-pfu-PCR Master Mix, 1.0 μL of 10 μm / L upstream and downstream primers, 2.0 μL of template, and add deionized water to 25 μL.

[0126] PCR reaction...

Embodiment 3

[0141] Embodiment 3, melA (alpha-galactosidase) and nisI gene are the expression vector of double selectable marker

[0142] Construction of NN1 (pMG36e+p32+SPusp45+melA+nisI)

[0143] 1 Design and synthesis of the complete expression cassette sequence of the nisI gene

[0144] A complete expression cassette sequence was designed based on the coding sequence of the known nisI gene sequence (accession number: X76884.1) in GenBank and combined with the P32 promoter sequence. The sequence was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0145] 2 NisI (bacteriocin nisin immune) gene sequence amplification and cloning

[0146] 2.1 Design of primers for nisI gene expression cassette

[0147] Design primers according to the artificially synthesized gene sequence as shown in Table 3, and add enzyme cutting sites upstream and downstream of the primers. Primers were synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0148] table 3

[0149]

[0150] 2.2 PCR ...

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Abstract

The invention relates to a lactococcus lactis food-level double-screening marker secretory expression vector and a construction method thereof. The vector comprises a melA gene, a nisI fused gene double selection marker containing a P32 promoter, a replicor of a plasmid pWV01, a multiple cloning site, a P32 promoter for secretory expression and a part of sequence of enterococcus faecalis Usp45 secretory protein. The invention also discloses an application of NN1-1 as an exogenous protein expression vector, including recombinant lactococcus lactis of endoglucanase (end-1) constructed for secretory expression of fibrobacter succinogenes with NN1-1 as a vector.

Description

[0001] 【Technical field】 [0002] The invention belongs to the field of bioengineering and technology, and relates to a double screening marker secretion expression vector of exogenous genes that can be secreted and expressed in Lactococcus lactis, an engineering strain containing the vector, its construction method and application. [0003] 【Background technique】 [0004] Lactic acid bacteria (LAB) are Gram-positive bacteria that can ferment sugars to produce lactic acid [De VosW M. Gene expression systems for lactic acid bacteria. Current Opinion in Microbiology, 1999, 2(3): 289~295], It is recognized as a safe food-grade microorganism. It has important biological functions such as antibacterial, lowering cholesterol, maintaining the balance of micro-ecosystem, suppressing cancer and enhancing immunity. It is more and more widely used in food, feed, pharmaceutical and other fields [ELMADFA I, KLEIN P, MEYER A L. Immune-stimulating effects of lactic acid bacteria in vivo and i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/65C12N15/66C12N1/21C12R1/01
CPCC12N9/2437C12N15/65C12N15/66C12N15/746C12N2800/101C12Y302/01004
Inventor 呼和苏少锋刘红葵萨初拉王藴华吴青海其布日
Owner INNER MONGOLIA AUTONOMOUS REGION ACAD OF AGRI & ANIMAL HUSBANDRY SCI
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