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A kind of hyposensitivity omega-5-gliadin and its preparation method and application

A gliadin, hypoallergenic technology, applied in the protein field, can solve problems such as poor effect

Active Publication Date: 2022-03-04
ZHEJIANG GONGSHANG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

but it doesn't work well

Method used

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  • A kind of hyposensitivity omega-5-gliadin and its preparation method and application
  • A kind of hyposensitivity omega-5-gliadin and its preparation method and application
  • A kind of hyposensitivity omega-5-gliadin and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Preparation of hypoallergenic omega-5-gliadin:

[0037] (1) Extraction and purification of natural ω-5-prolamin: take the raw wheat, pulverize it with a pulverizer at a speed of 3000 rpm for 45 seconds to obtain wheat flour; Centrifuge at 10,000 rpm and 4°C for 15 min, discard the supernatant, and obtain the first precipitate. Next, add 10 times the volume of 1 mol / L sodium chloride to the first precipitate, sonicate for 15 minutes, then centrifuge at 10,000 rpm and 4°C for 15 minutes, discard the supernatant, and obtain the second precipitate . Then, add 5 times the volume of the 60% ethanol solution of the second precipitate to the second precipitate, sonicate for 15 min, centrifuge at 10,000 rpm and 4°C for 15 min, collect the supernatant, and obtain the supernatant; Finally, after the supernatant was blown with nitrogen, it was redissolved with 10% acetonitrile solution, purified by AKTA pure 25 (the purification steps were carried out according to the instruction...

Embodiment 2

[0040] Enzyme-linked immunosorbent assay:

[0041] The purified natural ω-5-gliadin and hyposensitivity ω-5-gliadin obtained in Example 1 were respectively dissolved in 100mmol / L sodium carbonate buffer (pH 10.0) containing 10% acetonitrile, diluted to 5 μg / mL to form sample 1 (natural ω-5-gliadin), sample 2 (hyposensitive ω-5-gliadin) and sample 3 (natural ω-5-gliadin + hypoallergenic ω -5-gliadin). When coating, 6 parallel wells were made for each sample, and 100 μL of sample was added to each well of the microtiter plate, and placed at 4°C for overnight coating. Wash 5 times with ultrapure water, 3 min each time, and pat dry. Add 200 μL of blocking solution (PBS buffer containing 1% BSA) to each well, block at 37°C for 1 h, wash and pat dry, add 100 μL of primary antibody (serum from wheat allergy patients diluted 10 times with blocking solution) to each well , incubate at 37°C for 2h, wash and pat dry, add secondary antibody (HRP-labeled streptavidin) diluted 100 times ...

Embodiment 3

[0047] β-Hexosaminidase Release Assay from LAD 2 Human Mast Cells:

[0048] In vitro assays of mast cell degranulation, such as the release of β-hexosaminidase, can be used to detect hypersensitivity.

[0049] LAD 2 mast cells (1×106 cells / well) were seeded on a 96-well plate, 50 μL per well, and cultured at 37° C. for 24 h. 50 μL of samples were added to each well (the samples were sample 1, sample 2 and sample 3 in Example 2), and three parallels were made for each sample, and incubated at 37° C. for 1 h. Add 100 μL of wheat allergy serum (diluted 10 times), incubate for 1 h, centrifuge at 1000 rpm for 5 min, take a new 96-well plate, add 30 μL of supernatant to each well, and then add 50 μL of 1 mmol / L PNAG solution (1mmol PNAG dissolved in 0.1mol / L citric acid buffer, pH 4.5), incubated at 37°C for 1h. Add 100 μL of 0.2 mol / L glycine solution (pH 10.7) to each well, and measure the OD value at 450 nm. Wherein, β-hexosaminidase release rate (%)=absorbance (OD) of the sam...

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Abstract

The present invention relates to a low-sensitivity ω-5-prolamin and its preparation method and application. The preparation method comprises: dissolving the purified ω-5-prolamin in trifluoromethanesulfonic acid, incubating on ice, Shaking from time to time during this period to obtain the first mixed solution; adding indicator to the first mixed solution, and adding pyridine solution drop by drop, shaking while dripping until the color of the first mixed solution is blue, to obtain the second mixed solution The second mixed solution; desalting the second mixed solution through gel filtration chromatography, collecting the eluate, and freeze-drying to obtain the low-sensitivity ω-5-prolamin. The hyposensitivity ω-5-gliadin prepared by the invention has reduced allergenicity, can inhibit or block wheat allergy information transmission, and can be used as the basis for developing a drug for treating wheat-dependent-exercise-induced allergic reactions.

Description

technical field [0001] The invention relates to the technical field of proteins, in particular to a low-sensitivity ω-5-gliadin and its preparation method and application. Background technique [0002] As one of the eight common allergic foods, wheat can cause severe allergic reactions, and wheat allergy has become a global food safety problem. Common wheat allergic reactions include atopic dermatitis, rhinitis, "baker's" asthma, celiac disease, leprosy skin disease, and exercise-induced allergic reactions. [0003] Glamins are the main constituents of wheat gluten, accounting for 40% to 50% of the total storage protein. Glamins mainly exist as monomers, and are divided into four categories based on their mobility in low-pH acidic polyacrylamide gel electrophoresis or sodium dodecyl sulfate polyacrylamide gel electrophoresis: ω-5-gliadins, ω-1,2-Prolamins, α-β-Prolamins and γ-Prolamins, and have a molecular weight of about 30 kDa to 75 kDa. Omega-5-gliadin is the main all...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C07K1/14C07K1/34A23L33/185A61K38/16A61P37/08
CPCC07K14/415A23L33/185A61P37/08A61K38/00A23V2002/00A23V2250/5486A23V2200/304
Inventor 傅玲琳王彦波王翀朱城波
Owner ZHEJIANG GONGSHANG UNIVERSITY
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