Application of a Bifidobacterium longum protein to enhance the susceptibility of Listeria monocytogenes to antibiotics

A technology of Listeria and antibiotics, applied in the field of microbiology, can solve problems such as the limitation of the application range of Bifidobacterium longum protein, and achieve the effect of improving microbial drug sensitivity, sensitivity, and broad application prospects

Active Publication Date: 2019-02-01
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The present invention aims at the technical defects of the prior art, and provides an application of a Bifidobacterium longum protein for improving the susceptibility of Listeria monocytogenes to antibiotics, so as to solve the problems in the application range of the Bifidobacterium longum protein in the prior art. limited technical issues

Method used

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  • Application of a Bifidobacterium longum protein to enhance the susceptibility of Listeria monocytogenes to antibiotics
  • Application of a Bifidobacterium longum protein to enhance the susceptibility of Listeria monocytogenes to antibiotics
  • Application of a Bifidobacterium longum protein to enhance the susceptibility of Listeria monocytogenes to antibiotics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039]Embodiment 1 (recombinant escherichia coli expression strain that prepares Bifidobacterium longum protein)

[0040] 1.1 Experimental materials

[0041] 1.1.1 Strains and vectors

[0042] Bifidobacterium longum, Escherichia coli DH5α, and vector pGEX-4T-1 were all purchased from the market.

[0043] 1.1.2 Commonly used reagents

[0044] (1) TE buffer (pH 8.0): 10mmol / L Tris-HCl (pH 8.0), 1mmol / L EDTANa 2 (pH8.0). After configuration, autoclave and store at room temperature.

[0045] (2) 10mg / mL lysozyme: 100mg lysozyme dissolved in 10mL ddH 2 O, stored at –20°C for later use.

[0046] (3) 10% SDS: 5g SDS plus ddH 2 O was dissolved and the volume was adjusted to 50 mL.

[0047] (4) 3M KAc: 14.72g KAc dissolved in ddH 2 O and make up to 50mL.

[0048] (5) 3M NaAc: 12.35g NaAc dissolved in ddH 2 O and make up to 50mL.

[0049] (6) 0.1M CaCl 2 : 11.1g CaCl 2 Dissolve in 1000mL ddH 2 In O, autoclave at 121°C for 15 minutes, and store at 4°C.

[0050] (7) 50×TAE...

Embodiment 2

[0158] Example 2 (Induced expression and purification of Bifidobacterium longum adhesion protein)

[0159] Step 1: Induced expression of Bifidobacterium longum adhesion protein

[0160] Take the Bifidobacterium longum adhesion protein expression strain E.coli DH5αpGEX-4T-AIP2 prepared in Example 1 and use IPTG to induce expression, the specific steps are as follows:

[0161] (1) Pick a single colony of the strain that has been streaked on the plate, inoculate it into 3ml LB liquid medium, add Amp to the test tube to a final concentration of 100μg / ml, and culture it at 37°C with a constant temperature shaker at 180r / m for 8h .

[0162] (2) Transfer to 20ml low-salt LB liquid medium according to the inoculum size of 1%, add Amp to a final concentration of 100μg / ml, and cultivate at 37°C with a constant temperature shaker at 180r / m.

[0163] (3) When the cell density OD600nm=0.6-0.9, add the inducer IPTG to a final concentration of 1 mM, incubate at 37° C. with a constant tempe...

Embodiment 3

[0184] Embodiment 3 (a kind of aminoacid sequence is the protein preparation method of SEQ ID NO:1)

[0185] 1) Culture the recombinant Escherichia coli DH5α pGEX-4T-AIP2, add the inducer IPTG to a final concentration of 1.2mM when the OD600nm of the culture solution is 0.9, and then induce culture at 39°C for 5 hours under shaking conditions;

[0186] 2) collecting the bacteria, crushing, and collecting the supernatant;

[0187] 3) Take buffer A, equilibrate the glutathione agarose resin column with a flow rate of 3.5mL / min; take the supernatant from step 2), load the sample with a flow rate of 2mL / min; take buffer B, The flow rate of min is eluted, and the solution at the elution peak is collected as the eluent; the eluent is collected by ultrafiltration and concentrated, then passed through a desalting column, and then eluted with pure water at a flow rate of 3.5mL / min, and the eluent at the elution peak is collected. The solution is the second eluent, and then freeze-drie...

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Abstract

The invention provides attachment protein derived from bifidobacterium longum. The amino acid sequence of the protein is defined. On the basis, it is found through experiments that the attachment protein can be attached to enterocytes and has the effect of improving the sensitivity of microorganisms to the antibiotics, and the synergistic effect on the antibiotics is particularly outstanding especially for mononuclear Lister monocytogenes. Based on the beneficial discovery, application of the protein as an antibacterial synergist for the mononuclear Lister monocytogenes is confirmed, the application range of the protein is expanded accordingly, and meanwhile a new way for improving the drug sensitivity of the microorganisms is provided. An outstanding technical effect is achieved based on a strict experiment measure, and wide application prospects are achieved.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to the application of a Bifidobacterium longum protein for improving the sensitivity of Listeria monocytogenes to antibiotics. Background technique [0002] Bifidobacteria are widely known as intestinal probiotics. In recent years, studies on their intestinal probiotics have found that certain metabolites of Bifidobacteria can inhibit the colonization of other microorganisms on the intestinal wall, thereby reducing the risk of pathogenic microorganisms. This feature has become an important basis for screening Bifidobacterium strains and developing functional probiotic agents. [0003] Studies in the prior art have found that the inhibitory mechanism of bifidobacteria on the colonization of other microorganisms on the inner wall of the intestinal tract is mainly through the secretion of organic acids to lower the pH value of the intestinal tract, regulate the intestinal micro-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/16A61K45/06A61P31/04A61K31/43A61K31/7036
CPCA61K31/43A61K31/7036A61K38/164A61K45/06A61K2300/00
Inventor 徐锋杨栋魏华武晓丽蔚晓敏吴姚平
Owner NANCHANG UNIV
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