Method for identifying dominant B cell epitope and T cell epitope in wheat omega-5 prolamin

Abstract

The invention belongs to the technical field of immunotherapy of allergic diseases, and provides a method for identifying dominant B cell epitopes and T cell epitopes in wheat omega-5 prolamin. According to the identification method provided by the invention, an immunoinformatics tool, a proliferation test, a degranulation test and the like are comprehensively applied, and the cell proliferation capacity, cell factor release, IgG / IgE binding capacity and degranulation capacity of T / B cell epitopes are evaluated. Results show that the T cell epitopes T1 and T2 sharing the same sequence are relatively strong in multiplication capacity, can up-regulate Th2 related cell factors, but do not have degranulation activity. Three B cell peptide fragments B3, B4 and B9 which share a common motif region QQXPQQQ (X = F, L) can promote IgG / IgE binding and degranulation capabilities. These findings would contribute to the development of epitope-based immunotherapies against wheat allergy individuals.

Description

Technical Field

[0001] This invention belongs to the field of immunotherapy technology for allergic diseases, and specifically relates to a method for identifying dominant B-cell epitopes and T-cell epitopes in wheat ω-5 prolysin. Background Technology

[0002] As an important staple food, wheat is an essential source of protein globally, but it is also a trigger for IgE-mediated food allergies, which may cause public health and food safety issues, resulting in a huge economic and health burden.

[0003] Wheat contains various allergenic proteins, among which gliadins are one of the main allergens. Gliadins are composed of three-dimensional single peptide chains, with a molecular weight ranging from 25 to 100 kDa. They are rich in proline and glutamine and exhibit alcohol-soluble characteristics. Based on their sequence, the monomeric gliadin family is divided into three groups: α / β-gliadins (… Tri a 21 ), γ-prolyzin ( Tri a 20 ) and ω-prolysin. Furthermore, as one of the subtypes of ω-prolysin, ω5-prolysin ( Tri a 19 It can lead to the most severe form of wheat allergy—wheat-dependent exercise-induced anaphylaxis (WDEIA), which can cause anaphylactic shock and even death.

[0004] One of the best ways to avoid allergic reactions is to avoid contact with allergens. However, due to the widespread use of wheat and its products worldwide, it is difficult to completely avoid wheat in daily life. In recent years, peptide-based immunotherapy has been considered a safe and effective treatment strategy, with numerous clinical trials demonstrating its successful application in house dust mite and birch pollen allergies. IgE-mediated hypersensitivity reactions mainly include a sensitization phase and an effector phase. In the sensitization phase, T cell epitopes can bind to major histocompatibility complex (MHC) molecules and be recognized by T lymphocytes, promoting their differentiation into Th2 subtypes. In the effector phase, B cell epitopes cross-link with allergen-specific IgE molecules, which are then recognized by FcεRI receptors on the surface of mast cells / basophils, triggering the release of allergic mediators.

[0005] Identifying epitopes in antigens is crucial for immune surveillance and the development of diagnostic methods. Computer simulations of epitope mapping can significantly reduce workload by decreasing the number of candidate epitopes, while immunological methods can further identify candidate peptides. Previous studies have reported various sensitizing epitopes in the prolactin family. Serological tools have identified two regions, QQPFP and PQQPF, in prolactin; immunoinformatics tools and overlapping peptide techniques have identified regions such as QQXPQQQ (X=I, F, S, Y, L), QQEFPQQQ, and QQQFPQQEFP in ω5 prolactin; and a combination of immunoinformatics tools, serological tools, and mouse models has identified regions such as QQPIPQQ, QQPFPQQ, FPTPQQQFPE, QQSFPLQPQQ, and QQLFPELQ in ω2 prolactin. On the other hand, most research focuses on celiac disease (CD)-related T-cell epitopes, which are classified into types such as DQ2.5, DQ2.2, DQ8, and DQ8.5. Studies targeting CD have identified two regions (α-glia-33 mer and α-glia-17 mer) in α-glia, DQ2-γ-I and DQ2-γ-II regions in γ-glia, and DQ2-ω-1 and DQ2-ω-2 regions in ω-glia. However, there are few reports on wheat allergy-related T-cell epitopes, and CD-related T-cell epitopes may differ from wheat allergy-related T-cell epitopes. Therefore, identifying wheat T-cell epitopes and analyzing the relationship between B-cell epitopes is of great significance. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention proposes a method for identifying dominant B-cell and T-cell epitopes in wheat ω-5 prolysin. Specifically, it discloses the use of peptides of 15 or 16 amino acid lengths to identify dominant T-cell and B-cell epitopes in ω-5 prolysin. The proliferative capacity or IgG / IgE binding activity of the synthesized peptides was analyzed using various immunoinformatics tools and serological methods. Furthermore, the degranulation capacity of the peptides was assessed using human basophils (KU812) passively sensitized from the serum of wheat-allergic patients.

[0007] The technical solution of this invention is: A method for identifying dominant B-cell epitopes and T-cell epitopes in wheat ω-5 gliadin, comprising the following steps: (1) T cell epitopes were predicted using a peptide window of 15 amino acid lengths, including LAMAMNIASASRLLS 10-24, AMNIASASRLLSPRG 13-27, ASRLLSPRGKELHTPQ 19-33, FPQQQQFLQQQQIPQ 67-81 and FPQQQFHQQQLPQQQF333-347; (2) Using DNAStar, BepiPred 3.0 server, ABCpred, and IEDB, B cell epitopes were predicted based on surface accessibility, fragment flexibility, and antigenic index. The predicted B cell epitopes included SASRLLSPRGKELHTP 18-33, HQIPQQPQQFPQQQQF 58-73, QQHQSPQQQFPQQQFP 105-120, FPQQQLPQQQQIPQQQ171-186, QQFPQQQFPQQQFPQQ 206-221, RQPQQLPQQQQIPQQP 241-256, SPQQQQFPQQQFPQQQ 270-285, PQPQQIPQQQQIPQQP 293-308, PQQQQFPQQQEFPQQQ 317-332, QQLPQQQFPQQQFPQQ 341-356, TQQQFPRPQQSPEQQQ 371-386 and QSEEPSPYQQYPQQQP 413-428.

[0008] Preferably, in (1), the tool used for T cell epitope prediction is selected from any one or more of NetMHC II, MHCPred, SYFPEITHI and IEDB.

[0009] Preferably, in (1), the predicted T cell epitopes include LAMAMNIASASRLLS 10-24 and AMNIASASRLLSPRG13-27. These two T cell epitopes have excellent proliferative capacity and can upregulate Th2-related cytokines, but do not have degranulation activity.

[0010] Preferably, in (2), the predicted B cell epitopes include QQHQSPQQQFPQQQFP 105-120, FPQQQLPQQQQIPQQQ 171-186, and PQQQQFPQQQEFPQQQ 317-332. These three B cell epitopes can promote IgG / IgE binding and degranulation.

[0011] The present invention has the following advantages and effects compared with the prior art: (1) This invention integrates immunoinformatics tools (such as NetMHC II, BepiPred 3.0) with functional experiments (proliferation assay, degranulation assay, ELISA) to systematically identify the core characteristics of T / B cell epitopes in wheat ω-5 gliadin for the first time. The results show that T1 and T2 epitopes (with the same AMNIASASRLLS core sequence) have strong T cell proliferation capacity and Th2 bias (upregulation of IL-4 / IL-13 and downregulation of Th1 factors), but no degranulation activity; while B3, B4, and B9 epitopes (containing the QQXPQQQ motif) have both high IgG / IgE binding capacity and degranulation activity (release of histamine and trypsin). This discovery reveals the immunological differences of T / B cell epitopes in wheat allergy, provides molecular markers for distinguishing between sensitization and tolerance mechanisms, and deepens the understanding of the immunopathology of wheat allergy. (2) The epitopes screened in this invention provide key targets for the precise treatment of wheat allergy. For example, T1 / T2, due to their strong proliferative capacity and lack of degranulation activity, can be used as candidate peptides for low-sensitivity vaccines to induce immune tolerance. The high sensitivity of B3 / B4 / B9 suggests that they should be avoided as vaccine components or their activity should be reduced through modification. In addition, the epitope sequences are highly consistent with previously reported sensitization motifs (such as QQXPQQQ), which verifies the common sensitization mechanism of trans-subtype prolysins and provides a theoretical basis for the development of broad-spectrum epitope vaccines. (3) This invention integrates multiple technologies such as MHC II binding prediction, CFSE proliferation assay, KU812 cell degranulation assay and patient serum ELISA, forming a reproducible standardized method. This method not only improves the accuracy of epitope identification (such as reducing false positives through consensus prediction), but also verifies the specific IgE binding ability of epitopes through clinical samples (33 wheat allergy serum samples), ensuring the clinical relevance of the results. Attached Figure Description

[0012] Figure 1 The T cell epitope activity of wheat ω5-gliadin is represented by A, where A represents T cell proliferation, B represents IL-2 concentration, T1-T5 represent predicted T cell epitopes, and "CR" represents wheat gliadin. The culture medium is used as a negative control, and Con A and CR are used as positive controls. Figure 2 The cytokine response of spleen cells is represented by A, where A is the concentration of IL-4, B is the concentration of IL-13, C is the concentration of IL-10, D is the concentration of IFN-γ, T1-T5 represent predicted T cell epitopes, and "CR" represents wheat gliadin. The culture medium is used as a negative control, and Con A and CR are used as positive controls. Figure 3The IgG / IgE binding of peptides is represented by A, which represents rabbit IgG binding activity, B, which represents mouse IgE binding activity, C, which represents IgE binding in the serum of individual patients with wheat allergy, B1-B12 represent predicted B cell epitopes, "CR" represents wheat gliadin, and BSA is used as a negative control. Figure 4 Mediator release from KU812 cells, where A represents β-hexosidase release, B represents histamine content, C represents trypsin-like enzyme content, C48 / 80 is the positive control, and NC is the negative control. Detailed Implementation

[0013] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0014] The materials and reagents used in the embodiments of this invention are as follows: Material: The wheat was purchased from a local supermarket (RT-Mart supermarket in Jinan, Shandong). ω5 prolysin was obtained by purification from wheat flour using existing technology; Anti-wheat gliadin IgG was obtained from the serum of mice immunized with gliadin using a protein A agarose column; Serum from immunized mice containing antibodies against wheat gliadin IgE was used as the primary antibody; Goat anti-mouse IgG and goat anti-mouse IgE were purchased from Zhongshan Jinqiao Company (Beijing, China). KU812 cell line was purchased from Pronosei Life Sciences Co., Ltd. (Wuhan, Hubei, China); IgE for multiple myeloma was purchased from Sigma-Aldrich; Enhanced chemiluminescence reagent kit (ECL) and alum adjuvant were purchased from Thermo Fisher; The ELISA kits for histamine, trypsin, IL-2, IL-4, IL-13, IL-10, and interferon (IFN)-γ were purchased from R&D systems. All other reagents were of analytical grade.

[0015] Wheat-specific IgE serum: Serum samples from 33 individuals with documented clinical history of wheat allergy were obtained from the Affiliated Hospital of Qingdao University (Qingdao, Shandong, China). Total IgE and wheat-specific IgE levels were determined using Phadia ImmunoCAP. All serum samples were stored at -80°C prior to use, and donors provided institutionally approved clinical consent forms.

[0016] Example 1: T cell epitope prediction The ω5-prolysin sequence (Genbank accession number: 73912496, Triticum aestivum) was obtained from the NCBI database. T-cell epitopes were predicted using a 15-amino acid peptide window based on its binding affinity to major histocompatibility complex (MHC) class II molecules. Epitope prediction was performed using various tools, including NetMHCII, MHCPred, SYFPEITHI, and IEDB (Table 1).

[0017] Table 1. T-cell epitope prediction of ω5 prolysin.

[0018] The results showed that potential T-cell epitopes of ω5-prolysins were predicted based on the binding affinity of peptides to MHC class II molecules as predicted by different online tools. Six alleles (DRB1-0101, DRB1-0301, DRB1-0401, DRB1-0701, DRB1-1101, and DRB1-1501) were typically used as templates to predict peptide interaction parameters. The predicted T-cell epitopes included T1 (LAMAMNIASASRLLS, 10-24), T2 (AMNIASASRLLSPRG, 13-27), T3 (ASRLLSPRGKELHTPQ, 19-33), T4 (FPQQQQFLQQQQIPQ, 67-81), and T5 (FPQQQFHQQQLPQQQF, 333-347).

[0019] Example 2: B-cell epitope prediction B-cell epitopes of ω5-prolysin were predicted using various immunoinformatics tools based on the hydrophilicity, flexibility, accessibility, and antigenicity of the amino acid sequence, including DNAStar, BepiPred 3.0, ABCpred, and IEDB (Table 2). The final consensus epitope results, synthesized from these tools, are listed in Table 3.

[0020] Table 2. Prediction of B-cell epitopes of ω5-prolysin using different bioinformatics tools. tool Epitope location DNAstar 19-40, 107-154, 164-173, 178-220,224-249, 254-303, 305-371, 377-403, 411-434 BepiPred 43-50, 54-68, 73-80, 84-98, 102-120, 124-136, 140-171, 200-218,240-266, 270-282, 304-328, 376-381, 411-419 ABCpred 3-18, 18-33, 26-41, 32-47, 38-53,49-64, 58-73, 79-94, 88-103, 98-113, 105-120, 113-128, 123-138,136-151, 142-157, 160-175,171-186,177-192, 183-198, 190-205, 199-214, 209-224, 219-234, 229-244,241-256, 254-269, 260-275, 271-286, 279-294, 293-308, 302-317,311-326,317-332, 327-342, 344-359,353-368, 359-374, 376-391, 382-397, 391-406, 404-419, 413-428,422-437 IEDB 55-77, 80-112, 135-160, 178-205,230-237, 247-256, 260-279, 282-290, 299-307, 335-345, 362-372,382-389

[0021] Table 3. Final prediction results of B-cell epitopes of ω5-prolysin. polypeptide Sequence (C→N) Location length B1 SASRLLSPRGKELHTP 18-33 16 B2 HQIPQQPQQFPQQQQF 58-73 16 B3 QQHQSPQQQFPQQQFP 105-120 16 B4 FPQQQLPQQQQIPQQQ 171-186 16 B5 QQFPQQQFPQQQFPQQ 206-221 16 B6 RQPQQLPQQQQIPQQP 241-256 16 B7 SPQQQQFPQQQFPQQQ 270-285 16 B8 PQPQQIPQQQQIPQQP 293-308 16 B9 PQQQQFPQQQEFPQQQ 317-332 16 B10 QQLPQQQFPQQQFPQQ 341-356 16 B11 TQQQFPRPQQSPEQQQ 371-386 16 B12 QSEEPSPYQQYPQQQP 413-428 16

[0022] B cell epitopes were predicted using DNAStar, BepiPred 3.0 server, ABCpred, and IEDB based on surface accessibility, fragment flexibility, and antigenic index. Table 2 shows the prediction results for ω5 prolysins using each tool, and Table 3 shows the final results combining the immunoinformatics tools. Predicted B cell epitopes include B1 (SASRLLSPRGKELHTP, 18-33), B2 (HQIPQQPQQFPQQQQF, 58-73), B3 (QQHQSPQQQFPQQQFP, 105-120), B4 (FPQQQLPQQQQIPQQQ, 171-186), B5 (QQFPQQQFPQQQFPQQ, 206-221), B6 ​​(RQPQQLPQQQQIPQQP, 241-256), B7 (SPQQQQFPQQQFPQQQ, 270-285), B8 (PQPQQIPQQQQIPQQP, 293-308), B9 (PQQQQFPQQQEFPQQQ, 317-332), B10 (QQLPQQQFPQQQFPQQ, The predicted T / B cell epitope sequence regions were B11 (TQQQFPRPQQSPEQQQ, 371-386) and B12 (QSEEPSPYQQYPQQQP, 413-428). These predicted T / B cell epitope sequence regions were synthesized using a solid-phase method with 98% purity and were used for further characterization.

[0023] Reports indicate that immunoinformatics tools and overlapping peptide technology have identified sensitizing epitope regions of ω5-proteases as QQFPQQQ and QQLPQQQ, findings highly consistent with our predictions of ω5-protease B-cell epitopes using online tools. Interestingly, the QQX1X2X3Q motif region (X1=L, S, Q, P, F; X2=L, V, F, I, Q, P; X3=P, G, Q, E) was observed in different proteases (e.g., α-, α / β-, γ-, ω2-proteases). Furthermore, regions such as QQQFLQ, QQQIPQ, and QQLPQQ also appeared in our predicted T-cell epitopes. On the other hand, multiple CD-associated T-cell epitopes have been reported in different subtypes of gliadin, including six epitopes in α-gliadin (DQ 2.5-glia-α1a / 1b / 2 / 3, DQ 8-glia-α1 and DQ 8.5-glia-α1), eleven epitopes in γ-gliadin (DQ 2.5-glia-γ1 / 2 / 3 / 4a / 4b / 4c / 4d / 5, DQ 8-glia-γ1a / 1b, DQ 8.5-glia-γ1), and two epitopes in ω-gliadin (DQ 2.5-glia-ω1, DQ 2.5-glia-ω2). However, the sequences of our predicted wheat allergy-associated T-cell epitopes show almost no overlap with those of CD-associated T-cell epitopes, suggesting a distinct immune recognition mechanism between these two common wheat-induced diseases.

[0024] Example 3 Peptide Synthesis and Coupling The predicted T-cell epitopes (T1-T5) and B-cell epitopes (B1-B12) were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The purity was confirmed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) to be higher than 98%. The synthesized peptides were dissolved in 0.2 M M E3 buffer and mixed with BSA (10 mg / mL) overnight at 4°C. The solution was placed in a dialysis bag and dialyzed three times in PBS buffer (0.1 M, pH 7.4). The concentration of the conjugates was detected by the BCA method and analyzed by SDS-PAGE.

[0025] Example 4: Wheat gliadin-mouse sensitization All protocols and procedures used in this study complied with the National Research Council's Guidelines for the Care and Use of Laboratory Animals and were conducted in accordance with the guidelines of the Laboratory Animal Ethics Committee of the Shandong Academy of Agricultural Sciences (SAAS-2022-SD06). In short, six-week-old female BALB / c mice were purchased from the Jinan Laboratory Animal Center (Jinan, Shandong) and housed in a specific pathogen-free environment, fed a wheat protein-free diet. Mice were boosted with intraperitoneal injections of 50 μg of alcohol-soluble protein dissolved in alum adjuvant on days 1, 7, 13, 19, 25, and 31, followed by injections of 100 μg of alcohol-soluble protein dissolved in PBS buffer on days 34, 37, 40, and 43. Finally, mice were challenged with an injection of 1 mg of alcohol-soluble protein dissolved in PBS buffer. All mice were anesthetized and euthanized by cervical dislocation after an overnight fast. Blood and spleen were collected under aseptic conditions for subsequent analysis.

[0026] Example 5: T cell proliferation assay CD4+ T cells were isolated from the spleen of immunized mice using the Easysep CD4+ T Kit. Lymphocytes (5 × 10⁻⁶) were then isolated. 6 -10×10 6 Cells / mL were stained with 5 μM carboxyfluorescein diacetate succinimide at 37°C in the dark for 15 minutes. CFSE labeling was terminated by dilution with RPMI-1640 medium containing 10% fetal bovine serum and incubation on ice for 5 minutes. Subsequently, the washed cells were inoculated at 2 × 10⁻⁶ cells / mL. 6 Cells were seeded at 24-well plates at 1 / mL density and stimulated with 200 μg / mL peptides (T1-T5 and B1-B12), 20 μg / mL prolysin, or 20 μg / mL concanavalin A (Con A), respectively. After incubation at 37°C and 5% CO2 for 72 hours, the cells were centrifuged at 400g for 10 minutes. The supernatant was collected, and IL-2, IL-4, IL-10, IL-13, and IFN-γ were measured using appropriate commercial ELISA kits. After washing, cells were stained with anti-mouse CD4 APC and analyzed by flow cytometry.

[0027] Due to their ability to bind MHC class II molecules, T cell epitopes can stimulate T cell proliferation and promote IL-2 release. Splenic lymphocytes were isolated from sensitized mice under sterile conditions, and the proliferative capacity of predicted T cell epitopes was assessed using a lymphocyte proliferation assay based on carboxyfluorescein diacetate succinimide (CFSE). Significantly higher proliferation rates were observed for T1 and T2-induced epitopes. p <0.01). The proliferation levels of other peptides T3, T4, and T5 were similar to those of the negative control ( Figure 1 (A) In this invention, Figures 1-4 The "*" in the text indicates p<0.05 level is significant, "**" indicates p <0.01 level is significant, "***" indicates p The level <0.001 was significant. Furthermore, the predicted T-cell epitope-induced IL-2 release level showed a similar trend to the lymphocyte proliferation assay. The IL-2 levels produced by peptides T1 and T2 were significantly higher than those in the negative control. p <0.05), while the IL-2 values ​​of peptides T3, T4 and T5 were similar to those of the negative control (Figure 1B).

[0028] Therefore, based on high T cell proliferation rate and IL-2 levels, the predicted peptides T1 (LAMAMNIASASRLLS, 10-24) and T2 (AMNIASASRLLSPRG, 13-27) can be considered T cell epitopes of ω5 prolysin. The presence of the shared sequence AMNIASASRLLS (13-24) in both T cell epitopes indicates that this core sequence possesses T cell epitope characteristics that induce cell proliferation.

[0029] Example 6 Indirect ELISA test The ELISA test is performed according to the following method: 15 μg of peptide and 3 μg of prolysin (positive control) or 3 μg of bovine serum albumin (BSA, negative control) were coated onto 96-well plates and incubated overnight at 4°C. After blocking at 37°C for 2 hours, primary antibodies were added to the wells, including anti-mouse IgG (1:5000 dilution), serum from immunized mice containing specific IgE (1:20 dilution), or serum from allergic patients (1:10 dilution). Then, HRP-labeled goat anti-mouse IgG (1:5000), HRP-labeled goat anti-mouse IgE (1:5000), or HRP-labeled goat anti-human IgE (1:2000) were incubated. After washing with PBST buffer, TMB Turbo ELISA substrate was added for color development. Finally, the absorbance was measured at 450 nm using a microplate reader.

[0030] The results showed that, to further analyze triggered cell proliferation, Th1 (IL-10 and IFN-γ) and Th2 (IL-4 and IL-13) related cytokines in the supernatant of spleen lymphocytes were measured using a commercially available ELISA kit. Figure 2 As shown, compared with the negative control, peptide T1 and T2 stimulation were significantly downregulated ( p <0.05) Th1-related cytokine levels. Th2-related cytokine levels produced by T3, T4, and T5 stimulation were similar to those in the negative control, while peptide T1 and T2 stimulation significantly upregulated ( p <0.05) Th2-related cytokine levels. On the other hand, for T1, T2, Tri a 19The proliferation response of Con A was strongly correlated with the production of Th2-related cytokines. These results suggest that only T1 and T2 can be considered as... Tri a 19 Potential T-cell epitopes that alter the Th1 / Th2 balance by increasing Th2-related cytokine levels and decreasing Th1-related cytokine levels.

[0031] To verify the IgG and IgE binding capacity for predicting B cell epitopes, ELISA was performed using serum from mice immunized with anti-mouse IgG, wheat gliadin, and wheat-allergic individuals. Figure 3 As shown in Figure A, compared to the negative control, all twelve synthetic peptides (B1-B12) showed significantly higher IgG binding (…). p <0.01). Regarding IgE binding, peptides B7, B10, and B12 showed significantly higher IgE binding compared to the negative control. p <0.01), B1-B6, B9, and B11 also showed significantly higher IgE binding ( p <0.05), while the IgE binding level of B8 was similar to that of the negative control ( Figure 3 B). These results indicate that peptides B7, B10, and B12 exhibit strong IgG and IgE binding capacity, while B8 only shows strong IgG binding capacity when mouse serum is immunized with prolysin.

[0032] Subsequently, the specific IgE binding capacity predicting B-cell epitopes was assessed using serum from 33 individuals with wheat allergies (75.8% from children). Figure 3 C). Based on these individual serum samples, the predicted IgE binding of peptides showed significant differences. Compared with peptides B2, B6, B7, and B12, the other eight peptides showed significantly higher ( p The specific IgE binding capacity was <0.05. In addition, peptides B3 and B4 showed high specific IgE binding capacity, with average absorbance values ​​exceeding 1.0.

[0033] In summary, the seven peptides B1 (SASRLLSPRGKELHTP, 18-33), B3 (QQHQSPQQQFPQQQFP, 105-120), B4 (FPQQQLPQQQQIPQQQ, 171-186), B5 (QQFPQQQFPQQQFPQQ, 206-221), B9 (PQQQQFPQQQEFPQQQ, 317-332), B10 (QQLPQQQFPQQQFPQQ, 341-356), and B11 (TQQQFPRPQQSPEQQQ, 371-386) exhibited strong IgG and IgE binding capacity.

[0034] Table 3 summarizes the previously reported results of wheat gliadin sensitization epitopes. Overlapping peptides QQFPQQQ, QQLPQQQ, and QQIPQQQ were observed to occur frequently, consistent with the QQX1X2QQQ motif regions found in different gliadin subtypes. Furthermore, the peptide QQLPQQQ, present in B4 and B10, also appeared in the predicted T-cell epitope T5 (333-347).

[0035] Example 7 KU812 Cell Mediator Release Assay KU812 cells were cultured in complete RPMI-1640 medium containing 2 mM 90% L-glutamine, 10% FBS, and 1% penicillin-streptomycin, and placed in a 37°C, 5% CO2 environment, as described below: Cells (2×10) 6 Cells (per mL) were seeded in 96-well plates and incubated with 0.5 μg / mL human multiple myeloma IgE for 7 days to stimulate FcεRI expression. Subsequently, cells were passively sensitized with wheat-allergic serum (1:10) and stimulated with a synthetic peptide (200 μg / mL), wheat gliadin (20 μg / mL, positive control), compound 48 / 80 (20 μg / mL, positive control), or Tyrode's buffer (negative control). After centrifugation, the supernatant was collected, and histamine and trypsin levels were measured using a commercially available ELISA kit. β-hexosidase activity was detected colorimetrically based on the hydrolysis of p-nitrophenyl-N-acetyl-β-D-glucosinolate.

[0036] The degranulation capacity of sensitized epitopes plays a crucial role, as it can release inflammatory mediators, including histamine, and induce allergic reactions. Furthermore, degranulation capacity is an effective method for assessing the safety of T-cell epitopes. Figure 4 As shown, all synthesized T-cell and B-cell epitope peptides were screened using KU812 cells, with prolysin and compound 48 / 80 serving as positive controls. Compared to the negative control (Tyrode's buffer), T-cell epitopes T4 and T5, and B-cell epitopes B2, B3, B4, and B9 were significantly increased. p <0.001) The release of β-hexosidase, histamine, and trypsin was observed. However, the levels of β-hexosidase, histamine, and trypsin produced by peptides T1-T3, B1, B5-B8, and B10-B12 were similar to those in the negative control.

[0037] In summary, peptides B3, B4, and B9 exhibit strong IgG / IgE binding and degranulation capabilities, suggesting they are potential B-cell epitopes of ω5 prolysin. Furthermore, the overlapping sequence QQX1PQQQ (X1=F,L) appearing in these three epitopes can be presumed to be a core region triggering allergic reactions. The sequence QQLPQQQ also appears in T5, which exhibits strong degranulation capabilities, consistent with previous reports that the T-cell epitopes located at 49-69 in Anis 1 share the NRFTTIKN core region with the IgE and IgG4 epitopes. Additionally, peptides T1 and T2 share the common sequence AMNIASASRLLS (13-24), which may be considered a core region stimulating cell proliferation. Due to their lack of degranulation capabilities, T1 and T2 could be considered for peptide-based immunotherapy.

[0038] In summary, these findings support the T1 (LAMAMNIASASRLLS, 10–24) and T2 (AMNIASASRLLSPRG, 13–27) peptides as T-cell epitopes, and the B3 (QQHQSPQQQFPQQQFP, 105–120), B4 (FPQQQLPQQQQIPQQQ, 171–186), and B9 (PQQQQFPQQQEFPQQQ, 317–332) peptides as B-cell epitopes of ω5 prolysin. These peptides could be considered for designing immunotherapies for patients with wheat allergy. Tri a 19 Candidate peptides for vaccines.

[0039] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of the patent. All equivalent changes and modifications made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. A method for identifying dominant B-cell epitopes and T-cell epitopes in wheat ω-5 gliadin, characterized in that, The steps include the following: (1) T cell epitopes were predicted using a peptide window of 15 amino acid lengths, including LAMAMNIASASRLLS 10-24, AMNIASASRLLSPRG 13-27, ASRLLSPRGKELHTPQ 19-33, FPQQQQFLQQQQIPQ 67-81 and FPQQQFHQQQLPQQQF 333-347; (2) Using DNAStar, BepiPred 3.0 server, ABCpred, and IEDB, B cell epitopes were predicted based on surface accessibility, fragment flexibility, and antigenic index. The predicted B cell epitopes included SASRLLSPRGKELHTP 18-33, HQIPQQPQQFPQQQQF 58-73, QQHQSPQQQFPQQQFP 105-120, FPQQQLPQQQQIPQQQ 171-186, QQFPQQQFPQQQFPQQ 206-221, RQPQQLPQQQQIPQQP 241-256, SPQQQQFPQQQFPQQQ 270-285, PQPQQIPQQQQIPQQP 293-308, PQQQQFPQQQEFPQQQ 317-332, QQLPQQQFPQQQFPQQ 341-356, TQQQFPRPQQSPEQQQ 371-386 and QSEEPSPYQQYPQQQP 413-428.

2. The identification method as described in claim 1, characterized in that, In (1), the tool used for T cell epitope prediction is selected from any one or more of NetMHC II, MHCPred, SYFPEITHI and IEDB.

3. The identification method as described in claim 1, characterized in that, In (1), the predicted T cell epitopes include LAMAMNIASASRLLS 10-24 and AMNIASASRLLSPRG 13-27.

4. The identification method as described in claim 1, characterized in that, In (2), the predicted B cell epitopes include QQHQSPQQQFPQQQFP 105-120, FPQQQLPQQQQIPQQQ 171-186, and PQQQQFPQQQEFPQQQ 317-332.

Images