Recombinant horse allergen and mutant and preparation methods and applications thereof

A technology of allergens and mutants, applied in the field of genetic engineering, can solve problems such as limitations in the application of immunotherapy, induction of allergies, slow effect, etc., and achieve the effect of reducing allergenicity and reducing binding performance

Inactive Publication Date: 2012-08-01
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, traditional immunotherapy relies on allergen extracts, because its components are complex and cannot be standardized and quantified, so there are many disadvantages such as slow effect, long-term persistence of patients, and the risk of inducing allergies. application is limited

Method used

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  • Recombinant horse allergen and mutant and preparation methods and applications thereof
  • Recombinant horse allergen and mutant and preparation methods and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Cloning and Expression of Recombinant Equine Allergen

[0035] (1) Determination of the nucleic acid sequence of the recombinant equine allergen: Obtain the nucleic acid sequence of the natural equine allergen Equ-c1 (GenBank accession number U70823.1) from the NCBI database, and add affinity for purification to the C-terminus of the sequence Tag 6*HIS, STREPII or S protein, and then add restriction sites such as Nde I, Eco RI, Xho I, and Pst I to the N-terminus and C-terminus of the sequence, respectively, to obtain the target gene fragment Q9PL of the recombinant horse allergen.

[0036] (2) According to the codon preference of Escherichia coli, the codon optimization of the nucleic acid sequence of the target gene Q9PL made 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.

[0037] (3) Artificially synthesize...

Embodiment 2

[0044] Example 2 Antigen Epitope Analysis and Mutation Site Determination of Recombinant Equine 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 horse allergens for analysis. Based on the analysis of various software, the results showed that the antigenic value of the 45-75, 113-136, 146-169 regions in the amino acid sequence of the recombinant horse 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.

Embodiment 3

[0047] Example 3 Construction of recombinant horse allergen mutants

[0048] (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.

[0049] (2) According to the requirements of the mutation kit, the recombinant expression plasmid of the recombinant horse allergen was used as a template for mutation PCR.

[0050] (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 horse allergen and a mutant and preparation methods and applications thereof. The recombinant horse allergen is a non-natural mutant derived from a natural allergen, and the amino acid sequence of the recombinant horse allergen is shown as SEQ ID NO:1. An encoding gene of the recombinant horse allergen is artificially synthesized after codon optimization by using a gene engineering technology, the epitope of the encoding gene is analyzed by adopting bioinformatics software, and orientated displacement is performed specific to one or more high-antigenicity loci through a site-specific mutagenesis method to lower the allergenicity, so that a recombinant horse allergen mutant with low allergenicity is constructed. Protein expression is performed under an artificial control condition, so that a high-purity recombinant horse allergen and a mutant protein thereof are obtained. Compared with a natural horse allergen, the recombinant protein mutant has remarkably-lowered bonding capability with specific IgE (Immunoglobulin E), and can be safely applied to diagnosis and treatment of anaphylactic diseases and even judgment of the level of the allergenicity of other proteins.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a recombinant horse allergen and mutant, a preparation method and application thereof. Background technique [0002] With the development of social industry, the incidence of allergic diseases is increasing day by day. The reason is that people have more and more opportunities to be exposed to allergens, or they are stimulated by different stimuli during the development of the human immune system, or they may be affected by external factors. environmental impact. People can be sensitized by varying degrees of irritation from animal allergens and limited exposure to animal dander and hair. Not only pet cats and dogs can cause allergic diseases in animals, but horses are also a major culprit. [0003] Allergens from horses include Equ-c1, Equ-c2, Equ-c4 and Equ-c5, among which Equ-c1 protein (molecular weight 21.5KD, 187 amino acids, pI=4.5) has been identified as a major aller...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N15/70C12N15/81C12N15/82A61K39/35A61P37/08G01N33/68
Inventor 陶爱林彭俊玲何颖邹泽红陈惠芳刘雪婷
Owner THE SECOND AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV
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