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Sword prawn allergen protein and preparation and application in medicine thereof

A technology for new prawns and allergens, which is applied in the fields of natural biological active substances and their preparation technology and medical application, can solve the problems of less types of detection reagents and desensitization preparations, etc.

Inactive Publication Date: 2009-03-11
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the labeling and content limits of allergen ingredients in my country's food standards are still blank, and there are no detection reagents for food allergens in food safety monitoring, which are clinically used for the diagnosis and treatment of food-borne allergic diseases. There are fewer types of detection reagents and desensitization preparations
Isolation and purification of major allergens from the tissues of Penaeus clovenae for preparation of diagnostic reagents and desensitization preparations for clinical shrimp allergies have not been reported at home and abroad

Method used

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  • Sword prawn allergen protein and preparation and application in medicine thereof
  • Sword prawn allergen protein and preparation and application in medicine thereof
  • Sword prawn allergen protein and preparation and application in medicine thereof

Examples

Experimental program
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Effect test

example 1

[0019] 1. Extraction of the tissue protein of Penaeus prawns. Remove the head and shell of the fresh Penaeus prawns, take the pure muscle tissue and homogenize it with a homogenizer, add acetone at a ratio of 1:15, put it in the refrigerator at 4°C to degrease, and use it every day Stir for 2 hours and change the acetone solution once. After 48 hours, filter out acetone, weigh after natural drying, add 15ml PBS (0.01mol / L, pH7.4) per gram of sample, extract at 4°C for 48 hours, stir for 2 hours every day during this period, and then at 4°C 5000rpm× Centrifuge for 15 minutes and collect the supernatant.

[0020] 2. Separation of protein components of Penaeus penaeidus. Adjust the pH value of the crude protein solution to 7.4, and use 30%, 50%, 70%, and 90% ammonium sulfate for segmental salting out. Slowly add ammonium sulfate under stirring to achieve the required saturation, then slowly stir for 1 hour, stand at 4°C for 4 hours, centrifuge at 12000rpm×15min at 4°C, collect t...

example 2

[0024] After the homogenate products of Penaeus lanceolata were subjected to segmental salting out with ammonium sulfate, the allergen activity of the products of each salting segment was detected by Dot-ELISA.

[0025] Take 5 μl of ammonium sulfate segmental salting-out product and spot it on the center of nitrocellulose membrane (1cm×1cm), dry it and wash it three times with 0.01M PBST; immerse the spotted nitrocellulose membrane in blocking solution, and put it in a wet box Sealed overnight at 4°C, washed three times with 0.01MPBST after taking out, and dried in the air.

[0026] For Dot-ELISA detection, add a sample-printed nitrocellulose membrane into the reaction well, with the sample-printing side facing up, dilute the serum of patients with shrimp allergy 5 times with antibody diluent (blocking solution) and add it to the reaction well. Place at 4°C overnight, take it out and wash it three times with 0.01MPBST, then put the membrane into 1:1000 diluted goat anti-human ...

example 3

[0029] Eight allergen components were obtained from tissue protein of Penaeus penaeus lanceolata by DEAE-52 ion exchange chromatography, and ELISA was used to detect 11 sera of patients with shrimp allergy.

[0030] Take the isolated Penaeus lanceolata allergen protein solution, dilute it to 5 μg / ml with carbonate buffer (pH 9.6), and coat a 96-well enzyme-labeled reaction plate, 50 μl / well (the coating amount of the allergen is 0.25 μg / well), incubate at 37°C for 1 hr, overnight at 4°C; add blocking solution 250 μl / well the next day after washing, incubate at 37°C for 3 hr, overnight at 4°C, and wash.

[0031] Take the enzyme-labeled reaction plate coated with the new Penaeus prawn allergen, add the serum of patients with shrimp allergy to the 1st to 11th column respectively, 50 μl / well, add 1 column for each serum sample, a total of 11 serums; Set as blank, add 50 μl / well of sample diluent, incubate at 37°C for 1 hr, wash; add 1:500 diluted goat anti-human IgE-HRP, 50 μl / wel...

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Abstract

The invention discloses a group of proteins with allergen activity from metupcllu ensis, a preparation technology thereof and application thereof in medicine, and belongs to the field of natural bioactive substances and preparation technologies and medical applications thereof. The invention provides a group of proteins with allergen activity in a tissue of the metupcllu ensis, and molecular weights of the proteins are 21.6KDa, 36KDa, 46.2KDa, 60KDa, 64.8KDa, 68KDa, 78KDa and 88.5KDa, respectively. The operation according to the invention can obtain main allergen protein compositions of the metupcllu ensis which have high-purity and maintain good immunobiologic activity. The main allergen protein compositions of the metupcllu ensis produced by the invention can be used to prepare diagnostic reagents and desensitizing preparations for clinical shrimp-induced anaphylaxis and prepare reagents used to detect shrimp food allergen.

Description

technical field [0001] The invention relates to a group of proteins with allergen activity derived from Penaeus penaeus prawns, a preparation process and medical application thereof, and belongs to the field of natural biologically active substances, its preparation technology and medical application. Background technique [0002] New prawns (Metapenaeus ensis), commonly known as hemp shrimp, base shrimp, tiger shrimp, sand shrimp, red tail shrimp, used to be wild in the ocean, but now they are mainly cultivated species. It is the most common and popular in my country's aquatic food market. It is a favorite crustacean aquatic food, and it is also one of the common foods that cause food-borne allergic diseases. [0003] Allergic reactions caused by food are a large class of common food-borne diseases, and its clinical manifestations are various, ranging from skin allergies and urticaria in mild cases to diarrhea, vomiting, indigestion, fever, acute angioedema, etc. in severe c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C07K1/18C07K1/14G01N33/68A61K38/17A61P37/08
Inventor 陈伟张华汤鲁宏邓超洪林龙
Owner JIANGNAN UNIV
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