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Extraction and separation purification methods for main allergenic protein Bet v 8 from birch pollen and application

An extraction method and a separation and purification technology are applied in the field of separation and purification of the main allergenic protein Betv8 of birch pollen, can solve problems such as no specific reports are given, and achieve the improvement of the skin test positive rate, the high IgE positive reaction rate, and the improvement of detoxification. Sensitivity effect

Active Publication Date: 2018-06-08
PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no specific reports have been given in the prior art for the identification and isolation and purification of other major allergenic proteins in birch pollen

Method used

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  • Extraction and separation purification methods for main allergenic protein Bet v 8 from birch pollen and application
  • Extraction and separation purification methods for main allergenic protein Bet v 8 from birch pollen and application
  • Extraction and separation purification methods for main allergenic protein Bet v 8 from birch pollen and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Extraction of the main allergenic protein (about 70kDa) rich in birch pollen in embodiment 1

[0029] 1.1 Pollen raw material

[0030] Chinese birch pollen was collected from birch forest in Chifeng, Inner Mongolia during the birch pollen season in April 2014, and was stored at -80°C after collection.

[0031] 1.2 Experimental steps

[0032] 1.2.1 Dissolve 2 g of birch pollen in 20 mL of 20 mM sodium acetate (pH 4.5), add 250 μL of compound protease inhibitors, vortex for 1 minute, shake overnight at 4°C; centrifuge the pollen suspension at 6000 g for 30 minutes at 4°C , keep the supernatant, filter the supernatant with a 0.2μm filter, and concentrate to 5mL with a 3kDa Millipore ultrafiltration tube. Store at -20°C.

[0033] 1.2.2 Use BCA kit to detect protein concentration in birch pollen protein extract

[0034]Mix reagent A and reagent B in the BCA kit at a ratio of 50:1 to make BCA working solution. Take 25 μL of each diluted concentration of protein standard ...

Embodiment 2

[0039] Example 2 Birch pollen water-soluble protein immunoblotting (Western blot)

[0040] 2.1 SDS-PAGE electrophoresis of the birch pollen protein crude extract (the crude extract prepared in step 1.2.1 in Example 1) extracted with acetate buffer: the operation is the same as before. After electrophoresis, assemble the transfer membrane "sandwich", the sequence is negative electrode - 2 layers of filter paper - separation gel - nylon membrane - 2 layers of filter paper - positive electrode, and transfer the protein from the gel to the nylon membrane by semi-dry transfer method. The transfer buffer was 250mM Tris pure, 1.92M glycine. The nylon membrane was stained with Ponceau S. According to the staining results, the nylon membrane was cut into strips and numbered for incubation with different sera. Put the nylon membrane cut strips into the incubation box, add blocking solution, place on a side-swing shaker, and block at room temperature for 1 hour. The blocking solution i...

Embodiment 3

[0043] Embodiment 3 cation exchange chromatography

[0044] 3.1 Solution preparation

[0045] 3.1.1Cation A: 20mM sodium acetate, pH 4.5, filter the solution with 0.2μm filter paper, store at room temperature.

[0046] 3.1.2Cation B: 20mM sodium acetate, 1M NaCl, pH 4.5, filter the solution with 0.2μm filter paper, store at room temperature.

[0047] 3.2 Protein sample preparation: The birch pollen crude extract prepared in step 1.2.1 in Example 1 was concentrated 3 times using a 3kDa Millipore ultrafiltration tube, and set aside.

[0048] 3.3 Packing: Wash the column with Milli-Q water before use. Assemble the column, fix it on the tripod, and connect it to the peristaltic pump. First clean all the pipelines with Milli-Q water, and check for leakage at the same time, then add 1 / 3 volume of Milli-Q water to the column, and then pour about 3mL of cation exchanger (SP-sepherose) to make The column fills up.

[0049] 3.4 Sample loading, elution and collection: first clean th...

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Abstract

The invention provides extraction and separation purification methods for main allergenic protein Bet v 8 from birch pollen and application. The extraction method for Bet v 8 comprises the steps of performing extraction on birch pollen allergenic protein by using an acetate buffer with pH of 4.5 as an extraction liquid. The separation purification method comprises the steps of firstly preparing abirch pollen water-soluble protein extraction solution, and further performing purification separation on the extraction solution through cation exchange chromatography and Con A sepharose 4B chromatography so as to finally obtain the purified main allergenic protein Bet v 8. The extraction solution or purified protein prepared by the methods of the present invention can be used for detection of main allergenic proteins in serum samples of patients, and can also be further used to prepare allergen detection reagents or desensitizing preparations, and is widely applied to aspects of increasingthe positive rate of skin tests of birch pollen and desensitization therapeutic effect.

Description

technical field [0001] The invention relates to a protein separation and purification method and application, in particular to a separation and purification method and application of birch pollen main allergenic protein Bet v8. Background technique [0002] Allergies are caused by the body's immune response to normally harmless macromolecular (eg, protein) substances. With more than 25% of the population in industrialized countries suffering from type I allergy and the number is steadily increasing, birch hay fever is a very common form of type I anaphylaxis. In China, birch pollen is one of the main allergenic pollen in spring in the northern region (Ye Shitai, Survey of Allergenic Pollen in China, 1991). [0003] There are currently seven named birch pollen allergenic proteins, namely Bet v 1, Bet v 2, Bet v3, Bet v4, Bet v 6, Bet v 7, and Bet v 8, among which Bet v 1 is birch pollen Bet v 1 can be recognized by the serum of 62-98% birch allergy patients in different reg...

Claims

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Application Information

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IPC IPC(8): C07K14/415C07K1/18C07K1/16C07K1/14C07K1/36A61K38/16A61P37/08G01N33/68
CPCA61K38/168C07K14/415G01N33/68
Inventor 徐迎阳尹佳
Owner PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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