Method for expressing and purifying soluble dermatophagoides pteronyssinus major allergen Der p 2 protein

Abstract

The invention relates to a method for performing high-solubility expression and purification on dermatophagoides pteronyssinus major allergen Der p 2 protein in Escherichia coli. According to the method, the Escherichia coli chaperonin trigger factor (TF) is used as a fusion tag to implement high-solubility expression of Der p 2 and obtain the optimized high-efficiency purification technique; and the purity of the sample obtained by the method is higher than 98%. The verification proves that the purified recombinant Der p 2 protein has a consistent secondary structure folding state with the naturally extracted Der p 2 protein and has the identical combination activity with specific IgE in the serum of the dermatophagoides-pteronyssinus-allergic patient. The fusion expression method used in the invention can implement soluble expression of the Der p 2 protein, has the advantages of cuttable fusion tag, high recombinant protein expression level and simple purification technique, is suitable for large-scale industrial production, and can maintain the natural activity of the product.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for highly soluble expression and purification of Der p 2 protein, the main allergen of house dust mite, in Escherichia coli. Background technique [0002] Allergy is defined as an adverse immune overreaction to harmless substances in the environment known as allergens. Allergic diseases (also known as "atopic diseases"), such as hay fever, allergic bronchial asthma, atopic dermatitis, eczema, urticaria, angioedema and anaphylaxis, are global health problems affecting over 25% of the world's population. Since 1960, the global prevalence of asthma and allergic rhinitis has increased significantly. Allergic diseases are still the most common chronic diseases, which greatly affect the health and quality of life of patients, and increase the social and economic burden and medical expenses. Allergies can be caused by many substances from both outdoor and indoor sources. Polle...

AI Technical Summary

Problems solved by technology

[0005] (1) The allergen extraction components are complex, including proteins, sugars, and nucleic acids, making it difficult to standardize;

[0006] (2) The presence of proteolytic enzymes in the extract leads to the degradation of the allergen, or the degradation produces unnecessary harmful substances that cause stability problems;

[0007] (3) It is difficult to detect a small amount of allergenic molecules in the total extract;

[0008] (4) It is difficult to detect whether a patient is allergic to a single substance or to a series of unrelated substances;

[0009] (5) False positives due to cross-reaction of carbohydrate epitopes;

[0015] (5) It is difficult to standardize the amount of effective immunotherapeutic allergens, especially minor allergens in extracts;

[0016] (6) Current immunotherapy produces local, systemic, and even fatal reactions in some patients;

Obtaining biologically active proteins from inclusion bodies must go through a tedious and complicated protein renaturation process. Generally speaking, the renaturation efficiency of proteins is extremely low. Therefore, in industry, the production of recombinant proteins should try to avoid the form of inclusion bodies. Express

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