T cell epitope peptide of tropomyosin allergen and application of T cell epitope peptide

Abstract

The invention provides T cell epitope peptide of tropomyosin sensitizers and application of the T cell epitope peptide. The amino acid sequence of the T cell epitope peptide is shown as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. According to the invention, 9 and 10 T cell epitopes of Fujian oyster TM and Oratosquilla oratoria TM are predicted by using a bioinformatics technology, wherein 4 T cell epitopes of Fujian oyster TM and 4 T cell epitopes of Oratosquilla oratoria TM are successfully butted with MHC-II molecules; an RBL-2H3 cell degranulation experiment is adopted, and the anti-allergic activity of the T cell epitope peptides is further verified. The obtained epitope peptide can be used for developing a hybrid peptide preparation and a desensitization preparation for treating allergy of oysters and mouth mantis shrimps, and a new strategy is provided for researching specific immunotherapy of allergens 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 T cell epitope peptide of a tropomyosin allergen 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] In recent years, aquatic food allergies have gradually received attention. Among them, the aquaculture volume and consumption of Fujian oysters (Crassostrea angulata) and mantis shrimps (Oratosquilla oratoria) have been increasing, and the food allergy problems caused by them cannot be ignored. At present, peptide immunotherapy based on T cell epitope peptides has the effect of inducing the body to produce immune tolerance and is considered to be a safe and effective food allergy treatment strategy for food allergies. 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. The exploration of T cell epitopes of Fujian oysters and mantis shrimp allergens is expected to achieve specific immunotherapy for aquatic food allergies. Summary of the invention

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

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

[0006] A tropomyosin sensitizer T cell epitope peptide, the amino acid sequence of which is shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0007] Optionally, the tropomyosin-sensitizing pro-T cell epitope peptide can inhibit the release of β-hexosaminidase from RBL-2H3 cells.

[0008] A mixed peptide for desensitization therapy is a mixture of any of the following multiple epitope peptides, wherein the amino acid sequences of the epitope peptides are shown in SEQ ID NOs: 1-4.

[0009] Application of the tropomyosin allergen T cell epitope peptide and the mixed peptide for desensitization therapy in the preparation of desensitization preparations.

[0010] A polypeptide vaccine contains the above-mentioned tropomyosin T cell epitope peptide or the above-mentioned mixed peptide.

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

[0012] Compared with the prior art, the present invention provides a tropomyosin allergen T cell epitope peptide, which has the following beneficial effects:

[0013] The present invention uses bioinformatics technology to predict 9 and 10 T cell epitopes of Fujian oysters and mantis shrimp TM, among which 4 T cell epitopes of Fujian oyster TM and 4 T cell epitopes of mantis shrimp successfully docked with MHC-Ⅱ molecules; the RBL-2H3 cell degranulation experiment was used to further verify the anti-allergic activity of these T cell epitope peptides. The obtained epitope peptides can be developed into mixed peptide preparations and desensitization preparations for the treatment of oyster and mantis shrimp allergies, providing a new strategy for studying allergen-specific immunotherapy and biological preparations that simultaneously exert anti-allergic effects.

[0014] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The molecular docking simulation analysis of the T cell epitopes of the identified Fujian oyster and mantis shrimp TM with MHC-Ⅱ, Figure, where (A) is the molecular docking diagram of Fujian oyster TM and MHC-Ⅱ, (B) is the molecular docking diagram of mantis shrimp TM and MHC-Ⅱ;

[0016] Figure 2 The mast cell degranulation inhibition rate analysis of Fujian oyster and mantis shrimp TM T cell epitopes, wherein (A) is the degranulation inhibition rate analysis of Fujian oyster TM T cell epitopes, and (B) is the degranulation inhibition rate analysis of mantis shrimp TM T cell epitopes;

[0017] Figure 3 This is the primary sequence alignment of TM T cell epitopes in Fujian oyster and mantis shrimp;

[0018] Figure 4The tertiary structures of the T cell epitopes of the Fujian oyster and the mantis shrimp TM are shown in Figure 1, where (A) shows the localization of the T cell epitope on the Fujian oyster TM and (B) shows the localization of the T cell epitope on the mantis shrimp TM. DETAILED DESCRIPTION

[0019] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0020] The experimental methods in the embodiments described below are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in this area or according to the product specifications. The materials, reagents, etc. used in the embodiments described below, unless otherwise specified, can be obtained from commercial sources. In addition, unless otherwise specified, the implementation described below will adopt the traditional techniques in the fields of chemistry, molecular biology, etc. within the capabilities of those skilled in the art. In this article, the amino acid sequence is written from left to right in the direction from the amino terminal to the carboxyl terminal.

[0021] The present invention is described below by means of illustrative specific examples, which are not intended to limit the scope of the present invention in any way. It is particularly noted that the reagents used in the present invention are commercially available unless otherwise specified.

[0022] Example 1 Prediction of tropomyosin T cell epitopes

[0023] The amino acid sequence of Fujian oyster TM (UST29548.1) was obtained from GenBank, with a total of 286 amino acids. The amino acid sequence of mantis shrimp TM (AB3038320.1) was obtained from GenBank, with a total of 284 amino acids. NetMHCII-2.3, SYFPEITHI, ProPred, Rankpep, and TepiTool were used to predict the simulated T cell epitopes of tropomyosin. The simulated T cell epitope information predicted by the above five bioinformatics software was combined with the obtained tropomyosin simulated T cell epitope amino acid region (as shown in Table 1), and the common amino acid region predicted by three or more software was selected as the simulated T cell epitope of tropomyosin, and finally 9 simulated T cell epitopes of tropomyosin were obtained. The 9 predicted tropomyosin simulated T cell epitopes were sent to Jiangsu GenScript Biotechnology Co., Ltd. for synthesis, and a total of 9 simulated T cell epitope peptides were obtained, which were named P1 to P9 (as shown in Table 2). In the early stage of the laboratory, the mantis shrimp TM T cell epitopes were predicted (application number 202411222268.X), and the predicted mantis shrimp T cell epitopes were sent to Jiangsu GenScript Biotechnology Co., Ltd. for synthesis. A total of 10 simulated T cell epitope peptides were obtained, which were named P10~P19 (as shown in Table 3).

[0024] Table 1 T cell epitopes of Fujian oyster TM predicted by five bioinformatics software

[0025]

[0026] Table 2 Sequence information of Cra a 1 mimic T cell epitopes predicted by bioinformatics software

[0027]

[0028] Note: Bold comments indicate T cell epitopes that are docked in Fujian oyster TM

[0029] Table 3 Sequence information of mimicking T cell epitopes of mantis shrimp tropomyosin predicted by bioinformatics software

[0030]

[0031] Note: Bold comments indicate T cell epitopes identified in Mantis shrimp TM

[0032] Example 2 Molecular Docking of Tropomyosin T Cell Epitope and MHC-Ⅱ

[0033] AlphaFold was used to simulate the tertiary structure of the T cell epitopes of Fujian oysters and mantis shrimp TM, and the amino acid sequences of the T cell epitopes of Fujian oysters and mantis shrimp TM were uploaded to the ColabFold (https: / / colab.research.google.com / ) online software in the AlphaFold database for tertiary structure simulation. The amino acid sequence information of the T cell epitopes is shown in the bold marks in Tables 1 and 2. Then, the obtained MHC-Ⅱ molecule PDB file was used to perform molecular docking between the T cell epitope of Cra a 1 and MHC-Ⅱ in the ClusPro 2.0 (https: / / cluspro.org / ) online software, and 4 were successfully obtained and the docking results were visualized by PyMol software. The results are shown in the figure. Figure 1 As shown, the T cell epitope peptides P1, P3, P4, and P8 of Fujian oyster TM can bind to MHC-Ⅱ molecules ( Figure 1 A) The T cell epitope peptides P10, P17, P18, and P19 of the mantis shrimp TM can bind to MHC-Ⅱ molecules, indicating that these peptides can be recognized by MHC-Ⅱ molecules on the surface of dendritic cells to form stable MHC-Ⅱ-peptide complexes, and then presented to T cell surface receptors.

[0034] Example 3 Analysis of the anti-allergic activity of tropomyosin T cell epitopes

[0035] The anti-allergic activity was determined using RBL-2H3 cells. The specific steps are as follows:

[0036] RBL-2H3 cell suspension was mixed with Anti-DNP-IgE at a final concentration of 400 ng / mL and plated in a 96-well plate, with 5×10 4 cells, divided into positive group, negative group, lysis group and sample group (P1~P19), three parallel wells in each group, the system is 100μL, and incubated for 16h. Then discard the supernatant culture medium from each well, add 80μL HBSS buffer, add 5μg Cra a1 and simulated T cell epitope peptide of mantis shrimp TM to the sample group, with a final concentration of 300μg / mL, and add equal amount of HBSS buffer to the positive group, negative group and lysis well. After incubation in the cell culture incubator for 1h, 2μL of 500ng / mL DNP-BSA was added to the positive group and sample group (DNP-BSA was not added to the negative group and lysis group). After incubation in the cell culture incubator for 1h, 10μL of 1% Triton was added to the lysis well for lysis for 5min, and 50μL of supernatant from each well was taken to a new well and 50μL PNAG was added. After incubation in a 37℃ incubator for 90min, glycine buffer was added and the OD was detected. 405 The degranulation inhibition rate was calculated according to the following formula:

[0037]

[0038] The results of screening the simulated T cell epitope peptides of Fujian oyster and mantis shrimp TM are as follows Figure 2 The epitope information of Fujian oyster TM is shown in Table 2. The inhibition rates of P1, P3, P6, P7, and P8 in Fujian oyster TM on RBL-2H3 cell degranulation exceeded 50%, among which P1 (AA 2～18 )、P3(AA 82～104 )、P8(AA 251～256 ) is a T cell epitope with anti-allergic activity. The epitope information of Mantis shrimp TM is shown in Table 2, among which P10, P12, P13, P14, P15, and P16 in Mantis shrimp TM were screened and the inhibition rate of RBL-2H3 cell degranulation was more than 50%, among which P10 (AA 4～24 ) is a T cell epitope with anti-allergic activity.

[0039] Example 4 Localization of the anti-allergic active peptide of tropomyosin T cell epitope

[0040] According to the amino acid sequence of Fujian oyster TM (UST29548.1) and the amino acid sequence of mantis shrimp TM (AB308320.1) retrieved from GenBank, they were input into DAMMAN software for T cell epitope primary sequence alignment, as shown in Figure 1. Figure 3 As shown, Figure 3 The underlined part is the identified T cell epitope, and the yellow part is the T cell epitope with anti-allergic activity. Pymol software was used to locate the T cell epitopes of Fujian oyster and mantis shrimp TM at the third level. Figure 4 As shown in the results, it was found that there were 3 T cell epitopes with anti-allergic activity in Fujian oyster TM and 1 T cell epitope with anti-allergic activity in mantis shrimp TM, among which AA 2～24 The region is a T cell epitope region shared by both, and the peptide segment composed of this region has anti-allergic activity.

[0041] In summary, the present invention predicts the T cell epitopes of Fujian oyster and mantis shrimp TM by bioinformatics software, simulates the predicted T cell epitope structures of Fujian oyster and mantis shrimp TM by using AlphaFold software, then uses Cluspro 2.0 to perform molecular docking between T cell epitopes and MHC-Ⅱ, and further verifies the anti-allergic activity of T cell epitopes of Fujian oyster and mantis shrimp TM by using RBL-2H3 degranulation experiment, and obtains 3 T cell epitopes of Fujian oyster TM and 1 T cell epitope with anti-allergic activity of mantis shrimp TM, among which AA 2～24 The two TMs have similar amino acid composition in the T cell epitope region (such as Figure 4These epitopes can be used to develop mixed peptide preparations and desensitization preparations for the treatment of oyster and mantis shrimp allergies, providing a new strategy for studying allergen-specific immunotherapy and biological preparations that also exert anti-allergic effects, which is expected to reduce the side effects of reactions during the treatment process and improve the safety index during the treatment process.

[0042] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms should not be understood as necessarily being directed to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification.

[0043] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A tropomyosin allergen T cell epitope peptide, characterized in that: The amino acid sequence thereof is shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:

4.

2. The tropomyosin allergen T cell epitope peptide according to claim 1, characterized in that: The tropomyosin-sensitized pro-T cell epitope peptide can inhibit the release of β-hexosaminidase by RBL-2H3 cells.

3. A mixed peptide for desensitization therapy, characterized in that: It is a mixture of any of the following multiple epitope peptides, and the amino acid sequences of the epitope peptides are shown in SEQ ID NOs: 1-4.

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

5. A polypeptide vaccine, characterized in that: Contains the tropomyosin 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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