Dermtophagoides farinae allergens Der f 29 and Der f 30, and genes and applications thereof
A technology for allergens and dust mites, applied in the field of biomedicine, can solve problems such as hindering the standardization and use of dust mites allergen reagents
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Embodiment 1
[0049] Example 1: Identification of Dust Mite Allergen and Determination of its Amino Acid Sequence
[0050] 1. Breeding and collection of dust mites and preparation of dust mite homogenate
[0051] 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.
[0052] 2. Isolation and purification of dust mite allergens
[0053] 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 automatic collec...
Embodiment 2
[0072] Example 2: Gene Cloning of Dust Mite Allergens
[0073] 1. Extraction of dust mite total RNA
[0074] 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.
[0075] 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.
[0076] 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. .
[0077] 2. Purification of dust mite mRNA
[0078] Dust mite mRNA was isolated and purified using PolyATtract from PROMEGA, USA ? mRNA Isolation Systems Kit.
[0079] 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
[0112] Example 3: Detection of Allergenicity of Dust Mite Allergen Der f 29
[0113] The isolated and purified dust mite allergen Der f 29 was tested for allergenicity as follows.
[0114] 1. Western blot experiment
[0115] Prepare an SDS-PAGE gel with a gel concentration of 12%, and perform electrophoresis under reducing conditions. After electrophoresis, the Der f 29 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 the washing solution, and incubated overnight at 4°C with the primary antibody (the primary antibody was used for patients allergic to Yaofly). 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.
[0116] 2. Elisa experiment ...
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