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Recombinant lactobacillus rhamnosus engineering strain and preparation method thereof

A technology of Lactobacillus rhamnosus and engineering strains, which is applied in the field of recombinant Lactobacillus rhamnosus engineering strains and their preparation to achieve the effect of improving antioxidant capacity

Inactive Publication Date: 2012-06-20
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

People have transferred the CAT gene and SOD gene from Lactobacillus plantarum, Lactobacillus sake, Bacillus subtilis and Streptococcus thermophilus into various lactic acid bacteria recipients with weak oxygen tolerance, and successfully improved the antioxidant levels of these lactic acid bacteria. It has not been reported in Lactobacillus rhamnosus

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1 carries the construction of the plasmid vector of purpose gene katA

[0026] 1.1 Genomic DNA extraction of Lactobacillus sake

[0027] The genome was extracted using a bacterial genome DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.), the method is as follows:

[0028] (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;

[0029] (2) Add 180 μl lysozyme (20mg / ml), and treat in a water bath at 37°C for 1 hour;

[0030] (3) Add 20 μl RNase (10mg / ml), shake slightly for 15 seconds, and place at room temperature for 5 minutes;

[0031] (4) Add 20 μl proteinase K solution and mix gently;

[0032] (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

[0074] Embodiment 2 carries the construction of the plasmid vector of purpose gene katA and sodA

[0075] 2.1 Genomic DNA extraction of Streptococcus thermophilus

[0076] Method is with embodiment 1.

[0077] 2.2PCR amplification of catalase gene fragment sodA

[0078] Upstream primer: 5'-CCG CTCGAG CAAGATTTTGTAAG-3'

[0079] Downstream primer: 5'-GG GGTACC TGAGGATGATTCTAGAC-3'.

[0080] Xho I and Kpn I restriction sites were respectively introduced into the upstream and downstream primers, that is, the underlined part in the sequence.

[0081] The PCR program is as follows:

[0082] Component Addition (unit: μl)

[0083] Genomic DNA of Streptococcus thermophilus 1

[0084] Upstream primer (10μM) 0.5

[0085] Downstream primer (10μM) 0.5

[0086] dNTPs (10mM) 0.5

[0087] Ex Taq enzyme 0.5

[0088] 10× Reaction Buffer 2

[0089] Sterile double distilled water 15

[0090] The PCR reaction conditions are as follows:

[0091] Pre-denaturation at 95°C for 5min, d...

Embodiment 3

[0105] Example 3 Preparation of recombinant Lactobacillus rhamnosus engineering strain

[0106] 3.1 Preparation of Lactobacillus rhamnosus competent

[0107] Culture Lactobacillus rhamnosus with MRSS (MRS, 0.3M sucrose, 1% glycine (W / W)) medium and 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 at 4°C for 8min, discarded the supernatant; added 2ml of 30% (W / W) PEG-1500 resuspended cells, centrifuged at 6000rpm at 4°C for 10min, discarded the supernatant, and washed with 200μl of 30% Use after resuspension in PEG-1500.

[0108] 3.2 Electrotransformation of Lactobacillus rhamnosus with recombinant plasmid

[0109] Take 40 μl of Lactobacillus rhamnosus competent cells, mix with 2 μl of recombinant plasmid pSIPCS, transfer to a 2mm electroporation cup, use Bio-Rad Gene Pulser Xcell TM Type electric conversion ins...

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Abstract

The invention relates to a recombinant lactobacillus rhamnosus engineering strain and a preparation method thereof. The lactobacillus rhamnosus engineering strain carries expression vectors of a catalase gene and a superoxide dismutase gene which are connected in series. The method for preparing the engineering strain comprises the following steps of: 1) amplifying catalase gene segments and superoxide dismutase gene segments from genomes of lactobacillus sake and streptococcus thermophiles by a polymerase chain reaction (PCR) method; 2) designing a specific restriction enzyme site, wherein the restriction enzyme site is connected in series with an escherichia coli-lactobacillus shuttle plasmid vector pSIP502; and 3) transforming a recombinant plasmid into lactobacillus rhamnosus by an electroporation method. The recombinant lactobacillus rhamnosus engineering strain prepared by the method can remarkably enhance oxidation 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 rhamnosus engineering strain with improved antioxidant capacity and a preparation method thereof. Background technique [0002] Lactic acid bacteria (LAB) is a general term for Gram-positive bacteria that can ferment sugars to produce lactic acid. It is an internationally recognized large class of probiotics. Lactic acid bacteria fermentation products have become the most important functional food. Lactic acid bacteria are mostly facultative anaerobic bacteria, generally do not need oxygen in the growth process, and the presence of oxygen may cause harm to them. 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 me...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N1/21C12N15/53C12N15/74A23L1/00C12R1/225A23L5/00
Inventor 郝彦玲罗云波安浩然
Owner CHINA AGRI UNIV
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