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Bifidobacterium longum protein as well as preparation method and application thereof

A bifidobacterium longum and protein technology, applied in the field of microorganisms, can solve problems such as singleness and lack of ingredients, and achieve the effects of expanding the scope of use, improving sensitivity, and improving microbial drug sensitivity

Inactive Publication Date: 2016-06-08
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 application, so as to solve the lack of a bifidobacterium adhesion protein with single composition and clear structure in the prior art question

Method used

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  • Bifidobacterium longum protein as well as preparation method and application thereof
  • Bifidobacterium longum protein as well as preparation method and application thereof
  • Bifidobacterium longum protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1 (recombinant escherichia coli expression strain that prepares Bifidobacterium longum protein)

[0044] 1.1 Experimental materials

[0045] 1.1.1 Strains and vectors

[0046] Bifidobacterium longum, Escherichia coli DH5α, and vector pGEX-4T-1 were all purchased from the market.

[0047] 1.1.2 Commonly used reagents

[0048] (1) TE buffer (pH8.0): 10mmol / LTris-HCl (pH8.0), 1mmol / LEDTANa 2 (pH8.0). After configuration, autoclave and store at room temperature.

[0049] (2) 10mg / mL lysozyme: 100mg lysozyme dissolved in 10mLddH 2 O, stored at –20°C for later use.

[0050] (3) 10% SDS: 5gSDS plus ddH 2 O was dissolved and the volume was adjusted to 50 mL.

[0051] (4) 3MKAc: 14.72gKAc dissolved in ddH 2 O and make up to 50mL.

[0052] (5) 3MNaAc: 12.35gNaAc dissolved in ddH 2 O and make up to 50mL.

[0053] (6) 0.1MCaCl 2 : 11.1gCaCl 2 Dissolve in 1000mLddH 2 In O, autoclave at 121°C for 15 minutes, and store at 4°C.

[0054] (7) 50×TAE electrophor...

Embodiment 2

[0161] Example 2 (Induced expression and purification of Bifidobacterium longum adhesion protein)

[0162] Step 1: Induced expression of Bifidobacterium longum adhesion protein

[0163] The Bifidobacterium longum adhesion protein expression strain E.coliDH5αpGEX-4T-Aip3 prepared in Example 1 was induced and expressed with IPTG, and the specific steps were as follows:

[0164] (1) Pick a single colony of the strain that has been streaked on the plate, inoculate it into 3ml of 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.

[0165] (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.

[0166] (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 tem...

Embodiment 3

[0187] Embodiment 3 (a kind of aminoacid sequence is the protein preparation method of SEQIDNO:1)

[0188] 1) Culture the recombinant Escherichia coli E. coli DH5αpGEX-4T-Aip3, 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;

[0189] 2) collecting the bacteria, crushing, and collecting the supernatant;

[0190] 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...

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Abstract

The invention provides an adhesive protein sourced from Bifidobacterium longum. An amino acid sequence of the protein is determined, on the basis, an expression strain of the protein is obtained with a genetic engineering technology, innovative design is performed in the aspects of induction expression, purification by chromatography, ultrafiltration concentration and the like aiming at characteristics of the strain and the adhesive protein, and an efficient preparation method of the adhesive protein is obtained. Furthermore, experiments find that the adhesive protein not only can adhere to an enterocyte, but also has a function of improving antibiotics sensibility of microorganisms, on the basis of the beneficial discovery, an application of the protein as an anti-bacterial synergist for antibiotics is determined, an application range of the protein is extended, and a new path for improving the drug sensitivity of the microorganisms is provided. Prominent technological effects are obtained on the basis of strict experimental measures, and broad application prospect is provided.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to a bifidobacterium longum protein, its preparation method and application. 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 same time secre...

Claims

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Application Information

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IPC IPC(8): C07K14/195C07K1/22C12N15/70A61K38/16A61P37/04A61P31/04
CPCA61K38/00C07K14/195Y02A50/30
Inventor 徐锋杨栋魏华武晓丽蔚晓敏吴姚平
Owner NANCHANG UNIV
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