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Dust mite allergen and application thereof

An allergen and dust mite technology, applied in the field of biomedicine, can solve problems such as complex components, affecting repeatability and safety, and pollution

Active Publication Date: 2016-11-23
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the complex components of the natural allergen extract, it is very difficult to keep its components constant, and it is easily contaminated by exogenous toxic substances and pathogenic microorganisms, which affects its repeatability and safety.

Method used

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  • Dust mite allergen and application thereof
  • Dust mite allergen and application thereof
  • Dust mite allergen and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1. Discovery of Dust Mite Allergen Translation Elongation Factors

[0031] The gene sequence of the translation elongation factor of D. farinae was obtained by analyzing and comparing the whole genome sequence of D. farinae obtained by cooperation between Shenzhen University and Chinese University of Hong Kong.

Embodiment 2

[0032] Example 2. Molecular cloning of dust mite allergens

[0033] 1. Extraction of total RNA of dust mite

[0034] Pick clean live D. farinae, and use the RNeasy Mini Kit from Qicgen Company to extract total RNA. The operation steps are carried out according to the instructions.

[0035] 2. Der f 35 full-length cDNA clone

[0036] Using the extracted total RNA as a template, cDNA was reverse-transcribed and PCR amplification was performed. The reaction system is as follows (50 μL): 5 μL of 10×Ex Taq Buffer; 0.25 μL of TaKaRa ExTaq; 4 μL of dNTP Mixture; 2 μL of upstream and downstream primers, 1 μL of cDNA as template; add deionized water to 50 μL. PCR reaction conditions: denaturation at 94°C for 1 min; annealing at 50°C for 1 min; extension at 72°C for 1 min; 35 cycles. PCR products were verified by 1% agarose electrophoresis and photographed.

[0037] 3. Recombinant plasmid construction and enzyme digestion identification

[0038] After the above PCR product was conne...

Embodiment 3

[0043] Example 3: Detection of Allergenicity of Dust Mite Allergens

[0044] 1. Elisa experiment

[0045] Dilute the allergen to 10ug / ml with coating solution, coat with 50ul overnight at 4°C, dilute the patient’s serum at 1:5, dilute the secondary antibody at 1:2000, and finally measure the light absorption value of OD450nm.

[0046] The Elisa results of the effect of Der f 35 on serum IgE of dust mite allergic patients are as follows: image 3 as shown, image 3 Among them, P1-P3 are the results of different asthmatic patients, and C1-C2 are the results of different healthy people.

[0047] 2. Western blot experiment

[0048] Western blotting was performed with the serum of patients allergic to dust mite as the primary antibody, and goat anti-human IgE labeled with streptavidin-horseradish peroxidase (HRP) was used as the secondary antibody to incubate, and then immunoblotting chemiluminescence reagent ( ECL), autoradiography film exposure and film processing.

[0049] ...

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PUM

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Abstract

The invention provides a dust mite allergen and an application thereof. The dust mite allergen belongs to the TEF (translation elongation factor) protein family, can be used for diagnosing, treating and preventing allergic diseases, particularly dust mite allergic diseases, and is particularly used for diagnosing, treating and preventing the allergic disease caused by a thirty-fifth component of the dust mite allergen. Recombinant protein of the dust mite allergen is prepared through gene cloning and protein expression, the protein purity is high, the specificity is better, and the yield is high. The dust mite allergen is used for preparing a reagent for diagnosing, preventing or treating the allergic disease caused by the dust mite allergen, has the characteristics of high specificity and low cost, and particularly can be used for effectively diagnosing whether a patient is allergic to the thirty-fifth component of the dust mite allergen.

Description

Technical field: [0001] The invention belongs to the field of biomedicine, in particular to a dust mite allergen and its application. Background technique: [0002] Among the many allergens that cause allergic diseases, dust mites are the most important allergens. The positive rate of dust mites in specific immunodiagnosis of patients with allergic diseases is about 70-80%. Because the components of the natural allergen extract are very complex, it is very difficult to keep its components constant, and it is easily contaminated by exogenous toxic substances and pathogenic microorganisms, which affects its repeatability and safety. Recombinant allergen vaccines have the advantages of high purity, no impurities, easy standardization, and no exogenous toxic substances and pathogenic microorganisms. They have been used in clinical immunotherapy. [0003] Since dust mites are medical arthropods with complex structures and components, although 24 allergen components have been in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12C12Q1/68G01N33/68A61K39/35A61P37/08
Inventor 林建立刘晓宇刘志刚
Owner SHENZHEN UNIV
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