Recombinant peanut allergen and mutant and preparation method and application of recombinant peanut allergen and mutant

A technology of peanut allergens and mutants, applied in the field of genetic engineering, can solve the problems of large manpower and financial resources consumption, wide application limitations, complex extract components, etc., and achieve the effect of reducing the binding ability

Inactive Publication Date: 2012-07-18
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 peanut allergen and mutant and preparation method and application of recombinant peanut allergen and mutant
  • Recombinant peanut allergen and mutant and preparation method and application of recombinant peanut allergen and mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0035] (1) Determination of the nucleic acid sequence of the recombinant peanut allergen: Obtain the nucleic acid sequence of the natural peanut allergen from the NCBI database, add an affinity tag for easy purification to the C-terminus of the sequence, and then add an affinity tag to the N-terminus and C-terminus of the sequence respectively Adding restriction sites, the target gene fragment XH8 of the recombinant peanut allergen was obtained.

[0036] (2) According to the codon preference of Escherichia coli, the nucleic acid sequence of the target gene XH8 was codon-optimized. After optimization, the GC content was 35-65%, and the original amino acid sequence remained unchanged, as shown in SEQ ID NO:1.

[0037] (3) Synthesize the optimized XH8 gene sequence.

[0038] (4) After double digestion, clone the target fragment XH8 into the prokaryotic expression vector to construct the recombinant expres...

Embodiment 2

[0044] Example 2 Epitope Analysis and Mutation Site Determination of Recombinant Peanut Allergen

[0045] (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 peanut 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 peanut allergen was higher.

[0046] (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.

[0047]

Embodiment 3

[0048] Example 3 Construction of recombinant peanut allergen mutants

[0049] (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.

[0050] (2) According to the requirements of the mutation kit, the recombinant expression plasmid of the recombinant peanut allergen was used as a template for mutation PCR.

[0051] (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 peanut allergen and a mutant and a preparation method and application of the recombinant peanut allergen and the mutant. The recombinant peanut allergen is a non-natural mutant derived from a natural allergen, and the amino acid sequence of the recombinant peanut allergen is shown as SEQ ID NO: 1. A coding gene of the recombinant peanut allergen is artificially synthesized after codon optimization by using a gene engineering technology; and the epitope of the coding gene is analyzed by adopting biological informatics software, and directional displacement is performed on one or more sites with high antigenicity by a site-directed mutagenesis method to reduce the allergenicity of the allergen, and thus the recombinant peanut allergen mutant with low allergenicity is constructed. The recombinant peanut allergen with high purity and a mutant protein of the allergen are obtained by protein expression under an artificial controlled condition. Compared with a natural peanut allergen, the invention has the advantages that the bonding capacity of the recombinant protein mutant bonded with specific immunoglobulin E (IgE) is obviously reduced, and the recombinant protein mutant can be safely applied to diagnosis and treatment of allergic diseases and even judgment of other protein allergenicity.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a recombinant peanut allergen and mutant, a preparation method and application thereof. Background technique [0002] Peanuts belong to leguminous plants with rich nutritional value and relatively balanced amino acid content. It is reported that peanuts contain 45-53% oil and 24-29% protein. In recent years, allergic diseases have received attention from all walks of life, and peanut is one of the eight major allergic foods announced in the report of the Food and Agriculture Organization of the United Nations (FAO), and it is also a very important food allergen. 10%-47% of the total number of allergic cases, and the clinical symptoms of peanut allergy are severe, even eating a small amount of peanut allergen food can trigger a strong allergic reaction, which has attracted much attention in the fields of public health and food safety. On the other hand, due to the prevalence an...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/70C12N15/81C12N15/82C12N15/29C12N15/10A61K39/35A61P37/08G01N33/68
Inventor 陶爱林夏宏林邹泽红何颖陈惠芳
Owner THE SECOND AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV
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