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London plane tree pollen allergen extract, extracting solution and preparation method thereof

A technology of allergen and British Wu, applied in the biological field, can solve the problems of lower positive rate, uncertain composition, poor stability, etc., and achieve the effect of short cycle, quick effect and good safety

Active Publication Date: 2019-07-30
PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, the allergen used for sycamore pollen as a subcutaneous injection immune preparation has poor stability and uncertain composition. When the reagent is stored for a certain period of time and used to diagnose allergic diseases induced by sycamore pollen, the positive rate decreases and the accuracy is low.

Method used

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  • London plane tree pollen allergen extract, extracting solution and preparation method thereof
  • London plane tree pollen allergen extract, extracting solution and preparation method thereof
  • London plane tree pollen allergen extract, extracting solution and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] The identification of embodiment 1 English sycamore pollen

[0068] Since the identification and purity of raw materials are extremely important for subsequent allergen diagnosis and therapeutic preparations, this example uses DNA-specific sequence detection and microscopic examination to perform double identification and quality control of sycamore raw materials.

[0069] 1. DNA extraction from sycamore pollen

[0070] Tiangen Rapid DNA Extraction and Amplification Kit (Tiangen Biochemical KG203) was used.

[0071] Extraction method: Weigh 5mg of sycamore pollen sample into a 1.5mL centrifuge tube, add 100μl of Buffer 1, grind the sample thoroughly with a disposable grinding pestle, add 100μl of Buffer 2, shake and mix, centrifuge at 12000r / min for 5min Then take the supernatant and put it in a new centrifuge tube as a DNA template for later use.

[0072] 2. Primer design and synthesis

[0073] The primer sequences are as follows:

[0074] Pla ITS2-F (SEQ ID NO.1):...

Embodiment 2

[0093] Example 2 Degreasing and extraction of sycamore pollen Degreasing and Determination of Key Parameters of Key Process Steps

[0094] 1. Degreasing process parameters of sycamore pollen

[0095] (1) Sycamore pollen (g) and acetone (ml) were degreased at a weight-to-volume ratio (w / v) of 1:2. The fat content of different batches (different collection time) of sycamore pollen with different defatting time samples was tested to determine the optimal defatting time.

[0096] Haineng SOX500 fat analyzer was used to measure the fat content in sycamore pollen. Through Soxhlet extraction and dry weighing method, the change of pollen weight before and after extraction was compared to obtain the corresponding fat content. According to the results of fat content, after defatting Pollen for quality control.

[0097] see results figure 2 , it can be seen that after the acetone degreasing time is 30min, the fat content hardly decreases with the prolongation of the degreasing time, ...

Embodiment 3

[0108] Embodiment 3 English sycamore pollen raw material technology

[0109] 1. Pollen collection

[0110] Natural shedding method to collect sycamore pollen. Normal temperature or vacuum or fluidized bed drying, until the pollen no longer sticks. Pass the dried pollen through a 150-250 mesh sieve, preferably a 180 mesh sieve in this embodiment.

[0111] 2. Dry

[0112] Spread the pollen in a ventilated and dry place to dry naturally, or vacuum dry, or dry in a fluidized bed for 6-48 hours, until the pollen no longer adheres.

[0113] 3. Degreasing

[0114] The pollen (g) obtained above and acetone (ml) are subjected to degreasing treatment at a weight-to-volume ratio (w / v) of 1:5-1:1. In this embodiment, it is preferably 1:2. Stir or oscillate to degrease for 30 minutes, let stand to separate layers, and observe the clarification of the upper liquid. Pour out the upper layer, add new acetone, and repeat degreasing until the upper layer is clear.

[0115] 4. Dry again ...

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Abstract

The invention relates to a London plane tree pollen allergen extract and an extracting solution and a preparation method thereof. The amino acid sequences of London plane tree pollen contained in theLondon plane tree pollen allergen extract are shown in SEQ ID NO.4 and SEQ ID NO.5, and the extracting solution can be prepared by London plane tree pollen collection, drying, degreasing, extracting,ultra-filtration concentrating, freeze-drying, re-dissolving and other process methods. The London plane tree pollen allergen extracting solution has the characteristic of high specificity, and the London plane tree pollen component is fully extracted, the total biological titer is stable, the validity period is long, and the aseptic effect is good. Its stock solution can be effectively used for skin prick test diagnosis of allergic reaction diseases, can be used for in-vitro basophil activation test diagnosis, intradermal test diagnosis and specific immunotherapy after proper dilution (10-2 to 10-20), and can effectively diagnose and carry out specific immunotherapy on allergic reaction diseases induced by London plane tree pollen.

Description

Technical field [0001] The invention relates to the field of biotechnology, and specifically relates to a standardized allergen extract of British plane tree pollen, its extract and a preparation method thereof. Background technique [0002] People's understanding of allergic diseases began with "hay fever", also known as allergic rhinitis. In 1911, Noon and Freeman first used pollen extract to treat hay fever, and the history of allergenic disease treatment began. At present, allergic diseases are one of the major global health problems. More than 25% of the population in industrialized countries is troubled by allergic asthma, allergic rhinoconjunctivitis and allergic dermatitis, among which allergic asthma is the most common. Inhalation of allergenic pollen is the most important factor in triggering allergic asthma and other respiratory allergic diseases. VRTA-L.A and other surveys found that nearly 50% of patients with allergic diseases are allergic to grass pollen. ...

Claims

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Application Information

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IPC IPC(8): A61K39/36A61K9/19A61K47/10A61K49/00A61P37/08A61P11/02A61P17/00A61P11/06C07K14/415C07K1/14C07K1/34
CPCA61K39/36A61K9/19A61K47/10A61K49/0006A61P37/08A61P11/02A61P17/00A61P11/06C07K14/415
Inventor 周俊雄尹佳顾建青王瑞琦王茜高冬傲
Owner PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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