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Dust mite allergens Derf26 and Derf27 and genes and application thereof

An allergen and dust mite technology, applied in the field of biomedicine, can solve the problems that hinder the standardization and use of dust mite allergen reagents

Inactive Publication Date: 2013-04-24
KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the crude extract of dust mite is still clinically used for the diagnosis and desensitization treatment of patients with dust mite allergy, which contains a large number of non-specific allergens and other impurities to patients, which seriously hinders the standardization of dust mite allergen reagents. formulation and its clinical use

Method used

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  • Dust mite allergens Derf26 and Derf27 and genes and application thereof
  • Dust mite allergens Derf26 and Derf27 and genes and application thereof
  • Dust mite allergens Derf26 and Derf27 and genes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1: Identification of Dust Mite Allergen and Determination of its Amino Acid Sequence

[0076] 1. Breeding and collection of dust mites and preparation of dust mite homogenate

[0077] The pure strain of D. farinae was reared in batches under the condition of 25 ℃ and 75% relative humidity with powdered rat food. Because dust mites have the life characteristics of avoiding light, dust mites can be separated from feed by incandescent lighting. For the isolated dust mites, an appropriate amount of 20mM pH 7.8 Tris-HCL can be added for thorough grinding, and then centrifuged at 12000 × g for 30 min to obtain the supernatant.

[0078] 2. Separation and purification of dust mite homogenate, two-dimensional electrophoresis and Western blot detection

[0079] Using the supernatant collected above as a raw material, the sample was loaded on Sephadex-G75 molecular sieve gel chromatography equilibrated with 20 mM pH7.8 Tris-HCL buffer in advance, and eluted with the same...

Embodiment 2

[0095] Example 2: Gene Cloning of Dust Mite Allergens

[0096] 1. Extraction of dust mite total RNA

[0097] A. Weigh about 80 mg of live dust mites, add pre-cooled 1 mL Trizol extract (Invitrogen, USA), and add liquid nitrogen to fully homogenize.

[0098] B. Add 1 / 5 volume of Trizol in chloroform, mix vigorously for about 15 seconds, leave at room temperature for 5 minutes, centrifuge at 12000 rpm for 10 minutes at 4°C, and take the supernatant.

[0099] C. Add an equal volume of isopropanol to the supernatant, leave it at room temperature for 10 minutes, centrifuge at 12,000 rpm for 10 minutes at 4°C, wash the precipitate once with 75% ethanol, and dry it in the air. .

[0100] 2. Purification of dust mite mRNA

[0101] Dust mite mRNA was isolated and purified using PolyATtract from PROMEGA, USA ? mRNA Isolation Systems Kit.

[0102] A. Dissolve 500 μg of dust mite total RNA in 500 μL of DEPC water, put in a water bath at 65°C for 10 min, add 3 μL of Oligo(dT) probe a...

Embodiment 3

[0134] Example 3: Detection of allergenicity of dust mite allergens Der f 26-F and Der f 27-F

[0135] For the same sample in Example 1, it is necessary to run two pieces of two-dimensional electrophoresis, one of which is used for direct staining, and the other is used for Western blot to detect its allergenicity. The specific process is as follows:

[0136] 1: Membrane transfer: Transfer the protein on the 2-D-SDS-PAGE gel to the PVDF membrane under the condition of 200 mA for 2 h using a transfer tank.

[0137] 2: Blocking: The PVDF membrane transferred with protein was blocked with 5% skimmed milk powder at room temperature for 2 hours.

[0138] 3: Primary antibody incubation: Wash the blocked PVDF membrane with PBS, dilute the primary antibody (serum from dust mite allergic patients) 1:20 with 5% skimmed milk powder, and incubate overnight at 4 °C.

[0139] 4: Secondary antibody incubation: The PVDF membrane incubated with the primary antibody was washed with PBS, and the ...

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Abstract

The invention relates to dust mite allergens Derf26 and Derf27, and genes and an application thereof, and belongs to the field of biomedicine. The dust mite allergens Derf26 and Derf27 are respectively composed of amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:3; the dust mite allergens Derf26 and Derf27 are encoded; the dust mite allergens Derf26 and Derf27 are respectively composed of nucleotide sequences shown in SEQ ID NO:2 and SEQ ID NO:4; and the dust mite allergens Derf26 and Derf27 are applied to preparation of drugs for treating dust mite anaphylactic diseases. The dust mite allergens Derf26 and Derf27 have the beneficial effects that the dust mite allergens Derf26 and Derf27, and genes thereof are provided; and the dust mite allergens Derf26 and Derf27 can be applied to preparation of drugs for treating dust mite anaphylactic diseases.

Description

Technical field: [0001] The invention relates to dust mite allergens Der f 26 and Der f 27 and their genes and applications, belonging to the technical field of biomedicine. Background technique: [0002] Allergic diseases (such as asthma, allergic rhinitis, etc.) are common clinical diseases and frequently-occurring diseases. The total incidence of allergic diseases in the world is as high as 10-30%, which is a major health problem in the world. There are more than 20 million patients with asthma and more than 50 million patients with allergic rhinitis, and their morbidity and mortality are still on the rise, and the incidence rate has almost doubled in the past two decades. Among the many inhalant allergens that cause allergic diseases, dust mites are the most important factor leading to respiratory allergic diseases. Since Voorhorst and Spieksma (1964) confirmed for the first time that dust mites and their metabolites are the main allergens in house dust, allergists from...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/12A61K39/35A61P37/08
Inventor 赖仞安输容明强李东升
Owner KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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