Preparation method and use of peanut allergen reference material
A technology for peanut allergens and standards, applied in the biological field, can solve the problems of not being able to prepare allergens at the same time, achieve the effect of improving purity and immune activity, and having great application value
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Embodiment 1
[0021] Embodiment 1: the preparation of peanut allergen standard substance, comprises the steps:
[0022] (1) Degreasing
[0023] Peanut seeds are crushed into powder after peeling in liquid nitrogen, degreased with acetone, carry out shaking extraction at room temperature for 1 h (hour) according to the mass of peanut powder: acetone volume ratio of 1:10 (W / V), centrifuge at 5000 r / m for 10 min at 4 °C, discard The supernatant was blown with nitrogen for 10 minutes to obtain defatted peanut protein powder, which was stored at 4°C.
[0024] (2) Leaching
[0025] Dissolve peanut protein defatted powder in 1.0 mol / L pH 7.0 phosphate buffer (containing 0.2 mol / L sodium chloride) pre-cooled at 4°C for 2 hours according to the weight-to-volume ratio of 1:10 (W / V). , while adding an equal volume of 0.01mol / L β-mercaptoethanol and 0.5g / L ethylenediaminetetraacetic acid disodium (EDTA·2Na) solution to mix and extract, and stand overnight at 4°C. Then the extract was centrifuged at ...
Embodiment 2
[0032] Embodiment 2: Electrophoretic identification of peanut allergen protein
[0033] The peanut protein extract was divided into dialysis bags and dialyzed with double distilled water for 48 hours (4°C). After the protein solution was fully dialyzed in the dialysis bags, the AKTA system was used for purification and preparation. Conditions: Superdex200 (10×300mm) gel Column chromatography, eluting with pH 7.0 50mmol / L phosphate buffer containing 0.15mol / L NaCl, peristaltic pump flow rate 25ml / h, automatic collection at 10min intervals, monitoring 280nm absorption, collecting required peak samples, combined Distilled water was dialyzed at 4°C for 24 hours, and the sample was freeze-dried to obtain the pure product of peanut allergen protein. The protein was eluted by the AKTA system to obtain two elution peaks of the allergenic protein, namely the main peanut allergen Arah1 and the peanut allergen Arah2, and the main allergen components were identified by SDS-polyacrylamide ...
Embodiment 3
[0034] Embodiment 3: the application of peanut allergen standard substance
[0035] The purified peanut antigen was used to immunize 6 healthy male New Zealand white rabbits respectively, and the rabbits were numbered and divided into 2 groups with 3 rabbits in each group. Blood was collected from the ear vein one week before the immunization as a negative control; the first immunization was carried out by subcutaneous multiple spots on the back Injection, booster immunization after one month, 4 times of booster immunization, each interval of 2 weeks; after the titer is qualified, 20% urethane 5ml / Kg is used to anesthetize the rabbit intravenously, the rabbit carotid artery is bled, and the collected blood is stored at room temperature After standing still, put the blood into a 37°C incubator to stand for 1 hour after blood coagulation, and let it stand overnight at 4°C. After centrifuging at 3000r / m for 20min at 4°C, the supernatant obtained is the antiserum.
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