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Application of lactobacillus acidophilus S-layer protein to intestinal cell adhesion and macrophage proliferation

A technology of Lactobacillus acidophilus and macrophages, which is applied in the field of microbial technology application to achieve the effect of enhancing the cell's own immunity

Pending Publication Date: 2021-02-02
INNER MONGOLIA PUZE BIOLOGICAL PROD CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the immune regulation function of Lactobacillus S-layer protein is mainly reflected in slowing down the process of apoptosis, repairing damaged cells, and inducing immune cells to secrete inflammatory cytokines. The mechanism of immune function regulation needs to be further studied

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1: Selection of Lactobacillus acidophilus

[0018] The selected Lactobacillus acidophilus is: the strain number is NM, the classification is named Lactobacillus acidophilus, the preservation number is: CGMCC No 4472, the preservation date is December 14, 2010, and the preservation unit: China Microorganism Culture Collection Management Committee Ordinary Microbiology Center, address of preservation unit: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0019] The above strains were activated with MRS liquid medium before use, and cultured at 37° C. for 15 hours with 1% inoculum size for experimental use.

Embodiment 2

[0020] Embodiment 2: the extraction of Lactobacillus acidophilus S-layer protein

[0021] Cultivate the expanded bacterial solution to the stationary phase, collect the bacterial cells by centrifugation (5000r / min, 15min), wash the medium with 0.1M PBS buffer (pH=7.4), collect the bacterial solution and precipitate with 4-5mol / L chlorine Resuspend lithium chloride, incubate at 37°C for 60min, collect the supernatant (12000r / min, 15min), dialyze in 0.01mol / L PBS buffer (pH=7.4) at 4°C to remove all salt ions, and centrifuge the dialyzed inner solution (5000r / min, 15min), the supernatant was collected, filtered through a microporous membrane (0.22μm), and freeze-dried to obtain the surface crude protein of Lactobacillus acidophilus, which was stored at -20°C for later use.

[0022] After the surface crude protein was dissolved, it was purified by Sephadex G-57 gel filtration chromatography, equilibrated with 0.025mol / L Tris-HCL buffer, and the loading volume was 1mL. The eluent...

Embodiment 3

[0025] Example 3: Experiment of Lactobacillus acidophilus adhering to colon cancer cell HT-29

[0026] The cultured HT-29 cells were trypsinized and passaged in a six-well plate. When the cell density was close to 80%, the Lactobacillus acidophilus adhesion test was carried out.

[0027] Culture Lactobacillus acidophilus at 37°C to OD 590 1.000, collect 5mL of bacterial suspension (5000g, 3min), wash the culture medium with PBS (5000g, 3min), resuspend the collected bacterial solution with 1mL of fresh cell culture medium, add 500μL of bacterial suspension to each well of a six-well plate solution, the control group was untreated Lactobacillus acidophilus, and the experimental group was Lactobacillus acidophilus treated in Example 2 to remove the S-layer protein, and each group had at least 4 repetitions. The control group and the experimental group were co-incubated with HT-29 cells for 2 h (37°C, 5% CO 2 ).

[0028] Using a microplate reader to measure the changes in the ...

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Abstract

The invention relates to an application of lactobacillus acidophilus S-layer protein in intestinal cell adhesion and macrophage proliferation. An extraction method of the lactobacillus acidophilus S-layer protein comprises the following steps of destroying hydrogen bonds on the surface of lactobacillus acidophilus cell walls by using neutral 4-5mol / L lithium chloride, and purifying by gel filtration or ion exchange chromatography, to obtain the lactobacillus acidophilus S-layer protein. The lactobacillus acidophilus S-layer protein is beneficial to adhesion of lactobacillus acidophilus to intestinal cells, the adhesion function of lactobacillus acidophilus to intestinal cells is reduced along with extraction and separation of the S-layer protein, and the lactobacillus acidophilus S-layer protein is also beneficial to macrophage proliferation and macrophage lysosome secretion within a proper concentration range, and cell autoimmunity enhancement is realized.

Description

technical field [0001] The invention relates to the application of Lactobacillus acidophilus S-layer protein to adhere to intestinal cells and to proliferate macrophages, and belongs to the field of microbial technology applications. Background technique [0002] In recent years, a surface layer protein (S layer protein) has been found on the surface of the cell wall of Gram-positive bacteria, Gram-negative bacteria and archaea. This protein is generally arranged in a single molecular crystal with square or hexagonal shape. Symmetrical structure, chemical analysis S-layer protein is composed of a single protein or glycoprotein, the molecular weight is about 40-200kD, a large number of studies have shown that S-layer protein may be related to the adhesion of bacteria. [0003] It is generally believed that S-layer protein is one of the adhesins of lactic acid bacteria. S-layer protein (S-layer proteins, Slps) exists in some Lactobacillus, such as Lactobacillus acidophilus L....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/335C07K1/18C07K1/16
CPCC07K14/335
Inventor 高杰张玉龙
Owner INNER MONGOLIA PUZE BIOLOGICAL PROD CO LTD
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