Dust mite allergen Derf28 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 lar...

Method used

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  • Dust mite allergen Derf28 and genes and application thereof
  • Dust mite allergen Derf28 and genes and application thereof
  • Dust mite allergen Derf28 and genes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1: Identification of dust mite allergen Der f 28 and determination of its amino acid sequence

[0064] 1. Breeding and collection of dust mites and preparation of dust mite homogenate

[0065] 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.

[0066] 2. Isolation and purification of dust mite allergen Der f 28

[0067] 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 buffer. The flow rate was 0.3ml / min, and an...

Embodiment 2

[0086] Example 2: Cloning of dust mite allergen Der f 28 gene

[0087] 1. Extraction of dust mite total RNA

[0088] 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.

[0089] 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.

[0090] 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. .

[0091] 2. Purification of dust mite mRNA

[0092] Dust mite mRNA was isolated and purified using PolyATtract from PROMEGA, USA ? mRNA Isolation Systems Kit.

[0093] 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)...

Embodiment 3

[0191] Example 3: Detection of Allergenicity of Dust Mite Allergen Der f 28

[0192] The isolated and purified dust mite allergen Der f 28 was tested for allergenicity as follows.

[0193] 1. Western blot experiment of Der f 28

[0194] Prepare an SDS-PAGE gel with a gel concentration of 12%, and perform electrophoresis under reducing conditions. After electrophoresis, the Der f 28 on the PAGE gel was transferred to PVDF using a transfer tank, blocked with 5% skimmed milk powder for 2 hr, washed 3 times with washing solution, and incubated overnight at 4°C with the primary antibody (the primary antibody was used for patients with dust mite allergy). Serum, diluted 1:80). After washing 3 times with washing solution, add secondary antibody and incubate at room temperature for 1 hr (the secondary antibody is goat anti-human IgE). After washing 3 times again, the horseradish peroxidase substrate connected to the secondary antibody can be used for development.

[0195] 2. Elisa ...

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Abstract

The invention relates to dust mite allergen Derf28 and genes and an application thereof, and belongs to the field of biomedicine. A new natural allergen Derf28 in a manner of separating and purifying from the dust mites is obtained, and encoding genes of the allergen are cloned. The dust mite allergen Derf28 disclosed by the invention comprises 659 amino acids. The first-stage structure of the amino acid sequence of the allergen is shown in SEQ ID No.1. The sequences of the encoding genes of the allergens comprise 1980 nucleotides, wherein the serial number of the gene nucleotide sequence in GenBank is KC305502. The dust mite allergen has the following beneficial effects that the dust mite allergen Derf28 and the genes thereof are provided, and the dust mite allergen Derf28 can be applied to preparation of drugs for treating dust mite anaphylactic diseases.

Description

Technical field: [0001] The invention relates to a dust mite allergen Der f 28 and its gene and application, 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 all over the wo...

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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