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Isolation and purification method of main allergic protein of humulus pollen

A technology for separating and purifying pollen, which can be applied in the biological field and can solve the problems of limited research on allergenic proteins of Humulus pollen

Active Publication Date: 2009-03-25
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

[0011] Research on Humulus pollen allergens is limited

Method used

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  • Isolation and purification method of main allergic protein of humulus pollen
  • Isolation and purification method of main allergic protein of humulus pollen
  • Isolation and purification method of main allergic protein of humulus pollen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Determination of major allergenic proteins in pollen of Humulus japonicus

[0038] 1. Collection of serum from Humulus pollinosis patients and healthy volunteers: There are strict selection criteria for the collection of serum from Humulus pollinosis patients and healthy volunteers. A total of 28 patients with humulus pollinosis were collected in this study. The selection criteria for patients are as follows: 1) Typical history of pollen allergy in summer and autumn: seasonal rhinitis, conjunctivitis and / or bronchial asthma, during summer and autumn (July to October) ) disease; 2) Intradermal test of Humulus pollen extract ≥ ++ At the same time, Pharmacia CAP system RAST FEIA detected Humulus pollen sIgE ≥ grade II (0.70kU / l). At the same time, the serum of 10 healthy volunteers was collected in this study. The inclusion criteria were: no history of allergic diseases (including drug, food allergy, allergic rhinitis, bronchial asthma, and other allergic disease...

Embodiment 2

[0041] Example 2 Separation and purification of main allergenic proteins

[0042] 1. Gel size exclusion chromatography

[0043] Chromatography system: Purifier10 (Amersham Company)

[0044] Chromatographic column: Sephacryl 100 prepacked column (Amersham company)

[0045] Column volume 120ml

[0046] Length×Diameter: 60cm×16mm

[0047] Separation range: 10-100kDa

[0048] 1) Sample preparation:

[0049] a. Degrease the freshly collected Humulus pollen with acetone and dry.

[0050] b. Extract (1 / 20w / v) with 0.01M PBS (pH8.0) magnetic force and gentle stirring for 24hr at 4°C.

[0051] c. Centrifuge at 10000 g for 30 min (4° C.) to obtain a supernatant.

[0052] d. Concentrate the supernatant to a protein concentration of at least 1 mg / ml, carry out 80% saturation ammonium sulfate salting out precipitation, centrifuge at 5000g×30min, discard the supernatant and redissolve the precipitated protein in 0.01M PBS (pH8. 0) buffer solution, and use the buffer solution for ...

Embodiment 3

[0070] Example 3 Preparation of Monoclonal Antibody to Major Allergenic Protein of Humulus pollen

[0071] For specific operations, refer to Chapter 11: Hybridoma Technology and Monoclonal Antibody Preparation in "Contemporary Immunology Technology and Application" edited by Bardenian. The operation process is briefly described as follows:

[0072] 1) Immunization of animals Three 6-week-old female healthy BALB / c mice were immunized at the same time. For the first immunization, each mouse was injected with 10-15 μg of antigen. Mix Freund's complete adjuvant with 100 μl of antigen in an equal volume, mix thoroughly with a micro-stirrer, and inject intraperitoneally.

[0073] 2) The second immunization is carried out 2 to 4 weeks later, also known as booster immunization. The amount of antigen for booster immunization was halved, and Freund's incomplete adjuvant was used instead, but the injection volume and method remained unchanged. A total of 4 booster immunizations were pe...

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Abstract

The invention provides a method for separating and purifying the main sensitization protein of humulus pollen. The humulus pollen is taken as raw material and prepared into crude extract and then go through gel molecular exclusion chromatography and strong anion exchange chromatography to obtain the main sensitization protein. The protein can be combined with over 89 percent of the serological specificity IgE (sIgE) of patients allergic to the humulus pollen. The apparent molecular weight of the protein measured by SDS-PAGE is about 12kDa, the isoelectric point is 4.7 and the N-terminal sequence is NH4<+>-MDNPFENGMKA-COO<->. The separation and purification of the protein lay the foundation for realizing the standardization of the allergenic preparation of the protein.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for separating and purifying the main allergenic protein of humulus pollen. Background technique [0002] The incidence of hay fever in various countries in the world ranges from 1% to 25%. [1] , a number of epidemiological surveys show that hay fever patients account for about 10% to 15% of the world's total population [2～5] , but there is no epidemiological data on the incidence of hay fever in the general population in my country. 30 years ago, an epidemiological investigation of hay fever among a small number of ordinary people in some areas showed that the incidence rate could reach 5%. [6] . [0003] In the 1980s, Ye Shitai and Qiao Bingshan led dozens of hospitals across the country to complete the airborne allergenic pollen survey in China, which showed that the content of Humulus pollen in the air in summer and autumn in many areas is second only to A...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/14C07K1/18C07K4/10
Inventor 尹佳程璇
Owner PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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