Recombinant lactobacillus casei engineering strain and preparation method thereof

A technology of Lactobacillus casei and engineering strains, which is applied in the field of recombinant Lactobacillus casei engineering strains and its preparation, can solve the problems of cell death product quality, strain survival rate decline, decline, etc., to improve antioxidant and bile salt resistance Ability, the effect of improving the ability to withstand GDCA

Inactive Publication Date: 2013-02-13
CHINA AGRI UNIV
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AI Technical Summary

Problems solved by technology

Lactobacillus casei does not contain the CAT gene on the chromosome, and its ability to resist oxidative stress is weak. As a commonly used lactic acid bacteria, it will be harmed by various oxidative conditions during food processing, resulting in the death of the bacteria and the decline in product quality.
[0004] When Lactobacillus casei is ingested into the human body with food, the bile salt stress in the intestine may lead to a decrease in the survival rate of the strain, thereby limiting the probiotic effect of lactic acid bacteria

Method used

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  • Recombinant lactobacillus casei engineering strain and preparation method thereof
  • Recombinant lactobacillus casei engineering strain and preparation method thereof
  • Recombinant lactobacillus casei engineering strain and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Embodiment 1 carries the construction of the plasmid vector of purpose gene katA

[0028] 1.1 Genomic DNA extraction of Lactobacillus sake

[0029] The genome was extracted using a bacterial genome DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.), the method is as follows:

[0030] (1) Take 5ml of Lactobacillus sake cultured to the logarithmic phase, centrifuge at 12000rpm for 1 minute, discard the supernatant, add 200μl TES to resuspend once, centrifuge at 12000rpm for 1 minute, and suck off the supernatant as much as possible;

[0031] (2) Add 180 μl lysozyme (20mg / ml), and treat in a water bath at 37°C for 1 hour;

[0032] (3) Add 20 μl RNase (10mg / ml), shake slightly for 15 seconds, and place at room temperature for 5 minutes;

[0033] (4) Add 20 μl proteinase K solution and mix gently;

[0034] (5) Add 220 μl buffer GB, shake for 15 seconds, treat in a 70°C water bath for 10 minutes, and briefly centrifuge to remove water droplets on the t...

Embodiment 2

[0075] Example 2 Construction of a double-gene plasmid vector carrying the target gene bsh1 and katA

[0076] 2.1 Genomic DNA extraction of Lactobacillus plantarum

[0077] The genomic DNA of plantarum lactobacillus was extracted, and the method was the same as in Example 1.

[0078] 2.2PCR amplification of bile salt hydrolase gene

[0079] Design specific primers to amplify bile salt hydrolase gene bsh1 and its upstream promoter sequence from the genome of Lactobacillus plantarum. The primer sequences are as follows:

[0080] Upstream primer: 5'-CG GAATTC TATATCGGTTATAAGGG-3'

[0081] Downstream primer: 5'GG GGTACC TTAGTTAACTGCATAGT-3'

[0082] EcoR I and Kpn I restriction sites are introduced into the primer sequence, that is, the underlined part in the sequence.

[0083] The PCR reaction procedure is the same as in Example 1, wherein the template is the genomic DNA of Lactobacillus plantarum.

[0084] The PCR program is as follows:

[0085] Pre-denaturation at 95°...

Embodiment 3

[0087] Embodiment 3 prepares recombinant Lactobacillus casei engineering strain

[0088] 3.1 Preparation of competent cells of Lactobacillus casei

[0089] Cultivate Lactobacillus casei with MRSS (MRS, 0.3M sucrose, 1% glycine) medium, grow to OD 600 =0.4~0.6, take 10ml of bacterial liquid, centrifuge at 6000rpm, 4°C for 8min, collect bacterial cells; add 2ml of rinse solution (0.3M sucrose, 1mM MgCl 2 ) resuspended twice, centrifuged at 6000rpm, 4°C for 8min, discarded the supernatant; added 2ml 30% PEG-1500 resuspended cells, centrifuged at 6000rpm, 4°C for 10min, discarded the supernatant, and re-suspended with 200μl 30%PEG-1500 Suspended, 40μl aliquots for use.

[0090] 3.2 Electrotransformation of Lactobacillus casei with recombinant plasmid

[0091] Take 40 μl of competent cells of Lactobacillus casei, mix with 2 μl of recombinant plasmid pSIPCB, transfer to a 2mm electroporation cup, use Bio-Rad Gene Pulser Xcell TM Type electric conversion instrument, the electric ...

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Abstract

The invention relates to a recombinant lactobacillus casei engineering strain and a preparation method thereof. The lactobacillus casei engineering strain carries an expression vector of a catalase gene and a bile salt hydrolase gene which are connected in series. The preparation method of the engineering strain comprises the following steps of: 1) respectively amplifying segments of the catalase gene and the bile salt hydrolase gene in genomes of lactobacillus sake and lactobacillus plantarum by a PCR method; 2) designing a specific restriction enzyme cutting site and constructing the specific restriction enzyme cutting site in an Escherichia coli-lactobacillus shuttle plasmid vector pSIP502 by series connection; and 3) converting a recombinant plasmid to lactobacillus casei by an electroporation method. The recombinant lactobacillus casei engineering strain prepared by the method can obviously improve oxidation resistance and bile salt resistance and can be applied to the fermented food industry.

Description

technical field [0001] The invention relates to the field of microbial molecular biology, in particular to a recombinant Lactobacillus casei engineering strain and a preparation method thereof for improving the ability of resisting oxidative stress and bile salt stress. Background technique [0002] Lactobacillus casei is widely used in the food industry. It is a common strain of dairy product fermentation and one of the earliest recognized probiotics. Lactobacillus casei is a facultative anaerobic bacteria, and the oxygen in the growth process may cause harm to it. Oxidative stress mainly comes from various reactive oxygen species (Reactive oxygen spicies, ROS), including superoxide anion (O 2 - ), hydrogen peroxide (H 2 o 2 ), hydroxyl radicals (·OH), etc. ROS can damage biological macromolecules such as proteins, nucleic acids, and cell membranes, leading to cell death. [0003] Catalase (CAT) can remove H 2 o 2 Prevent the accumulation of OH and protect cells from...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N1/21C12N15/53C12N15/55C12N15/74A23L1/00C12R1/245A23C9/123A23L5/00
Inventor 郝彦玲罗云波王国宏陈尚武
Owner CHINA AGRI UNIV
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