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Preparation method and application of recombination ryegrass pollen allergen and mutant thereof

A technology for pollen allergy and ryegrass, applied in the field of genetic engineering, can solve the problems of complex components and increase the possibility of allergic reactions, and achieve the effect of reducing binding performance and reducing allergenicity

Inactive Publication Date: 2012-09-19
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

However, some new problems have also been exposed: since the allergens used clinically are derived from the crude extract of pollen, the composition is very complex, including major, minor allergens and other non-allergenic proteins
This will inevitably increase the possibility of causing a new allergic reaction

Method used

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  • Preparation method and application of recombination ryegrass pollen allergen and mutant thereof
  • Preparation method and application of recombination ryegrass pollen allergen and mutant thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Cloning and expression of recombinant ryegrass pollen allergen

[0036] (1) Determination of the nucleic acid sequence of the recombinant ryegrass pollen allergen: Obtain the nucleic acid sequence of the natural ryegrass pollen allergen (GenBank accession number: L13083.1) from the NCBI database, and add a conveniently purified parent And tag 6*HIS, STREPII or S protein. Then Nde I, Eco RI, Xho I, Pst I and other enzyme cutting sites were added to the N-terminus and C-terminus of the sequence respectively to obtain the target gene fragment Q4DF of the recombinant ryegrass pollen allergen.

[0037] (2) According to the codon preference of Escherichia coli, the nucleic acid sequence of the target gene Q4DF 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 remained unchanged. , as shown in SEQ ID NO:1.

[0038] (3) Synthesize the op...

Embodiment 2

[0045] Example 2 Antigen epitope analysis and mutation site determination of recombinant ryegrass pollen allergen

[0046] (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 amino acid sequence of the recombinant ryegrass pollen allergen Antigen index was analyzed. Based on the analysis of various software, the results showed that the antigenic value of the 1-25, 69-89, 301-323 regions in the amino acid sequence of the recombinant ryegrass pollen allergen was higher.

[0047] (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

[0048] Example 3 Construction of recombinant ryegrass pollen 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 ryegrass pollen 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 specifically discloses a preparation method and application of a recombination ryegrass pollen allergen and a mutant thereof. The recombination ryegrass pollen allergen disclosed by the invention is a mutant which exists in a non-natural manner and is derived by an allergen naturally existing; and the amino acid sequence of the recombination ryegrass pollen allergen is shown as SEQIDNO.1. According to the preparation method and application disclosed by the invention, a coding gene of the recombination ryegrass pollen allergen is artificially synthesized by optimizing a codon by using a genetic engineering technology; an antigenic epitope of the coding gene is analyzed by adopting bioinformatics software; and the directional displacement for one or more loci with high allergen is realized by site-specific mutagenesis, and thus the allergic endogenous property of the loci is reduced and the mutant of the recombination ryegrass pollen allergen with low allergic endogenous property is constructed. The protein expression is carried out by artificial control, and thus the recombination ryegrass pollen allergen with high purity and a mutant protein of the recombination ryegrass pollen allergen are obtained. Compared with natural allergen, the recombination ryegrass pollen allergen has the characteristic that the combining capacity between the recombination protein mutant and specific IgE (Immunoglobulin E) is remarkably reduced; and the recombination ryegrass pollen allergen can be safely applied to diagnosis and treatment of allergic diseases even the judgment for the levels of other allergic endogenous properties.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a preparation method and application of a recombinant ryegrass pollen allergen and a mutant. Background technique [0002] In recent decades, with the advancement of industrialization and urbanization, the incidence of allergic diseases has gradually increased. In developed countries, nearly 15% - 30% of people are allergic to pollen. In my country, there are at least 5-10 million patients allergic to pollen, and the incidence of pollen allergic patients has continued to rise in recent years. Therefore, the molecular biology research of plant pollen allergens is one of the hotspots in the field of allergy research in the world. Pollen allergy is not only related to the person's own constitution but also to the type and concentration of pollen in the air. The amount of pollen in the air is mainly affected by the geographical environment, season, climate and the life of the pla...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/63A61K39/35A61P37/08G01N33/68
Inventor 陶爱林董慧峰邹泽红何颖陈惠芳刘雪婷
Owner THE SECOND AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV
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