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Recombinant lactobacillus rhamnosus engineering strain and preparation method thereof

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

Inactive Publication Date: 2011-01-19
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 The construction of the plasmid vector carrying the target gene katA

[0026] 1.1 Lactobacillus sake genomic DNA extraction

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

[0028] (1) Take 5ml of Lactobacillus sake cultured to the logarithmic phase, centrifuge at 12,000 rpm for 1 minute, discard the supernatant, add 200 μl of TES to resuspend once, centrifuge at 12,000 rpm for 1 minute, and aspirate the supernatant as much as possible;

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

[0030] (3) Add 20 μl RNase (10 mg / 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 of buffer GB, shake for 15 seconds, treat in a water bath at 70°C for 10 minutes, and briefly centrifuge to remove water beads on the c...

Embodiment 2

[0080] Embodiment 2 The construction of the plasmid vector carrying the target genes katA and sodA

[0081] 2.1 Extraction of genomic DNA from Streptococcus thermophilus

[0082] The method is the same as in Example 1.

[0083] 2.2 PCR amplification of catalase gene fragment sodA

[0084] Upstream primer: 5'-CCG CTCGAG CAAGATTTTGTAAG-3’

[0085] Downstream primer: 5'-GG GGTACC TGAGGATGATTCTAGAC-3'.

[0086] Xho I and Kpn I restriction sites were introduced into the upstream and downstream primers respectively, that is, the underlined part in the sequence.

[0087] The PCR procedure is as follows:

[0088]

[0089] Component Addition (unit: μl)

[0090]

[0091] The genomic DNA of Streptococcus thermophilus 1

[0092] Upstream primer (10μM) 0.5

[0093] Downstream primer (10μM) 0.5

[0094] dNTPs (10mM) 0.5

[0095] Ex Taq enzyme 0.5

[0096] 10× Reaction Buffer 2 ...

Embodiment 3

[0117] Example 3 Preparation of recombinant Lactobacillus rhamnosus engineering strain

[0118] 3.1 Preparation of Lactobacillus rhamnosus competent

[0119] Lactobacillus rhamnosus was cultured in MRSS (MRS, 0.3M sucrose, 1% glycine (W / W)) medium and grown to OD 600 =0.4~0.6, take 10ml of bacterial liquid, centrifuge at 6000rpm, 4℃ for 8min, collect the bacteria; add 2ml of rinse solution (0.3M sucrose, 1mM MgCl) 2 ) for 2 times, centrifuge at 6000 rpm for 8 min at 4°C, discard the supernatant; add 2 ml of 30% (W / W) PEG-1500 to resuspend the cells, centrifuge at 6000 rpm for 10 min at 4°C, discard the supernatant, and use 200 μl of 30% Use after resuspending in PEG-1500.

[0120] 3.2 Electrotransformation of Lactobacillus rhamnosus with recombinant plasmids

[0121] Take 40 μl of Lactobacillus rhamnosus competent cells, mix with 2 μl of recombinant plasmid pSIPCS, transfer it to a 2mm electroporation cup, use Bio-Rad Gene Pulser Xcell TM Type electrotransformer, electroco...

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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. Most of the lactic acid bacteria belong to facultative anaerobic bacteria, the growth process generally does not require oxygen, 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 destroy biological macromolecules such as proteins, nucleic acids, and cell membranes, leading to cell death, of which OH is the most harmful. [0003] In the process of evolution, various antioxidant enzymes have app...

Claims

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Application Information

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Patent Type & Authority Applications(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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