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Humulus japonicas pollen allergen extract, infusion liquid and preparation method thereof

A technology for allergens and extracts, which is applied in the field of extracts and their preparation, and standardized allergen extracts from Humulus pollen, which can solve the problems of low accuracy, decreased positive rate, decreased protein content and biological potency, etc. question

Pending Publication Date: 2019-08-16
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

Currently, the Humulus pollen allergen used as a subcutaneous injection immune preparation has poor stability, and the protein content and biological potency will decrease with time. The reagents with uncertain components can be used to diagnose allergic diseases induced by Humulus pollen after being stored for a certain period of time. When , the positive rate decreases and the accuracy is low

Method used

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  • Humulus japonicas pollen allergen extract, infusion liquid and preparation method thereof
  • Humulus japonicas pollen allergen extract, infusion liquid and preparation method thereof
  • Humulus japonicas pollen allergen extract, infusion liquid and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1 Identification of Humulus pollen raw material

[0080] 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 microscopic examination for double identification and quality control of Humulus japonicus raw materials.

[0081] 1. DNA extraction from Humulus pollen

[0082] Tiangen Rapid DNA Extraction and Amplification Kit (Tiangen Biochemical KG203) was used.

[0083] Extraction method: Weigh 5 mg Humulus pollen sample and place it in a 1.5 mL centrifuge tube, add 100 μl of Buffer 1, grind the sample thoroughly with a disposable grinding pestle, add 100 μl 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.

[0084] 2. Primer design and synthesis

[0085] The primer sequences are as follows:

[0086] Hum ITS2-F (SEQ...

Embodiment 2

[0101] Example 2 Degreasing and extraction of Humulus pollen Degreasing and Determination of Key Parameters of Key Process Steps

[0102] 1. Degreasing parameters of humulus pollen degreasing process step

[0103] (1) Degrease Humulus 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 humulus pollen samples with different defatting time was tested to determine the optimal defatting time.

[0104] Haineng SOX500 fat analyzer was used to measure the fat content in Humulus pollen. By 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 degreasing Pollen for quality control.

[0105] 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 4

[0131] Example 4 Preparation process of humulus pollen allergen stock solution (lyophilized product)

[0132] 1. Prepare humulus pollen raw material according to the Humulus pollen raw material process in Example 3.

[0133] 2. Leaching

[0134] Add the degreased and dried Humulus pollen to 10 L at a weight-to-volume ratio of 1:50 to 1:10, preferably 1:20 in this example, and stir in pH 7.9 to 8.2 phosphate-saline buffer solution at 2-8°C for 22 ~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.

[0135] Table 4 Phosphate-saline buffer (pH 8.2) formulation

[0136]

[0137] 3. Solid-liquid separation

[0138] Take the extracted extract, add all the extract into the centrifuge bucket, adjust the balance, centrifugal force 8000-12000g, centrifuge temperat...

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Abstract

The invention provides a Humulus japonicas pollen allergen extract, infusion liquid and a preparation method thereof. The Humulus japonicas pollen allergen extract contains an amino acid sequence of the Humulus japonicas pollen allergen protein shown as SEQ ID NO. 4-SEQ ID NO. 20. The infusion liquid is prepared by the processes of collecting of Humulus japonicas pollen, drying, degreasing, extracting, ultrafiltration concentrating, freeze drying, and redissolving. The Humulus japonicas pollen allergen infusion liquid has the characteristics of high specificity, the Humulus japonicas allergenic protein component is fully extracted, the total biological titer is stable, the effective period is long, and the sterilizing effect is good; an original solution can be effectively used for skin prick test diagnosis of allergic diseases, and can be used for in-vitro basophilic granulocyte activation test diagnosis, intradermal test diagnosis and specific immunotherapy after appropriate dilution(10-2-10-20), The infusion liquid can effectively diagnose allergic diseases induced by Humulus japonicas pollen and performs specific immunotherapy.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a humulus pollen standardized allergen extract, an infusion 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 ...

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
CPCA61K39/36A61K9/19A61K47/10A61K49/0006A61P37/08A61P11/02A61P17/00A61P11/06C07K14/415A61K47/02A61P37/00
Inventor 周俊雄尹佳马武强顾建青李欣
Owner PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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