AlphaS1-casein IgG epitope and effect thereof in relieving allergy

Abstract

The invention belongs to the fields of bioinformatics, immunology and food, and particularly relates to an alpha S1-casein IgG epitope obtained through bioinformatics analysis and screening and an effect of the alpha S1-casein IgG epitope in allergy alleviation, a polypeptide fragment from alpha S1-casein is obtained through bioinformatics analysis and screening, and after the polypeptide is administrated to a mouse, the alpha S1-casein IgG epitope can be used for preparing the alpha S1-casein IgG epitope. The allergy caused by the alpha S1-casein can be effectively relieved.

Description

Technical Field

[0001] The present 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 Art

[0002] Cow's milk protein allergy (CMPA) is the most common food allergy in infants and young children (also reported in adults), affecting 2% to 3% of the total population. In addition, the incidence rate in my country is about 0.83%-3.50%. The allergic proteins in milk mainly include αS1-casein. The main clinical manifestations are vomiting, diarrhea, and constipation. Severe cases can lead to atopic dermatitis and anaphylactic shock, which seriously endanger human health.

[0003] Cow's milk protein allergy (CMPA) is divided into IgE-mediated, non-IgE-mediated and mixed-mediated according to the immune-mediated mode, among which IgE-mediated milk allergy is the main mode. Figure 1 As shown: When α S1 When -casein enters the small intestine after being digested in the stomach, the allergen activates immature B and T cells. Under the action of Th2 cells and the IL-4 and IL-5 they secrete, B cells differentiate into memory B cells and plasma cells and secrete IgE; mast cells and basophils bind to IgE through the IgE-Fc receptors on the membrane and become sensitized. When exposed to the same allergen again, sensitized mast cells and basophils will degranulate and release bioactive mediators, leading to smooth muscle spasm, increased vascular permeability, increased secretion of mucosal glands, sensitive nerve endings, and eosinophil activation, leading to clinical symptoms.

[0004] There are many treatments for CMPA. For example, strictly avoiding contact with and eating allergens may lead to malnutrition. For allergic symptoms, antihistamines, leukotriene inhibitors, mast cell stabilizers, etc. are usually used, but they can cause dizziness, drowsiness, anxiety and heart problems. In addition, because adrenaline can relax smooth muscles, constrict blood vessels, and inhibit the release of allergic mediators, it is usually used in combination with the above drugs.

[0005] At present, the most in-depth research is allergen-specific immunotherapy (AIT), the basic mechanism of which is to increase the tolerance of the patient's immune system to allergens by gradually increasing the inoculation amount of allergens. Oral immunotherapy (OIT), sublingual immunotherapy (SLIT), and epicutaneous immunotherapy (EPIT) have been derived and applied to the treatment of protein allergies in peanuts, milk, eggs, and pollen. Although AIT has advantages such as high clinical efficacy and cost-effectiveness compared to traditional treatments, it still has disadvantages such as side effects (such as the difficulty in determining the dosage of allergens) and poor persistence of immune tolerance. Therefore, it is imperative to develop a treatment plan with good persistence of immune tolerance and few side effects.

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

[0007] Currently, there are two main methods for screening antigenic epitopes. One is to identify antigenic epitopes based on specific antibodies, including X-ray crystallography and nuclear magnetic resonance (NMR), peptide scanning technology, amino acid site-directed mutagenesis technology, and immunoaffinity mass spectrometry technology, etc. The other is to predict antigenic epitopes based on bioinformatics methods. Previously, due to the limitations of informatics development, the screening of antigenic epitopes was mainly based on the first type of method. However, although this type of method has high accuracy, it often has defects that are difficult to make up for. For example, the operation process of NMR is very complicated, the sample purity requirements are extremely high, and the equipment is extremely expensive, making it difficult to promote widely; although the other types of methods are constantly improving, it is still difficult to perform high-throughput antigenic epitope identification.

[0008] With the rapid progress of bioinformatics and immunoinformatics, a variety of tools have been developed for epitope-based vaccine design in the post-genomic era. Generally speaking, the steps of vaccine design using informatics methods include searching the antigen protein database, characterizing the epitopes recognized by T cells and B cells, analyzing protein interactions (molecular docking and molecular dynamics simulation), and analyzing antigenicity and homology, and experimentally verifying the obtained antigen epitopes. At present, a considerable number of studies have shown the feasibility and effectiveness of using informatics to predict antigen epitopes. As this study shows, 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 the prediction of B cell epitopes is relatively poor, the AUC can reach 0.7 (the AUC for complete molecular matching is 1, and the random binding is 0.5); Moten et al., for Grass Group 1 Allergens, used immunoinformatics to screen out T cells that can activate but not be bound by IgE antibodies, providing preliminary data support for tolerance vaccines for the treatment of allergies. Similar methods have been applied to the design of vaccinia virus vaccines, the screening of T cell epitopes restricted by pollen allergen MHC molecules, and allergic asthma caused by house dust mites (HDM), and similar results have been achieved. Therefore, it is also feasible to use immunoinformatics to screen T / B cell antigens in the milk allergy protein αs1-casein. Summary of the invention

[0009] In order to overcome the defects of the prior art, the present invention provides the following technical solutions: The first aspect of the present invention is to provide a polypeptide extracted from αs1-casein, characterized in that the amino acid sequence of the polypeptide comprises NSAEERLHSM and KHPIKHOGLP, and the length of the polypeptide does not exceed 20 amino acids; Further, the polypeptide comprises NSAEERLHSM, and the polypeptide length does not exceed 20 amino acids; Furthermore, the polypeptide is NSAEERLHSM.

[0010] 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.

[0011] Furthermore, the composition is prepared as follows: 0.8-1.2 mg alum and 80-120 ug polypeptide are dissolved in 200 μL PBS; preferably, the composition is prepared as follows: 1 mg alum and 100 μg polypeptide are dissolved in 200 μL PBS.

[0012] 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.

[0013] Furthermore, the composition is prepared as follows: 0.8-1.2 mg alum and 80-120 ug polypeptide are dissolved in 200 μL PBS; preferably, the composition is prepared as follows: 1 mg alum and 100 μg polypeptide are dissolved in 200 μL PBS.

[0014] The second aspect of the present invention provides use of the polypeptide described in the first aspect in preparing 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; Furthermore, the allergy is caused by the milk protein αs1-casein; Furthermore, the composition is prepared as follows: 0.8-1.2 mg alum and 80-120 ug polypeptide are dissolved in 200 μL PBS; preferably, the composition is prepared as follows: 1 mg alum and 100 μg polypeptide are dissolved in 200 μL PBS.

[0015] The third aspect of the present invention provides the use of the polypeptide described in the first aspect in preparing 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; Furthermore, the allergic diarrhea is allergic diarrhea caused by the milk protein αs1-casein; Furthermore, the composition is prepared as follows: 0.8-1.2 mg alum and 80-120 ug polypeptide are dissolved in 200 μL PBS; preferably, the composition is prepared as follows: 1 mg alum and 100 μg polypeptide are dissolved in 200 μL PBS.

[0016] The beneficial effects of the present invention are: 1) Obtain the B cell receptor-binding peptide fragment from αs1-casein through bioinformatics analysis; obtain the peptide fragment that can induce high concentration of IgG but low concentration of IgE through experimental verification; 2) Mouse model experiments confirmed that the peptides screened can protect α S1 -casein-sensitized mice developed allergic diarrhea. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Mechanism of IgE-mediated milk allergy; Figure 2 α S1 -Screening results of B cell epitopes of casein; Figure 3 Validation results of the Blg mouse sensitization model; Figure 4 Screening of IgE levels in polysaccharide-induced mouse models; Figure 5 Fecal status of mice after challenge; Figure 6 Serum IgE levels in mice after challenge. DETAILED DESCRIPTION

[0018] The following is a further description of the concept of the present invention and the technical effects produced in conjunction with specific embodiments, so as to fully understand the purpose, features and effects of the present invention. The methods are conventional methods unless otherwise specified. The materials can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations of the present invention. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0019] Example 1 α S1 Screening of B cell epitopes of -casein The amino acid sequence of αS1-casein was obtained from NCBI, and the linear B cell antigen epitope of αS1-casein was predicted using the Immune Epitope Database (IEDB) analysis resource. That is, the amino acid sequence of αS1-casein was input, the prediction method was selected as BepipredLinear Epitope Prediction 2.0, and Submit was clicked for prediction. In the result panel, the window size and threshold were set to the default settings (i.e., Windows Size: 7; Threshold: 0.35), and peptides above the threshold red line were selected for subsequent experiments. In addition, 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; in addition, 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 a receptor for batch docking with Autodock Vina to screen out B cell peptides with low binding energy for subsequent experiments.

[0020] Prediction of B cell linear epitopes of αS1-casein protein Figure 2As shown, according to the parameters in the method, a total of 3 long B cell epitopes were obtained. Referring to the method of Cong et al. and combined with the IEDB prediction results, the 3 long peptide sequences in Table 1 were all obtained in a 10-peptide-5 overlapping manner to obtain several peptides. When performing Autodock batch molecular docking, peptides with lower binding free energy were selected, where "-" indicates that the peptide has no possibility of docking with BCR. In summary, a total of 8 peptides that may bind to BCR were obtained for subsequent stimulation of sensitized mice for verification.

[0021] Table 1 Short peptides predicted from B cell linear epitopes of αS1-casein protein

[0022] Example 2 Verification of the B cell epitope of αS1-casein The BALB / c mice used were purchased from Beijing Huafukang Biotechnology Co., Ltd. and were in good health.

[0023] 1) Build α S1 -casein-sensitized Babl / c mice: 4-week-old Balb / c mice were purchased and adaptively fed for one week, and then randomly divided into a control group and a sensitized group. The entire experimental animal process was as follows: On day 0 and day 7, mice in the sensitized group and control group were intraperitoneally injected with 200 μL PBS (mixed with 100 μg α S1 -casein and 1 mg alum) and 200 μl PBS (mixed with 1 mg alum); on the 14th day of the experiment, the sensitized group and the control group were intraperitoneally injected with 200 μL PBS (mixed with 100 μg α S1 -casein) and 200 μL PBS until day 21. Due to the limitation of the number of mice, we randomly selected mice from the sensitized group and the control group for stimulation to verify whether the allergy model was successfully established.

[0024] 2) Using the peptide fragments obtained in Example 1 and α S1 -casein was used to stimulate the validated sensitized animal model, and the serum IgE and IgG1 / 2a / 2b / 3 levels were measured to select peptides that produced low IgE levels and high IgG levels.

[0025] The results of the Blg mouse sensitization model validation are as follows Figure 3 As shown, it indicates that the model was successfully constructed ( P<0.001 ). Mice in the PBS control group and sensitized group were selected and treated with α S1 -casein protein and the peptide fragment in Example 1 were stimulated, and the results were as follows Figure 4 As shown, the IgE level in the positive group was still significantly higher than that in the PBS group ( P<0.05), further indicating the successful construction of the sensitization model, while the peptides NSAEERLHSM, KHPIKHOGLP, LRLKKYKVPO and STEDOAMEDI had no significant difference with the control group ( P> 0.05 ), but only the peptides NSAEERLHSM and KHPIKHOGLP were significantly lower than those in the positive group; at the IgG level, there was no clear pattern between the 8 peptides and the positive group, but most of them had no significant differences from the positive group ( P<0.05 ). In summary, the peptides NSAEERLHSM and KHPIKHOGLP met the screening conditions of low IgE and high IgG levels and were used in subsequent experiments.

[0026] Example 3 In α S1 Evaluation of the protective effect of the peptide NSAEERLHSM in casein-allergic mice Four-week-old Babl / c mice were purchased and raised for one week, and then randomly divided into a control group, a prevention group, and a sensitization group. Each mouse in the prevention group was first intraperitoneally injected twice with 200ul of the peptide preparation (preparation formula: 1mg alum and 100ug of the peptide obtained in Example 2 dissolved in 200uL PBS), and then the sensitization process in Example 2 was performed. Finally, α S1 -casein was used to stimulate three groups of mice, and the phenotypes (diarrhea) and IgE levels of the mice were observed within one hour.

[0027] The fecal conditions and serum IgE levels of the three groups of mice after challenge Figure 5 As shown in the figure, refer to the allergic diarrhea scoring standard of Bramdt et al. The feces of mice in the control group and peptide treatment group were solid, and no allergic diarrhea occurred, while the feces of mice in the sensitized group were serous, that is, allergic diarrhea; in addition, the serum IgE level of the sensitized group was significantly higher than that of the control group and peptide treatment group, while there was no significant difference between the control group and the peptide treatment group (such as Figure 6 ). Taken together, this suggests that the peptide NSAEERLHSM can protect α S1 -casein-sensitized mice developed allergic diarrhea.

[0028] The embodiments described above are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work shall all fall within the scope of protection of the present invention.

Claims

1. A polypeptide extracted from αs1-casein, characterized in that The amino acid sequence of the polypeptide comprises NSAEERLHSM and KHPIKHOGLP, and the length of the polypeptide does not exceed 20 amino acids.

2. The polypeptide according to claim 1, characterized in that The polypeptide amino acid sequence comprises NSAEERLHSM, and the polypeptide length does not exceed 20 amino acids.

3. The polypeptide according to claim 2, characterized in that The amino acid sequence of the polypeptide is NSAEERLHSM.

4. A composition for preventing milk protein allergy, characterized in that: The composition comprises the polypeptide according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

5. The composition according to claim 4, characterized in that The composition is prepared as follows: 0.8-1.2 mg alum and 80-120 ug polypeptide are dissolved in 200 μL PBS.

6. A composition for preventing milk protein allergic diarrhea, characterized in that: The composition comprises the polypeptide according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

7. Use of the polypeptide according to any one of claims 1 to 3 in preparing a composition for preventing milk protein allergy, characterized in that: The composition also includes a pharmaceutically acceptable carrier.

8. The use according to claim 7, wherein the allergy is caused by the cow's milk protein αs1-casein.

9. Use of the polypeptide according to any one of claims 1 to 3 in the preparation of a preparation for preventing milk protein allergic diarrhea, characterized in that: The composition also includes a pharmaceutically acceptable carrier.

10. The use according to claim 9, characterized in that The allergic diarrhea is allergic diarrhea caused by milk protein αs1-casein.

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