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Destructive beta-conglycinin beta subunit processing antigen area and screening method based on phage display technological orientation

A conglycinin, phage display technology, applied in the fields of molecular biology, immunology, bioinformatics

Inactive Publication Date: 2018-10-30
HENAN UNIVERSITY OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the continuous development and improvement of phage technology, this technology has been widely used in the fields of medicine, food and agriculture, including the application of antibiotic analysis, the application of biological toxins, the application of small molecules of agricultural and veterinary drugs, and the application of phage display technology for soybean allergy The research on the localization of the original β-conglycinin β subunit epitope has not been reported, especially the localization of the allergen epitope destroyed by ultra-high pressure treatment, and there is no related literature

Method used

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  • Destructive beta-conglycinin beta subunit processing antigen area and screening method based on phage display technological orientation
  • Destructive beta-conglycinin beta subunit processing antigen area and screening method based on phage display technological orientation
  • Destructive beta-conglycinin beta subunit processing antigen area and screening method based on phage display technological orientation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1. Preparation of allergen epitope-specific antibodies destroyed by ultra-high pressure treatment

[0026] 1. Ultra-high pressure treatment of β-conglycinin:

[0027] Put β-conglycinin with a concentration of 15 mg / mL in a sterile homogeneous bag, seal and vacuumize, place the sealed homogeneous bag in the processing chamber (23°C) of the ultra-high pressure processing device, and start Boost the pressure at a rate of 250MPa / min. When the pressure rises to 455MPa, keep the pressure for 18 minutes and then release the pressure at a rate of 300MPa / min. The inhibition rate of β-conglycinin antigen after treatment was 49.59%.

[0028] 2. Preparation of allergen epitope-specific antibodies (antigen-absorbed serum) destroyed by ultra-high pressure treatment:

[0029] New Zealand white rabbits were fed with natural β-conglycinin to prepare β-conglycinin polyclonal antibody (product 1). Add excess β-conglycinin treated by ultra-high pressure to the prepared polyclona...

Embodiment 2

[0030] Example 2, β-conglycinin β subunit gene overlapping (overlapping) segmental cloning

[0031] 1. Cloning of β-conglycinin β subunit gene

[0032] The amino acid sequence of β-conglycinin β subunit (LOC547465) was retrieved from NCBI GenBank database. Login to the PDB database to search for homologous proteins of β-conglycinin β subunit as templates for tertiary structure prediction. Log in to the SWISS-MODEL homology modeling server to predict the tertiary structure of the β-conglycinin β subunit. According to the tertiary structure model of β-conglycinin β subunit, its conformational epitope was predicted in DiscoTope 2.0 web server. According to the predicted position of protein tertiary structure and B cell conformation epitope, the amino acid sequence of β-conglycinin β subunit is divided into 3 segments, and each sequence overlaps with the previous sequence by about 40 amino acid residues (underlined) . The overlapping fragments of the β-conglycinin β subunit se...

Embodiment 3

[0064] Example 3, Phage Surface Presentation of Overlapping Fragments

[0065] 1. Enzyme digestion and recovery of β-conglycinin β subunit gene and its fragments

[0066] Use EcoRI and HindIII to perform double enzyme digestion on the recombinant plasmid, and cut out the β-conglycinin β subunit and its fragments. The method is the same as in Example 2. The enzyme digestion products are separated with 2% agarose gel, and the target DNA gel is cut out. Afterwards, the target fragment was recovered with a gel recovery kit.

[0067] 2. Phage packaging

[0068] Ligate the recovered and purified target fragment to the carrier arm at a molar ratio of 1:1-3:1 (insert:vector), and the connection system is as follows:

[0069]

[0070] Mix by pipetting up and down, then incubate at 16°C for 3-16h or store at 4°C until use. Add 5 times the volume of packaging extract to the ligation product, mix gently with the pipette tip, incubate at room temperature (22°C) for 2 h, add 9 times t...

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Abstract

The invention discloses a destructive beta-conglycinin beta subunit processing antigen area and a screening method based on phage display technological orientation. The amino acid sequence of the antigen area is as shown in SEQ ID NO. 15. By the aid of a series of bioinformatics software and reference of an analyzed beta-conglycinin three-dimensional crystalline structure of a PDB (protein database), the antigen area of ultrahigh-pressure destructive beta-conglycinin is researched by a phage display technology, a beta-conglycinin beta subunit and overlapping protein thereof are represented onthe surface of a phage, and an area with the beta-conglycinin beta subunit with antigenicity reduced by an ultrahigh-pressure is accurately positioned. A theoretical basis for screening processing methods is provided for food industry, and an application product for rapidly detecting the desensitization effect of processed food can be further developed.

Description

technical field [0001] The invention relates to the fields of molecular biology, immunology, and bioinformatics, in particular to an antigenic region that processes and destroys the β-conglycinin β subunit located based on phage display technology. Background technique [0002] Soybean is rich in high-quality protein and has a wide range of sources. It is a more edible and cheap plant protein resource. It has been planted in my country for thousands of years and is one of the main food crops in my country. There are as many as 18 kinds of soybean amino acids, especially rich in lysine, and eating together with cereals can basically meet the types and quantities of amino acids required by the human body. The oil content in soybean is second only to protein, which is rich in high-quality unsaturated fatty acids, which is of great benefit to the health of human cardiovascular system. Soy isoflavones, soybean oligosaccharides, and rich vitamins and calcium contained in soybeans...

Claims

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Application Information

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IPC IPC(8): C07K14/415G01N33/68
CPCC07K14/415G01N33/68
Inventor 席俊皮江一贺梦雪李爽于秋荣
Owner HENAN UNIVERSITY OF TECHNOLOGY
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