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A kind of bifidobacterium longum protein, its preparation method and medical application

A bifidobacterium longum and protein technology, applied in the field of microorganisms, can solve the problems of lack of ingredients, singleness, etc., and achieve the effects of improving sensitivity, expanding the scope of use, 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 a Bifidobacterium longum protein, its preparation method and medical application, so as to solve the problem of the lack of a bifidobacterium adhesion protein with a single component and a clear structure in the prior art. technical problem

Method used

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  • A kind of bifidobacterium longum protein, its preparation method and medical application
  • A kind of bifidobacterium longum protein, its preparation method and medical application
  • A kind of bifidobacterium longum protein, its preparation method and medical application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1 (recombinant escherichia coli expression strain that prepares Bifidobacterium longum protein)

[0043] 1.1 Experimental materials

[0044] 1.1.1 Strains and vectors

[0045] Bifidobacterium longum, Escherichia coli DH5α, and vector pGEX-4T-1 were all purchased from the market.

[0046] 1.1.2 Commonly used reagents

[0047] (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.

[0048] (2) 10mg / mL lysozyme: 100mg lysozyme dissolved in 10mL ddH 2 O, stored at –20°C for later use.

[0049] (3) 10% SDS: 5g SDS plus ddH 2 O was dissolved and the volume was adjusted to 50 mL.

[0050] (4) 3M KAc: 14.72g KAc dissolved in ddH 2 O and make up to 50mL.

[0051] (5) 3M NaAc: 12.35g NaAc dissolved in ddH 2 O and make up to 50mL.

[0052] (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.

[0053] (7) 50×TA...

Embodiment 2

[0160] Example 2 (Induced expression and purification of Bifidobacterium longum adhesion protein)

[0161] Step 1: Induced expression of Bifidobacterium longum adhesion protein

[0162] 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:

[0163] (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 culture shaker at 180r / m for 8h .

[0164] (2) Transfer to 20ml low-salt LB liquid medium according to 1% inoculum size, add Amp to a final concentration of 100μg / ml, and culture at 37°C with a constant temperature shaker at 180r / m.

[0165] (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 temp...

Embodiment 3

[0186] Embodiment 3 (a kind of aminoacid sequence is the protein preparation method of SEQ ID NO:1)

[0187] 1) Culture the recombinant Escherichia coli DH5αpGEX-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;

[0188] 2) collecting the bacteria, crushing, and collecting the supernatant;

[0189] 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-dried...

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Abstract

The invention provides an attachment protein derived from bifidobacterium longum, and discloses the amino acid sequence thereof; on the basis, experiments discover that the attachment protein not only can attach to enterocyte, but also has the function of improving microorganism antibiotic sensitivity, and especially for escherichia coli O157:H7, the antibiotic synergy is particularly prominent. Based on the beneficial discovery, the application of the protein as an anti-bacteria synergist, escherichia coli O157:H7, is determined, so that the application range of the protein is expanded, and at the same time, a new approach is provided for improving microorganism drug sensitivity. The prominent technological effect is achieved on the basis of strict experimental measures, and a broad application prospect is obtained.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to a bifidobacterium longum protein, a preparation method and a medical application thereof. 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-ecological environment, and at the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/195C07K1/34C07K1/22C12N15/70A61K38/16A61P31/04
CPCA61K38/00C07K14/195
Inventor 徐锋杨栋魏华武晓丽蔚晓敏吴姚平
Owner NANCHANG UNIV
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