Cypress pollen allergen educt, immersion liquid thereof and preparation method of immersion liquid

A technology for allergens and extracts, which is applied in the field of standardized allergen infusion of juniper pollen and its preparation, can solve the problems of uncertain composition, reduced positive rate, poor stability and the like, achieves long validity period and improved stability , the effect of short cycle

Active Publication Date: 2019-05-21
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

[0007] At present, the allergens used for juniper pollen as subcutaneous injection immune preparations have poor stability and uncertain components. When the reagents are stored for a certain period of time and used to diagnose allergic diseases induced by juniper pollen, the positive rate decreases and the accuracy is low.

Method used

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  • Cypress pollen allergen educt, immersion liquid thereof and preparation method of immersion liquid
  • Cypress pollen allergen educt, immersion liquid thereof and preparation method of immersion liquid
  • Cypress pollen allergen educt, immersion liquid thereof and preparation method of immersion liquid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] The identification of embodiment 1 juniper pollen raw material

[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 plus microscopy to perform double identification and quality control of Juniper raw materials.

[0069] 1. DNA extraction from juniper pollen

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

[0071] Extraction method: Weigh 5mg juniper pollen sample into a 1.5mL centrifuge tube, add 1100μl of Buffer, grind the sample thoroughly with a disposable grinding pestle, add 100μl of Buffer 2, shake and mix, centrifuge at 12000r / min for 5min Take the supernatant in a new centrifuge tube and use it as a DNA template for later use.

[0072] 2. Primer design and synthesis

[0073] The primer sequences are as follows:

[0074] Sab c ITS2-F (SEQ ID NO.1): 5'-atgcgatacttggt...

Embodiment 2

[0089] Example 2 Degreasing and extraction of juniper pollen key parameters determination

[0090] 1. Determination of process parameters of juniper pollen degreasing process

[0091] (1) Degreasing juniper pollen (g) and acetone (ml) at a weight-to-volume ratio (w / v) of 1:2. The fat content of different batches (different collection time) of juniper pollen with different degreasing time samples was tested to determine the optimal degreasing time.

[0092] The fat content in the juniper pollen was measured by the Sea Energy SOX500 fat analyzer, and the weight change of the pollen before and after extraction was compared by the method of Soxhlet extraction and dry weighing to obtain the corresponding fat content, and according to the results of the fat content, the fat content was compared after degreasing Pollen for quality control.

[0093] see results figure 2 , it can be seen that after the acetone degreasing time is 30min, the fat content hardly decreases with the prol...

Embodiment 4

[0105] Example 4 Preparation process of juniper pollen allergen stock solution (lyophilized product)

[0106] 1. Prepare juniper pollen raw material according to the raw material process of juniper pollen in Example 3.

[0107] 2. Leaching

[0108] Add the degreased and dried juniper pollen to 10L at a weight-to-volume ratio of 1:50 to 1:10, which is 1:10 in this example, and stir in pH 7.9 to 8.2 phosphate-saline buffer solution at 2-8°C for 22 to 26h for leaching. Wherein, the formula of preparing 1000ml phosphate-saline buffer (pH 8.2) as an example is shown in Table 4. Phosphate-saline buffer solution is fully dissolved, sterilized and filtered, placed at 2-8°C, and the storage period does not exceed 30 days.

[0109] Table 4 Phosphate-saline buffer (pH 8.2) formulation

[0110]

[0111] 3. Solid-liquid separation

[0112] Take the extracted extract, add all the extract into the centrifuge bucket, adjust the balance, centrifugal force 8000-12000g, centrifuge temper...

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Abstract

The invention provides a cypress pollen allergen educt, an immersion liquid thereof and a preparation method of the immersion liquid. The cypress pollen allergen educt contains an amino acid sequenceof cypress pollen allergen protein shown as SEQ ID NO.4-SEQ ID NO.7. The immersion liquid of the educt can be prepared by cypress pollen collection, drying, degreasing, extraction, ultrafiltration andconcentration, freeze drying, redissolving and other process methods. The cypress pollen allergen educt has the advantages of high specificity, complete extraction of allergenic cypress protein components, stable total biological potency, long validity period and good aseptic effect. The immersion liquid of the educt can be effectively used for skin pricking test diagnoses of allergic reaction diseases and can also be used for in-vitro basophil activation test diagnoses, intradermal test diagnoses and specific immunotherapy after proper dilution (10-2)-(10-20); the allergic reaction diseasesinduced by cypress pollen can be effectively diagnosed, and specific immunotherapy can be effectively carried out on the allergic reaction diseases.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a standardized allergen infusion of juniper pollen and a preparation method thereof. Background technique [0002] People's understanding of allergic diseases began with "hay fever", which is also known as allergic rhinitis. In 1911, Noon and Freeman used pollen extract to treat hay fever for the first time, and the history of allergenic disease treatment began from then on. 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. Surveys such as VRTA-L.A found that nearly 50% of patients with allergic diseases were allergic to grass pollen. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/36A61K49/00A61K9/19A61K9/08A61K47/10A61P37/08A61P11/06A61P11/02A61P17/00
Inventor 尹佳周俊雄关凯马武强王良录文利平李宏
Owner PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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