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Recombinant soybean allergen, mutant and preparation methods and application of recombinant soybean allergen and mutant

A soybean allergen and mutant technology, applied in the field of genetic engineering, can solve the problems of consuming a lot of manpower and financial resources, affecting the therapeutic effect and clinical application specifications, complex purification process, etc., and achieving the effect of reducing the binding ability.

Inactive Publication Date: 2012-07-25
THE SECOND AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the specific immunotherapy mainly uses allergen extracts, so another problem is very prominent: due to the complex composition of the extracts, and also contains a large number of non-specific antigens, it is difficult to standardize, which seriously affects the therapeutic effect and Clinical application norms; at the same time, due to the existence of allergens, even the use of simple standardized allergens for immunotherapy has certain risks
Pure allergens can be obtained by biochemical separation and purification technology, but the purification process of this method is complicated and the yield is low, and it consumes a lot of manpower and financial resources, so its wide application is greatly limited

Method used

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  • Recombinant soybean allergen, mutant and preparation methods and application of recombinant soybean allergen and mutant
  • Recombinant soybean allergen, mutant and preparation methods and application of recombinant soybean allergen and mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Cloning and expression of recombinant soybean allergen

[0035] (1) Determination of the nucleic acid sequence of the recombinant soybean allergen: Obtain the nucleic acid sequence (X60043) of the natural soybean allergen Gly m4 from the NCBI database, and add an affinity tag 6*HIS, STREPII or S protein, and then add Nde I, Eco RI, Xho I, Pst I and other enzyme cutting sites to the N-terminal and C-terminal of the sequence respectively to obtain the target gene fragment Gly m4 of the recombinant soybean allergen.

[0036] (2) According to the codon preference of Escherichia coli, the nucleotide sequence of the target gene Gly m4 was codon-optimized to make the target gene more suitable for expression in the host bacteria. After optimization, the GC content was 35-65%, and the original amino acid sequence was not Change, as shown in SEQ ID NO:1.

[0037] (3) Artificially synthesized optimized Gly m4 gene sequence.

[0038] (4) After double digestion, the ta...

Embodiment 2

[0043] Example 2 Antigen epitope analysis and mutation site determination of recombinant soybean allergen

[0044] (1) Using online software platforms including SYFPEITHI, MHCPred, SYFPEITHI, BIMAS, NetMHCⅡ, MHC-THREAD, EpiPredict, HLA-DR4 binding, ProPred, RankPep, SVMHC, PREDEP, PREDICT, etc. to analyze the antigenic index of amino acid sequences of recombinant soybean allergens for analysis. Based on the analysis of various software, the results showed that the antigenic value of the 54-73, 79-98, 135-157 regions in the amino acid sequence of the recombinant soybean allergen was higher.

[0045] (2) Carry out amino acid substitutions for one or more sites with relatively high antigenic value to weaken the antigenicity. The above-mentioned antigenicity analysis is performed on the amino acid sequence after antigenic modification, and so on, and the mutation sites with significantly reduced antigenicity are screened out.

Embodiment 3

[0046] Example 3 Construction of recombinant soybean allergen mutants

[0047] (1) For the identified mutation site, design mutant primers according to the method provided by the mutation kit. The Tm value is generally required to be greater than 78°C, and the primer length is more suitable between 25-45 bases.

[0048] (2) According to the requirements of the mutation kit, the recombinant expression plasmid of the recombinant soybean allergen was used as a template for mutation PCR.

[0049] (3) After digesting the PCR product with the enzyme provided in the kit, transform the competent cells, pick positive clones, and sequence to check whether the mutation is successful.

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Abstract

The invention discloses a recombinant soybean allergen, a mutant and preparation methods and application of the recombinant soybean allergen and the mutant. The amino acid sequence of the recombinant soybean allergen disclosed by the invention is shown as SEQ ID NO:1. According to the invention, by a genetic engineering technology, a coding gene of the recombinant soybean allergen is artificially synthesized after codon optimization is carried out; an antigenic epitope of the recombinant soybean allergen is analyzed by adopting bioinformatics software; and by a site-specific mutagenesis method, directional replacement is carried out for one or a plurality of sites with high antigenicity to reduce the allergen property of the recombinant soybean allergen so as to construct the recombinant soybean allergen mutant with low allergen property. The high-purity recombinant soybean allergen and a mutant protein thereof are obtained by carrying out protein expression under the condition of manual control. Compared with a natural soybean allergen, the combination capacity of the recombinant protein mutant with the specificity IgE is obviously reduced and the recombinant soybean allergen and the mutant can be safely applied to diagnosis and treatment of allergic diseases and judgment on the allergen property of other proteins.

Description

Technical field [0001] The present invention involves the field of genetic engineering, which specifically involves a reorganized soybean allergen and mutant and its preparation methods and applications. Background technique [0002] Due to the characteristics of high protein, high energy, and low fiber, soybeans have become the most commonly used food and feed ingredients in the world.Soybean -by -products -Soybean meal, as an excellent plant -based feed protein source, can generally reach 25%to 30%of the diet, which is a high -quality plant protein source.Soybean is one of the main protein sources of human and animals, but it is also one of the eight major allergenic foods stipulated by the United Nations.The widespread application of soy protein in the food processing industry has brought certain food safety problems to soybean sensitive people.The survey found that about 2%of adults and 6%to 8%of children suffer from food allergies.Most food allergens caused a type I allergic...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/63A61K39/35A61P37/08G01N33/68
Inventor 陶爱林高洁荣邹泽红何颖刘雪婷陈惠芳
Owner THE SECOND AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV
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