Engineering strain for expressing dust mite allergen derived protein and application thereof

Abstract

The invention relates to the technical field of biology, in particular to an engineering strain for expressing dermatophagoides pteronyssinus allergen derived protein and application of the engineering strain. The invention provides engineering bacteria such as ECDer1, ECDer2, SEAgr-Der1, SEAgr-Der2 and the like by adopting an authenticated probiotic chassis strain, and the engineering bacteria are used for safely and effectively treating the allergic diseases, particularly allergic skin diseases mediated by dust mite related inhalation allergens and respiratory tract allergic diseases.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to the application of an engineering strain expressing dust mite 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 influenced 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 multiple comorbidities, including food allergies, dust mite allergies, asthma, allergic rhinitis, and mental health disorders.

[0003] Dust mites are one of the most common allergens that cause exogenous AD. They can invade the skin barrier, promote the polarization of helper T cells 2 (Th2), cause the Th1 / Th2 balance to drift, and lead to and aggravate AD. Studies have shown that there is a certain correlation between dust mites and AD. Some scholars have found that AD patients are more sensitive to some allergens of dust mites than those with respiratory allergic diseases. Studies have found that dust mites are the most important type of inhaled specific immunoglobulin E (sIgE) in AD patients. Studies have shown that the positive detection rate of inhaled allergens increases with the age of infants and children with AD, and patients gradually change from food allergies to inhaled allergies.

[0004] There are many types of dust mites, but the three most common ones are house dust mites (Dermatophagoides pteronyssinus, Der p), dust mites (Dermatophagoides farina, Der f) and house dust mites (Euroglyphus maynei, Euro m). According to current research, there are 31 allergens for house dust mites, 36 for dust mites, and 5 for house dust mites. There are certain differences in the biochemical properties of different allergens. At present, Der p 1, Der f 1, Der p 2, Der f 2, and Der p 23 are considered to be the main dust mite allergens. Based on their different biochemical properties, current research still has some ambiguity in their respective biological functions. Skin barrier damage caused by dust mite invasion and Th1 / Th2 immune imbalance are the two main causes of AD.

[0005] There is an urgent need to develop a treatment method that can 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.

[0006] 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 the original 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 diet 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 2-3 peanut allergens, which can avoid IgE and T cell-mediated side effects and induce the production of allergen-specific IgG and inhibit specific IgE.

[0007] At present, the treatment of atopic dermatitis mainly focuses on conventional topical medication (topical corticosteroids, calcineurin inhibitors, antibiotics, topical antipruritic agents, emollients, etc.), oral medication (antihistamines, systemic corticosteroids, immunosuppressants, etc.), and biological agents. Long-term use of topical corticosteroids (TCS) is associated with risks such as skin atrophy, abnormal pigmentation, acne-like rashes, and systemic absorption-related risks (such as hypothalamic-pituitary axis effects and Cushing's disease), so the long-term use of TCS is not safe enough. 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 the drug unsafe. Repeated use of any topical medication over a long period of time or use 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 biologics against IL-4Rα (dupirumab) show certain therapeutic effects, but their process of blocking T helper cell response signals may be accompanied by the adverse consequences of immunosuppression.

[0008] At present, there is dust mite allergen-specific immunotherapy for AD with combined allergies. Currently, there are five main methods of SIT: subcutaneous injection, sublingual administration, oral administration, transcutaneous immunotherapy and lymphatic immunotherapy. Studies have shown that sublingual administration of dust mite drops and house dust mite allergen lymphatic immunotherapy are both effective. However, the risk of local and systemic adverse reactions still exists in clinical practice, including itching, redness, swelling, nodules, necrosis at the injection site, and even shock, laryngeal edema, urticaria, vasculitis, bronchial asthma, etc. in severe cases. Summary of the invention

[0009] In view of this, the present invention provides an application of an engineered strain expressing dust mite allergen-derived protein in allergic diseases, adopts a certified probiotic chassis strain, safely and effectively treats allergic diseases, and expands allergic skin diseases and respiratory allergic diseases mediated by dust mite-related inhalation allergens.

[0010] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0011] The present invention provides the use of antigenic epitopes in the preparation of products for preventing and / or treating allergy-related diseases;

[0012] The antigenic epitope has:

[0013] (1) an amino acid sequence as shown in any of SEQ ID NOs: 1 to 14; or

[0014] (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

[0015] (3) an amino acid sequence having at least 90% homology to the amino acid sequence shown in (1) or (2);

[0016] The plurality is 2 to 5;

[0017] The products include pharmaceuticals, biological products, skin care products or cosmetics.

[0018] In some specific embodiments of the present invention, the antigenic epitope used above includes a combination of two or more of SEQ ID NOs: 1 to 14.

[0019] In some specific embodiments of the present invention, the allergy-related disease described above is allergic dermatitis.

[0020] In some specific embodiments of the present invention, the allergic dermatitis described above is atopic dermatitis.

[0021] In some specific embodiments of the present invention, the atopic dermatitis described in the above application is atopic dermatitis caused by dust mites.

[0022] The present invention also provides a polypeptide having the antigenic epitope in the above application.

[0023] In some specific embodiments of the present invention, the above polypeptide is polypeptide 1 and / or polypeptide 2

[0024] The sequence of polypeptide 1 is divided into 21 parts, namely SEQ ID NO: 2, SEQ ID NO: 20, SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 5, SEQ ID NO: 20, SEQ ID NO: 6, SEQ ID NO: 20, SEQ ID NO: 7, SEQ ID NO: 20, SEQ ID NO: 1, SEQ ID NO: 20, SEQ ID NO: 3, SEQ ID NO: 20, SEQID NO: 8, SEQ ID NO: 20, SEQ ID NO: 9 or 10, SEQ ID NO: 20, SEQ ID NO: 11 or 12, SEQID NO: 20, SEQ ID NO: 13 or 14.

[0025] The sequence of polypeptide 2 is divided into 21 parts, namely SEQ ID NO: 9 or 10, SEQ ID NO: 20, SEQ ID NO: 7, SEQ ID NO: 20, SEQ ID NO: 6, SEQ ID NO: 20, SEQ ID NO: 1, SEQ ID NO: 20, SEQ ID NO: 5, SEQ ID NO: 20, SEQ ID NO: 13 or 14, SEQ ID NO: 20, SEQ ID NO: 8, SEQ ID NO: 20, SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 2, SEQ ID NO: 20, SEQ ID NO: 3, SEQ ID NO: 20, SEQ ID NO: 11 or 12.

[0026] In some specific embodiments of the present invention, the amino acid sequence of polypeptide 1 of the above-mentioned polypeptide is SEQ ID NO: 21.

[0027] In some specific embodiments of the present invention, the amino acid sequence of polypeptide 2 of the above-mentioned polypeptide is SEQ ID NO: 22.

[0028] The present invention also provides a nucleic acid molecule having a nucleotide sequence encoding the above polypeptide.

[0029] The present invention also provides an expression vector comprising the above nucleic acid molecule.

[0030] In some specific embodiments of the present invention, the backbone of the above-mentioned expression vector is pRMC2_Agr or pET-22b.

[0031] The present invention also provides a host cell, comprising at least one of the above polypeptide, the above nucleic acid molecule or the above expression vector;

[0032] The host cell may be Escherichia coli and / or Staphylococcus epidermidis.

[0033] In some specific embodiments of the present invention, the Escherichia coli in the above-mentioned host cell includes Escherichia coli BL21 (DE3);

[0034] In some specific embodiments of the present invention, the Staphylococcus epidermidis in the above host cell includes Staphylococcus epidermidis ATCC12228.

[0035] The present invention also provides the use of the above polypeptide or the above host cell in any of the following items:

[0036] (a) Reduce the body's IgE and IgG1;

[0037] (b) Increase the body's serum IgG2a;

[0038] (c) Relieve skin allergy symptoms;

[0039] (d) Prevention and / or treatment of allergic diseases;

[0040] (e) Desensitization therapy;

[0041] (f) preparing a product that achieves at least one of (a), (b), (c), (d), and (e);

[0042] The skin diseases include allergic dermatitis;

[0043] The products include pharmaceuticals, biological products, skin care products or cosmetics.

[0044] The present invention also provides a product, comprising the above polypeptide and / or the above host cell, and an acceptable auxiliary material or adjuvant;

[0045] The product includes a drug, a biologic, a skin care product, or a cosmetic;

[0046] The administration routes of the drug include oral administration or external application.

[0047] The present invention also provides a method for preparing the host cell, which is obtained by introducing the expression vector into chassis cells.

[0048] The present invention also provides a medicine, comprising the above polypeptide and / or the above host cell and an acceptable auxiliary material or adjuvant.

[0049] 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.

[0050] The present invention also provides cosmetics, comprising the above polypeptide and / or the above host cell and acceptable auxiliary materials or additives.

[0051] 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.

[0052] 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.

[0053] The present invention also provides a method for preventing and / or treating a disease, comprising:

[0054] Oral or external administration of the host cells; or

[0055] Oral or external use of the above products;

[0056] The diseases include allergic diseases;

[0057] The allergic disease includes allergic dermatitis and / or allergic gastroenteritis;

[0058] The allergic dermatitis may be at least one of atopic dermatitis, eczema, urticaria or psoriasis.

[0059] The present invention has the following effects:

[0060] The present invention develops sustainably colonized engineered bacteria that express dust mite allergens, which can be taken orally or applied externally to safely and continuously prevent dust mite-related allergic skin diseases and allergic respiratory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] 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.

[0062] Figure 1 The schematic diagram of the improved pRMC2_Agr plasmid and its DNA gel verification are shown, wherein A shows the schematic diagram of pRMC2_Agr (5.5k+330 bp), and B shows the DNA gel verification of the cloning site KpnI / pstI;

[0063] Figure 2 The hybrid peptides of dust mite allergens are described, wherein A represents Der1 and B represents Der2;

[0064] Figure 3A Shows the map of the dust mite allergen hybrid peptide plasmid pET-22b-Der1;

[0065] Figure 3B Shows the map of the dust mite allergen hybrid peptide plasmid pET-22b-Der2;

[0066] Figure 3C Shows the map of the dust mite allergen hybrid peptide plasmid SE_Agr-Der1;

[0067] Figure 3D Shows the map of the dust mite allergen hybrid peptide plasmid SE_Agr-Der2;

[0068] Figure 4 The figures show the therapeutic effect of the engineered bacteria on the dust mite allergy mouse model, wherein A shows total serum IgE after stimulation, B shows serum dust mite-specific IgE after stimulation, C shows serum dust mite-specific IgG1 after stimulation, and D shows serum dust mite-specific IgG2α after stimulation. * represents p<0.05, ** represents p<0.01, *** represents p<0.001, and **** represents p<0.0001. n=5. DETAILED DESCRIPTION

[0069] The present invention discloses the use of engineered strains expressing dust mite 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.

[0070] Specific immunotherapy for allergic diseases such as atopic dermatitis has limitations, mainly allergic side effects, inconvenient administration and uncertainty in achieving long-term tolerance. The application of the engineered strain expressing dust mite allergen-derived protein in allergic diseases of the present invention is to expand the indications of the original invention "application of engineered strain expressing peanut allergen-derived protein in allergic diseases": ① increase the indications of allergic diseases related to inhaled allergens such as allergic asthma and allergic rhinitis, with respiratory allergy as the main clinical manifestation; ② increase the indications of allergic skin diseases such as atopic dermatitis and allergic dermatitis combined with allergies to inhaled allergens such as dust mites.

[0071] The sequence information involved in the present invention is as follows.

[0072] Der p 1 Tepi se1: ESYYRYVAREQSCRR (SEQ ID NO: 1);

[0073] Der p 1 Tepi se2:AQTHSAIAVIIGIKD (SEQ ID NO: 2);

[0074] Der p 5 Tepi se1:RKDLDIFEQYNLEMA (SEQ ID NO: 3);

[0075] Der p 3 Tepi se1:NAKAVGLPAKGSDVK (SEQ ID NO: 4);

[0076] Der p 3 Tepi se2: CNELYSKANAEVTDN (SEQ ID NO: 5);

[0077] Der p 3 Tepi se3: SIRYNSLKHSLGGEK (SEQ ID NO: 6);

[0078] Der f 21 Tepi se1:LAELAKKVKAVKSDD (SEQ ID NO: 7);

[0079] Der f 13 Tepi se1: VKIIREFNGDEVVVT (SEQ ID NO: 8);

[0080] Der p 1 Bepi se1 Adjust peptide:

[0081] IEYIQHNGVVQESYYRYVAR(SEQ ID NO: 9);

[0082] Der p 1 Bepi se1 Peptide core:

[0083] IQHNGVVQESYYRYV(SEQ ID NO: 10);

[0084] Der p 1 Bepi se2 Adjust peptide:

[0085] QSCRRPNAQRFGISNYCQIY(SEQ ID NO: 11);

[0086] Der p 1 Bepi se2 Peptide core:

[0087] RRPNAQRFGISNYC (SEQ ID NO: 12);

[0088] Der p1 Bepi se3 Adjust peptide:

[0089] VNIVGYSNAQGVDYWIVRNS (SEQ ID NO: 13);

[0090] Der p1 Bepi se3 Peptide core:

[0091] SNAQGVDYW (SEQ ID NO: 14).

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] The present invention will be further described below in conjunction with the embodiments.

[0098] Example 1: Optimization of the inducible expression element of plasmid pRMC2

[0099] 1. Plasmid design:

[0100] All Tet-on elements in pRMC2 were replaced with the RBS-P2-P3-RBS promoter region of the auxiliary gene regulatory system of Staphylococcus epidermidis ATCC12228, the specific sequence of which is: AGTTGTTTACTACTCTCCTCAAGTGTCATTATACAATTTTGCGCAACATTTTTTAGAAAGCATGCCTAACTGTTAAAAAAATATACCTAAGTGTTTTAATTAAGTACTATTAGATATTTTACCATATTTAGTTTTACAGTTGAGTACTAAATATTGCTATTTACGAAATTTTAATCTTTAAATGGAAAAATCATGTTTTAATAGACTCATATCACAGAGATGTGATTGAAAGATAGTTGAAAAATTTGCTTAATCTAGTCGAGTGAATGTTAAATTCATTCGTATCCATTACCTTAATTCGAAAGGAGTGAAGTTATAATGGTAC (SEQ ID NO: 23). After eliminating unnecessary restriction endonucleases in the recombinant gene sequence and connecting it to pUC57, the recombinant plasmid pUC_Agr was obtained.

[0101] 2. PCR amplification, recovery and purification of target gene fragments and expression vectors:

[0102] Plasmid pUC57_Agr was used as a template and upstream and downstream primers were used, including Agr-fwd: GGTACCATTATAACTTCACT (SEQ ID NO: 15); Agr-rev: CTCATCATTTGTTGACGTC (SEQ ID NO: 16) for PCR amplification. The obtained PCR product was detected by 1% agarose gel electrophoresis, and the target fragment was recovered by gel excision. Follow the steps below:

[0103] After the PCR experiment, take out 2~3 μL of sample from each PCR tube for electrophoresis detection, prepare a gel with a concentration of 1%, a voltage of 100V, and a low molecular weight nucleic acid standard as the marker. The desired band can be used for the next step. Run the remaining product on 1% agarose gel electrophoresis, cut the target band from the UV detector with a blade, place the band in plastic wrap and crush it by hand, place it in a centrifuge tube, use a gel purification kit to add the binding solution to dissolve it, and then transfer it to the recovery column to remove impurities through the rinsing step. The purified DNA is eluted from the recovery column by adding the elution solution.

[0104] 3. Double restriction enzyme digestion of vector and target gene fragment:

[0105] Taking KpnI and BamHI double enzyme linearization as an example, the reaction system is: substrate DNA (1 μg), KpnI (1 μL), BamHI (1 μL), 10× buffer (2 μL), ddH2O to 20 μL. Flick to mix, reaction temperature 37℃, reaction for 1 h, and use DNA fragment purification kit to purify the reaction solution.

[0106] 4. Connect the target gene fragment to the linearized vector:

[0107] The vector and target fragment were ligated at a molar ratio of 1:10 using a DNA ligation kit at 16°C for 30 minutes.

[0108] 5. Transformation and plate culture of ligation products:

[0109] Take out 100 μL of E. coli DH5α competent cells from the -80℃ refrigerator, place on ice and wait for thawing, transfer to a 2 mL centrifuge tube, add 10 μL of the above ligation product, mix well, and place on ice for 30 min. Heat shock at 42℃ for 1.5 min, place on ice for 2 min. Add 1 mL of SOC liquid culture medium, shake at 37℃ and 180 rpm for 1 h. Centrifuge at 5000 rpm for 1 min, discard the supernatant, resuspend with 100 μL of culture medium, spread on an ampicillin LB plate, and invert and culture at 37℃ overnight.

[0110] 6. Plasmid extraction and transformation DNA gel and sequencing verification:

[0111] Pick a single colony into LB medium with ampicillin resistance, shake at 220 rpm and 37℃ for about 12 hours, use Takara's plasmid extraction kit to extract the plasmid according to the kit instructions, perform DNA gel verification with the same method as 3 and 4, and send it for sequencing. Sequencing primer: M13f, unidirectional sequencing. The correctly sequenced plasmid is named pRMC2_Agr. Figure 1 shown.

[0112] Example 2: Bioinformatics method to predict dust mite allergen protein epitopes

[0113] 1. Included allergens:

[0114] The gene sequences and protein sequences of dust mite allergens: Derp1_EL (GenBank: M35523), Der p 3 (GenBank: M84364), Der p 5 (GeneBank: U27081), Der f 2 (GenBank: M35524), Der f 13 (GenBank: AF441292), and Der f 21 (GenBank: AY283292) were retrieved from the NCBI protein database.

[0115] 2. Removal of signal peptide:

[0116] Signal peptide analysis The online server SignalP 5.0 was used to analyze the sequence signal peptides, and the signal peptide fragments were deleted from the sequence, and the remaining sequence was saved in FASTA format for further analysis.

[0117] 3. Identification of helper T cell epitopes using two bioinformatics approaches:

[0118] A data-driven prediction method 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. The above sequences were first analyzed and identified using the MHC-IIbinding prediction software of the Immune Epitope Database (IEDB-AR). 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-DQA1*05:05 / DQB1*03:01, HLA-DPA1*01:03 / DPB1*02:01, HLA-DPA1*02:01 / DPB1*05:01 (targeting MHC class II DP, DQ, and DR, types commonly found in China) and mouse MHC-II H2 I Ab, H2 I Ad, and H2 I Ed were selected to restrict T cell epitopes. 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 was the final choice. Net MHC II pan 4.0 was used to verify the T cell epitope results. The software uses artificial neural network (ANN) to predict the binding ability of peptides to MHC II molecules, and sets the thresholds (% Rank scores) of strong binding ability and weak binding ability to 2% and 10%, respectively. Then, the random forest method Bepipred 2.0 was used to predict whether there were linear B cell epitopes in the T cell epitopes (the threshold was set to 0.55, at which 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.

[0119] After the above process, the T cell epitope peptide screening method:

[0120] (1) IEDB MHCII predicted percentile ranking value ≤ 10, stable matrix method IC50 ≤ 500 nM, neural network method IC50 ≤ 500 nM, and NetMHCIIpan2.0 method rank EL% ≤ 2, and IC50 ≤ 500 nM. Finally, IL-4Pred was used to exclude peptides that may induce IL-4 response;

[0121] (2) When selecting epitopes for hybrid vaccines, IgPred or Bepipred is preferred to identify peptides with B cell epitopes, as shown in Table 1.

[0122] Table 1: Bioinformatics methods to predict T cell epitope information of dust mite allergens

[0123]

[0124] 4. Identification of linear B cell epitopes using two bioinformatics approaches:

[0125] First, Bepipred 2.0 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 EpiBuilder 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.

[0126] After the above process, the B cell epitope peptide screening method:

[0127] (1) The scores predicted by Bepipred 2.0 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 of >0.55 were used for subsequent comparison;

[0128] (2) Expand peptides less than 20 aa and then analyze their accessibility, antigenicity, and hydrophilicity;

[0129] (3) The selected peptides were subjected to IgPred subunit analysis. The peptides that were epitopes of both IgPred and Bepipred methods were preferred, as shown in Table 2.

[0130] Table 2: Bioinformatics methods to predict B cell epitope information of dust mite allergens

[0131]

[0132] 5. Predict the ability of epitopes to induce IL-4:

[0133] The IL4 pred tool was used to calculate the ability of epitopes to induce IL-4 secretion by Th2 cells, and a hybrid prediction method consisting of a support vector machine and a motif was selected. In order to increase the probability that true negatives were indeed identified, the support vector machine threshold was selected as 0.6 (at this threshold, the sensitivity was 53.43 and the specificity was 95.01).

[0134] 6. Linker for screening epitopes:

[0135] The candidate epitopes were connected through different linkers (no linker, GGGGS [SEQ ID NO: 17], GGGSGGG [SEQ ID NO: 18], EAAAK [SEQ ID NO: 19], GPPGG [SEQ ID NO: 20], AAY, KK) to obtain different candidate sequences. The Expasy ProtParam tool was used to predict the half-life and stability of the candidate proteins. Finally, through comprehensive analysis, the best candidate sequence was screened out as: GPPGG.

[0136] 7. Construction of recombinant hybrid allergens:

[0137] ExPASy ProtParam software analyzes the amino acid composition, secondary structure, and solvent accessibility of hybrid peptides and screens out hybrid peptides with high stability, such as Figure 2 As shown, the final protein sequence was named and reverse-translated into a DNA sequence using Sequence Manipulation Suite version 2. A 6*his tag was attached to its C-terminus and optimized for expression in Escherichia coli DE3 and Staphylococcus epidermidis ATCC12228 using the OPTIMIZER online codon optimization tool. The optimized DNA sequence was sent to gene synthesis to obtain pET-22b-Der1, pET-22b-Der2, pRMC2_Agr-Der1, and pRMC2_Agr-Der2 ( Figure 3A , Figure 3B , Figure 3C , Figure 3D ).

[0138] Among them, the amino acid sequence of Der1 is:

[0139] AQTHSAIAVIIGIKDGPPGGNAKAVGLPAKGSDVKGPPGGCNELYSKANAEVTDNGPPGGSIRYNSLKHSLGGEKGPPGGLAELAKKVKAVKSDDGPPGGESYYRYVAREQSCRRGP PGGRKDLDIFEQYNLEMAGPPGGVKIIREFNGDEVVVTGPPGGIEYIQHNGVVQESYYRYVARGPPGGQSCRRPNAQRFGISNYCQIYGPPGGVNIVGYSNAQGVDYWIVRNS (SEQ ID NO: 21);

[0140] The amino acid sequence of Der2 is:

[0141] IEYIQHNGVVQESYYRYVARGPPGGLAELAKKVKAVKSDDGPPGGSIRYNSLKHSLGGEKGPPGGESYYRYVAREQSCRRGPPGGCNELYSKANAEVTDNGPPGGVNIVGYSNAQGV DYWIVRNSGPPGGVKIIREFNGDEVVVTGPPGGNAKAVGLPAKGSDVKGPPGGAQTHSAIAVIIGIKDGPPGGRKDLDIFEQYNLEMAGPPGGQSCRRPNAQRFGISNYCQIY (SEQ ID NO: 22).

[0142] Example 3: Expression of dust mite allergen-derived proteins by Escherichia coli DE3 and Staphylococcus epidermidis ATCC12228

[0143] 1. Plasmid transformation and verification:

[0144] (1) Plasmid transformation and verification of E. coli DE3: Same as 5 and 6 in Example 1. The verified correct strains were named ECDer1 and ECDer2.

[0145] (2) Plasmid transformation and verification of Staphylococcus epidermidis ATCC12228: First, establish Staphylococcus competent cells, resuscitate Staphylococcus epidermidis ATCC12228, inoculate into 5 mL of TSB medium at a ratio of 1:200, and culture at 37°C overnight; pipette 1 mL of culture into 100 mL of freshly prepared TSB culture medium and shake gently at 37°C; culture in TSB medium for about 3.5 h, and the OD 600=0.6~0.8; The following operations were all performed on ice. The bacterial solution was divided into 2 50 mL centrifuge tubes, centrifuged at 4℃, 4000 rpm for 10 min, and the supernatant was discarded; 25 mL of pre-cooled sterile ddH2O was added to resuspend the bacteria, centrifuged at 4℃, 4000 rpm for 10 min, and the supernatant was discarded; 20 mL of pre-cooled sterile ddH2O was added to resuspend the bacteria, centrifuged at 4℃, 4000 rpm for 10 min, and the supernatant was discarded; Repeat the above washing steps twice; Discard the supernatant, resuspend with 25 mL of pre-cooled 10% glycerol; Ice bath for 5 min, 4℃, 4000 rpm, centrifuged for 10 min, discard the supernatant, repeat once; Discard the supernatant, add 500 μL of 10% glycerol to the residual liquid in the tube and resuspend it, mix well and divide into new EP tubes at 80~90 μL per tube, and store at -80℃. Then the recombinant plasmid was electrotransformed and verified. The electrotransformed competent cells stored at -80℃ were taken out and placed at room temperature for 20 min. 10 µg of the recombinant plasmid was added to 100 μL competent epi-glucose and mixed. The above mixture was transferred to a 0.1 cm electric shock cup and electroporated (resistance 100Ω, voltage 2.0kV, capacitance 25pF). Immediately, 1mL of B2 medium preheated at 37℃ was added to the electric shock cup, mixed and aspirated, and transferred to a sterile test tube for shaking culture for 3 h. 4000g, centrifuged for 5min, discarded most of the supernatant, resuspended the bacteria and plated TSA (chloramphenicol resistance), and cultured at 37℃ for 24 h. Single colonies were selected and grown on TSA (chloramphenicol) plates at 37℃ overnight. The transformants obtained after transformation were activated by shaking and plasmid extraction, and sent for sequencing. The sequencing primer was M13f. Finally, the verified epi-glucose strains were named SE_Agr-Der1 and SE_Agr-Der2.

[0146] 2. Induced expression of proteins:

[0147] 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 0.5 mM IPTG and continue to culture, induce overnight at 20°C and culture for 6 h at 37°C, respectively. The negative control is without IPTG addition; centrifuge at 12000 rpm for 3 min, discard the supernatant and collect the bacteria; fully break the bacterial precipitate with an ultrasonic disruptor and centrifuge at 6500 rpm for 10 min; dissolve the precipitate after centrifugation with TE solution for SDS-PAGE detection.

[0148] Example 4: Effectiveness of using engineered bacteria to prevent dust mite allergy in mice models

[0149] 1. Preparation of recombinant bacteria:

[0150] The recombinant strains cultured overnight were transferred into fresh liquid containing the corresponding antibiotics at a ratio of 2% (v / v) and cultured at the respective temperatures until OD 600 When the concentration is about 0.6, add the inducer for induction (if necessary). After 6 h of induction at the corresponding temperature, take 100 μL of bacterial solution for gradient dilution, and take bacterial solution of different dilutions (10 5 , 10 6 , 10 7 , 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.

[0151] 2. Animal Experimental Procedures:

[0152] (1) Dust mite allergy mouse sensitization procedure:

[0153] 6-8 weeks old male Balb / c mice, 5 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.

[0154] Prevention stage: ECDer1, ECDer2, and ECDH5α groups: 0.2 mL of resuspended bacterial solution (2×10 9 SE_Agr-Der1, SE_Agr-Der2, SE_ATCC12228 groups: 0.2 mL of resuspended bacterial solution (2×10 9 CFU); Naïve group used 0.2 mL PBS, Positive group used 0.2 mL PBS;

[0155] Sensitization stage: On days 17, 24, 31, and 38, mice were intraperitoneally injected with 10 μg of dust mite extract (calculated based on total protein) + 2 mg of alum sensitization adjuvant (dissolved in 200 μL PBS) for sensitization;

[0156] Challenge phase: On days 43, 44, and 45, for three consecutive days, 0 μg of dust mite extract (calculated based on total protein) was used for nasal challenge (dissolved in 50 μL PBS).

[0157] (2) Sample collection:

[0158] Serum samples:

[0159] After the stimulation, the mice were anesthetized with isoflurane, and the eyeballs of the mice were removed to collect blood, about 1 mL of blood was collected. The blood sample was left to stand at room temperature for 1-2 hours, and after the serum was precipitated, it was centrifuged at 4000g for 10 minutes, and the upper serum was carefully aspirated and stored at -80℃.

[0160] (3) ELISA test of total serum IgE antibody:

[0161] Coating capture antibody: Equilibrate the ELISA reagents to room temperature, dilute the rat anti-mouse 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 three times with PBST, 5 minutes each time;

[0162] Blocking: 200 μL 1% BSA solution at 37°C for 30 minutes; wash 3 times with PBST, 5 minutes each time;

[0163] Add samples: Leave the first column as a blank control and add 100 μL of blocking solution, and add 100 μL of samples to the rest. 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;

[0164] 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℃ for 1 h; wash 6 times with PBST, 5 minutes each time;

[0165] HRP coupling: add Streptavidin HRP diluted 1:1000, incubate at 37℃ for 30 min; wash 6 times with PBST, 5 min each time;

[0166] Color development: TMB method, used according to the instructions, detected at 450 nm.

[0167] (4) ELISA method for detection of serum dust mite-specific IgE, IgG1, and IgG2α:

[0168] Biotin coupling with dust mites: Take 500 μL of the 3 mg / mL dust mite protein to be coupled and add it to a 1.5 mL centrifuge tube; add 250 μL of biotin coupling solution and 250 μL of dissolved 1 mg 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 1 min, remove the top and bottom covers of the desalting column, put it into a 15 mL centrifuge tube, centrifuge at 1000g for 2 min, and remove the resin preservation solution; add 5 times the column volume of biotin coupling solution (25 mL) 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 to the center of the resin, and centrifuge at 1000g for 8 min; collect the liquid in the centrifuge tube as the labeled product.

[0169] The capture antibody coating, blocking, and sample addition were the same as (3). The detection antibody incubation used Biotin-coupled CPE (diluted to 8 μg / mL), coupled with HRP, and color development was the same as (3).

[0170] (5) ELISA test results:

[0171] After completing the sensitization procedure, serum was collected for ELISA testing, and it was found that the total IgE of the sensitized engineering bacteria group (ECDer1, ECDer2, SE_Agr-Der1, SE_Agr-Der2 group) was significantly lower than that of the model group (Positive group), the pure bacteria control group (SE_ATCC12228 group, ECDH5α group) and the blank control group (Naïve group). There was no difference between the pure bacteria control group and the model group, indicating that the preventive ability comes from the secretory protein of the engineering bacteria rather than the chassis strain. The dust mite-specific IgG1 and IgG2α also conform to this conclusion ( Figure 4 ).

[0172] Among them, the total IgE and specific IgE of ECDer1 group and SE_Agr-Der1 group were the lowest, the specific IgG1 was reduced most significantly in ECDer1 group and SE_Agr-Der1 group, and the content of specific IgG2α was significantly increased in ECDer1 group and SE_Agr-Der1 group. In summary, in the dust mite sensitization model, engineered bacteria can significantly improve the allergic phenotype.

[0173] 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 of SEQ ID NOs: 1 to 14; 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 products include pharmaceuticals, biological products, skin care products or cosmetics.

2. The use according to claim 1, characterized in that The antigenic epitope includes a combination of two or more of SEQ ID NOs: 1 to 14.

3. A polypeptide, characterized in that Having the antigenic epitope in the use according to claim 1 or 2.

4. The polypeptide according to claim 3, characterized in that It is polypeptide 1 and / or polypeptide 2; The amino acid sequence of the polypeptide 1 is SEQ ID NO: 21; The amino acid sequence of polypeptide 2 is SEQ ID NO:

22.

5. A nucleic acid molecule, characterized in that A nucleotide sequence encoding the polypeptide according to claim 3 or 4.

6. An expression vector, characterized in that Comprising the nucleic acid molecule of claim 5.

7. A host cell, characterized in that It comprises at least one of the polypeptide according to claim 3 or 4, the nucleic acid molecule according to claim 5 or the expression vector according to claim 6.

8. Use of the polypeptide according to claim 3 or 4 or the host cell according to claim 7 in any of the following: (a) Reduce the body's IgE and IgG1; (b) Increase the body's serum IgG2a; (c) Relieve skin allergy symptoms; (d) Prevention and / or treatment of allergic diseases; (e) Desensitization therapy; (f) preparing a product that achieves at least one of (a), (b), (c), (d), and (e); The allergic diseases include allergic dermatitis; The products include pharmaceuticals, biological products, skin care products or cosmetics.

9. A product, characterized in that Comprising the polypeptide according to claim 3 or 4 and / or the host cell according to claim 7, and acceptable adjuvants or auxiliary agents; The product includes a drug, a biologic, a skin care product, or a cosmetic; The administration routes of the drug include oral administration or external application.

10. The method for preparing a host cell according to claim 7, characterized in that: The expression vector according to claim 6 is introduced into chassis cells to obtain the result.

Images