A preparation method of peanut allergenic protein arah2 standard
A technology of peanut protein and allergen protein, applied in chemical instruments and methods, biological testing, material inspection products, etc., can solve the problems of failing to meet the requirements of large-scale preparation, achieve the effect of improving purity and immune activity, and retaining biological activity
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Embodiment 1
[0036] Example 1: Preparation of a peanut allergenic protein Arah2 standard
[0037] 1) Degreasing
[0038] Take peanut seeds and bake in an oven at 60°C for 6 hours, remove the red skin, freeze and thaw with liquid nitrogen, and crush them with a pulverizer. According to the ratio of peanut powder mass: n-hexane volume 1:2, carry out overnight extraction at -20°C, at 4°C Centrifuge at 10000r / m for 10min, pour off the n-hexane, air-dry the degreased powder, pass through a 60-mesh sieve, store at 4°C for later use, and obtain peanut powder.
[0039] 2) Preparation of crude extract
[0040] Add 10ml / g of peanut powder to Tris-HCL (containing 1mol / L NaCl, 8mol / L urea, 0.07% β-mercaptoethanol 0.5mg / ml EDTA) with pH 8.0 and a concentration of 0.5M, and mix with a vortex mixer. Sonicate in a disruptor for 100s, 5s each time, with an interval of 1s (in order to break the cells), and then ultracentrifuge (10000-12000r, 4°C, 10-15min) to remove fat and obtain the peanut protein super...
Embodiment 2
[0061] Example 2: SDS-PAGE identification of peanut protein allergen Arah2
[0062] The peanut allergenic protein Arah2 standard obtained in Example 1 was analyzed by SDS-PAGE electrophoresis to identify the main allergen components. The result is as figure 1 shown.
[0063] figure 1 The relative molecular weight of the peanut protein was obvious at 19Kda, which was consistent with the literature report, and the protein was determined to be the peanut protein allergen Arah2.
Embodiment 3
[0064] Example 3: Determination of antibody titer by ELISA
[0065] 1. Coating: Add 100 μl of the prepared allergen protein solution (with 0.05mol / L, pH9.6 NaCO 3 -NaHCO 3 Buffer diluted from 1g / L), 4°C, let stand overnight. Take it out, wash with 0.1mol / L, pH7.4 PBSTween buffer solution (containing 0.5mol / LTween-20) full well 3 times, and blot dry.
[0066] 2. Blocking: Add 250 μl of 10g / LBSA-PBS solution (dissolved in 0.01mol / L, PBS, pH7.4) to each well for blocking, incubate at 37°C for 2 hours, discard the blocking solution, wash 3 times with PBST, 1 minute each time, Dry;
[0067] 3. Adding antibodies: Dilute the antibodies with 1g / LBSA-PBS solution, add 100μl of different dilutions of antibodies to each well, make 3 parallels, and incubate at 37°C for 1h. Wash with PBSTween and button dry 3 times.
[0068] 4. Add enzyme-labeled secondary antibody: add 100 μl goat anti-rabbit IgG-HRP conjugate (diluted with 1 LBSA-PBS solution at a volume ratio of 1:6000) to each wel...
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