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Dust mite allergen and application thereof

A technology of allergens and dust mites, applied in the field of biomedicine, to achieve high protein purity, high specificity, and good specificity

Active Publication Date: 2015-09-09
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, other dust mite allergens belonging to CTSD homologues have not been reported

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

[0041] Example 1. Identification of Dust Mite Allergen and Determination of its Amino Acid Sequence

[0042] 1. Breeding of dust mite and extraction of total protein of dust mite

[0043] Grind Dermatophagoides farinae into powder with liquid nitrogen, weigh 2g sample and add 1ml lysate (9M urea, 4% CHAPS, 60mM DTT, 2% IPG buffer), stir on ice for 5min, centrifuge at 15000g for 50min, carefully avoid the upper layer The floating lipid layer and the supernatant absorbed are the total protein of D. farinae. The total protein concentration was quantified by Bradford method, and the remaining supernatant was used for protein electrophoresis analysis.

[0044] 2. Separation of D. farinae protein by two-dimensional electrophoresis

[0045] 1) First dimension isoelectric focusing electrophoresis (IEF) Transfer the hydrated IPG strips to the universal cup loading strip groove, cover the surface of the IPG strips with an appropriate amount of mineral oil, and then separate the two st...

Embodiment 2

[0058] Example 2. Molecular cloning of dust mite allergens

[0059] 1. Extraction of total RNA of dust mite

[0060] 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.

[0061] 2. Der f 34 full-length cDNA clone

[0062] 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.

[0063] The PCR results of Der f 34 gene of dust mite are as follows Pic 4-1 Shown; M is the DNA marker, and lane 1 is the PC...

Embodiment 3

[0069] Example 3: Detection of Allergenicity of Dust Mite Allergens

[0070] 1. Elisa experiment

[0071] Dilute the allergen to 10ug / ml with coating solution, coat with 100ul at 4°C overnight, dilute the patient’s serum at 1:5, and dilute the secondary antibody at 1:2000, and finally measure the light absorption value of OD450nm. The Elisa results of the effect of Der f 34 on the serum IgE of dust mite allergic patients are as follows: Figure 5 As shown, among them, p1-p3 is the experimental group, c1-c2 is the control group, P1-P3 is the serum of patients with positive skin test, and C1-C2 is the serum of healthy people; Figure 5 It can be seen that the absorption value at 450 nm of the Der f 34 positive group is more than 4 times that of the negative control group.

[0072] 2. Skin acupuncture experiment

[0073] The allergen dissolved in normal saline was dropped on the skin of the palm side of the patient’s forearm, and the skin was punctured with a special pricking ...

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Abstract

The invention provides a dust mite allergen and application thereof. The dust mite allergen provided by the invention belongs to a congener of cathepsin D, can be used for diagnosis, treatment and prevention of allergic diseases and especially dust mite allergic diseases, and is especially used for diagnosis, treatment and prevention of allergic diseases caused by the 34th component of the dust mite allergen. The dust mite allergen recombinant protein is prepared by gene cloning and protein expression, and has the advantages of high protein purity, high specificity and high yield. When being used for preparing reagents for diagnosing, preventing or treating allergic diseases caused by the dust mite allergen, the dust mite allergen provided by the invention has the characteristics of high specificity and low cost. Especially, the dust mite allergen can be efficiently used for diagnosing whether the patient is allergic to the 34th 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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IPC IPC(8): C12N9/64C12N15/57C12N15/63C12Q1/68A61K39/35A61P37/08
CPCA61K39/00C12N9/6413C12Q1/6888C12Y304/23005
Inventor 刘晓宇林建立王媛媛梁志林邬玉兰刘志刚
Owner SHENZHEN UNIV
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