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Beta-1,3-glucan recognition protein as well as preparation method and application thereof

A technology for recognizing proteins and glucans, which is applied in the field of β-1,3-glucan recognition proteins and its preparation and application, and can solve problems such as not seeing

Active Publication Date: 2014-05-14
SHENYANG PHARMA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are no research reports on the preparation method and biological function of β-1,3-glucan recognition protein-b

Method used

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  • Beta-1,3-glucan recognition protein as well as preparation method and application thereof
  • Beta-1,3-glucan recognition protein as well as preparation method and application thereof
  • Beta-1,3-glucan recognition protein as well as preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0082] Separation and purification of natural β-1,3-glucan recognition protein

[0083] 1. Stir the pre-cooled (0°C) saturated ammonium sulfate and slowly add it to the hemolymph until the concentration of ammonium sulfate is 30%, centrifuge at 12,000 rpm at 4°C for 15 minutes to take the supernatant. The supernatant was continuously added dropwise with pre-cooled saturated ammonium sulfate (0° C.) until the ammonium sulfate concentration was 50%, centrifuged at 12,000 rpm at 4° C. for 15 minutes to collect the precipitate. The precipitate was washed with 50% ammonium sulfate and centrifuged to collect the precipitate, which was repeated 5 times.

[0084] 2. Dissolve the precipitate obtained in the previous step in a buffer solution (50mmol / L citric acid buffer, 5mmol / L EDTA, pH5.0) and remove salt by ultrafiltration.

[0085] 3. Load the sample solution obtained in the previous step to SP Sepharose F.F, equilibrate the column with 0mol / L~1mol / L NaCl (50mmol / L citric acid buf...

Embodiment 2

[0090] Separation and purification of natural β-1,3-glucan recognition protein

[0091] 1. Will be collected in anticoagulant buffer solution (15mmo1 L -1 NaCl; 136mmol L -1 Sodium citrate; 26mmol·L -1 Citric acid; 20mmol·L -1 EDTA, pH 5.0) hemolymph raw material solution, loaded to heparin-Sepharose, 0mol / L~1mol / LNaCl anticoagulant buffer solution gradient elution. Collect each fraction and detect the target protein by Western blot.

[0092] 2. Ultrafilter the sample components obtained in the previous step to desalt with anticoagulant buffer solution, centrifuge to take the supernatant and load it to SP Sepharose F.F. After equilibrating the chromatographic column, use pH5.0~pH8.0, 1mol / L NaCl anticoagulant buffer solution gradient elution. Collect each fraction and detect the target protein by Western blot.

[0093] 3. Load the sample components obtained from the analysis in the previous step onto an octyl hydrophobic column, and elute with a gradient of 1mol / L-0mol...

Embodiment 3

[0096] Separation and purification of natural β-1,3-glucan recognition protein

[0097] 1. Stir the pre-cooled saturated ammonium sulfate and slowly add it to the raw material solution until the concentration of ammonium sulfate is 30%, centrifuge at 12,000 rpm at 4°C for 15 minutes to take the supernatant. The supernatant was continuously added dropwise with pre-cooled saturated ammonium sulfate (0° C.) until the ammonium sulfate concentration was 80%, centrifuged at 12,000 rpm at 4° C. for 15 minutes to collect the precipitate. The precipitate was dissolved in 20mmol / L PBS, pH 7.0 for later use.

[0098] 2. Load the sample components separated in the previous step to a β-1,3-glucan affinity chromatography column, and perform gradient elution with 0mol / L-3M NaCl, 20mmol / L PBS, and pH 7.0. Collect each fraction and detect the target protein by Western blot.

[0099] 3. Use 20mmol / L PBS, pH 7.0 buffer solution to desalt the sample fraction separated by ultrafiltration, centri...

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Abstract

The invention relates to the technical field of a biological medicine, and relates to a beta-1,3-glucan recognition protein which is obtained from a lepidoptera insect body in manners of extraction, separation and purification, and a structure thereof, a preparation method of the obtained beta-1,3-glucan recognition protein in manners of separation and purification, a recombinant beta-1,3-glucan recognition protein which is obtained by using a genetic engineering technology, and structures of a derivative, an analogue, and an active fragment thereof, and a preparation method in manners of separation and purification. In addition, the invention also relates to an antibody which is generated by the process of utilizing natural and recombinant beta-1,3-glucan recognition proteins, and derivatives, analogues and active fragments thereof as antigens to stimulate an organism, and an antibody obtaining method. Meanwhile, the invention also relates to the natural and recombinant beta-1,3-glucan recognition proteins, and the derivatives, the analogues and the active fragments, and the antibodies thereof. The beta-1,3-glucan recognition protein can be widely applied to the fields of prevention and treatment drugs aiming at microorganisms, corresponding detection and diagnosis, and the like.

Description

Technical field: [0001] The present invention relates to the technical field of biomedicine, and relates to the structure of β-1,3-glucan recognition protein and its obtaining method and application. Specifically, the present invention relates to β-1,3-glucan recognition protein and its derivatives Structures of substances, analogues, active fragments and their production methods, as well as their detection in microorganisms and related molecular patterns, induction of insects to produce antimicrobial peptides, preparation of β-1,3-glucan recognition proteins and their derivatives, similar Drugs, active fragment antibodies and other applications. Background technique: [0002] Innate immunity is an ancient host defense mechanism. The recognition strategy for "non-self" is not that they can recognize every possible antigen, but that they can recognize highly conserved shared structures that are ubiquitous in microorganisms (pathogens). This recognition method, which is diffe...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K1/34C07K1/22C07K1/20C07K1/18C07K1/16C07K16/18C07K16/06C12N15/12C12N15/63G01N33/68
CPCC07K14/43586C07K16/18C07K2319/20C07K2319/21C07K2319/23C07K2319/50
Inventor 张嵘张景海吴春福
Owner SHENYANG PHARMA UNIVERSITY
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