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
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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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