Application of engineered strains expressing peanut allergen-derived proteins in allergic diseases

Abstract

The present invention relates to the field of biotechnology, and in particular to the use of engineered strains expressing peanut allergen-derived proteins in allergic diseases. The present invention provides allergen T cell and B cell epitopes, plasmid pRMC2-Agr, and engineered strains ECArahA, ECArahB, SEArah2, and ECArah2. The present invention changes the drug administration route and designs low-sensitivity allergen peptides, thereby reducing the incidence of side effects; and uses genetic engineering to transform bacteria combined with allergen proteins, which is convenient for drug administration and colonization.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the application of an engineered strain expressing peanut allergen-derived protein in allergic diseases. Background Art

[0002] Atopic dermatitis (AD) is a chronic inflammatory skin disease that seriously affects the quality of life of patients. It is characterized by recurrent eczematous dermatitis and intense itching. The pathogenesis of AD is complex and is affected by multiple genetic and environmental factors, including a complex interaction between a dysfunctional epidermal barrier, a disordered microbiome, and type 2 immune responses. AD also increases the risk of a variety of comorbidities, including food allergies, asthma, allergic rhinitis, and mental health disorders. AD and food allergy (FA) are closely related. One-third of AD children also suffer from FA. Epithelial barrier dysfunction is a common pathogenesis of both. Loss-of-function mutations in the FLG gene in AD patients lead to a reduction in FLG, resulting in a weak skin barrier. Barrier dysfunction leads to the penetration of foreign allergens, activation of Th2 response, promotion of B cell class switching and IgE secretion, and upregulation of the secretion of mediators that induce and maintain Th2 response by damaged epithelial cells, aggravating AD. Peanut allergy is the most common type of food allergy associated with AD. Peanut allergy is the most severe and persistent FA and is a class I allergic disease caused by IgE. Peanut allergens are highly stable and can exist stably under acidic, heated and protease conditions. They are not easily destroyed by natural digestion and usually cause sensitization through the skin or intestinal mucosa. Among the 18 peanut allergens registered by the World Health Organization / International Union of Immunological Societies (WHO / IUIS), those that can bind specific IgE in more than 50% of peanut allergy patients are the main serum allergens, including Arachis hypogaea 1 (Ara h 1), 2, 3, and 6. Ara h 2 is the most dangerous, and Ara h 6 has 59% sequence homology with Ara h 2.

[0003] A treatment method is needed to simultaneously regulate the flora, induce tolerance to allergens, and regulate immunity. Specific immunotherapy (SIT) meets this expectation. By repeatedly exposing patients to gradually increasing amounts of allergen extracts, SIT can improve patients' tolerance to the allergen, thereby achieving causal treatment and alleviating allergic symptoms. SIT can induce a shift from Th2 to Th1 immune response mode, reduce the release of mediators by mast cells, and produce blocking antibodies IgG4 to change the natural course of the disease. In recent years, more and more studies have used SIT to treat AD patients. SIT can be divided into subcutaneous immunotherapy (SCIT), sublingual immunotherapy (SLIT), etc. according to different administration methods. In one study, after 2 years of treatment, the eczema severity score of the dust mite subcutaneous immunotherapy group was reduced by half compared with the control group. After treatment, the serum s-IgG4 level increased and the IL-4 level decreased.

[0004] The current limitations of SIT are mainly allergic side effects, inconvenient administration, and uncertainty in achieving long-term tolerance, and the next generation of SIT therapies aims to overcome these limitations. First, optimize the method of administration, such as oral immunotherapy (OIT) and epidermal immunotherapy (EPIT), whose effectiveness and safety have been verified in clinical trials. Second, use the method of combining engineered bacteria with allergen proteins. Engineered bacteria can interact closely with resident flora and immune cells in the human body through local delivery, and can respond quickly to a series of diseases. In previous studies, engineered bacteria have been used in cancer, colitis, infectious diseases and other fields. Lactobacillus expressing CXCL12 can promote wound healing in mice; E. coli engineered bacteria can metabolize cruciferous diets into products that fight colorectal cancer, and can also secrete phenylalanine metabolic enzymes to treat phenylketonuria, and express enzymes on the ammonia metabolic pathway to treat hyperammonemia. Third, design hypoallergenic allergen peptides, such as the hypoallergenic peptides developed for the single allergen Ara h 2 in previous studies, or hybrid peptides formed by the fusion of 2 to 3 peanut allergens, which can avoid IgE and T cell-mediated side effects and induce the production of allergen-specific IgG, thereby inhibiting specific IgE.

[0005] At present, the treatment of atopic dermatitis mainly focuses on conventional topical drug treatment (topical corticosteroids, calcineurin inhibitors, antibiotics, topical antipruritic agents, emollients, etc.), oral drug treatment (antihistamines, systemic corticosteroids, immunosuppressants, etc.), biological agent treatment and other means. Long-term use of topical corticosteroids (TCS) has risks such as skin atrophy, abnormal pigmentation, acne-like rash, and risks related to systemic absorption (such as hypothalamic-pituitary axis effect, Cushing's disease), so the safety of long-term use of TCS is insufficient. Topical calcineurin inhibitors (TCIs) are usually effective and safe when used for short-term treatment, but concerns about skin malignancies and increased risk of lymphoma make long-term use of drugs insufficient. Any topical drug treatment that is repeatedly used over a long period of time or used over a large area of ​​the body will also lead to reduced patient compliance. Oral immunosuppressants and glucocorticoids are sometimes accompanied by severe toxicity and side effects, so they are limited to short-term treatment and / or intermittent therapy. Systemic corticosteroids are associated with side effects such as diabetes, hypertension, and osteoporosis, and there is a risk of rebound after discontinuation of the drug. Small molecule biological agents against IL-4Rα (duplimab) show certain therapeutic effects, but their process of blocking T helper cell response signals may be accompanied by adverse consequences of immunosuppression.

[0006] Specific immunotherapy for AD combined with allergies is currently limited to the field of dust mites, and there is still a gap in the field of specific immunotherapy for AD combined with peanut allergy. Summary of the invention

[0007] In view of this, the present invention provides the use of engineered strains expressing peanut allergen-derived proteins in allergic diseases, changing the route of administration and designing low-allergenic allergen peptides to reduce the incidence of side effects; using genetically engineered bacteria in combination with allergen proteins to facilitate administration and colonization.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides the use of antigenic epitopes in the preparation of products for preventing and / or treating allergy-related diseases;

[0010] The antigenic epitope has:

[0011] (1) an amino acid sequence as shown in any one of SEQ ID NOs: 18 to 47; or

[0012] (2) an amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (1), and having the same or similar function as (1); or

[0013] (3) an amino acid sequence having at least 90% homology to the amino acid sequence shown in (1) or (2);

[0014] The plurality is 2 to 5;

[0015] The allergy-related diseases include peanut allergy-related diseases;

[0016] The allergy-related diseases also include one or more of allergic dermatitis, allergic gastroenteritis or atopic dermatitis;

[0017] The product includes one or more of a drug, a biological product or a cosmetic.

[0018] The present invention also provides a polypeptide having an amino acid sequence of the antigenic epitope described in the above application;

[0019] or

[0020] have:

[0021] (4), the amino acid sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8; or

[0022] (5) An amino acid sequence obtained by replacing, deleting or adding one or more residues of the amino acid sequence shown in (4), and having the same or similar function as (4); or

[0023] (6) an amino acid sequence having at least 90% homology to the amino acid sequence shown in (4) or (5);

[0024] The plurality is 2 to 30.

[0025] The present invention also provides a nucleic acid molecule having a nucleotide sequence encoding the above polypeptide;

[0026] or

[0027] have:

[0028] (7), a nucleotide sequence as shown in any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7; or

[0029] (8) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (7), and having the same or similar function as (7); or

[0030] (9) a nucleotide sequence having at least 90% homology to the nucleotide sequence shown in (7) or (8);

[0031] The plurality is 2 to 100.

[0032] The present invention also provides an expression vector comprising the above nucleic acid molecule.

[0033] In some specific embodiments of the present invention, the backbone of the above expression vector is pRMC2_Agr or pET-22b;

[0034] The preparation method of pRMC2_Agr is to replace all the Tet-on inducible elements of the pRMC2 plasmid with the mutually inverse P2 and P3 promoter regions of the auxiliary gene regulatory system Agr of Staphylococcus epidermidis ATCC12228;

[0035] The mutually reversed P2 and P3 promoter regions of the auxiliary gene regulatory system Agr of Staphylococcus epidermidis ATCC12228 have:

[0036] (10), the nucleotide sequence shown in SEQ ID NO: 9; or

[0037] (11) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (10), and having the same or similar function as (10); or

[0038] (12) A nucleotide sequence having at least 90% homology to the nucleotide sequence shown in (10) or (11);

[0039] The plurality is 2 to 30.

[0040] The present invention also provides a host cell, comprising the above polypeptide, the above nucleic acid molecule or the above expression vector;

[0041] The host cells include Escherichia coli and / or Staphylococcus epidermidis.

[0042] In some specific embodiments of the present invention, the Escherichia coli in the above-mentioned host cell includes Escherichia coli BL21 (DE3);

[0043] In some specific embodiments of the present invention, the Staphylococcus epidermidis in the above host cell includes Staphylococcus epidermidis ATCC12228.

[0044] The present invention also provides the use of the above host cell in any of the following items:

[0045] (a) Reduce the body's IgE and IgG1;

[0046] (b) Increase serum IgG2a;

[0047] (c) reducing one or more of IL-4, IL-33 or IL-13 in the body;

[0048] (d) Promote the body's Th2 cell response to shift toward Th1 type;

[0049] (e) Alleviate gastrointestinal allergy symptoms and / or skin allergy symptoms;

[0050] (f) Prevention of allergic gastroenteritis and / or skin diseases;

[0051] (g) Treatment of allergic gastroenteritis and / or skin diseases;

[0052] (h) Maintaining gastrointestinal tract and / or skin health;

[0053] (i) prevention and / or treatment of allergic diseases;

[0054] (j) desensitization therapy;

[0055] (k) preparing a product that achieves one or more of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j) or (k);

[0056] The skin disease includes allergic dermatitis, atopic dermatitis, eczema, urticaria or psoriasis;

[0057] The product includes one or more of medicines, biological products, skin care products or cosmetics.

[0058] The present invention also provides a product, comprising the above polypeptide and / or the above host cell and an acceptable auxiliary material or adjuvant;

[0059] The product includes medicine, biological product, skin care product or cosmetic;

[0060] The administration routes of the drug include oral administration or external application.

[0061] The present invention also provides a medicine, comprising the above polypeptide and / or the above host cell and an acceptable auxiliary material or adjuvant.

[0062] The present invention also provides a biological product, comprising the above polypeptide and / or the above host cell and an acceptable auxiliary material or adjuvant.

[0063] The present invention also provides cosmetics, comprising the above polypeptide and / or the above host cell and acceptable auxiliary materials or additives.

[0064] In some specific embodiments of the present invention, the auxiliary materials or adjuvants in the above products include protective agents, wetting agents, emollients, abrasives, salts and / or surfactants.

[0065] The present invention also provides a method for preparing the host cell, comprising introducing the expression vector into chassis cells to obtain the host cell.

[0066] The present invention also provides a method for preventing and / or treating a disease, comprising:

[0067] Oral or external administration of the host cells; or

[0068] Oral or external use of the above products;

[0069] The diseases include allergic diseases;

[0070] The allergic disease includes one or more of allergic dermatitis, allergic gastroenteritis, atopic dermatitis, eczema, urticaria or psoriasis.

[0071] The application of the engineered strain expressing peanut allergen-derived protein in allergic diseases has the following effects:

[0072] The present invention incorporates 6 major serum peanut allergens and uses bioinformatics methods to screen T cell epitopes and B cell epitopes, which are connected into new fusion proteins, reducing allergenicity and improving safety. The cloning vector is constructed, and oral immunotherapy with engineered Escherichia coli and transdermal immunotherapy with engineered Staphylococcus epidermidis are used. The efficacy in the intestinal peanut sensitization mouse model and transdermal sensitization AD mouse model is explored. The mechanism is deeply excavated and it is found that ECArahB and SEArah2 can prevent the occurrence of peanut allergy in the CPE intestinal sensitization mouse model, and ECArahB can reduce dermatitis, allergic symptoms and serum phenotype in the CPE transdermal sensitization AD model. The mechanism is to promote the bias of Th2 cell response to Th1 type. Engineered bacteria can be applied to diseases related to peanut allergy and combined atopic dermatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0074] Figure 1 pRMC2_Tet1 (6k+112bp, cloning site kpnI / BamHI);

[0075] Figure 2 pRMC2_Tet2 (6k+110bp, cloning site kpnI / BamHI);

[0076] Figure 3 pRMC2_Agr (5.5k+330bp, cloning site kpnI / pstI);

[0077] Figure 4Figure 2 shows the gel verification of the improved pRMC2 plasmid DNA; wherein A shows the gel verification of pRMC2_Tet1; B shows the gel verification of pRMC2_Tet2; C shows the gel verification of pRMC2_Agr;

[0078] Figure 5 Shows the genetic design of Arah 2.02;

[0079] Figure 6 The expression and properties of Arah 2.02 derived proteins are shown; A shows the results of SDS-PAGE; B shows Western blot, anti-his-tag; C shows Western blot, anti-Arah2-MA1C4; the yield is about 10 mg / ml;

[0080] Figure 7 It shows the linear B cell epitopes predicted by Bepipred2.0 to exist on the protein surface;

[0081] Figure 8 The plasmid enzyme digestion verification is shown; A shows pET_22b_ArahA; B shows pET_22b_ArahB; C shows pET_22b_Arah2EC; Lane M is 1kb DNA Marker; Lane 1 is NdeI-XhoI enzyme digested plasmid; Lane 2 is the original plasmid;

[0082] Fig. 9 The SDS-PAGE and Western blot analysis of the expression test of ECArahA, ECArahB, and ECArah2, and the expression test of the fusion protein are shown; wherein, A shows ECArahA; B shows ECArahB; C shows ECArah2; lane M is ProteinMarker; lane 1 is the total protein before induction; lane 2 is the supernatant at 20°C; lane 3 is the precipitate at 20°C; lane 4 is the supernatant at 37°C; lane 5 is the precipitate at 37°C;

[0083] Fig.10 The figure shows the SDS-PAGE analysis of the purification of ECArahA, ECArahB and ECArah2 proteins and the small-scale expression and purification test of the nickel agarose affinity layer system; wherein, A shows ECArahA; B shows ECArahB; C shows ECArah2; lane M is the protein marker; lane 1 is the loading; lane 2 is the outflow; lane 3 is 20mM Imidazole; lane 4 is 50mM Imidazole; lane 5 is 500mM Imidazole;

[0084] Fig.11 Figure 4 shows the SDS-PAGE analysis of extracted CPE;

[0085] Fig.12 The therapeutic effect of the engineered bacteria on the CPE allergic mouse model; A shows the allergic symptom score after stimulation; B shows the degree of body temperature drop in each group after stimulation; C shows the total serum IgE after sensitization; D shows the total IgE after stimulation; E to G show the CPE-specific total IgE, IgG1, and IgG2α after sensitization; H to J show the CPE-specific total IgE, IgG1, and IgG2α after stimulation; * represents p<0.05, ** represents p<0.01, *** represents p<0.001, and **** represents p<0.0001, n=6;

[0086] Fig.13 Shows the gross photograph of mice in the transdermal sensitization procedure;

[0087] Fig.14 The phenotypes of mice sensitized with MC903 and CPE are shown; A shows the ear thickness on the 7th day after sensitization; B shows the ear thickness on the 1st day after sensitization; C shows the disease score on the 1st day after sensitization; D shows the ear thickness on the 1st day before challenge; E shows the disease score on the 1st day before challenge; F shows the ear thickness at challenge; G shows the disease score at challenge; H shows the temperature difference after challenge; I shows the allergic reaction score; * represents p<0.05, ** represents p<0.01, *** represents p<0.001, **** represents p<0.0001, n=6;

[0088] Fig.15 Shows HE slice scans of mice sensitized with MC903 and CPE and the thickness of the ears measured by the slices; A shows the first day after sensitization; B shows the day of stimulation; * represents p<0.05, ** represents p<0.01, *** represents p<0.001, **** represents p<0.0001, n=6;

[0089] Fig.16 The expression of ear cytokines in the mouse model sensitized with MC903 and CPE is shown; A shows IL-4; B shows IL-13; C shows IL-10; D shows TSLP; E shows IL-33; * represents p<0.05, ** represents p<0.01, *** represents p<0.001, **** represents p<0.0001, n=3;

[0090] Fig.17 The serological changes in the MC903 combined with CPE sensitized mouse model are shown; wherein, A shows total IgE after sensitization; B shows total IgE after challenge; C shows CPE-specific IgE after sensitization; D shows IgG1; E shows IgG2α; F shows CPE-specific IgE after challenge; G shows IgG1; H shows IgG2α; * represents p<0.05, ** represents p<0.01, *** represents p<0.001, **** represents p<0.0001, n=6. DETAILED DESCRIPTION

[0091] The present invention discloses the use of engineered strains expressing peanut allergen-derived proteins in allergic diseases. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the purpose. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0092] Specific immunotherapy for allergic diseases such as atopic dermatitis has limitations, mainly allergic side effects, inconvenient administration, and uncertainty in achieving long-term tolerance. Based on the current state of the art, the purpose of the present invention is to effectively treat peanut allergy and atopic dermatitis combined with peanut allergy using a certified probiotic chassis strain.

[0093] The invention aims to overcome the aforementioned limitations when the engineered strain expressing peanut allergen-derived proteins is used in allergic diseases: 1) changing the route of administration and designing low-allergenic allergen peptides to reduce the incidence of side effects; 2) using genetically engineered bacteria in combination with allergen proteins to facilitate administration and colonization.

[0094] The gene sequence information of the engineering bacteria involved in the present invention is as follows:

[0095] SEArah2 nucleotide sequence (SEQ ID NO: 1):

[0096]

[0097] SEArah2 amino acid sequence (SEQ ID NO: 2):

[0098]

[0099] ECArah2 nucleotide sequence (SEQ ID NO:3):

[0100]

[0101]

[0102] ECArah2 amino acid sequence (SEQ ID NO:4):

[0103]

[0104] ECArahA nucleotide sequence (SEQ ID NO:5):

[0105]

[0106] ECArahA amino acid sequence (SEQ ID NO:6):

[0107]

[0108] ECArahB nucleotide sequence (SEQ ID NO:7):

[0109]

[0110] ECArahB amino acid sequence (SEQ ID NO:8):

[0111]

[0112] pRMC2-Agr promoter region (SEQ ID NO:9):

[0113]

[0114] The concentration, action time and frequency of the engineering bacteria used in the present invention can be changed according to different inflammatory skin diseases and allergic diseases and individual specific situations, and are interchangeable.

[0115] The protein products obtained by culturing the engineered strain of the present invention at different times, their dilution multiples, external application concentrations, action time, and frequency can vary according to different inflammatory skin diseases and individual specific situations, and are interchangeable.

[0116] Various external preparations, including protective agents, moisturizers, emollients, abrasives, thickened topical preparations containing engineered strains, salts and / or surfactants, can be used in the context of the present invention.

[0117] Various external preparations including protective agents, moisturizers, emollients, abrasives, thickened topical preparations of salts and / or surfactants containing the recombinant proteins of the engineered strains can be used in the context of the present invention.

[0118] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0119] The present invention will be further described below in conjunction with embodiments:

[0120] Preparation Example 1: Preparation of engineered Staphylococcus epidermidis strains

[0121] 1. Experimental strains and plasmids: Staphylococcus epidermidis ATCC12228 and plasmid pRMC2 were purchased from the Miaoling plasmid platform (pRMC2 product and sequence information: https: / / www.addgene.org / 68940 / ), and Escherichia coli DH5α competent cells and Escherichia coli HST04 (dam-dcm-) competent cells were purchased from TAKARA and frozen in Huashan Hospital Affiliated to Fudan University.

[0122] 2. Design of pRMC2 plasmid and optimization of Arah2.02 gene: All Tet-on elements were replaced with the RBS-P2-P3-RBS promoter region of the auxiliary gene regulatory system of Staphylococcus epidermidis ATCC12228 (derived from Nucleotide (GenBank): AE015929 Staphylococcus epidermidis ATCC 12228, complete genome). After eliminating the restriction endonucleases that were not used in the recombinant gene sequence, the recombinant plasmid pRMC2_Agr was synthesized by Shanghai Bioengineering Co., Ltd. The amino acid sequence of Arah2.02 (genbank: AAN77576.1) was predicted using the signal peptide prediction software SignalP (http: / / www.cbs.dtu.dk / services / SignalP / ). The gene sequence corresponding to the amino acid sequence of Arah 2.02 (genbank: AY158467.1) was 519 bp in total. The predicted signal peptide sequence was deleted, and the total length after removal was 456 bp. The Ara h 2.02 gene sequence was codon optimized using the tool JCAT (http: / / www.jcat.de / ), and the low-frequency codons in the Ara h 2.02 gene were replaced with the preferred codons of Staphylococcus epidermidis (ATCC12228). In order to better express it in Staphylococcus epidermidis, the YSIRK / GXXS signal peptide sequence of Staphylococcus epidermidis ATCC12228 glycerol hydrolase precursor MKTRQNKYSIRKFSVGASSILIAALLFMGGGSAQAAE (SEQ ID NO: 10) was added to the N-terminus, and a 6*his tag was added to its C-terminus. According to the codon preference of Staphylococcus, the gene sequence used was: CATCATCATCATCATCATCAT (SEQ ID NO: 11) to form the recombinant fusion protein sigara2SE. After excluding the useless restriction endonucleases in the recombinant gene sequence, it was synthesized by Jinweizhi Company and connected to pUC57 to obtain the recombinant plasmid pUC57_sigara2SE.

[0123] 3. Construction of engineered Staphylococcus epidermidis: Using plasmid pUC57_sigara2SE as template, PCR amplification was performed using upstream and downstream primers, the vector and target gene fragment were double-digested, the target gene fragment was connected with the linearized vector pRMC2_Agr, and transformed into E.coli DH5α competent state, inverted and cultured at 37℃ overnight. The plasmid was extracted and transferred into dam-dcm- E. coli HST04. Resuscitate the competent cells of Staphylococcus epidermidis ATCC12228, take 10μg of recombinant plasmid and add it to 100μl competent epidermidis, mix well; transfer the above mixture to a 0.1cm electric shock cup, shock (resistance 100Ω, voltage 2.0kV, capacitance 25pF), immediately add 1ml B2 medium preheated at 37℃ to the electric shock cup, aspirate after mixing, transfer to a sterile test tube and shake and culture at 37℃ for 3h; centrifuge at 4000g for 5min, discard most of the supernatant, resuspend the bacteria and plate TSA (chloramphenicol resistance), and culture at 37℃ for 24h. Select a single colony and grow it on a TSA (chloramphenicol) plate at 37℃ overnight, activate the transformant after transformation, extract the plasmid, and send it to Biotech for sequencing, the sequencing primer is M13f, and finally the verified correct epidermidis strain is named SEAgrArah2.

[0124] 4. Expression of peanut allergen Ara2.02 derivative protein in Staphylococcus epidermidis ATCC12228: Pick a single colony of recombinant bacteria and shake it in 2ml chloramphenicol medium at 37℃ overnight. At the same time, S.epi ATCC12228 containing an empty plasmid was used as a control. Transfer the inoculum to a fresh 1.5mL chloramphenicol medium with a 1% volume, culture for 24h, collect the fermentation broth and bacteria after centrifugation at 6000g for 10min, and filter the fermentation broth with a 0.22mm microporous filter; ultrasonic method was used to extract intracellular protein. Purify the protein according to the standardized protocol of Sangon, and the eluted protein was used for SDS-PAGE analysis. If it is fermentation broth, directly add 40μL supernatant + 20μL 5×SDS-PAGE loading buffer; 20μL of purified protein or ultrasonically extracted protein + 5μL 5×SDS-PAGE loading buffer, heat at 98℃ heating block for 10min; add 10μL sample into the electrophoresis tank, 120v, 60min; stain with Coomassie Brilliant Blue for 10min, stand in ddH20 for decolorization, and stain with WB.

[0125] Preparation Example 2: Construction of engineered Escherichia coli

[0126] 1. Experimental strains and plasmids: Escherichia coli BL21 (DE3) and pET-22b plasmid were purchased from Shanghai Sangon Biotechnology Co., Ltd.

[0127] 2. Bioinformatics methods to predict peanut allergen epitopes:

[0128] 1) T cell epitope prediction method: The gene sequences and protein sequences of peanut allergens Ara h 2.02 (GenBank: AAN77576.1), Ara h 6.01 (GenBank: AAD56337.1), Ara h 1 (GeneBank: AAB00861), Ara h 3.01 (GenBank: AAC63045), Ara h 7.0101 (GenBank: AAD56719), and Ara h8.0101 (GenBank: AAQ91847) were retrieved from the NCBI protein database. A data-driven prediction approach was used to identify T cell epitopes by distinguishing between MHC binders and non-binders and predicting the binding affinity of peptides to MHC molecules. Human HLA DRB1*09:01, DRB1*15:01, DRB1*07:01, DRB1*12:02, DRB1*08:03, DRB1*11:01, DRB1*03:01; HLA-DQA1*05:01 / DQB1*03:01, HLA-DQA1*05:03 / DQB1*03:01, HLA-DQA1*05:05 / DQB1*03:01, HLA-DPA1*01:03 / DPB1*02:01, HLA-DPA1*02:01 / DPB1*05:01 and mouse MHC-II H2 I Ab, H2I Ad and H2 I Ed restrict T cell epitopes. The four methods recommended by IEDB were selected, including NN-align (artificial neural network), SMM-align (stabilization matrix), CombLib (combinatorial library) and Sturniol method, of which Sturniol method was the final choice. Net MHCII pan 4.0 was used to verify the T cell epitope results. Then, the random forest method Bepipred2.0 was used to predict whether there were linear B cell epitopes in the T cell epitopes (the threshold was set to 0.55, the specificity was 0.81, the sensitivity was 0.3, and there were more than 7 amino acid residues). Finally, IgPred was used to identify subunit vaccines such as IgG, IgM, and IgE.

[0129] After the above process, the screening method of T cell epitope peptides is as follows:

[0130] 1) IEDB MHCII predicted percentile ranking value ≤ 10, stable matrix method IC50 ≤ 500nM, neural network method IC50 ≤ 500nM, and NetMHCIIpan2.0 method rankEL% ≤ 2 and IC50 ≤ 500nM. Finally, IL-4Pred was used to exclude peptides that may induce IL-4 response; 2) When selecting epitopes for hybrid vaccines, IgPred or Bepipred was preferred to identify peptides with B cell epitopes.

[0131] 2) B cell epitope prediction method

[0132] Bepipred2.0 was used to predict linear B cell epitopes present on the protein surface (the threshold was set to 0.55, with a specificity of 0.81 and a sensitivity of 0.3 at this threshold, with more than 7 amino acid residues). Next, the Kolaskar&Tongaonkar Antigenicity tool in EpiBuilde was used to score the antigenicity of the screened antigenic peptides. A higher antigenicity score indicated that it had a stronger potential to initiate an immune response, and the window size was kept at 7. To identify the surface availability and hydrophilicity of the epitope, Parker Hydrophilicity Prediction was used, and the window was set to 7. Finally, IgPred was used to identify the subunit vaccine.

[0133] After the above process, the screening strategy for B cell epitope peptides is as follows:

[0134] 1) The scores predicted by Bepipred2.0 were calculated progressively with 0.50, 0.55, 0.60, and 0.70 as nodes to screen out the core peptides with the highest scores and the least redundant ones. The core peptides with an average score > 0.55 were used for subsequent comparison; 2) Peptides less than 20aa were expanded, and then accessibility, antigenicity, and hydrophilicity were analyzed; 3) The selected peptides were subjected to IgPred subunit analysis, and peptides that were epitopes by both IgPred and Bepipred were preferred.

[0135] The candidate epitopes were connected through different linkers (no linker, GGGGS (SEQ ID NO: 12), GGGSGGG (SEQ ID NO: 13), EAAAK (SEQ ID NO: 14), GPPGG (SEQ ID NO: 15), AAY, KK) to obtain different candidate sequences. The Expasy ProtPara tool was used to predict the half-life and stability of the candidate protein. Finally, the best candidate sequence was screened out through comprehensive analysis.

[0136] The final protein sequence was optimized for E. coli DE3 expression using the OPTIMIZER online codon optimization tool. The optimized DNA sequence was sent to gene synthesis to obtain pET-22b-ArahA, pET-22b-ArahB, and pET-22b-Arah2.

[0137] 4. Expression of Peanut Allergen-derived Proteins in E. coli DE3

[0138] Activate the engineered bacteria, pick a single strain and culture it in LB liquid medium with ampicillin resistance; when the OD value reaches 0.6, add the inducer 0.5mM IPTG and continue to culture, induce overnight at 20°C and culture for 6h at 37°C, respectively, and the one without adding IPTG is a negative control; centrifuge at 12000rpm for 3min, discard the supernatant, and collect the bacteria; fully crush the bacterial precipitate with an ultrasonic disruptor, centrifuge at 6500rpm for 10min; dissolve the precipitate after centrifugation with TE solution for SDS-PAGE detection and western blot verification.

[0139] Effect Example 1

[0140] According to the design of the pRMC2 plasmid, there is a lack of effective ribosome binding site sequence in the Tet-on inducible element and the multiple cloning site used in the present invention. Therefore, in order to increase the yield of allergen protein, the RBS sequence of the hld gene -AGAAGGAGA- is inserted into this region. The modified plasmid is named pRMC2_tet1 (eg Figure 1 The RBS sequence of the top10 gene -AGGAGG- was inserted into this region, and the modified plasmid was named pRMC2_tet2 (as shown in Figure 2 shown).

[0141] The quorum sensing system (QS) is a type of density-dependent communication between bacteria. Autoinducers bind to related gene promoters and induce cascade reactions. In the QS system, the concentration of autoinducers increases with the proliferation of bacteria, inducing related proteins that can regulate the physiological characteristics of microbial populations. The accessory gene regulator (Agr) is a QS system between Staphylococcus species. The promoters P3 and P2 at this site are regulated by the auto-inducing peptide (AIP) produced by the bacteria themselves. Therefore, all the Tet-on inducible elements of the pRMC2 plasmid were replaced with the mutually opposite P2 and P3 promoter regions of the accessory gene regulator system (Agr) of Staphylococcus epidermidis ATCC12228, which has its own ribosome binding site. The modified plasmid was named pRMC2_Agr (such as Figure 3As shown), M13rev single-end sequencing and DNA gel verification were performed to confirm that the plasmid sequence was connected correctly (as shown Figure 4 shown).

[0142] Considering that the Tet-on inducible element of pRMC2 needs to be induced by adding dehydrotetracycline, it is not convenient in practical application and dehydrotetracycline may be harmful to organisms. Therefore, pRMC2_Agr was selected as the subsequent expression vector.

[0143] Genetic codons are degenerate and have codon preference in use. When foreign genes are expressed in heterologous hosts, the host prefers different codons, resulting in low expression or even no expression, which limits the expression of recombinant proteins in prokaryotes. Therefore, the codons were modified to ATCC12228 preferred codons using JCAT software with codon adaptation index (CAI) as the main reference index. The closer the CAI is to 1, the stronger the adaptability (e.g. Figure 5 , as shown in Table 1). Finally, the original signal peptide of Arah2.02 was removed, and the signal peptide sequence of the ATCC12228 glycerol hydrolase precursor sec pathway was added to the N-terminus, and extracellular secretory expression was expected. After sequencing verification, the pRMC2_Agr_sigArah2SE plasmid was successfully transferred into S.epi ATCC12228. The fermentation broth supernatant, bacterial lysis supernatant and precipitate were extracted and tested by western blot. Figure 6 As shown, the results show that the recombinant protein is expressed intracellularly, and the molecular weight is consistent with the expected molecular weight. Since the result is a single band, BCA protein quantification is directly performed, and the yield is about 10 mg / L. Arah2.02 has a B cell conformational epitope, which is a repeated DPYSPOHS (SEQ ID NO: 16) sequence (the second proline is hydroxylated). This domain is the main IgE binding epitope. The conformational epitope antibody was used to detect whether this epitope still exists when it is expressed in S.epi. Finally, it was found that the recombinant protein cannot fold into a conformational epitope, which means lower allergenicity.

[0144] Table 1

[0145]

[0146]

[0147] Effect Example 2

[0148] The immunogenicity of T cell epitopes is reflected in: (1) antigen processing; (2) binding to MHC molecules; (3) cognate TCR recognition. Among them, the binding of MHC molecules to antigenic peptides is the most selective, and predicting the binding of peptides to MHC molecules is the basis for predicting T cell epitopes. MHC II binding peptides have different lengths (9 to 22 residues), but the same is that there is a 9-residue core located in the binding groove. Peptide-MHC II binding prediction technology focuses on these peptide binding cores.

[0149] First, the MHC-II binding prediction software of the immune epitope database (IEDB-AR) and the NetMHC II pan 4.0 software were used to identify and screen T cell epitopes of Arah1, 2, 3, 6, 7, and 8 (such as Figure 7 As shown in the table 2), epitopes with the top 2% binding capacity were retained; Bepipred2.0 was used to predict whether there were linear B cell epitopes in the screened T cell epitopes, and those with B cell epitopes were given priority; IgPred was then used to identify immunoglobulin subunits; finally, IL4pred software was used to exclude epitopes that might have the ability to induce Th2 cells to secrete IL-4 in the long term, and finally 12 T cell epitopes were screened out (as shown in Table 2).

[0150] Methods for identifying linear B cell epitopes include amino acid scales and machine learning-based methods. Combining these two methods, Bepipred2.0 was first used to predict linear B cell epitopes present on the protein surface, and then the epitope properties were verified again using the scale: Kolaskar & Tongaonkar Antigenicity tool scored the antigenicity of the screened antigenic peptides, and a higher antigenicity score indicated that it had a stronger potential to initiate an immune response; Parker Hydrophilicity Prediction described the surface availability and hydrophilicity of the epitope, and higher surface availability and hydrophilicity represented a more likely B cell epitope. IgPred was also used to identify immunoglobulins, and a total of 8 B cell linear epitopes were finally screened (as shown in Table 3).

[0151] The recombinant protein was constructed by using 12 T cell epitopes plus 4 B cell epitopes. The peptides were connected with GPGPG (SEQ ID NO: 17) linkers, which can stimulate the helper T cell immune response and retain the immunogenicity of the epitopes. The amino acid composition, secondary structure, and solvent accessibility of the hybrid peptide were described by ExPASy ProtParam software. The ArahA and ArahB sequences formed were codon optimized and gene synthesized together with the Arah2.02 sequence without the signal peptide, and finally cloned into the NdeI-XhoI site of the pET-22b (+) vector.

[0152] Table 2: The 12 T cell epitopes finally obtained

[0153]

[0154] Table 3: 8 B cell linear epitopes finally screened

[0155]

[0156] pET_22b_ArahA, pET_22b_ArahB, and pET_22b_Arah2EC plasmids were transferred into E. coli BL21, and the plasmids were extracted and verified by restriction digestion and single-end sequencing (e.g. Figure 8 Then, a small expression test was carried out, the bacteria were lysed by ultrasonication, and the protein was induced to express at two culture temperatures. The SDS-PAGE results showed that the recombinant protein was expressed intracellularly, mainly in the bacterial lysis precipitate, and a small amount in the supernatant. Western blot also showed obvious bands at the corresponding positions, further confirming that the protein was the target protein. The molecular weight of ArahA was about 40kDa, the molecular weight of ArahB was about 40kDa, and the molecular weight of Arah2 was about 20kDa (as shown in Fig. 9 The protein was purified by nickel agarose affinity chromatography and the protein content was determined by BCA method. After purification, the yield of ArahA was about 20 mg / L, the yield of ArahB was about 20 mg / L, and the yield of Arah2 was about 30 mg / L (as shown in Fig.10 Effect Example 3: Application of engineered bacteria in mouse models

[0157] 1. Extraction of crude peanut protein

[0158] Weigh 100g of fresh unsalted peanut powder, add 250mL of 20mmol / L pH7.2 Tris buffer, shake and mix, place on a shaker, low speed, 24h, 4℃. Centrifuge at 3000g for 30min to collect the aqueous phase, centrifuge repeatedly to remove the upper fat and lower particles, and finally retain the clarified middle layer of peanut crude protein, and perform SDS-PAGE electrophoresis and protein concentration determination.

[0159] 2. Preparation of recombinant bacteria

[0160] Transfer the recombinant strain cultured overnight to fresh liquid containing the corresponding antibiotic at a ratio of 2% (v / v), and culture at the respective temperature until OD600 is about 0.6, and then add the inducer for induction (if necessary). After 6 hours of induction at the corresponding temperature, take 100 μL of the bacterial solution for gradient dilution, and take different dilutions of bacterial solution (10 5 , 10 6 , 107 , 10 8 The cells were spread on antibiotic plates at different dilutions and cultured overnight at 37°C. The colonies of each dilution were counted and the bacterial concentration was calculated. The induced cells were collected by centrifugation, washed twice with sterile PBS solution, and resuspended in an appropriate amount of PBS solution to adjust the bacterial concentration to 10 10 CFU / mL.

[0161] 3. Animal Experimental Procedures

[0162] 1) CPE allergy mouse model

[0163] Sensitization procedure: 6-8 week old male Balb / c mice, 6 mice in each group, 8 groups in total. On day 0, the back of the mice was shaved with a shaver, leaving the vellus hair. The entire animal experiment process was divided into 3 stages, as shown below.

[0164] Prevention phase: SEArah2 group, SE group EPIT: 0.2 mL of resuspended bacterial solution (2×10 9 CFU); ECArahA, ECArahB, ECArah2, EC group OIT: 0.2 mL of resuspended bacterial solution (2×10 9 CFU), group used 0.2 ml PBS, and the Positive group used 0.2 ml PBS;

[0165] Sensitization phase: 2 mg CPE and 15 μg CT were administered intragastrically on days 17, 24, 31, and 38;

[0166] Challenge phase: 7 days after the last oral gavage, i.e., on day 45, 10 mg CPE was injected intraperitoneally for challenge.

[0167] 2) MC903 combined with CPE transcutaneous sensitization AD model

[0168] Female Balb / c mice of 6 to 8 weeks old were divided into 6 groups, with 6 mice in each group.

[0169] EPIT group: On day 0, the back of mice was depilated using a shaver, leaving vellus hair, and the depilated area was 2 cm × 3 cm.

[0170] The entire animal experiment process is divided into 3 stages, as shown below.

[0171] Prevention phase: EPIT in the SEArah2 group and SE group, 0.2 mL of the induced bacterial solution was applied to the back continuously from day 1 to day 7. 9 CFU; ECArahB group, EC group OIT, intragastric administration of 0.2 mL bacterial solution 1×10 9CFU; 0.2 mL PBS was used in the blank control group (naive group) and the model group (positive group);

[0172] Transdermal sensitization stage: group with EtOH + PBS 38 μL, and the remaining groups with 0.25 mg CPE + 28 μL MC903 / ear;

[0173] Challenge phase: 28 μL MC903 was applied continuously per ear starting from the 7th day after the last gavage, and CPE was challenged by intraperitoneal injection on the 32nd day.

[0174] 3) Sample collection

[0175] Serum samples: In the experimental procedure, blood was collected from the eye socket. After the mouse was anesthetized with isoflurane, a sterilized capillary tube was inserted from the inner canthus of the mouse to collect about 100 μL of blood. After stimulation, the mouse was anesthetized with isoflurane, and the eyeball of the mouse was removed to collect blood, and about 1 ml of blood was collected. The blood sample was left at room temperature for 1 to 2 hours. After the serum was precipitated, it was centrifuged at 4000g for 10 minutes, and the upper serum was carefully aspirated and stored at -80°C.

[0176] Ear skin samples: The ear skin was collected from the MC903 combined with CPE percutaneous sensitization AD model. After the mice were anesthetized with isoflurane, the ear skin was cut about 6 mm from the edge with sterile scissors and divided into 2 parts. One part was immediately placed in paraformaldehyde for subsequent HE detection; the other part was frozen in a -80℃ refrigerator for subsequent detection.

[0177] 4) Allergic reaction score and temperature curve

[0178] Scoring of allergic reactions: 30 min after stimulation, the peanut allergy symptoms of mice in each group were evaluated. The reaction scoring system is as follows: 0 points - no symptoms; 1 point - scratching the nose and head; 2 points - increased edema around the eyes, hair erection, diarrhea, decreased activity frequency and increased respiratory rate; 3 points - cyanosis around the mouth and tail, difficulty breathing; 4 points - only mild or no reaction after stimulation, tremor and spasm; 5 points - death. The cage position information was hidden in the evaluation of allergic symptoms. Rectal temperature was measured with a rectal probe.

[0179] 5) ELISA test for total serum IgE antibody

[0180] Coating capture antibody: Equilibrate the ELISA reagents to room temperature, dilute the rat anti-moust IgE capture antibody to 2 μg / ml with coating solution (50 mM sodium carbonate / sodium bicarbonate, pH 9.6). Add 100 μL to each well of a 96-well plate, shake the plate, and incubate overnight at 4°C; wash with PBST three times, 5 minutes each time;

[0181] Blocking: 200 μL 1% BSA solution at 37°C for 30 minutes; wash 3 times with PBST, 5 minutes each time;

[0182] Add samples: The first column is reserved as a blank control and 100 μL of blocking solution is added. The rest are added with 100 μL of sample. The dilution of IgE blocking solution is 1:20, IgG1 is 1:1000, and IgG2a is 1:1000. Incubate overnight at 4°C; wash 3 times with PBST, 5 minutes each time;

[0183] Detection antibody incubation: dilute biotin rat anti-mouse IgE to 2 μg / ml with blocking solution, add 100 μL to each well; incubate at 37°C for 1 hour; wash 6 times with PBST, 5 minutes each time;

[0184] HRP coupling: add Streptavidin HRP diluted 1:1000, incubate at 37℃ for 30min; wash 6 times with PBST, 5min each time;

[0185] Color development: TMB method, used according to the instructions, detected at 450nm.

[0186] 6) ELISA for detection of serum CPE-specific IgE, IgG1, and IgG2α

[0187] Biotin coupling CPE: Take 500μL of 3mg / ml CPE protein to be coupled and add it to a 1.5ml centrifuge tube; add 250μl biotin coupling solution and 250μl dissolved 1mg activated biotin at the same time. On a rotating suspension instrument, slowly react at room temperature for 2 hours; centrifuge the desalting column at 1000g for 1min, remove the top and bottom covers of the desalting column, put it into a 15ml centrifuge tube, centrifuge at 1000g for 2min, and remove the resin preservation solution; add 5 times the column volume of biotin coupling solution (25ml) to balance the desalting column, and centrifuge to remove the coupling solution after each balance: 1000g for 2 minutes; transfer the desalting column to a new centrifuge tube, slowly transfer the coupling product of step 2 to the center of the resin, centrifuge at 1000g for 8min; collect the liquid in the centrifuge tube as the labeled product.

[0188] The capture antibody coating, blocking, and sample addition were the same as in step 5). The detection antibody incubation used Biotin coupled to CPE (diluted to 8 μg / ml), coupled to HRP, and color development was the same as in step 5).

[0189] 7) HE staining of ear skin and cytokine qRT-PCR

[0190] 5. Results Analysis

[0191] In order to explore the functional effects of engineered bacteria and their secreted proteins, a CPE intestinal sensitization mouse model and a MC903 combined with CPE transdermal sensitization AD model were established, and four engineered bacteria, SEArah2, ECArah2, ECArahB, and ECArahA, were used for preventive treatment.

[0192] 5.1 CPE intestinal sensitization mouse model

[0193] Extract the peanut protein (CPE) from the unsalted peanut powder and perform SDS-PAGE analysis. The bands of Ara h1, Ara h 2, Ara h 3, and Ara h 6 can be clearly seen (e.g. Fig.11 The protein concentration measured by BCA method was 25 mg / mL.

[0194] After the sensitization procedure was completed on the 38th day, serum was collected for ELISA test, and it was found that the total IgE of the engineered bacteria group after sensitization was significantly lower than that of the model group (Positive group), pure bacteria control group (SE group, EC group) and blank control group ( The ECArahB group and the SEArah2 group had the lowest total IgE, indicating that the prevention effect was the best. The CPE-specific IgE also conformed to this conclusion. The ECArahB group and the SEArah2 group had the most obvious reduction in specific IgG1 and significantly increased specific IgG2α (such as Fig.12 The corresponding data are shown in Table 4).

[0195] On the 45th day, 10 mg of CPE was injected intraperitoneally to stimulate the allergic symptoms in the engineered bacteria group, and there was no obvious drop in body temperature. The trends of total IgE and specific IgE after stimulation were similar to those after sensitization. The total IgE and specific IgE in the ECArahB group and the SEArah2 group were the lowest. The ECArahB group reduced specific IgG1 most significantly, and the ECArahB group and the ECArah2 group were able to significantly increase the amount of specific IgG2α. In summary, in the model of intestinal sensitization to peanut protein, it was found that engineered bacteria can significantly improve allergic symptoms and allergic phenotypes. The mechanism may be to promote the bias of Th2 cell response toward Th1 type. Among the four groups of engineered bacteria, the ECArahB group and the SEArah2 group had the most significant preventive treatment effects, so these two strains were selected for subsequent animal experiments (such as Fig.12 The corresponding data are shown in Table 4).

[0196] Table 4: Therapeutic effects of engineered bacteria on CPE allergy mouse model

[0197]

[0198]

[0199] 5.2 MC903 combined with CPE transcutaneous sensitization AD model

[0200] Gross photos of mice during the transdermal sensitization procedure Fig.13 As shown in the statistical graph Figure 14 to Figure 17 The detailed data are shown in Tables 5 to 8. The ear thickness was measured manually during sensitization. The ear thickness of the SEArah2 group and the ECArahB group was thinner than that of the model group and the pure bacteria group, with statistically significant differences. After completing the sensitization procedure on the 25th day, the ear thickness was measured manually and the EASI disease score was performed. The ear thickness of the ECArahB group was significantly thinner than that of the model group and the EC group, and the EASI score was lower. On the HE section, the epidermal thickness of the ECArahB engineered bacteria group was significantly thinner. The ear cytokine detection showed that the SEArah2 group significantly reduced the expression of IL-4, IL-13, and IL-33. The serum was taken for ELISA detection and found that the total IgE of the engineered bacteria group after sensitization was significantly lower than that of the model group, the pure bacteria control group and the blank control group, and there was no difference between the pure bacteria control group and the model group; CPE-specific IgE also conformed to this conclusion, and the ECArahB group was lower than the SEArah2 group; the ECArahB group and the SEArah2 group significantly reduced specific IgG1 and significantly increased specific IgG2α.

[0201] On the 35th day, the allergic symptoms of the engineered bacteria group were significantly milder, and there was no obvious drop in body temperature. The trend of total IgE and specific IgE after stimulation was similar to that after sensitization. The level of specific IgG1 was significantly reduced in the ECArahB group and the SEArah2 group, and the level of specific IgG2α was significantly increased in the ECArahB group and the ECArah2 group.

[0202] It was concluded that the ECArahB engineered bacteria for oral immunotherapy and the SEArah2 engineered bacteria for transcutaneous immunotherapy can alleviate the symptoms and phenotypes of dermatitis caused by MC903 combined with CPE, and the mechanism may be to promote the bias of Th2 cell response towards Th1 type.

[0203] Table 5: Phenotype of mice sensitized by MC903 combined with CPE

[0204]

[0205]

[0206] Table 6: Ear thickness measured in sections of mice sensitized with MC903 and CPE

[0207] Day 1 HE ear thickness after sensitization Naive EC ECArahB SE SEArah2 Positive Number of values 30 30 32 32 30 30 Mean 162.7 497.7 263.4 395.5 427.1 437.8 Std. Deviation 24.93 34.96 61.14 55.51 60.01 77.06 Std.Error of Mean 4.551 6.383 10.81 9.813 10.96 14.07 HE ear thickness when excited: 1.2 Naive EC ECArahB SE SEArah2 Positive Number of values 20 29 27 30 30 30 Mean 278.6 506.3 452.1 520.5 479.3 490.3 Std. Deviation 60.96 80.9 59.16 88.01 71.6 95.14 Std.Error of Mean 13.63 15.02 11.38 16.07 13.07 17.37

[0208] Table 7: Expression of ear cytokines after sensitization of the mouse model sensitized by MC903 combined with CPE

[0209] IL-4 Naive EC ECA SE SEArah2 Positive Mean 1.615 9.43 4.218 11.43 3.706 9.089 Std. Deviation 1.515 6.026 3.192 4.893 2.532 4.049 Std.Error of Mean 0.6184 3.013 1.303 2.188 1.034 1.653 IL-33 Naive EC ECA SE SEArah2 Positive Mean 0.3495 8.433 3.006 4.421 1.119 3.828 Std. Deviation 0.2676 6.747 3.522 3.226 0.9814 1.785 Std.Error of Mean 0.1338 3.373 1.438 1.317 0.4007 0.7285 IL-13 Naive EC ECA SE SEArah2 Positive Mean 1.251 1.947 0.7993 0.8738 0.4758 1.208 Std. Deviation 0.7921 1.877 0.6537 0.497 0.4763 0.4377 Std.Error of Mean 0.3234 0.7664 0.2669 0.2029 0.1944 0.1787 TSLP Naive EC ECA SE SEArah2 Positive Mean 1.273 0.6927 1.942 1.564 1.515 2.341 Std. Deviation 1.011 0.4819 2.126 0.7512 1.535 1.535 Std.Error of Mean 0.5053 0.2409 1.228 0.4337 0.6865 0.6866 IL-10 Naive EC ECA SE SEArah2 Positive Mean 1.919 3.314 1.434 2.23 0.8005 1.842 Std. Deviation 3.319 2.965 1.477 1.41 0.5773 0.7083 Std.Error of Mean 1.484 1.326 0.6031 0.5757 0.2357 0.2892

[0210] Table 8: Serological changes in MC903 combined with CPE sensitized mouse model

[0211]

[0212]

[0213] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of antigenic epitopes in the preparation of products for the prevention and / or treatment of allergy-related diseases; The antigenic epitope has: (1) an amino acid sequence as shown in any one of SEQ ID NOs: 18 to 47; or (2) an amino acid sequence obtained by substituting, deleting or adding one or more residues of the amino acid sequence shown in (1), and having the same or similar function as (1); or (3) an amino acid sequence having at least 90% homology to the amino acid sequence shown in (1) or (2); The plurality is 2 to 5; The allergy-related diseases include peanut allergy-related diseases; The allergy-related diseases also include one or more of allergic dermatitis, allergic gastroenteritis or atopic dermatitis; The product includes one or more of a drug, a biological product or a cosmetic.

2. A polypeptide, characterized in that An amino acid sequence having the antigenic epitope in the use according to claim 1; or have: (4), the amino acid sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO: 8; or (5) An amino acid sequence obtained by replacing, deleting or adding one or more residues of the amino acid sequence shown in (4), and having the same or similar function as (4); or (6) an amino acid sequence having at least 90% homology to the amino acid sequence shown in (4) or (5); The plurality is 2 to 30.

3. A nucleic acid molecule, characterized in that Having a nucleotide sequence encoding the polypeptide according to claim 2; or have: (7), a nucleotide sequence as shown in any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7; or (8) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (7), and having the same or similar function as (7); or (9) a nucleotide sequence having at least 90% homology to the nucleotide sequence shown in (7) or (8); The plurality is 2 to 100.

4. An expression vector, characterized in that Comprising the nucleic acid molecule as claimed in claim 3.

5. The expression vector according to claim 4, characterized in that The backbone is pRMC2_Agr or pET-22b; The preparation method of pRMC2_Agr is to replace all the Tet-on inducible elements of the pRMC2 plasmid with the mutually inverse P2 and P3 promoter regions of the auxiliary gene regulatory system Agr of Staphylococcus epidermidis ATCC12228; The mutually reversed P2 and P3 promoter regions of the auxiliary gene regulatory system Agr of Staphylococcus epidermidis ATCC12228 have: (10), the nucleotide sequence shown in SEQ ID NO: 9; or (11) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (10), and having the same or similar function as (10); or (12) A nucleotide sequence having at least 90% homology to the nucleotide sequence shown in (10) or (11); The plurality is 2 to 30.

6. A host cell, characterized in that comprising the polypeptide according to claim 2, the nucleic acid molecule according to claim 3, or the expression vector according to claim 4 or 5; The host cells include Escherichia coli and / or Staphylococcus epidermidis.

7. Use of the host cell according to claim 6 in any of the following: (a) Reduce the body's IgE and IgG1; (b) Increase serum IgG2a; (c) reducing one or more of IL-4, IL-33 or IL-13 in the body; (d) Promote the body's Th2 cell response to shift toward Th1 type; (e) Alleviate gastrointestinal allergy symptoms and / or skin allergy symptoms; (f) Prevention of allergic gastroenteritis and / or skin diseases; (g) Treatment of allergic gastroenteritis and / or skin diseases; (h) Maintaining gastrointestinal tract and / or skin health; (i) prevention and / or treatment of allergic diseases; (j) desensitization therapy; (k) preparing a product that achieves one or more of (a), (b), (c), (d), (e), (f), (g), (h), (i), (j) or (k); The skin disease includes one or more of allergic dermatitis, atopic dermatitis, eczema, urticaria or psoriasis; The product includes one or more of medicines, biological products, skin care products or cosmetics.

8. A product, characterized in that Comprising the polypeptide according to claim 2 and / or the host cell according to claim 6, and acceptable adjuvants or auxiliary agents; The product includes medicine, biological product, skin care product or cosmetic; The administration routes of the drug include oral administration or external application.

9. The method for preparing a host cell according to claim 6, characterized in that: The expression vector according to claim 4 or 5 is introduced into chassis cells to obtain the protein.

10. A method for preventing and / or treating a disease, characterized in that include: Oral or external use of the polypeptide as claimed in claim 2; or Oral or external administration of the host cell according to claim 6; or The product as claimed in claim 8 for internal or external use; The diseases include allergic diseases; The allergic disease includes one or more of allergic dermatitis, allergic gastroenteritis, atopic dermatitis, eczema, urticaria or psoriasis.

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