An alpha s1 -casein igg epitope and its role in allergy mitigation

Abstract

The present application belongs to the field of bioinformatics, immunology and food, and particularly relates to an αS1-casein IgG epitope obtained through bioinformatics analysis and screening and the role of the αS1-casein IgG epitope in allergy relief. S1 -casein-induced allergy.

Description

Technical Field

[0001] This application belongs to the fields of bioinformatics, immunology, and food, and specifically relates to an αS1-casein IgG epitope obtained by bioinformatics analysis and screening and its role in allergy relief. Background Technology

[0002] Milk protein allergy (CMPA) is the most common food allergy in infants and young children (and has also been reported in adults), affecting 2% to 3% of the total population. Furthermore, the incidence rate in my country is approximately 0.83%-3.50%. The main allergenic protein in milk is αS1-casein. The main clinical manifestations are vomiting, diarrhea, and constipation; severe cases can lead to atopic dermatitis and anaphylactic shock, seriously endangering human health.

[0003] Milk protein allergy (CMPA) is classified into IgE-mediated, non-IgE-mediated, and mixed-mediated types according to the immune-mediated mechanism, with IgE-mediated milk allergy being the predominant mode. The mechanism of IgE-mediated milk protein allergy is as follows: Figure 1 As shown: when α S1 When casein enters the small intestine after digestion in the stomach, the allergen activates immature B and T cells. Under the influence of Th2 cells and their secreted IL-4 and IL-5, B cells differentiate into memory B cells and plasma cells, secreting IgE. Mast cells and basophils become sensitized by binding to IgE via IgE-Fc receptors on their membranes. Upon re-exposure to the same allergen, sensitized mast cells and basophils undergo degranulation, releasing bioactive mediators that lead to smooth muscle spasms, increased vascular permeability, increased mucosal gland secretion, nerve ending sensitivity, and eosinophil activation, ultimately resulting in clinical symptoms.

[0004] In response to the current situation of CMPA, various treatment methods are available. For example, strictly avoiding contact with and consumption of allergens can lead to malnutrition. For allergic symptoms, antihistamines, leukotriene inhibitors, and mast cell stabilizers are commonly used, but these can cause dizziness, drowsiness, anxiety, and heart problems. Furthermore, because adrenaline can relax smooth muscles, constrict blood vessels, and inhibit the release of allergy mediators, it is often used in combination with the above medications.

[0005] Currently, the most researched treatment is allergen-specific immunotherapy (AIT), whose basic mechanism is to gradually increase the dosage of allergens to increase the patient's immune system's tolerance to allergens. Oral immunotherapy (OIT), sublingual immunotherapy (SLIT), and epidermal immunotherapy (EPIT) have been developed and applied to treat allergies to proteins in peanuts, milk, eggs, and pollen. Although AIT has advantages in clinical efficacy and cost-effectiveness compared to traditional treatments, it still has disadvantages such as side effects (e.g., difficulty in determining the dosage of allergens) and poor persistence of immune tolerance. Therefore, developing a treatment regimen with good persistence of immune tolerance and fewer side effects is imperative.

[0006] According to existing research, for α S1 The IgG and IgE epitopes of allergic individuals have been characterized, which is significant because the use of IgG epitope peptides may provide guidance for the treatment of allergy patients. For example, studies have shown that allergen-specific IgG produced by the mother during pregnancy helps protect offspring from sensitization by this allergen; and in some epidemiological studies, researchers have found that raw milk containing allergen-specific IgG is associated with a reduced incidence of asthma, allergic rhinitis, and atopic sensitization. Based on these studies, a review proposed a mechanism by which allergen-specific IgG can reduce the incidence of allergies. On the one hand, the specific IgG in the mother forms a complex with the allergen, which is transferred to the offspring through the neonatal Fc receptor (FcRn) to induce the activation of Treg cells and exert an immune tolerance function; on the other hand, allergen-specific IgG has a high affinity for FcγRIIb on immune cells, and the activation of FcγRIIb inhibits the production of IgE by B cells; in addition, allergen-specific IgG competitively competes with IgE for allergens, preventing the formation of IgE-allergen complexes and inhibiting the activation of the allergic pathway. In summary, allergen-specific IgG can induce immune tolerance in the body.

[0007] Currently, there are two main types of methods for screening antigenic epitopes: one is based on specific antibodies to identify antigenic epitopes, including X-ray diffraction crystallography and nuclear magnetic resonance (NMR), peptide scanning technology, site-directed mutagenesis, and immunoaffinity mass spectrometry; the other is based on bioinformatics methods for antigenic epitope prediction. Previously, due to limitations in the development of bioinformatics, antigenic epitope screening mainly relied on the first type of method. However, while these methods are highly accurate, they often have insurmountable drawbacks. For example, NMR is extremely complex, requires very high sample purity, and involves very expensive equipment, making widespread adoption difficult. While the other type of method is constantly being improved, it still struggles to achieve high-throughput antigenic epitope identification.

[0008] With the rapid advancements in bioinformatics and immunoinformatics, various tools have been developed for epitope-based vaccine design in the post-genomic era. Generally, the steps for vaccine design using informatics methods include searching antigen protein databases, characterizing epitopes recognized by T cells and B cells, analyzing protein-protein interactions (molecular docking and molecular dynamics simulations), and analyzing antigenicity and homology, followed by experimental validation of the obtained antigenic epitopes. Currently, a considerable number of studies have demonstrated the feasibility and effectiveness of using informatics to predict antigenic epitopes. As this study demonstrates, the IEDB database predicts MHC class I binding and MHC class II binding with AUC values ​​between 0.9 and 0.76-0.87, respectively. Although B cell epitope prediction is relatively poor, the AUC still reaches 0.7 (1 for molecularly complete matching and 0.5 for random binding). Moten et al., using immunoinformatics, screened for GrassGroup 1 allergens that can activate T cells without being bound by IgE antibodies, providing preliminary data support for tolerable vaccines for treating allergies. Similar methods have been applied to vaccinia virus vaccine design, screening for MHC-restricted T cell epitopes in pollen allergens, and allergic asthma caused by house dust mites (HDM), achieving similar results. Therefore, using immunoinformatics to screen for T / B cell antigens in the milk allergy protein αs1-casein is also feasible. Summary of the Invention

[0009] To overcome the shortcomings of the prior art, the present invention provides the following technical solution:

[0010] The first aspect of the present invention is to provide a polypeptide truncated from αs1-casein, characterized in that the amino acid sequence of the polypeptide contains NSAEEERLHSM and KHPIKHOGLP, and the length of the polypeptide does not exceed 20 amino acids.

[0011] Furthermore, the polypeptide comprises NSAEERLHSM, and the length of the polypeptide does not exceed 20 amino acids.

[0012] Furthermore, the polypeptide is NSAEEERLHSM.

[0013] A composition for preventing milk protein allergy, characterized in that the composition comprises the polypeptide described in the first aspect and a pharmaceutically acceptable carrier.

[0014] Further, the composition is prepared as follows: 0.8~1.2mg alum and 80~120ug polypeptide are dissolved in 200μL PBS; preferably, the composition is prepared as follows: 1mg alum and 100μg polypeptide are dissolved in 200μL PBS.

[0015] A composition for preventing milk protein allergic diarrhea, characterized in that the composition comprises the polypeptide described in the first aspect and a pharmaceutically acceptable carrier.

[0016] Further, the composition is prepared as follows: 0.8~1.2mg alum and 80~120ug polypeptide are dissolved in 200μL PBS; preferably, the composition is prepared as follows: 1mg alum and 100μg polypeptide are dissolved in 200μL PBS.

[0017] A second aspect of the present invention is to provide the use of the polypeptide described in the first aspect in the preparation of a composition for preventing milk protein allergy, characterized in that the composition comprises the polypeptide described in the first aspect and a pharmaceutically acceptable carrier;

[0018] Furthermore, the allergy mentioned is an allergy triggered by the milk protein αs1-casein;

[0019] Further, the composition is prepared as follows: 0.8~1.2mg alum and 80~120ug polypeptide are dissolved in 200μL PBS; preferably, the composition is prepared as follows: 1mg alum and 100μg polypeptide are dissolved in 200μL PBS.

[0020] A third aspect of the present invention is to provide the use of the polypeptide described in the first aspect in the preparation of a composition for preventing milk protein allergic diarrhea, characterized in that the composition comprises the polypeptide described in the first aspect and a pharmaceutically acceptable carrier;

[0021] Furthermore, the aforementioned allergic diarrhea is allergic diarrhea caused by milk protein αs1-casein;

[0022] Further, the composition is prepared as follows: 0.8~1.2mg alum and 80~120ug polypeptide are dissolved in 200μL PBS; preferably, the composition is prepared as follows: 1mg alum and 100μg polypeptide are dissolved in 200μL PBS.

[0023] The beneficial effects of this invention are as follows:

[0024] 1) A polypeptide fragment that binds to the B cell receptor in αs1-casein was obtained through bioinformatics analysis; a polypeptide fragment that can induce high concentrations of IgG but low concentrations of IgE was obtained through experimental verification.

[0025] 2) Mouse model experiments confirmed that the selected peptides could protect α S1 - Casein sensitized mice exhibited allergic diarrhea. Attached Figure Description

[0026] Figure 1IgE-mediated milk allergy mechanism;

[0027] Figure 2 α S1 - Screening results of B-cell epitopes for casein;

[0028] Figure 3 Validation results of the Blg mouse sensitization model;

[0029] Figure 4 Screening for IgE levels in polysaccharide-induced mouse models;

[0030] Figure 5 Fecal matter in mice after challenge;

[0031] Figure 6 Serum IgE levels in mice after challenge. Detailed Implementation

[0032] The following detailed embodiments further illustrate the concept and technical effects of the present invention to fully understand its purpose, features, and effects. Unless otherwise specified, all methods described are conventional methods. Unless otherwise specified, all materials are available from publicly available commercial sources. The illustrative embodiments and descriptions of the present invention are used to explain the invention and do not constitute an undue limitation thereof. It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0033] Example 1 α S1 Screening for B-cell epitopes of casein

[0034] The amino acid sequence of αS1-casein was obtained from NCBI, and the linear B-cell antigenic epitopes of αS1-casein were predicted using the Immunological Epitope Database (IEDB). Specifically, the αS1-casein amino acid sequence was input, the prediction method was BepipredLinear Epitope Prediction 2.0, and the prediction was submitted. In the results panel, the window size and threshold were set to default (Window Size: 7; Threhold: 0.35), and peptides above the threshold line were selected for subsequent experiments. Furthermore, the obtained epitopes were overlapped with a certain number of amino acids to form several peptides; alphfold3 was used to predict the peptide structure and save the PDB file; additionally, the PDB file of Babl / c mouse BCR was downloaded from the RCSB PDB website; finally, several peptides were used as ligands, and Babl / c mouse BCR was used as the receptor for batch docking with Autodock Vina, screening out B-cell peptides with low binding energies for subsequent experiments.

[0035] Prediction of αS1-casein protein linear epitopes in B cells, such as Figure 2 As shown, following the parameters in the method, a total of three long B-cell epitopes were obtained. Referring to the method of cong et al. and combining the IEDB prediction results, the three long peptide sequences in Table 1 were obtained as several peptides in a 10-peptide-5 overlap pattern. During Autodock batch molecular docking, peptides with lower binding free energy were selected, where "-" indicates that the peptide has no possibility of docking with the BCR. In summary, a total of eight peptides that may bind to the BCR were obtained and used for subsequent challenge and sensitization in mice for validation.

[0036] Table 1. Short peptides obtained from B-cell linear epitope prediction of αS1-casein protein.

[0037]

[0038] Example 2: Validation of αS1-casein B-cell epitopes

[0039] The BALB / c mice used were purchased from Beijing Huafukang Biotechnology Co., Ltd., and were in good health.

[0040] 1) Construct α S1 - Casein-sensitized Balb / c mice: Four-week-old Balb / c mice were purchased and acclimatized for one week before being randomly divided into a control group and a sensitized group. The entire experimental animal procedure was as follows: On day 0 and day 7, mice in both the sensitized and control groups were intraperitoneally injected with 200 μL of PBS (mixed with 100 μg α-casein). S1 α-casein and 1 mg alum) and 200 μl PBS (mixed with 1 mg alum); on day 14 of the experiment, mice in the sensitized group and the control group were intraperitoneally injected daily with 200 μL PBS (mixed with 100 μg α-casein and 1 mg alum) and 200 μl PBS (mixed with 1 mg alum); S1 Mice were administered 200 μL of PBS and serotonin (-casein) until day 21. Due to limited mouse numbers, mice in the sensitized and control groups were randomly selected for challenge to verify the successful establishment of the allergy model.

[0041] 2) Using the peptide obtained in Example 1 and α S1 -Casein stimulation was used to create a well-established sensitized animal model. Serum IgE and IgG1 / 2a / 2b / 3 levels were measured, and peptides that produced low IgE and high IgG levels were selected.

[0042] The validation results of the Blg mouse sensitization model are as follows: Figure 3 As shown, this indicates the successful construction of the model ( P<0.001 Mice in the PBS control group and sensitized group were selected and treated with α-... S1 -casein protein and the peptide from Example 1 were stimulated, and the results were as follows: Figure 4As shown, the IgE level in the positive group was still significantly higher than that in the PBS group. P<0.05 This further demonstrates the successful construction of the sensitization model, while the peptides NSAEEERLHSM, KHPIKHOGLP, LRLKKYKVPO, and STEDOAMEDI showed no significant differences compared to the control group. P> 0.05 However, only the peptides NSAEEERLHSM and KHPIKHOGLP were significantly lower than those in the positive group; at the IgG level, there was no clear pattern between the eight peptides and the positive group, but most of them did not differ significantly from the positive group. P<0.05 In summary, the peptides NSAEEERLHSM and KHPIKHOGLP meet the screening criteria of low IgE and high IgG levels, and will be used in subsequent experiments.

[0043] Example 3 In α S1 Evaluation of the protective effect of the peptide NSAEEERLHSM in casein-allergic mice

[0044] Four-week-old Babl / c mice were purchased and fed for one week before being randomly divided into a control group, a prevention group, and a sensitization group. Each mouse in the prevention group was first injected intraperitoneally twice with 200 μL of a peptide formulation (formulation: 1 mg alum and 100 μg of the peptide obtained in Example 2 dissolved in 200 μL PBS), followed by the sensitization procedure described in Example 2. Finally, α-... S1 Three groups of mice were stimulated with casein, and their phenotype (diarrhea status) and IgE levels were observed within one hour.

[0045] The fecal condition and serum IgE levels of the three groups of mice after challenge were as follows: Figure 5 As shown, the anaphylactic diarrhea scoring criteria of Brandt et al. were used as a reference. The feces of mice in the control group and peptide-treated group were solid and no anaphylactic diarrhea occurred, while the feces of mice in the sensitized group were serous, indicating anaphylactic diarrhea. Furthermore, serum IgE levels were significantly higher in the sensitized group than in the control group and peptide-treated group, while there was no significant difference between the control group and the peptide-treated group (e.g., ...). Figure 6 In summary, this indicates that the peptide NSAEEERLHSM can protect α S1 - Casein sensitized mice exhibited allergic diarrhea.

[0046] The embodiments described above are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

Claims

1. A polypeptide truncated from αsl-casein, characterized in that, The amino acid sequence of the polypeptide is NSAEERLHSM.

2. A composition for preventing cow's milk protein allergy, characterized by comprising, The composition comprises the polypeptide of claim 1 and a pharmaceutically acceptable carrier.

3. The composition of claim 2, wherein, The composition is formulated as follows: 0.8-1.2 mg alum and 80-120 ug polypeptide are dissolved in 200 μL PBS.

4. Use of the polypeptide according to claim 1 for the manufacture of a composition for the prevention of cow's milk protein allergy, characterized in that, The composition further comprises a pharmaceutically acceptable carrier.

5. Use according to claim 4, characterized in that, The allergy is an allergy induced by αs1-casein in milk protein.

6. Use of the polypeptide of claim 1 for the manufacture of a preparation for the prevention of cow's milk protein allergic diarrhoea, characterised in that, The formulation further comprises a pharmaceutically acceptable carrier.

7. Use according to claim 6, characterized in that, The allergic diarrhea is an allergic diarrhea induced by αs1-casein in milk protein.

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