Fujian oyster allergen Craa2 T cell epitope peptide and application thereof

Abstract

The invention provides a Fujian oyster allergen Craa a2T cell epitope peptide and application thereof. The amino acid sequence of the Fujian oyster allergen Craa a2T cell epitope peptide is shown as SEQ ID NO: 1 or SEQ ID NO: 2. The method comprises the following steps: predicting seven simulated T cell epitopes of Craa2 by utilizing a bioinformatics technology; identifying two T cell epitopes of Craa2 by combining a CD4 + T cell proliferation experiment with MHC-II molecular docking simulation; an RBL-2H3 cell degranulation experiment is adopted, and the anti-allergic activity of the Cra a 2T cell epitope peptide is further verified. The obtained epitope peptide can be used for developing a hybrid peptide preparation and a desensitization preparation for treating oyster allergy, and a new strategy is provided for researching allergen specific immunotherapy and simultaneously exerting an anti-allergic effect.

Description

Technical Field

[0001] The invention belongs to the technical field of biological immunology, and in particular relates to a Fujian oyster allergen Cra a 2 T cell epitope peptide and application thereof. Background Art

[0002] Food allergy is an immune disease with recurrent, rapid adverse reactions and is considered an important public health and safety issue. Food allergy includes sensitization and effector stages. When the body first comes into contact with the allergen, the allergen is presented to CD4 in the form of peptides by dendritic cells. + T cells are induced to differentiate into Th2 cells, produce related cytokines (IL-4, IL-13, etc.), and then induce B cells to differentiate into plasma cells to produce allergen-specific IgE. When the body is exposed to the allergen again, it will cross-link with the IgE on the FcεRI receptor on the surface of mast cells and release allergic mediators, which will then cause severe allergic reactions in the body.

[0003] Fujian oyster (Crassostrea angulata) is an important aquatic product of aquaculture. With the increase of oyster aquaculture and consumption, the problem of food allergy caused by eating oysters is becoming more and more serious. At present, peptide immunotherapy based on T cell epitope peptides has the effect of inducing immune tolerance in the body and is considered to be a safe and effective food allergy treatment strategy. Mast cells play a key role in IgE-mediated allergic reactions. Studies have found that peptides corresponding to T cell epitopes have the effect of stabilizing mast cells and reducing the occurrence of allergies. Fujian oyster arginine kinase (systematically named Cra a 2) is one of the main allergens, and its T cell epitopes need to be studied. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems in the above-mentioned technologies to a certain extent, and provides a Fujian oyster allergen Cra a 2 T cell epitope peptide and its application, so as to develop a mixed peptide preparation and a desensitization preparation for treating oyster allergy.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A Fujian oyster allergen Cra a 2 T cell epitope peptide, the amino acid sequence of which is shown as SEQ ID NO: 1 or SEQ ID NO: 2.

[0007] Optionally, the Cra a 2 T cell epitope peptide can stimulate the proliferation of Cra a 2-specific T cells and inhibit the release of β-hexosaminidase from RBL-2H3 cells.

[0008] A hybrid peptide for desensitization therapy, which comprises a Cra a 2 T cell epitope peptide with the amino acid sequence shown in SEQ ID NO: 1 and a Cra a 2 T cell epitope peptide with the amino acid sequence shown in SEQ ID NO: 2.

[0009] Use of the above-mentioned Crassostrea angulata allergen Cra a 2 T cell epitope peptide and the above-mentioned hybrid peptide for desensitization therapy in the preparation of an oyster desensitization preparation.

[0010] A polypeptide vaccine containing the above-mentioned Crassostrea angulata allergen Cra a 2 T cell epitope peptide or the above-mentioned hybrid peptide.

[0011] Optionally, the polypeptide vaccine further comprises a pharmaceutically acceptable adjuvant.

[0012] Compared with the prior art, the present invention provides a Crassostrea angulata allergen Cra a 2 T cell epitope peptide, having the following

[0013] Beneficial effects:

[0014] The present invention uses bioinformatics technology to predict 7 simulated T cell epitopes of Cra a 2; 2 T cell epitopes of Cra a 2 are identified by CD4 + T cell proliferation experiment combined with MHC-II molecular docking simulation; the anti-allergic activity of the Cra a 2 T cell epitope peptide is further verified by RBL-2H3 cell degranulation experiment. The obtained epitope peptides can be developed into hybrid peptide preparations and desensitization preparations for the treatment of oyster allergy, providing a new strategy for the research of biological preparations that can simultaneously exert anti-allergic effects in allergen-specific immunotherapy.

[0015] The additional aspects and advantages of the present invention will be partly given in the following description, partly will become obvious from the following description, or will be understood through the practice of the present invention. Description of the Drawings

[0016] Figure 1 For the simulated T cell epitopes of Cra a 2;

[0017] Figure 2 For the identification of the T cell epitopes of Cra a 2, wherein (A) is a representative flow cytometry graph of the stimulation of CD4 + T cell proliferation by the predicted T cell epitope peptide, (B) statistical analysis graph of the stimulation of CD4 + T cell proliferation by the predicted T cell epitope peptide, (C) ELISA method for detecting the release level of IL-2 in the cell supernatant;

[0018] Figure 3Molecular docking simulation analysis of the identified T cell epitopes with MHC-II, where (A) is the molecular docking map of P3 with MHC-II and (B) is the molecular docking map of P7 with MHC-II;

[0019] Figure 4 Analysis of the predicted T cell epitope peptide inhibiting mast cell degranulation;

[0020] Figure 5 Localization of the Cra a 2 T cell epitope in the tertiary structure. Detailed implementation mode

[0021] The embodiments of the present invention will be described in detail below. The examples of the embodiments are shown in the drawings, where the same or similar reference numerals represent the same or similar elements or elements with the same or similar functions from beginning to end. The embodiments described below with reference to the drawings are exemplary and are intended to explain the present invention and should not be construed as limiting the present invention.

[0022] The experimental methods in the embodiments described below are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the embodiments described below can be obtained from commercial channels unless otherwise specified. Additionally, unless otherwise stated, the embodiments described below will employ traditional techniques in the fields of chemistry, molecular biology, etc. within the capabilities of those skilled in the art. In this article, amino acid sequences are written from left to right in the direction from the amino terminus to the carboxyl terminus.

[0023] The present invention will be described below through illustrative specific embodiments, and these embodiments do not limit the scope of the present invention in any way. It should be particularly noted that: the reagents used in the present invention are all commercially available unless otherwise specified.

[0024] Example 1 Prediction of Cra a 2 T cell epitopes

[0025] The amino acid sequence of Cra a 2 (QZP44316.1) was obtained from GenBank, with a total of 350 amino acids. Five bioinformatics software, namely NetMHCII-2.3, SYFPEITHI, ProPred, Rankpep, and TepiTool, were used to predict the mimotope T cell epitopes of Cra a 2. Based on the mimotope T cell epitope information predicted by the above five bioinformatics software, five alleles that are relatively common in allergic patients, namely DRB1-0101, DRB1-0301, DRB1-0401, DRB1-0701, DRB1-1101, and DRB1-1501, were selected for bioinformatics prediction (as shown in Table 1). Combining the amino acid regions of the Cra a 2 mimotope T cell epitopes obtained, the common amino acid regions predicted by three or more software were selected as the mimotope T cell epitopes of Cra a 2 (as Figure 1 shown), and finally 7 mimotope T cell epitopes of Cra a 2 were obtained (as shown in Table 2). The 7 predicted mimotope T cell epitopes of Cra a 2 were sent to Nanjing Genscript Biotech Co., Ltd. for synthesis, and a total of 7 mimotope peptides of Cra a 2 were obtained. Among them, P3 and P7 are the T cell epitope information identified for Cra a 2.

[0026] Table 1 T cell epitopes of Crassostrea angulata TM predicted by five bioinformatics software

[0027]

[0028]

[0029] Table 2 Sequence information of mimotope T cell epitopes of Cra a2 predicted by bioinformatics software

[0030]

[0031] Note: Bold annotations indicate the T cell epitopes identified in Cra a 2. Table 2 Prediction information of T cell epitopes of Cra a 2

[0032] Example 2 Identification of Cra a 2 T cell epitopes

[0033] After one week of adaptive feeding of SPF-grade female Balb / c mice, on the 7th day and the 14th day respectively, the mice were sensitized by intraperitoneal injection of Cra a 2 (induced and expressed by the pET-28a-Cra a 2 strain, the construction of which is recorded in the literature "Identification and characterization of Crassostrea angulata arginine kinase, a novel allergen that causes cross-reactivity among shellfish") to induce the body to differentiate and produce specific T cells against Cra a 2, and the mice were sacrificed on the 15th day. The spleens of the sensitized mice were treated with erythrocyte lysate to prepare single-cell suspensions, and 5×10 7 cells were stained with CFSE (final concentration of 5 μM). Subsequently, PBS, Cra a 2, and a simulated T cell epitope peptide were added to stimulate the cells (the final concentration of Cra a 2 in the positive group was 12 μg / mL, and the final concentration of the simulated T cell epitope peptide in the sample group was 300 μg / mL), and they were incubated in a 37°C incubator for 72 h. The supernatant was collected and the release of the cytokine IL-2 was detected using an ELISA kit. The spleen cells were resuspended in buffer, stained with APC / Cy7-anti-mouse CD3, APC-anti-mouse CD4, and Percp / cy5.5-anti-mouse CD44, and then the proliferation of CD4 + T cells was detected using a flow cytometer.

[0034] Flow cytometric results of the stimulation of CD4 + T cells by the simulated T cell epitope peptide are shown in Figure 2 (A), and statistical data analysis was performed on all flow cytometry results, and the results are shown in Figure 2 (B). Compared with the PBS group, there were obvious iterative peaks in the T cells incubated with Cra a 2 and peptide segments P3 (AA 185～212 ), P7 (AA 267～285 ), indicating that Cra a 2, P3, and P7 could all significantly stimulate the proliferation of CD4 + T cells and promote the release of IL-2, as shown in detail in Figure 2 (C). A total of 2 T cell epitopes of Cra a 2 were identified using the T cell proliferation experiment combined with the release of IL-2, and the amino acid sequence information of the T cell epitopes is shown in Table 1 (SEQ ID NO: 1, SEQ ID NO: 2).

[0035] Example 3 Molecular Docking of Cra a 2 T Cell Epitopes with MHC-II

[0036] Use I-TASSER to simulate the tertiary structure of the T-cell epitopes of Cra a 2 (upload the amino acid sequences of the T-cell epitopes of Cra a 2 to the online software I-TASSER (https: / / zhanglab.ccmb.med.umich.edu / I-TASSER / ) for tertiary structure simulation. The information of the amino acid sequences of the T-cell epitopes is shown in Table 1 (SEQ ID NO: 1, SEQ ID NO: 2). Subsequently, obtain the PDB file of the MHC-II molecule, use the online software Clus Pro 2.0 (https: / / cluspro.org / ) to perform molecular docking of the two obtained T-cell epitopes of Cra a 2 with MHC-II, and visualize the docking results through PyMol software. The results are as Figure 3 shown that the T-cell epitope peptides P3 and P7 can bind to the MHC-II molecule, indicating that P3 and P7 can be recognized by the MHC-II molecule on the surface of dendritic cells to form a stable MHC-II-peptide complex, and then presented to the T-cell surface receptor.

[0037] Example 4 Analysis of the anti-allergic activity of Cra a 2 T-cell epitopes

[0038] Use RBL-2H3 cells to measure the anti-allergic activity. The specific steps are as follows:

[0039] Mix the RBL-2H3 cell suspension with Anti-DNP-IgE at a final concentration of 400 ng / mL and spread it into a 96-well plate, with 5×10 4 cells per well. It is divided into a positive group, a negative group, a lysis group, and a sample group (P1 - P7), with three parallel wells in each group. The system is 100 μL and incubated for 16 h. Subsequently, discard the supernatant medium in each well, add 80 μL of HBSS buffer, add 5 μg of the Cra a 2 simulated T-cell epitope peptide to the sample group, with a final concentration of 300 μg / mL, and add an equal amount of HBSS to the positive group, negative group, and lysis wells. After incubating in a cell culture incubator for 1 h, add 2 μL of 500 ng / mL DNP-BSA to the positive group and the sample group (the negative group and the lysis group do not add DNP-BSA). After incubating in a cell culture incubator for 1 h, add 1% Triton to the lysis wells and lyse for 5 min, then take 50 μL of the supernatant from each well to a new well and add 50 μL of PNAG. Incubate in a 37°C incubator for 90 min and then add glycine buffer to detect OD 405 . Calculate the degranulation inhibition rate according to the following formula:

[0040]

[0041] Through the screening of the Cra a 2 simulated T-cell epitope peptides, the results are as Figure 4As shown, the inhibitory rates of epitope peptides P2, P3, P4, and P7 on the degranulation of RBL-2H3 cells exceeded 80%. Among them, P3 (AA 185～212 ) and P7 (AA 267～285 ) are T cell epitopes with anti-allergic activity, as Figure 5 shown.

[0042] In summary, in this application, bioinformatics software and T cell proliferation experiments were used to analyze the T cell epitopes of Cra a 2. A total of 2 T cell epitopes of Cra a 2 were identified. The degranulation experiment of RBL-2H3 cells further verified the anti-allergic activity of the epitope peptides. The epitope peptides and their compositions can be developed into mixed peptide preparations with the potential to induce immune tolerance in the body. The anti-allergic activity of the epitope peptides can reduce the side effects of the reaction during treatment and improve the safety index during treatment.

[0043] In the description of this specification, the description referring to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms should not be understood as necessarily referring to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, those skilled in the art can combine and combine the different embodiments or examples described in this specification.

[0044] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A Fujian oyster allergen Cra a 2 T cell epitope peptide, characterized in that: The amino acid sequence thereof is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. The Fujian oyster allergen Cra a 2 T cell epitope peptide according to claim 1, characterized in that: The Cra a 2 T cell epitope peptide can stimulate the proliferation of Cra a 2 specific T cells and inhibit the release of beta-hexosaminidase by RBL-2H3 cells.

3. A mixed peptide for desensitization therapy, characterized in that: The peptide comprises a Cra a 2 T cell epitope peptide having an amino acid sequence as shown in SEQ ID NO: 1 and a Cra a 2 T cell epitope peptide having an amino acid sequence as shown in SEQ ID NO:

2.

4. Use of the Fujian oyster allergen Cra a 2 T cell epitope peptide according to claim 1 or 2 and the mixed peptide according to claim 3 in the preparation of oyster desensitization preparations.

5. A polypeptide vaccine, characterized in that: Contains the Fujian oyster allergen Cra a 2 T cell epitope peptide according to claim 1 or 2 or the mixed peptide according to claim 3.

6. The polypeptide vaccine according to claim 5, characterized in that The polypeptide vaccine further comprises a pharmaceutically acceptable adjuvant.

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