Digestion-resistant linear epitope peptides of south american white shrimp allergens and applications thereof

Abstract

The application discloses a digestion-resistant linear epitope peptide of Penaeus vannamei allergic antigen and application thereof, and belongs to the technical field of immunology. The amino acid sequence of the digestion-resistant linear epitope peptide comprises DSGVGIYAPDAEA, EGELKGTYYPLTGM, GRQGDPHGKFDLPPGV, IFAWPHKDNNGIE and KSTESSVTVPDVPSIHD. The five digestion-resistant linear epitope peptides have IgE binding capacity and can be used as key allergic epitope peptides of Penaeus vannamei. The prepared allergic epitope peptides have good application in the preparation of shrimp and shrimp product allergy diagnosis reagents.

Description

Technical Field

[0001] This invention relates to digestible linear epitope peptides of allergens in Litopenaeus vannamei and their applications, belonging to the field of immunology technology. Background Technology

[0002] Food allergies, a prevalent food safety issue worldwide, are experiencing a rising incidence rate, drawing widespread attention. Food allergies are characterized by a short onset time, high incidence, and a wide affected population. They can trigger systemic, multi-system reactions such as asthma, diarrhea, urticaria, and allergic dermatitis, and can even lead to shock and death. Food allergies are a type I (immediate) hypersensitivity reaction mediated by immunoglobulin E (IgE). When a food allergen enters the body, it selectively induces the production of specific IgE antibodies, sensitizing the body. When the same allergen re-enters the body, it binds to the specific IgE antibodies, inducing cell degranulation and subsequently triggering a series of allergic symptoms. Currently, the most effective way to prevent food allergies is to avoid contact with food allergens. However, cross-reactions during the production process increase the likelihood of allergens in food, therefore, allergy sufferers need to be extra careful to avoid food allergies.

[0003] Currently identified major and minor shrimp allergens include tropomyosin, arginine kinase, myosin light chain, sarcoplasmic calcium-binding protein, hemocyanin, and triose phosphate isomerase. After oral ingestion, shrimp proteins are digested in the gastrointestinal tract and enter the body to exert sensitizing activity. Allergen epitopes are the immune material basis for triggering food allergic reactions. However, after food digestion, some resistant linear epitopes remain, which can stimulate allergic reactions. Therefore, identifying the linear epitopes that truly cause food allergies is crucial. Furthermore, research on linear epitopes is of great significance for the development of hypoallergenic and hypoallergenic foods and the study of allergic diseases. Summary of the Invention

[0004] This invention provides digestible epitope peptides of Litopenaeus vannamei allergens, which can be used to prepare diagnostic reagents for shrimp and shrimp product allergies. By combining digestomics and bioinformatics, food allergen epitope peptides can be predicted relatively accurately. Furthermore, by screening for the number of key amino acids and their binding affinity to MHC-II molecules, linear epitope peptides with strong sensitizing capabilities are identified. Validation of the screened digestible epitope peptides of Litopenaeus vannamei allergens can clearly identify the true epitopes that induce allergic reactions in Litopenaeus vannamei. These screened epitopes have the ability to recognize Litopenaeus vannamei, arginine kinase, and hemocyanin, and also exhibit high homology with other seafood allergens. Therefore, the screened allergen epitope peptides have promising applications in preparing diagnostic reagents for shrimp and shrimp product allergies.

[0005] This invention provides a digestible linear epitope peptide for allergens in Litopenaeus vannamei, wherein the amino acid sequence of the epitope peptide includes: (1) Asp-Ser-Gly-Val-Gly-Ile-Tyr-Ala-Pro-Asp-Ala-Glu-Ala, abbreviated as DSGVGIYAPDAEA, as shown in SEQ ID No:1, has a molecular weight of 1264.31 Da; (2) Glu-Gly-Glu-Leu-Lys-Gly-Thr-Tyr-Tyr-Pro-Leu-Thr-Gly-Met, abbreviated as EGELKGTYYPLTGM, as shown in SEQ ID No:2, has a molecular weight of 1558.77 Da; (3) Gly-Arg-Gln-Gly-Asp-Pro-His-Gly-Lys-Phe-Asp-Leu-Pro-Pro-Gly-Val, abbreviated as GRQGDPHGKFDLPPGV, as shown in SEQ ID No:3, has a molecular weight of 1676.85 Da; (4) Ile-Phe-Ala-Trp-Pro-His-Lys-Asp-Asn-Asn-Gly-Ile-Glu, abbreviated as IFAWPHKDNNGIE, as shown in SEQ ID No:4, has a molecular weight of 1540.69 Da; (5) Lys-Ser-Thr-Glu-Ser-Ser-Val-Thr-Val-Pro-Asp-Val-Pro-Ser-Ile-His-Asp, abbreviated as KSTESSVTVPDVPSIHD, as shown in SEQ ID No:5, has a molecular weight of 1797.93 Da.

[0006] This invention provides a method for screening digestible epitope peptides of allergens in Litopenaeus vannamei, the steps of which are as follows: (1) Screening of digestible linear epitope peptides based on digestomics and bioinformatics Litopenaeus vannamei protein was digested in the gastrointestinal tract using a conventional simulated digestion method. Resistant peptides from the digestion products of Litopenaeus vannamei protein allergens were collected using liquid chromatography-mass spectrometry (LC-MS). Allergen epitopes in Litopenaeus vannamei were predicted using five bioinformatics software programs: DNAStar, AntheProt, ABCpred, Immunomedicine Group, and SOPMA. Epitopes predicted by at least three of these software programs were designated as linear epitopes of Litopenaeus vannamei allergens. The resistant peptides in the digestion products of Litopenaeus vannamei were compared with the predicted linear epitopes, and peptides containing the predicted linear epitopes were screened as resistant linear epitope peptides of Litopenaeus vannamei allergens with sensitizing potential.

[0007] (2) Screening of digestible linear epitope peptides of Litopenaeus vannamei allergens with strong sensitizing ability By analyzing amino acid frequency and homology, the key amino acids in the linear epitope of Litopenaeus vannamei allergen in step (1) were predicted; by molecular docking technology, the digestible linear epitope peptide of Litopenaeus vannamei allergen with sensitization potential was docked with MHC-II molecules; and digestible linear epitope peptides containing more key amino acids and with strong binding ability were screened as digestible linear epitope peptides of Litopenaeus vannamei allergen.

[0008] The present invention also provides an antigen composition comprising two or more of the said epitope peptides.

[0009] The present invention also provides a shrimp allergy detection kit containing the said antigen composition.

[0010] In one embodiment, the kit further includes a coating solution, a blocking solution, a sample diluent, a washing solution, a detection antibody, a substrate chromogenic solution, and a stop solution.

[0011] The present invention also provides a method for detecting shrimp allergen antibodies using the aforementioned detection kit, comprising the following steps: (1) Coat the antigen composition with coating solution for at least 8 hours and block for at least 2 hours; (2) Add sample diluent to the antigen composition coated in step (1) and incubate for at least 2 hours; (3) Add biotin-labeled IgE secondary antibody and HRP-labeled streptavidin, and incubate for at least 1 hour; (4) Add the substrate color development solution and incubate for at least 15 min; (5) Add the stop solution and measure the absorbance.

[0012] In one embodiment, the coating solution in step (1) comprises sodium carbonate buffer.

[0013] In one embodiment, the sample diluent in step (2) includes a serum diluent.

[0014] In one embodiment, the substrate developing solution in step (4) includes TMB substrate developing solution.

[0015] In one embodiment, dot-blot analysis revealed that all five digestible linear epitope peptides of Litopenaeus vannamei allergens screened in this invention possess IgE binding capacity.

[0016] In one embodiment, enzyme-linked immunosorbent assay (ELISA) revealed that the epitope peptides screened in this invention have a significant IgE binding capacity with the whole protein-sensitized serum of Litopenaeus vannamei.

[0017] The present invention also provides a method for improving the accuracy of shrimp allergy detection, using the epitope peptide or antigen composition for detection.

[0018] The present invention also provides the use of the epitope peptide, or the antigen composition, in reagents or kits for the diagnosis of allergies to shrimp and shrimp products.

[0019] Beneficial effects: This invention utilizes a combined approach of digestomics and bioinformatics to screen for digestible linear epitopes of allergens in Litopenaeus vannamei and, for the first time, synthesizes five epitope peptides predicted to have strong sensitizing potential. All of these epitope peptides possess IgE binding capacity and can be used in the preparation of allergy diagnostic reagents for shrimp and shrimp products. The reagents exhibit high accuracy and specificity and have significant research value. Attached Figure Description

[0020] Figure 1 This invention provides the linear epitopes in Litopenaeus vannamei allergens predicted by digestomics and bioinformatics, and their localization in a 3D model.

[0021] Figure 2 This invention relates to the distribution frequency of allergens in Litopenaeus vannamei and predicted linear epitope amino acids.

[0022] Figure 3 This invention demonstrates the sequence conservation of linear epitopes predicted in allergens of Litopenaeus vannamei across different species.

[0023] Figure 4 This invention relates to the docking of epitope peptides of allergens in Litopenaeus vannamei with MHC-II molecules.

[0024] Figure 5 This is a secondary mass spectrum of the epitope peptide of the allergen in Litopenaeus vannamei, as described in this invention.

[0025] Figure 6 This invention relates to the IgE binding capacity of the epitope peptide of the allergen in Litopenaeus vannamei.

[0026] Figure 7 This invention describes the IgE binding capacity of the allergen epitope peptides (P1 and P2) derived from allergen arginine kinase, the allergen epitope peptides (P3, P4 and P5) derived from allergen hemocyanin, and the allergen epitope peptides (P1, P2, P3, P4 and P5) of Litopenaeus vannamei to Litopenaeus vannamei whole protein sensitized serum (experimental serum pool).

[0027] Figure 8 This invention relates to the binding capacity of the epitope peptide of the allergen in Litopenaeus vannamei to IgE in the total protein-sensitized serum (experimental serum pool) of Litopenaeus vannamei. Detailed Implementation

[0028] The present invention will be further illustrated below with specific examples, but the implementation and scope of protection of the present invention are not limited thereto. For process parameters not specifically specified, conventional techniques can be referred to.

[0029] This invention identified five digestible linear epitope peptides of Litopenaeus vannamei allergens, with the amino acid sequence Asp-Ser-Gly-Val-Gly-Ile-Tyr-Ala-Pro-Asp-Ala-Glu-Ala, abbreviated as DSGVGIYAPDAEA, as shown in SEQ ID No:1, with a molecular weight of 1264.31 Da; Glu-Gly-Glu-Leu-Lys-Gly-Thr-Tyr-Tyr-Pro-Leu-Thr-Gly-Met, abbreviated as EGELKGTYYPLTGM, as shown in SEQ ID No:2, with a molecular weight of 1558.77 Da; and Gly-Arg-Gln-Gly-Asp-Pro-His-Gly-Lys-Phe-Asp-Leu-Pro-Pro-Gly-Val, abbreviated as GRQGDPHGKFDLPPGV, as shown in SEQ ID No:1. As shown in SEQ ID No:3, the molecular weight is 1676.85 Da; Ile-Phe-Ala-Trp-Pro-His-Lys-Asp-Asn-Asn-Gly-Ile-Glu, abbreviated as IFAWPHKDNNGIE, as shown in SEQ ID No:4, has a molecular weight of 1540.69 Da; and Lys-Ser-Thr-Glu-Ser-Ser-Val-Thr-Val-Pro-Asp-Val-Pro-Ser-Ile-His-Asp, abbreviated as KSTESSVTVPDVPSIHD, as shown in SEQ ID No:5, has a molecular weight of 1797.93 Da.

[0030] The digestible linear epitope peptides of Litopenaeus vannamei allergens screened in this invention all have IgE binding ability.

[0031] Example 1: Identification of digestible peptides from Litopenaeus vannamei digestive products S1. Extraction of protein from Litopenaeus vannamei using alkali dissolution and acid precipitation method.

[0032] Whiteleg shrimp were placed in boiling water and heat-treated for 5 minutes. After cooling, the shells and heads were removed, and the shrimp paste was minced. The shrimp paste was then mixed with three times its volume of water and stirred until homogeneous. The pH was adjusted to 11.0, and the mixture was stirred at 4°C for 8 hours. After centrifugation at 10,000 rpm for 25 minutes at 4°C, the supernatant was collected. The pH of the supernatant was adjusted to 5.5, and the mixture was stirred at 4°C for 1.5–2 hours. After centrifugation at 10,000 rpm for 20 minutes at 4°C, the precipitate was collected. The precipitate was dissolved and homogenized, and the pH was adjusted to 7.0 to obtain whiteleg shrimp whole protein. The whole protein was freeze-dried and stored at -80°C until use. S2, in vitro simulation of stomach and gastrointestinal digestion Simulated gastric and intestinal fluids were pre-prepared according to standards and stored at -20°C. The recommended electrolyte concentration in the simulated gastric fluid was 7.8 mmol / L. + 72.2 mmol / L Na + 70.2 mmol / L Cl - 0.9 mmol / L H2PO4 - 25.5 mmol / L HCO3 - and CO3 2- 0.1 mmol / L Mg 2+ 1.0 mmol / L NH4 + and 0.15 mmol / L Ca 2+ The recommended electrolyte concentration in simulated intestinal fluid is 7.6 mmol / L. + 123.4 mmol / L Na + 55.5 mmol / L Cl - 0.8 mmol / L H2PO4 - 85 mmol / L HCO3 - and CO3 2- 0.33 mmol / L Mg 2+ and 0.6 mmol / L Ca 2+ Simulated gastric digestion: Total protein from Litopenaeus vannamei was mixed with the simulated gastric juice described in step S1, and the pH was adjusted to 3.0. Pepsin was added to achieve a final enzyme activity of 2000 U / mL (pepsin:total protein = 1:100 enzyme:substrate). The mixture was stirred at 37°C for 120 min to simulate gastric digestion, yielding simulated gastric digestate. The pH was adjusted to 7.0, and the mixture was centrifuged at 10000 g for 10 min. The supernatant was collected, representing the gastric digestion product. Simulated intestinal digestion: The above-mentioned gastric digestion products are mixed with the simulated intestinal fluid described in step S1, and the pH is adjusted to 7.0. Trypsin is added to make the final enzyme activity 100 U / mL. The mixture is stirred at 37°C for 120 min to simulate the intestinal digestion process, and simulated gastrointestinal digests are obtained. The enzymes are inactivated by heating in a water bath at 100°C for 5 min, and the mixture is centrifuged at 10000 g for 10 min. The supernatant is collected, which is the gastrointestinal digestion product. S3. Identification of resistant peptides in the whole protein digestion products of Litopenaeus vannamei: The peptide sequences of the gastric digestion products and gastrointestinal digestion products described in step S2 were identified using NanoLC-MS / MS mass spectrometry. Peptide sequences were retrieved from the established database using Peaks Studio software to obtain a series of resistant peptides.

[0033] Example 2: Identification of digestible epitopes with sensitizing potential in tropomyosin of Litopenaeus vannamei Tropomyosin (TM) from Litopenaeus vannamei is one of the main allergens of Litopenaeus vannamei. Its molecular weight is estimated to be 36 kDa and its isoelectric point is 4.5. It consists of two different α-helical peptide chains wrapped together to form a superhelical structure. The serum of more than 80% of shrimp allergy patients can interact with it.

[0034] This embodiment uses a combination of digestomics and bioinformatics to screen for digestible epitope peptides of tropomyosin from Litopenaeus vannamei, including the following steps: S1. Obtain the tropomyosin sequence of Litopenaeus vannamei from the NCBI database, and locate the tropomyosin peptides in the stomach and gastrointestinal digestive products of Litopenaeus vannamei in the protein sequence. S2. Use five bioinformatics software programs, DNAStar, AntheProt, ABCpred, ImmunomedicineGroup, and SOPMA, to predict epitopes of Litopenaeus vannamei tropomyosin. Take the epitopes predicted by ≥3 software programs as linear epitopes of Litopenaeus vannamei tropomyosin and locate the predicted linear epitopes of Litopenaeus vannamei tropomyosin in the Litopenaeus vannamei tropomyosin sequence described in step S1. S3. Compare the digestible peptides in the gastrointestinal digestion products of Litopenaeus vannamei tropomyosin described in step S1 with the predicted linear epitopes of Litopenaeus vannamei tropomyosin described in step S2, and screen out the digestible (intact) linear epitopes as the predicted digestible linear epitopes of Litopenaeus vannamei tropomyosin. At the same time, screen out the epitope peptides containing the predicted digestible linear epitopes of Litopenaeus vannamei tropomyosin as digestible epitope peptides of Litopenaeus vannamei tropomyosin.

[0035] S4. Using SWISS-MODEL software, a three-dimensional model of tropomyosin from Litopenaeus vannamei was established using the protein with the highest similarity (1C1G) as a template for homology modeling. S5. Using Pymol software, locate the predicted digestible linear epitopes of Litopenaeus vannamei tropomyosin in the three-dimensional model of Litopenaeus vannamei tropomyosin described in step S4. S6. The amino acid frequencies of tropomyosin and predicted tropomyosin digestible linear epitopes in Litopenaeus vannamei were calculated using ExPASy software. Amino acids with a higher proportion in the predicted tropomyosin digestible linear epitopes than those in tropomyosin were identified as key amino acids in the epitopes. S7. Collect all allergenic amino acid sequences of tropomyosin from the SDAP database, perform multiple sequence alignment using ClustalW software, and identify highly conserved amino acids as key amino acids in epitopes. S8. The key amino acids recorded in steps S6 and S7 are identified as potential key amino acids in the digestible linear epitopes of tropomyosin in Litopenaeus vannamei. S9. Using molecular docking technology, the digestible epitope peptide of tropomyosin from Litopenaeus vannamei screened in step S3 is docked with MHC-II molecules, and the digestible epitope peptide of tropomyosin from Litopenaeus vannamei with strong binding ability is screened by docking scoring.

[0036] Experimental results: such as Figure 1 As shown in (A), the digestible peptides of tropomyosin from the stomach and gastrointestinal digestion products of Litopenaeus vannamei were obtained by NanoLC-MS / MS mass spectrometry. Simultaneously, five linear epitopes of Litopenaeus vannamei tropomyosin were predicted using bioinformatics, as shown in Table 1. Tables 1 and 2 show the Litopenaeus vannamei tropomyosin digestible linear epitopes DLDQV and MQQLENDLDQV identified through combined digestomics and bioinformatics screening. These digestible linear epitopes were also mapped into a three-dimensional model of Litopenaeus vannamei tropomyosin, as shown in Table 1. Figure 1 As shown in (B). Figure 2 (A) and Figure 3 (A) Combining two methods, the key amino acid in the digestible linear epitope of tropomyosin from Litopenaeus vannamei was predicted. The figure shows that the linear epitope does not contain the key amino acid. Molecular docking revealed that the binding energy between the digestible epitope peptide MQQLENDLDQV and MHC-II molecules is -191.424 kcal / mol, indicating that the binding effect between the epitope peptide and MHC-II molecules is generally weak. Figure 4(A) and Table 3 show the binding sites of the epitope peptides with MHC-II molecules predicted by molecular docking. The epitope peptides form hydrogen bonds with αS53, αG58, βQ64, βK71, and βN82 in the MHC-II molecule, and have hydrophobic interactions with αF54, αN62, βF26, βY60, βW61, βD66, βL67, βA74, and βY78.

[0037] Example 3: Identification of digestion-resistant epitopes with sensitizing potential in arginine kinase of Litopenaeus vannamei Arginine kinase (AK) from Litopenaeus vannamei is also an important crustacean allergen, with a molecular weight of approximately 40 kDa and an isoelectric point of approximately 6.0–6.5. It consists of a C-terminal domain (7 α-helices surrounding 8 antiparallel β-sheets) and an N-terminal domain (α-helix).

[0038] This embodiment uses a combination of digestomics and bioinformatics to screen for digestion-resistant epitope peptides of arginine kinase in Litopenaeus vannamei, including the following steps: S1. Obtain the arginine kinase sequence of Litopenaeus vannamei from the NCBI database, and locate the arginine kinase peptides in the stomach and gastrointestinal digestive products of Litopenaeus vannamei in the protein sequence. S2. Use five bioinformatics software programs, DNAStar, AntheProt, ABCpred, ImmunomedicineGroup, and SOPMA, to predict arginine kinase epitopes in Litopenaeus vannamei. Use the epitopes predicted by ≥3 software programs as linear epitopes of arginine kinase in Litopenaeus vannamei, and locate the predicted linear epitopes of arginine kinase in Litopenaeus vannamei in the arginine kinase sequence described in step S1. S3. Compare the digestible peptides in the gastrointestinal digestion products of Litopenaeus vannamei arginine kinase described in step S1 with the predicted linear epitopes of Litopenaeus vannamei arginine kinase described in step S2, and screen out the digestible (intact) linear epitopes as the predicted digestible linear epitopes of Litopenaeus vannamei arginine kinase. At the same time, screen out the epitope peptides containing the predicted digestible linear epitopes of Litopenaeus vannamei arginine kinase as digestible epitope peptides of Litopenaeus vannamei arginine kinase.

[0039] S4. Using SWISS-MODEL software, a three-dimensional model of arginine kinase in Litopenaeus vannamei was established using the protein with the highest similarity (5ZHQ) as a template for homology modeling. S5. Using Pymol software, locate the predicted linear epitopes of Litopenaeus vannamei arginine kinase in the three-dimensional model of Litopenaeus vannamei arginine kinase described in step S4. S6. The amino acid frequencies of arginine kinase and predicted arginine kinase digestibility linear epitopes in Litopenaeus vannamei were calculated using ExPASy software. Amino acids with a higher proportion in the predicted arginine kinase digestibility linear epitopes than those in the arginine kinase were identified as key amino acids in the epitopes. S7. Collect all allergen amino acid sequences of arginine kinases from the SDAP database, perform multiple sequence comparison using ClustalW software, and select the amino acids with higher conservation as key amino acids in the epitopes. S8. The key amino acids recorded in steps S6 and S7 are identified as potential key amino acids in the digestion-resistant linear epitopes of arginine kinase in Litopenaeus vannamei. S9. Using molecular docking technology, the digestible epitope peptide of Litopenaeus vannamei arginine kinase selected in step S3 is docked with MHC-II molecules, and the digestible epitope peptide of Litopenaeus vannamei with strong binding ability is screened by docking scoring.

[0040] Experimental results: such as Figure 1 As shown in (C), the digestible peptides of Litopenaeus vannamei arginine kinase in the stomach and gastrointestinal digestion products were obtained by NanoLC-MS / MS mass spectrometry. Simultaneously, 10 linear epitopes of Litopenaeus vannamei arginine kinase were predicted using bioinformatics, as shown in Table 1. Tables 1 and 2 show that two linear epitopes of Litopenaeus vannamei arginine kinase with digestibility, APDAEA and KGTYYP, and two epitope peptides with digestibility, DSGVGIYAPDAEA and EGELKGTYYPLTGM, were screened using a combination of digestomics and bioinformatics. These linear epitopes were also mapped into a three-dimensional model of Litopenaeus vannamei arginine kinase, as shown in Table 1. Figure 1 As shown in (D). Figure 2 (B) and Figure 3 (B) Combining the two methods, key amino acids in the digestibility epitope of arginine kinase in Litopenaeus vannamei were predicted. The figure shows that the linear epitope contains a large number of key amino acids. Molecular docking revealed that the binding energies of the digestibility epitope peptides DSGVGIYAPDAEA and EGELKGTYYPLTGM to MHC-II molecules are -223.012 kcal / mol and -227.357 kcal / mol, respectively, demonstrating a good binding interaction between the epitope peptides and MHC-II molecules. Figure 4(BC) and Table 3 show the binding sites of the epitope peptides with MHC-II molecules predicted by molecular docking. Among them, the epitope peptide DSGVGIYAPDAEA forms hydrogen bonds with αS53, αF54, αN62, βQ70, βK71, and βN82 in the MHC-II molecule, and has hydrophobic interactions with αF24, αF32, αW43, βW61, βL67, βY78, βH81, and βV85. The epitope peptide EGElkGTYYPLTGM forms hydrogen bonds with αQ9, αN62, βN82, βY30, and βY47 in the MHC-II molecule, and has hydrophobic interactions with αQ9, αN62, βN82, βY30, and βY47.

[0041] Example 4: Identification of digestible epitopes with sensitizing potential in the myosin light chain of Litopenaeus vannamei Myosin light chain (MLC) from Litopenaeus vannamei is a novel allergen with a molecular weight of approximately 18–20 kDa. It exhibits high IgE reactivity with the serum of patients allergic to crustaceans.

[0042] This embodiment uses a combination of digestomics and bioinformatics to screen for digestible epitope peptides in the light chain of myosin from Litopenaeus vannamei, including the following steps: S1. Obtain the light chain sequence of tropomyosin from the NCBI database, and locate the myosin light chain peptides in the stomach and gastrointestinal digestive products of Litopenaeus vannamei in the protein sequence. S2. Five bioinformatics software programs, DNAStar, AntheProt, ABCpred, ImmunomedicineGroup, and SOPMA, are used to predict the myosin light chain epitopes of Litopenaeus vannamei. Epitopes predicted by ≥3 software programs are taken as linear epitopes of the myosin light chain of Litopenaeus vannamei, and the predicted linear epitopes of the myosin light chain of Litopenaeus vannamei are located in the myosin light chain sequence of Litopenaeus vannamei described in step S1. S3. Compare the digestible peptides in the gastrointestinal digestion products of the myosin light chain of Litopenaeus vannamei described in step S1 with the predicted linear epitopes of the myosin light chain of Litopenaeus vannamei described in step S2. Select digestible (intact) linear epitopes as predicted digestible linear epitopes of the myosin light chain of Litopenaeus vannamei. At the same time, select epitope peptides containing the predicted digestible linear epitopes of the myosin light chain of Litopenaeus vannamei as digestible epitope peptides of the myosin light chain of Litopenaeus vannamei.

[0043] S4. Using SWISS-MODEL software, a three-dimensional model of the myosin light chain of Litopenaeus vannamei was established using the protein with the highest similarity (5W1A) as a template for homology modeling. S5. Using Pymol software, locate the predicted digestible linear epitopes of the myosin light chain of Litopenaeus vannamei in the three-dimensional model of the myosin light chain of Litopenaeus vannamei described in step S4. S6. The amino acid frequencies of the myosin light chain and the predicted digestible linear epitopes of the myosin light chain of Litopenaeus vannamei were calculated using ExPASy software. Amino acids with a higher proportion in the predicted digestible linear epitopes of the myosin light chain than those in the myosin light chain were identified as key amino acids in the epitopes. S7. Collect all allergen amino acid sequences of myosin light chain from the SDAP database, perform multiple sequence comparison using ClustalW software, and identify highly conserved amino acids as key amino acids in epitopes. S8. The key amino acids recorded in steps S6 and S7 are identified as potential key amino acids in the digestible linear epitopes of myosin light chain in Litopenaeus vannamei. S9. Using molecular docking technology, the myosin light chain digestible epitope peptides of Litopenaeus vannamei screened in step S3 are docked with MHC-II molecules, and the Litopenaeus vannamei tropomyosin light chain digestible epitope peptides with strong binding ability are screened by docking scoring.

[0044] Experimental results: such as Figure 1 As shown in (E), the digestible peptides of the myosin light chain from the stomach and gastrointestinal digestion products of Litopenaeus vannamei were obtained by NanoLC-MS / MS mass spectrometry. Simultaneously, four linear epitopes of the myosin light chain from Litopenaeus vannamei were predicted using bioinformatics, as shown in Table 1. Tables 1 and 2 show the digestible linear epitopes of the myosin light chain from Litopenaeus vannamei, NLNPT, and ALNLNPTLA, which were screened using a combination of digestomics and bioinformatics. These digestible linear epitopes of the myosin light chain from Litopenaeus vannamei were also located in a three-dimensional model of the myosin light chain from Litopenaeus vannamei, as shown in Table 1. Figure 1 As shown in (F). Figure 2 (C) and Figure 3 (C) Combining the two methods, the key amino acid in the digestible linear epitope of myosin light chain from Litopenaeus vannamei was predicted. The figure shows that the linear epitope contains only one key amino acid. Molecular docking revealed that the binding energy between the digestible epitope peptide ALNLNPTLA of Litopenaeus vannamei myosin light chain and MHC-II molecules is -213.211 kcal / mol, demonstrating a good binding interaction between the epitope peptide and MHC-II molecules. Figure 4(D) and Table 3 show the binding sites of the epitope peptides with MHC-II molecules predicted by molecular docking. The epitope peptide ALNLNPTLA forms hydrogen bonds with αE11, αD66, αN69, αR76, and βW61 in the MHC-II molecule, and has hydrophobic interactions with αI72, βF26, βY47, βY60, βL67, βK71, and βY78.

[0045] Example 5: Identification of digestible epitopes with sensitizing potential in calcium-binding proteins of Litopenaeus vannamei muscle Sarcoplasmic calcium-binding protein (SCP) of Litopenaeus vannamei is a minor allergen with a molecular weight of 20-22 kDa. It is a calcium ion-binding protein and has been shown to have IgE binding activity at the molecular level in Litopenaeus vannamei.

[0046] This embodiment uses a combination of digestomics and bioinformatics to screen for digestible epitope peptides of calcium-binding protein in the sarcoplasm of Litopenaeus vannamei, including the following steps: S1. Obtain the sequence of calcium-binding protein in the sarcoplasm of Litopenaeus vannamei from the NCBI database, and locate the sarcoplasmic calcium-binding protein peptides in the stomach and gastrointestinal digestive products of Litopenaeus vannamei in the protein sequence. S2. Use five bioinformatics software programs, DNAStar, AntheProt, ABCpred, ImmunomedicineGroup, and SOPMA, to predict epitopes of calcium-binding protein in the myoplasm of Litopenaeus vannamei. Use epitopes predicted by ≥3 software programs as linear epitopes of calcium-binding protein in the myoplasm of Litopenaeus vannamei, and locate the predicted linear epitopes of calcium-binding protein in the myoplasm of Litopenaeus vannamei in the sequence described in step S1. S3. Compare the digestible peptides in the gastrointestinal digestion products of Litopenaeus vannamei sarcoplasmic calcium-binding protein described in step S1 with the predicted linear epitopes of Litopenaeus vannamei sarcoplasmic calcium-binding protein described in step S2. Select digestible (intact) linear epitopes as predicted digestible linear epitopes of Litopenaeus vannamei sarcoplasmic calcium-binding protein. At the same time, select epitope peptides containing predicted digestible linear epitopes of Litopenaeus vannamei sarcoplasmic calcium-binding protein as digestible epitope peptides of Litopenaeus vannamei sarcoplasmic calcium-binding protein.

[0047] S4. Using SWISS-MODEL software, a three-dimensional model of calcium-binding protein in the myoplasm of Litopenaeus vannamei was established using the protein with the highest similarity (2SAS) as a template for homology modeling. S5. Using Pymol software, locate the predicted digestible linear epitopes of Litopenaeus vannamei sarcoplasmic calcium-binding protein in the three-dimensional model of Litopenaeus vannamei sarcoplasmic calcium-binding protein described in step S4. S6. The amino acid frequencies of the sarcoplasmic calcium-binding protein and the predicted sarcoplasmic calcium-binding protein digestible linear epitopes were calculated using ExPASy software. Amino acids with a higher proportion in the predicted sarcoplasmic calcium-binding protein digestible linear epitopes than those in the sarcoplasmic calcium-binding protein were identified as key amino acids in the epitopes. S7. Collect all allergen amino acid sequences of sarcoplasmic calcium-binding proteins from the SDAP database, perform multiple sequence comparison using ClustalW software, and select highly conserved amino acids as key amino acids in epitopes. S8. The key amino acids recorded in steps S6 and S7 are identified as potential key amino acids in the digestible linear epitopes of calcium-binding protein in Litopenaeus vannamei. S9. Using molecular docking technology, the digestible epitope peptides of Litopenaeus vannamei muscle calcium-binding protein screened in step S3 are docked with MHC-II molecules, and the digestible epitope peptides of Litopenaeus vannamei muscle calcium-binding protein with strong binding ability are screened by docking scoring.

[0048] Experimental results: such as Figure 1 As shown in (G), the digestible peptides of Litopenaeus vannamei (S. spp.) myosin calcium-binding protein in the stomach and gastrointestinal digestion products were obtained by NanoLC-MS / MS mass spectrometry. Simultaneously, seven linear epitopes of Litopenaeus vannamei myosin calcium-binding protein were predicted using bioinformatics, as shown in Table 1. Tables 1 and 2 show the digestible linear epitopes NKDGE and SNPDESCSA, and the myosin calcium-binding protein digestible epitope peptides LADFNKDGEVTVDE and ISNPDESCSAC, which were screened using a combination of digestomics and bioinformatics. These linear epitopes were also mapped into a three-dimensional model of Litopenaeus vannamei myosin calcium-binding protein, as shown in Table 1. Figure 1 As shown in (H). Figure 2 (D) and Figure 3 (D) Combining the two methods, key amino acids in the digestible linear epitope of the calcium-binding protein in Litopenaeus vannamei were predicted. The figure shows that the linear epitope contains a small amount of key amino acids. Molecular docking revealed that the binding energies of the digestible epitope peptides LADFNKDGEVTVDE and ISNPDESCSAC to MHC-II molecules are -197.657 kcal / mol and -184.241 kcal / mol, respectively, indicating that the binding interaction between the epitope peptides and MHC-II molecules is generally weak. Figure 4(EF) and Table 3 show the binding sites of the epitope peptides with MHC-II molecules predicted by molecular docking. The epitope peptide LADFNKDGEVTVDE forms hydrogen bonds with αN62, βN82, and βY60 in the MHC-II molecule, and exhibits hydrophobic interactions with αF24, αF54, αK67, αE71, βD28, βY30, βY47, βW61, βK71, βA74, βY78, and βH81. The epitope peptide ISNPDESCSAC forms hydrogen bonds with αN69, αR76, βW61, and βN82 in the MHC-II molecule, and exhibits hydrophobic interactions with αF22, αF24, αF32, αW43, αA52, αF54, αN62, αV65, αD66, and αI72. K75, βD57, βQ70, βK71, βT77, βY78, and βV85 exhibit hydrophobic properties.

[0049] Example 6: Identification of digestible epitopes with sensitizing potential in hemocyanin of Litopenaeus vannamei Hemocyanin, a protein from the whiteleg shrimp, is generally composed of 2-8 different monomers. Each monomer (consisting of a polypeptide chain) contains 630-660 amino acid residues and has a molecular weight of approximately 70-80 kDa. Because hemocyanin circulates in the animal's body through the blood and lymph, allergic reactions to it are relatively common, and the resulting immune responses are often severe.

[0050] This embodiment uses a combination of digestomics and bioinformatics to screen for digestible epitope peptides of hemocyanin from Litopenaeus vannamei, including the following steps: S1. Obtain the hemocyanin sequence of Litopenaeus vannamei from the NCBI database, and locate the hemocyanin peptides in the stomach and gastrointestinal digestive products of Litopenaeus vannamei in the protein sequence. S2. Use five bioinformatics software programs, DNAStar, AntheProt, ABCpred, ImmunomedicineGroup, and SOPMA, to predict epitopes of hemocyanin in Litopenaeus vannamei. Use the epitopes predicted by ≥3 software programs as linear epitopes of hemocyanin in Litopenaeus vannamei, and locate the predicted linear epitopes of hemocyanin in Litopenaeus vannamei in the hemocyanin sequence described in step S1. S3. Compare the digestible peptides in the gastrointestinal digestion products of Litopenaeus vannamei hemocyanin described in step S1 with the predicted linear epitopes of Litopenaeus vannamei hemocyanin described in step S2, and screen out the digestible (intact) linear epitopes as the predicted digestible linear epitopes of Litopenaeus vannamei hemocyanin. At the same time, screen out the epitope peptides containing the predicted digestible linear epitopes of Litopenaeus vannamei hemocyanin as digestible epitope peptides of Litopenaeus vannamei hemocyanin.

[0051] S4. Using SWISS-MODEL software, a three-dimensional model of hemocyanin from Litopenaeus vannamei was established using the protein with the highest similarity (618S) as a template for homology modeling. S5. Using Pymol software, locate the predicted digestible linear epitopes of Litopenaeus vannamei hemocyanin in the three-dimensional model of Litopenaeus vannamei hemocyanin described in step S4. S6. The amino acid frequencies in the hemocyanin of Litopenaeus vannamei and the predicted digestible linear epitopes of hemocyanin of Litopenaeus vannamei were calculated using ExPASy software. Amino acids with a higher proportion in the predicted digestible linear epitopes of hemocyanin of Litopenaeus vannamei than those in hemocyanin of Litopenaeus vannamei were identified as key amino acids in the epitopes. S7. Collect all allergen amino acid sequences of hemocyanin from the SDAP database, perform multiple sequence comparison using ClustalW software, and identify highly conserved amino acids as key amino acids in epitopes. S8. The key amino acids recorded in steps S6 and S7 are identified as potential key amino acids in the digestible linear epitopes of hemocyanin in Litopenaeus vannamei. S9. Using molecular docking technology, the digestible epitope peptides of Litopenaeus vannamei hemocyanin selected in step S3 are docked with MHC-II molecules, and the digestible epitope peptides of Litopenaeus vannamei hemocyanin with strong binding ability are screened by docking scoring.

[0052] Experimental results: such as Figure 1As shown in (I), the digestible peptides of hemocyanin in the stomach and gastrointestinal tract of Litopenaeus vannamei were obtained by NanoLC-MS / MS mass spectrometry. At the same time, 21 epitopes of hemocyanin in Litopenaeus vannamei were predicted by bioinformatics as shown in Table 1. Tables 1 and 2 show the digestibility linear epitopes of Litopenaeus vannamei hemocyanin screened by combined digestomics and bioinformatics: SFDPVGN, LDPVG, ARPDNV, SLYSPNV, RQGDPHGKFDLPP, WPHKDNNG, IERKSTES, VPDVPS, TDGDADSAVPN, and PNFKH; and the digestibility epitope peptides of Litopenaeus vannamei hemocyanin: ATANSFDPVGNLGS, LSNYLDPVGE, KYGGQFPARPDNV, SLYSPNV, GRQGDPHGKFDLPPGV, IFAWPHKDNNGIE, FWVSLKGGKTSIERKSTESSVTVPDVPSIH, KSTESSVTVPDVPSIHD, VVAVTDGDADSAVPN, and VFEDLPNFKH. Simultaneously, the digestibility linear epitopes of Litopenaeus vannamei hemocyanin were located in a three-dimensional model of Litopenaeus vannamei hemocyanin, such as... Figure 1 As shown in (J). Figure 2 (E) and Figure 3 (E) Combining the two methods, the key amino acids in the digestible linear epitope of hemocyanin in Litopenaeus vannamei were predicted. As can be seen from the figure, the linear epitope contains a large number of key amino acids. Molecular docking revealed that the digestible epitope peptides ATANSFDPVGNLGS, LSNYLDPVGE, KYGGQFPARPDNV, GRQGDPHGKFDLPPGV, IFAWPHKDNNGIE, KSTESSVTVPDVPSIHD, VVAVTDGDADSAVPN, and VFEDLPNFKH of Litopenaeus vannamei hemocyanin have binding energies of -246.23 kcal / mol, -225.131 kcal / mol, -250.948 kcal / mol, -231.713 kcal / mol, -295.065 kcal / mol, -227.258 kcal / mol, -215.242 kcal / mol, and -249.28 kcal / mol with MHC-II molecules, respectively, demonstrating that the epitope peptides have a good binding effect with MHC-II molecules. Figure 4Tables (GN) and 3 show the binding sites of the epitope peptides with MHC-II molecules predicted by molecular docking. Epitope peptide ATANSFDPVGNLGS forms hydrogen bonds with αQ9, αS53, αN62, and βN82 in the MHC-II molecule, and exhibits hydrophobic interactions with αV65, βY47, βY60, βW61, βL67, βT77, and βH81. Epitope peptide LSNYLDPVGE forms hydrogen bonds with αS53, βY47, βY60, and βN82 in the MHC-II molecule, and exhibits hydrophobic interactions with αF24, αI31, αF32, αF54, αN62, αV65, βY30, βW61, βL67, βK71, and βY78. V85 exhibits hydrophobic interactions. The epitope peptide KYGGQFPARPDNV forms hydrogen bonds with αS53, βY60, βQ70, and βN82 in the MHC-II molecule, and also exhibits hydrophobic interactions with αF24, αI31, αF32, αA52, αA61, αV65, βQ64, βL68, and βH81. The epitope peptide GRQGDPHGKFDLPPGV forms hydrogen bonds with αE55, βY30, and βN82 in the MHC-II molecule, and also exhibits hydrophobic interactions with αQ9, αF22, αF54, αV65, αI72, αK75, βK65, βD66, βL67, βQ70, βK71, and βA74. Y78 exhibits hydrophobic interactions. The epitope peptide IFAWPHKDNNGIE forms hydrogen bonds with αS53, αN62, βW61, and βN82 in the MHC-II molecule, and also exhibits hydrophobic interactions with αQ9, αF51, αA52, αK67, αI72, βD57, βY60, βL67, βQ70, βK71, βT77, βH81, and βV85. The epitope peptide KSTESSVTVPDVPSIHD forms hydrogen bonds with αS53, αN62, βY60, and βW61 in the MHC-II molecule, and also exhibits hydrophobic interactions with αF22, αF24, αA52, αV65, αN69, βF26, βD28, βY47, βY78, and βH81. V85 exhibits hydrophobic interactions. The epitope peptide VVAVTDGDADSAVPN forms hydrogen bonds with αS53, βD57, βK71, and βN82 in the MHC-II molecule, and with αF22, αF24, αF54, αN62, αR76, βP56, βY60, βW61, βD66, βL67, βY78, and βH81.βV85 exhibits hydrophobic interactions. The epitope peptide VFEDLPNFKH forms hydrogen bonds with αN62, βQ70, and βK71 in the MHC-II molecule, and also exhibits hydrophobic interactions with αV65, βF26, βY47, βW61, βL67, βA74, βT77, and βY78.

[0053] Example 7: Identification of digestibility epitopes with sensitizing potential in triose phosphate isomerase of Litopenaeus vannamei Triose phosphate isomerase (TIM) in Litopenaeus vannamei is a novel allergen found in crustaceans. It is present in sarcoplasmic proteins and is a glycolytic enzyme composed of two identical subunits. It exists in dimer form with a molecular weight of 26-29 kDa and a sensitization rate of 15%-23%. This embodiment uses a combination of digestomics and bioinformatics to screen for digestible epitope peptides resistant to triose phosphate isomerase in Litopenaeus vannamei, including the following steps: S1. Obtain the triose phosphate isomerase sequence of Litopenaeus vannamei from the NCBI database, and locate the triose phosphate isomerase peptides in the stomach and gastrointestinal digestion products of Litopenaeus vannamei in the protein sequence. S2. Five bioinformatics software programs, DNAStar, AntheProt, ABCpred, ImmunomedicineGroup, and SOPMA, are used to predict the triose phosphate isomerase epitopes of Litopenaeus vannamei. Epitopes predicted by ≥3 software programs are taken as linear epitopes of Litopenaeus vannamei triose phosphate isomerase, and the predicted linear epitopes of Litopenaeus vannamei triose phosphate isomerase are located in the Litopenaeus vannamei triose phosphate isomerase sequence described in step S1. S3. Compare the digestible peptides in the gastrointestinal digestion products of Litopenaeus vannamei triose phosphate isomerase described in step S1 with the predicted linear epitopes of Litopenaeus vannamei triose phosphate isomerase described in step S2. Select digestible (intact) linear epitopes as predicted digestible linear epitopes of Litopenaeus vannamei triose phosphate isomerase. At the same time, select epitope peptides containing the predicted digestible linear epitopes of Litopenaeus vannamei triose phosphate isomerase as digestible epitope peptides of Litopenaeus vannamei triose phosphate isomerase.

[0054] S4. Using SWISS-MODEL software, a three-dimensional model of the triose phosphate isomerase of Litopenaeus vannamei was established using the protein with the highest similarity (6JOX) as a template for homology modeling. S5. Using Pymol software, locate the predicted linear epitopes of Litopenaeus vannamei triose phosphate isomerase in the three-dimensional model of Litopenaeus vannamei triose phosphate isomerase described in step S4. S6. The amino acid frequencies of the triose phosphate isomerase and the predicted triose phosphate isomerase digestibility linear epitopes of Litopenaeus vannamei were calculated using ExPASy software. Amino acids with a higher proportion in the predicted triose phosphate isomerase digestibility linear epitopes than those in the Litopenaeus vannamei triose phosphate isomerase were identified as key amino acids in the epitopes. S7. Collect all allergen amino acid sequences of triose phosphate isomerases from the SDAP database, perform multiple sequence comparison using ClustalW software, and identify the most conserved amino acids as key amino acids in the epitopes. S8. The key amino acids recorded in steps S6 and S7 are identified as potential key amino acids in the digestible linear epitopes of triose phosphate isomerase in Litopenaeus vannamei. S9. Using molecular docking technology, the digestible epitope peptide of Litopenaeus vannamei triose phosphate isomerase selected in step S3 is docked with MHC-II molecules, and the Litopenaeus vannamei triose phosphate isomerase digestible epitope peptide with strong binding ability is screened by docking scoring.

[0055] Experimental results: such as Figure 1 As shown in (K), the digestible peptides of the gastric and gastrointestinal digestion products of *Litopenaeus vannamei* triose phosphate isomerase were obtained by NanoLC-MS / MS mass spectrometry. Simultaneously, nine *Litopenaeus vannamei* triose phosphate isomerase epitopes were predicted using bioinformatics, as shown in Table 1. Tables 1 and 2 show the *Litopenaeus vannamei* triose phosphate isomerase digestibility linear epitopes GPLSPN and GAALK, and the *Litopenaeus vannamei* triose phosphate isomerase digestibility epitope peptides MKTGPLSPN and LVGGAALKPD, screened by combining digestomics and bioinformatics. Furthermore, the digestibility linear epitopes of *Litopenaeus vannamei* hemocyanin were localized in a three-dimensional model of *Litopenaeus vannamei* hemocyanin, as shown in Table 1. Figure 1 As shown in (L). Figure 2 (F) and Figure 3 (F) Combining the two methods, key amino acids in the digestible linear epitope of Litopenaeus vannamei triphosphate isomerase were predicted. The figure shows that the linear epitope contains a small amount of key amino acids. Molecular docking revealed that the binding energies of the digestible epitope peptides MKTGPLSPN and LVGGAALKPD of Litopenaeus vannamei hemocyanin to MHC-II molecules are -216.969 kcal / mol and -196.36 kcal / mol, respectively, proving that the epitope peptides bind to MHC-II molecules. Figure 4(OP) and Table 3 show the binding sites of the epitope peptides with MHC-II molecules predicted by molecular docking. Epitope peptide MKTGPLSPN forms hydrogen bonds with αQ9, αS53, βW61, βK71, and βN82 in the MHC-II molecule, and exhibits hydrophobic interactions with αF22, αF24, αF32, αF54, αV65, βV11, βH13, βY30, βY47, βY60, βQ64, βL67, βY78, βH81, and βV85. Epitope peptide LVGGAALKPD forms hydrogen bonds with αS53 and βY47 in the MHC-II molecule, and exhibits hydrophobic interactions with αF24, αI31, αF32, αF54, αE55, αN62, βF26, and βY30. β K71, β Y78, β H81, β N82, and β V85 exhibit hydrophobic properties.

[0056] Example 8: Screening of digestible linear epitope peptides for allergens in Litopenaeus vannamei S1. The digestibility linear epitopes of the above-mentioned potential allergens in Litopenaeus vannamei (tropomyosin, arginine kinase, myosin light chain, sarcoplasmic calcium-binding protein, hemocyanin, and triose phosphate isomerase) were summarized, and epitope peptides with ≥5 key amino acids in the epitopes were screened as digestibility linear epitope peptides of Litopenaeus vannamei allergens with strong allergenic potential. The results showed that the epitopes of arginine kinase AK-01, AK-02 and hemocyanin HEM-04, HEM-05, HEM-06, HEM-07, and HEM-08 contained a large number of key amino acids (≥5 aa), and could be used as potential digestibility linear epitope peptides of Litopenaeus vannamei allergens.

[0057] S2. The digestible linear epitope peptides of the above-mentioned potential allergens in Litopenaeus vannamei (tropomyosin, arginine kinase, myosin light chain, sarcoplasmic calcium-binding protein, hemocyanin, and triose phosphate isomerase) were summarized, and epitope peptides with strong docking ability with MHC-II molecules (docking score ≤ -200 kcal / mol) were screened as potential digestible linear epitope peptides of Litopenaeus vannamei allergens. The results showed that arginine kinases AK-01 and AK-02, myosin light chain MLC-01, hemocyanin HEM-01, HEM-02, HEM-03, HEM-05, HEM-06, HEM-08, HEM-09, HEM-10, and triose phosphate isomerase TIM-01 had strong docking ability with MHC-II molecules (docking score ≤ -200 kcal / mol) and could be used as potential digestible linear epitope peptides of Litopenaeus vannamei allergens.

[0058] S3. Summarize the potential Litopenaeus vannamei allergen-resistant linear epitope peptides predicted by the two methods in steps S1 and S2. Select the allergen epitope peptides present in both methods as Litopenaeus vannamei allergen-resistant linear epitope peptides, namely arginine kinase AK-01 (Asp-Ser-Gly-Val-Gly-Ile-Tyr-Ala-Pro-Asp-Ala-Glu-Ala), AK-02 (Glu-Gly-Glu-Leu-Lys-Gly-Thr-Tyr-Tyr-Pro-Leu-Thr-Gly-Met), and Blood Blue. Protein HEM-05 (Gly-Arg-Gln-Gly-Asp-Pro-His-Gly-Lys-Phe-Asp-Leu-Pro-Pro-Gly-Val), HEM-06 (Ile-Phe-Ala-Trp-Pro-His- Lys-Asp-Asn-Asn-Gly-Ile-Glu), HEM-08 (Lys-Ser-Thr-Glu-Ser-Ser-Val-Thr-Val-Pro-Asp-Val-Pro-Ser-Ile-His-Asp).

[0059] Example 9: Solid-phase synthesis of digestible linear epitope peptides for allergens in Litopenaeus vannamei Using polymer resin (Hefei Saimano Biotechnology Co., Ltd.), the amino acid sequences Asp-Ser-Gly-Val-Gly-Ile-Tyr-Ala-Pro-Asp-Ala-Glu-Ala (SEQ ID NO:1), Glu-Gly-Glu-Leu-Lys-Gly-Thr-Tyr-Tyr-Pro-Leu-Thr-Gly-Met (SEQ ID NO:2), Gly-Arg-Gln-Gly-Asp-Pro-His-Gly-Lys-Phe-Asp-Leu-Pro-Pro-Gly-Val (SEQ ID NO:3), and Ile-Phe-Ala-Trp-Pro-His-Lys-Asp-Asn-Asn-Gly-Ile-Glu (SEQ ID NO:3) were synthesized. (SEQ ID NO:4) and Lys-Ser-Thr-Glu-Ser-Ser-Val-Thr-Val-Pro-Asp-Val-Pro-Ser-Ile-His-Asp (SEQ ID NO:5). The allergen epitope peptides were purified using high-performance liquid chromatography, followed by liquid nitrogen flash freezing and freeze-drying. A purity exceeding 98% was required, and the actual purities were 98.10%, 98.87%, 98.41%, 99.37%, and 98.71%, respectively. Figure 5 The images show the secondary maps of five digestible linear epitope peptides that are allergens in Litopenaeus vannamei.

[0060] Example 10: IgE binding capacity of digestible linear epitope peptides to allergens in Litopenaeus vannamei S1. Construction of the sensitization model: Sixty female BALB / c mice were selected and divided into 6 groups of 10 mice each. Each group received an intraperitoneal injection of the digestible linear epitope peptides AK-01 (AK-01 group), AK-02 (AK-02 group), HEM-05 (HEM-05 group), HEM-06 (HEM-06 group), HEM-08 (HEM-08 group), and total protein from Litopenaeus vannamei (Litopenaeus vannamei sensitization group), respectively. The injections were administered every 7 days, with a dosage of 50 μg per mouse (the epitope peptides were mixed with alum adjuvant at a 1:3 volume ratio and pre-adsorbed for 30 min). A saline group (Saline group) and an adjuvant group (Alum group) were set up as control groups, with 10 mice in each group. Each control group received an equal volume of saline and adjuvant, respectively, for a total of 5 injections. On day 42 (14 days after the fifth injection), blood was collected from the orbital cavity. Serum samples were incubated at 4℃ with a 4000... Centrifuge at rpm for 10 min and collect the supernatant.

[0061] S2. Dot blot hybridization method for detecting the binding ability of epitope peptides to IgE: On a nitrocellulose membrane (NC) with a pre-drawn grid, add 2 μL of the epitope peptide sample or BSA (10 mg / mL) of each group to be tested in the middle of each grid, and air dry at room temperature (25 ℃); place the treated NC membrane in an incubation box, add blocking solution (TBST containing 1% BSA), and incubate at room temperature for 2 h; after washing with washing solution (TBST), place the above NC membrane in the serum (dilution 1:10) of the corresponding group in step S1, and incubate overnight at 4 ℃; after washing with TBST, incubate the NC membrane sequentially in biotin-labeled rat anti-mouse IgE and HRP-labeled streptavidin at 37 ℃ for 1 h; perform color development using an ECL chemiluminescence detection kit. S3. Enzyme-linked immunosorbent assay (ELISA) to detect the binding ability of epitope peptides to IgE: Epitope peptides from arginine kinase (mixed AK-01 and AK-02), epitope peptides from hemocyanin (mixed HEM-05, HEM-06, and HEM-08), epitope peptides from Litopenaeus vannamei resistant to digestive allergens (mixed AK-01, AK-02, HEM-05, HEM-06, and HEM-08), and a single target epitope peptide were diluted to a concentration of 10 μg / mL using coating buffer (0.05 mol / L sodium carbonate buffer). These solutions were then coated onto 96-well plates (100 μL / well) and incubated overnight at 4 °C. After washing, 150 μL of blocking buffer was added and incubated for 2 h. After washing, Litopenaeus vannamei whole protein (Litopenaeus vannamei sensitized group) mouse serum (1:10) from step S1 was added and incubated for 2 hours. h; after washing, biotin-labeled rat anti-mouse IgE and HRP-labeled streptavidin were added sequentially and incubated for 1 h; after washing, 100 μL TMB substrate chromogenic solution was added to each well and incubated at 37 ℃ for 15 min; 50 μL sulfuric acid stop solution was added to terminate the reaction; the absorbance of each well at 450 nm was measured.

[0062] Experimental results: such as Figure 6 As shown, all five linear epitope peptides of Litopenaeus vannamei allergens exhibited IgE binding capacity but showed no reaction with BSA. Among them, AK-01 and AK-02 showed strong positive reactions, indicating that AK-01 and AK-02 are relatively important linear epitope peptides of Litopenaeus vannamei.

[0063] like Figure 7 As shown, epitope peptides from arginine kinase (AK-01 and AK-02), epitope peptides from hemocyanin (HEM-05, HEM-06, and HEM-08), and epitope peptides from Litopenaeus vannamei resistant to digestive allergens (AK-01, AK-02, HEM-05, HEM-06, and HEM-08) exhibited significant IgE binding capacity with Litopenaeus vannamei whole-protein sensitized serum, showing a significant difference compared to the control group. Figure 8 As shown, the epitope peptides of Litopenaeus vannamei (AK-01, AK-02, HEM-05, HEM-06, and HEM-08) exhibited significant IgE binding ability with Litopenaeus vannamei total protein-sensitized serum, and all showed significant differences compared to the control group. These results indicate that the synthesized Litopenaeus vannamei epitope peptides can accurately identify the sensitization of Litopenaeus vannamei arginine kinase protein, Litopenaeus vannamei hemocyanin, and Litopenaeus vannamei total protein with high accuracy.

[0064] Example 11: Standard Curve for Serum Dilution Serum dilution was detected using enzyme-linked immunosorbent assay (ELISA): The antigen (episode peptide described in Example 9) was diluted to a concentration of 10 μg / mL with coating buffer (0.05 mol / L sodium carbonate buffer), and then coated into 96-well plates (100 μL / well) and incubated overnight at 4 °C. After washing, 150 μL of blocking buffer was added and incubated for 2 h. After washing, mouse serum of different dilutions (serially diluted from 1:5 to 1:200) was added to each group sequentially and incubated for 2 h. After washing, biotin-labeled rat anti-mouse IgE and HRP-labeled streptavidin were added sequentially and incubated for 1 h. After washing, 100 μL of TMB substrate chromogenic solution was added to each well and incubated at 37 °C for 15 min. 50 μL of sulfuric acid stop solution was added to terminate the reaction. The absorbance of each well at 450 nm was measured, and a standard curve was constructed.

[0065] Example 12: Detection of the specificity of the shrimp allergy test kit by verifying allergic serum from other species. Enzyme-linked immunosorbent assay (ELISA) was used to detect the binding capacity of IgE to allergic sera from other shrimp species: Epitopes from arginine kinase (AK-01 and AK-02), epitopes from hemocyanin (HEM-05, HEM-06, and HEM-08), and epitopes from Litopenaeus vannamei resistant to digestive allergens (AK-01, AK-02, HEM-05, HEM-06, and HEM-08) were diluted to a concentration of 10 μg / mL using coating buffer (0.05 mol / L sodium carbonate buffer) and coated into 96-well plates (100 μL / well), and incubated overnight at 4 °C. After washing, 150 μL of blocking buffer was added and incubated for 2 h. After washing, serum from allergic mice of other species (1:10) was added and incubated for 2 h. After washing, biotin-labeled rat anti-mouse IgE and HRP-labeled streptavidin were added sequentially and incubated for 1 h. After washing, 100 μL of blocking buffer was added to each well. Incubate μL of TMB substrate solution at 37 °C for 15 min; add 50 μL of sulfuric acid stop solution to terminate the reaction; measure the absorbance of each well at 450 nm.

[0066] Table 1. Linear epitopes of allergens in Litopenaeus vannamei predicted by five software programs.

[0067] The bold text in the table represents linear epitopes of Litopenaeus vannamei allergens. Table 2 Epitope peptides of allergens in Litopenaeus vannamei.

[0068] The bold text in the table represents epitope peptides that are allergens in Litopenaeus vannamei. The shaded areas represent key amino acids that have been predicted.

[0069] Table 3. Docking sites between epitope peptides of allergens in Litopenaeus vannamei and MHC-II molecules.

[0070] The more stable binding sites of epitopes to MHC-II are indicated in bold. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A digestible linear epitope peptide of Litopenaeus vannamei allergen, characterized in that, The amino acid sequence of the epitope peptide is IFAWPHKDNNGIE.

2. An antigen composition, characterized in that, The antigen composition comprises two or more of the epitope peptides of claim 1.

3. A shrimp allergy detection kit containing the antigen composition of claim 2.

4. The detection kit as described in claim 3, characterized in that, The kit also includes coating solution, blocking solution, sample diluent, washing solution, detection antibody, substrate chromogenic solution, and stop solution.

5. The use of the epitope peptide of claim 1, or the antigen composition of claim 2, in the preparation of reagents or kits for the diagnosis of allergies to shrimp and shrimp products.

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