Epitope polypeptide derived from peanut allergen Ara h 3 and application thereof

Abstract

The invention belongs to the technical field of food safety, and particularly relates to epitope polypeptide derived from peanut allergen Ara h 3 and application of the epitope polypeptide. The epitope polypeptide contains EYDEEDRRRGRGSR, EADEEDRRRGRGSR, EYDAEDRRRGRGSR, EYDEEARRRGRGSR, EYDEEDRRARGRGSR, EYDEEDRRRARGSR, EYDEEDRRRGAGSR, or EYDEEDRRRGRASR, and is characterized in that the epitope polypeptide contains EYDEEDRRRGRGSR, EYDEEDRRRGRGSR, EYDEEDRRRGRGSR, EYDEEDRRRGRGSR, EYDEEDRRRGRGSR, EYDEEDRRRGRGSR Wherein the EYDEEDRRRGRGSR is an epitope region of which the sensitization of the baked Ara h 3 is enhanced. A theoretical basis and a scientific basis are provided for developing low-sensitization peanut food, optimizing a food processing technology and establishing a more accurate allergen detection technology, so that the sensitization risk of the peanut product is effectively reduced.

Description

Technical Field

[0001] This invention belongs to the field of food safety technology, specifically relating to epitope peptides derived from peanut allergen Ara h3 and their applications. Background Technology

[0002] Peanuts belong to the legume family and are an important oilseed crop and source of plant protein. However, peanuts are also a significant food allergen, triggering allergic reactions in individuals with allergies. The Allergen Nomenclature Subcommittee of the World Health Organization / International Union of Immunological Societies (WHO / IUIS) (http: / / www.allergen.org) has published a list of 18 peanut allergens, named Ara h 1 to Ara h 18. Ara h 1, Ara h 2, Ara h 3, and Ara h 6 are the main peanut allergens. Among them, Ara h 3, although accounting for only 3.7% to 4.3% of the total protein content of peanuts, has a strong sensitizing effect and can be recognized by the serum of more than 44% of peanut allergy sufferers. Common peanut processing methods can be divided into thermal processing (including roasting, boiling, frying, and autoclaving) and non-thermal processing (including high pressure, radiation, plasma, and enzymatic methods). Roasting, as a traditional processing technique, not only extends the shelf life of peanut products but also improves their nutritional value and taste. Roasted peanut products, with their unique flavor and texture, are widely used in the baking industry. However, it is worth noting that roasting has a particularly significant impact on peanut allergens. Among various thermal and non-thermal processing methods, roasting deserves further investigation due to its widespread application in peanut processing and its unique ability to enhance allergenicity.

[0003] Of the food allergens, only a portion of their antigenic determinants, also known as epitopes, are truly associated with allergic reactions. Epitopes determine the antigenic specificity of food allergens, enabling them to be recognized by immune cells and trigger an immune response. Understanding the structure and sequence of these epitopes is crucial for developing diagnostic and therapeutic methods for food allergies, as well as for the development of hypoallergenic foods. Therefore, by precisely locating the regions and key amino acids in Ara h3 that enhance sensitization through baking, elucidating the molecular mechanism by which baking enhances Ara h3 antigenicity, can help develop targeted prevention strategies and reduce the risk of food allergies. Summary of the Invention

[0004] The purpose of this invention is to provide a region where roasting treatment enhances the allergenicity of peanut allergen Ara h3. This invention elucidates the molecular mechanism by which the allergenicity of peanut allergen Ara h3 is enhanced during the roasting process, providing a theoretical basis and scientific evidence for developing low-allergenic peanut foods, optimizing food processing technology, and establishing more accurate allergen detection technology, thereby effectively reducing the allergenic risk of peanut products.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] The present invention first provides epitope polypeptides derived from peanut allergen Ara h3, wherein the epitope polypeptides contain EYDEEDRRRGRGSR (SEQ ID NO:1), EYDEEDRRRGRGSR (SEQ ID NO:2), EYDAEDRRRGRGSR (SEQ ID NO:3), EYDEEARRRGRGSR (SEQ ID NO:4), EYDEEDRARGRGSR (SEQ ID NO:5), EYDEEDRRRARGSR (SEQ ID NO:6), EYDEEDRRRGAGSR (SEQ ID NO:7), or EYDEEDRRRGRASR (SEQ ID NO:8).

[0007] In a further embodiment, the amino acid sequence of the epitope polypeptide is EYDEEDRRRGRGSR (SEQ ID NO:1), EYDEEDRRRGRGSR (SEQ ID NO:2), EYDAEDRRRGRGSR (SEQ ID NO:3), EYDEEARRRGRGSR (SEQ ID NO:4), EYDEEDRARGRGSR (SEQ ID NO:5), EYDEEDRRRARGSR (SEQ ID NO:6), EYDEEDRRRGAGSR (SEQ ID NO:7), or EYDEEDRRRGRASR (SEQ ID NO:8).

[0008] GGNIFSGFTPEFLEQAFQVDDRQIVQNLRGETESEEEGAIVTVRGGLRILSPDRKRRADEEEE YDEDEYEYDEEDRRRGRGSRGRGNGIEETICTASAKKNIGRNRSPDIYNPQAGSLKTANDL NLLILRWLGPSAEYGNLYRNALFVAHYNTNAHSIIYR(SEQ ID NO:9), DRKRRADEEEEYDEDEYEYDEEDRRRGRGSRGRGNGIEETICTASAKKNIGRNRSPDIYNP QAG (SEQ ID NO:10) or EYDEEDRRRGRGSRGRGNGIEETICT (SEQ ID NO:11).

[0009] The present invention also provides the application of the above-mentioned epitope peptide in the preparation of a kit for detecting peanut allergen protein.

[0010] The present invention also provides a kit for detecting peanut allergen protein, the kit containing reagents for detecting the epitope peptides.

[0011] In a further embodiment, the reagent contains an antibody against the epitope polypeptide.

[0012] The present invention also provides an anti-peanut allergy product, wherein the product contains an antibody, which is obtained by screening the epitope polypeptide as a target.

[0013] In a further embodiment, the antibody is one of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, or a humanized modified antibody. The product targets peanuts or peanut products as the allergen. The peanuts are roasted peanuts.

[0014] This invention provides a method for locating an epitope polypeptide containing EYDEEDRRRGRGSR (SEQ ID NO: 1), wherein the epitope polypeptide is an epitope region that enhances the sensitization of baking Ara h3.

[0015] The positioning method includes the following steps:

[0016] (1) Baked Ara h3 rabbit-derived polyclonal antibodies were prepared by immunizing white rabbits with baked Ara h3;

[0017] (2) The amino acid sequence of Ara h 3 was segmented by overlap;

[0018] (3) The protein Ara h3 and its overlapping segments are presented on the surface of the phage;

[0019] (4) Using indirect enzyme-linked immunosorbent assay (ELISA), the epitope regions that enhance the sensitization of Ara h3 by baking were precisely located, and the amino acid sequence of the fragment that most significantly enhances the sensitization of Ara h3 by baking was obtained.

[0020] The beneficial effects of this invention are as follows:

[0021] (1) The prepared baked Ara h3 rabbit-derived polyclonal antibody exhibited high titer, good sensitivity, and strong specificity. After five immunizations, the serum titer reached 1:6.55×10⁻⁶. 6 The sensitivity of polyclonal antibody serum was assessed using an indirect competitive ELISA method. A linear regression model was constructed based on the inhibition rate (B / B0) and the logarithm of the baked Ara h3 concentration. The results showed that the half-maximal inhibitory concentration (IC50) of the polyclonal antibody serum was [value missing]. 50 The concentration was 673 ng / mL, indicating that the polyclonal antibody serum has high sensitivity. The polyclonal antibody serum specifically recognizes baked Ara h3, while the cross-reactivity rates for glycinin, β-conglycinin, bovine serum albumin, phycocyanin, and whey protein are all less than 0.1%. Lower cross-reactivity rates indicate stronger specificity of the polyclonal antibody serum.

[0022] (2) This invention uses Ara h3 treated under optimal baking conditions to immunize New Zealand rabbits, preparing baked Ara h3 rabbit-derived polyclonal antibodies. Based on this, the obtained antibodies are combined with phage display technology to successfully locate the epitope region where baking enhances the sensitization of Ara h3, and the key amino acid sites affecting the sensitization and antigenicity of this region. This research can contribute to a deeper understanding of the mechanism by which baking affects the sensitization of Ara h3 at the molecular level, providing a scientific basis and theoretical support for developing safer food processing methods.

[0023] (3) A series of bioinformatics software were used to predict potential linear and conformational epitopes of Ara h3. Based on the prediction results, overlapping was performed, and a cloning vector of the target fragment was constructed. By displaying Ara h3 and its overlapping protein on the surface of bacteriophages, the region and key amino acids of peanut allergen Ara h3 that enhances sensitization during roasting were precisely located. This invention elucidates the molecular mechanism by which peanut allergen Ara h3 enhances sensitization during roasting, provides a scientific basis for the localization of Ara h3 sensitization epitopes, and provides a theoretical foundation for establishing more accurate allergen detection technology. Attached Figure Description

[0024] Figure 1 Inhibition curve of Ara h 3 pairs of polyclonal antibody serum indirect competitive ELISA.

[0025] Figure 2PCR amplification diagram of Ara h3 and overlapping gene segments. Lanes 0, 1, 2, 3, and 4 contain the PCR amplification products of Ara h3, Ara h3-1, Ara h3-2, Ara h3-3, and Ara h3-4, respectively.

[0026] Figure 3 Blue-and-white screening chart.

[0027] Figure 4 (a) Plasmid PCR identification of Ara h 3 and overlapping gene segments; (b) Double enzyme digestion identification of Ara h 3 and overlapping gene segments. Lanes 0, 1, 2, 3, and 4 represent Ara h 3, Ara h 3-1, Ara h 3-2, Ara h 3-3, and Arah 3-4, respectively.

[0028] Figure 5 (a) Phage plaque image of Ara h 3 and overlapping gene segments; (b) PCR identification image of phage plaques of Ara h 3 and overlapping gene segments. Lanes 0, 1, 2, 3, and 4 represent Ara h 3, Ara h 3-1, Ara h 3-2, Ara h 3-3, and Ara h 3-4, respectively.

[0029] Figure 6 Figure: ELISA identification results of Ara h3 and overlapping segmented genes.

[0030] Figure 7 A diagram of Ara h 3-3 overlapping segment gene amplification. Lanes A, B, and C represent the Ara h 3-3 overlapping segment genes Arah 3-3-A, Ara h 3-3-B, and Ara h 3-3-C.

[0031] Figure 8 (a) Plasmid PCR identification of the Ara h 3-3 overlapping gene segments; (b) Plasmid double enzyme digestion identification of the Ara h 3-3 overlapping gene segments. Lanes A, B, and C represent the Ara h 3-3 overlapping gene segments Ara h 3-3-A, Ara h 3-3-B, and Ara h 3-3-C, respectively. Figure 9 Diagram identifying recombinant phages of the Ara h 3-3 overlapping segment genes. Lanes A, B, and C represent the Ara h 3-3-A, Ara h 3-3-B, and Ara h 3-3-C overlapping segment genes.

[0032] Figure 10 Figure: ELISA identification results of Ara h 3-3 and its overlapping gene segments.

[0033] Figure 11PCR identification diagram of Ara h 3-3-B and its overlapping segment genes. I, II, and III represent the overlapping segment genes Ara h 3-3-B-I, Ara h 3-3-B-II, and Ara h 3-3-B-III, respectively.

[0034] Figure 12 (a) Identification diagram of Ara h 3-3-B plasmid; (b) Identification diagram of Ara h 3-3-B double enzyme digestion. I, II, and III represent the overlapping gene segments of Ara h 3-3-B: Ara h 3-3-B-I, Ara h 3-3-B-II, and Ara h 3-3-B-III.

[0035] Figure 13 Diagram of Ara h 3-3-B and its segmented recombinant phages. I, II, and III represent the overlapping segments of the Ara h 3-3-B gene: Ara h 3-3-B-I, Ara h 3-3-B-II, and Ara h 3-3-B-III.

[0036] Figure 14 ELISA identification diagram of Ara h 3-3-B and its overlapping segments.

[0037] Figure 15 HPLC and MS spectra of the synthesized peptides. (a) HPLC identification of P1; (b) MS identification of P1; (c) HPLC identification of P2; (d) MS identification of P2; (e) HPLC identification of P3; (f) MS identification of P3.

[0038] Figure 16 : Diagram for identifying peptide coupling. Lanes B, 1, 2, and 3 represent BSA, P1-BSA complex, P2-BSA complex, and P3-BSA complex, respectively.

[0039] Figure 17 Dot-Blot identification of the peptides. A, II, B, 1, 2, 3, and P represent Ara h 3, phage-expressed protein Ara h 3-3-B-II, BSA, peptides P1, P2, P3, and PBS, respectively.

[0040] Figure 18 ELISA identification diagram of synthetic polypeptides.

[0041] Figure 19 HPLC and MS identification chromatograms of mutant polypeptide M1-14.

[0042] Figure 20 : Conjugation identification diagram of mutant polypeptide M1-14. Lanes B and 1-14 represent BSA, M1-BSA to M14-BSA, respectively. Figure 21Dot-Blot identification of mutant peptide M1-14. A, P1, B, P, and M1-14 represent Ara h3, peptide P1, BSA, PBS, and M1-14, respectively.

[0043] Figure 22 : Sensitization identification diagram of mutant peptide M1-14. 1-14 represents M1-14. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Unbaked natural Ara h3 was prepared according to (Wu Xiao, Xi Jun, Sun Fuyu, Yang Huanhuan, Shui Tianjiao, Huang Pengbo, Gao Yida. Effect of wet heat treatment on the antigenicity of peanut globulin Ara h3 [J / OL]. Journal of Henan University of Technology (Natural Science Edition). https: / / kns.cnki.net / kcms2 / detail / 41.1378.N.20230605.1036.002.html). Baked Ara h3 was obtained by baking natural Ara h3 at 140℃ for 20 min.

[0045] Example 1: Preparation and identification of baked Ara h3 rabbit polyclonal antibody

[0046] 1. Preparation of Ara h3 rabbit-derived polyclonal antibody by baking

[0047] Two healthy, 3-month-old female New Zealand White rabbits (from the Henan Academy of Agricultural Sciences) were acclimatized for one week, and blood was collected from their marginal ear veins as a negative control, which was stored at -20℃. The two rabbits were immunized with baked Ara h 3 as the antigen, according to the specific immunization protocol shown in Table 1. Freund's complete adjuvant (FCA) was used for the initial immunization, and Freund's incomplete adjuvant (FIA) was used for the booster immunization. During immunization, baked Ara h 3 was dissolved in sterile PBS and thoroughly emulsified with an equal volume of adjuvant (FCA or FIA) on ice at a speed of 21,000 rpm until the antigen solution remained stable and did not diffuse when dropped into water. The emulsified antigen solution was administered via subcutaneous injection at multiple sites in the neck. Blood was collected from the heart 4 days after the last immunization. The blood was incubated at 37°C for 2 hours, then at 4°C overnight. The solution was centrifuged at 3500 rpm for 15 minutes at 4°C. The supernatant was collected and stored at -20°C to obtain the baked Ara h 3 rabbit polyclonal antibody.

[0048] Table 1. Immunization Regimen

[0049] Number of immunizations Immunization time (d) Immune antigens Immunization dose (μg) Injection dose (mL) 1 1 Antigen + Complete Adjuvant 200 1 2 14 Antigen + Incomplete Adjuvant 300 1 3 24 Antigen + Incomplete Adjuvant 300 1 4 34 Antigen + Incomplete Adjuvant 300 1 5 44 antigen 300 1 / 48 Cardiac blood sampling / /

[0050] 2. Immunological characterization of baked Ara h3 rabbit polyclonal antibody

[0051] Experimental data showed that after five immunizations, the titer of rabbit serum No. 1 reached 1:6.55×10. 6 This indicates that the rabbit produced high-titer antibodies, allowing for large-scale blood collection; the indirect competitive ELISA inhibition curve ( Figure 1 The results showed that the half-maximal inhibitory concentration (IC50) of the serum was 673 ng / mL, indicating good sensitivity. The cross-reactivity of this serum with other proteins, such as soy globulin and bovine serum albumin, was less than 0.1%. These results demonstrate that a high-titer, highly sensitive, and specific rabbit-derived polyclonal antibody against baked Ara h3 was obtained by immunizing New Zealand white rabbits, providing support for subsequent localization of the epitope regions that enhance the antigenicity of Ara h3 during baking.

[0052] Example 2: Ara h3 gene overlapping segment cloning

[0053] 1. Cloning of Ara h3 and its overlapping segment genes

[0054] The amino acid residue sequence of Ara h 3 (507 amino acids, Gene ID: 112695262) ​​was retrieved using NCBI. A PDB format file of the three-dimensional structure of Ara h 3 was obtained from the PDB database. The PDB format file was then submitted to the DiscoTope 3.0 server for conformational epitope prediction. Based on the epitope prediction results, overlapping segments were created without compromising the integrity of the antigenic epitopes, ensuring partial overlap between each segment and the preceding segment. The Ara h 3 overlapping segmentation design is as follows (underlined parts represent overlapping fragments):

[0055] Ara h 3-1, 1~170aa (its encoding gene is 510bp):

[0056] RQQPEENACQFQRLNAQRPDNRIESEGGYIETWNPNNQEFECAGVALSRLVLRRNALRRPFYSNAPQEIFIQQGRGYFGLIFPGCPRHYEEPHTQGRRSQSQRPPRRLQGEDQSQQQRDS HQKVHRFDEGDLIAVPTGVAFWLY NDHDTDVVAVSLTDTNNNDNQLDQFP;

[0057] Ara h 3-2, 121~285aa (its encoding gene is 495bp):

[0058] HQKVHRFDEGDLIAVPTGVAFWLYNDHDTDVVAVSLTDTNNNDNQLDQFP RRFNLAGNTEQEFLRYQQQSRQSRRRSLPYSPYSPQSQPRQEEEREFSPRGQHSRRERAGQEEENE GGNI FSGFTPEFLEQAFQVDDRQIVQN LRGETESEEEGAIVTVRGGLRIL;

[0059] Ara h 3-3, 236~396aa (its encoding gene is 483bp):

[0060] GGNIFSGFTPEFLEQAFQVDDRQIVQNLRGETESEEEGAIVTVRGGLRIL SPDRKRRAD EEEEYDEDEYEYDEEDRRRGRGSRGRGNGIEETICTASAKKNIGRNRSPDIY NPQAGSLKTA NDLNLLILRWLGPSAEYGNLY RNALFVAHYNTNAHSIIYR;

[0061] Ara h 3-4, 347~507aa (its encoding gene is 483bp):

[0062] NPQAGSLKTANDLNLLILRWLGPSAEYGNLYRNALFVAHYNTNAHSIIYR LRGRAHVQ VVDSNGNRVYDEELQEGHVLVVPQNFAVAGKSQSENFEYVAFKTDSRPSIANLAGENSVID NLPEEVVANSYGLQREQARQLKNNNPFKFFVPPSQQSPRAVA.

[0063] Primers were designed for the gene encoding the segmented amino acid sequence. The reliability of the template and primers was analyzed using software such as Primer Premier 6.0 to ensure that they did not contain EcoRI and HindIII restriction sites. The primer structure was: 5′-protective base-restriction site-primer-3′.

[0064] Table 2. Primer design for segmented Ara h3

[0065]

[0066] Using the full-length encoding gene of Ara h 3 (SEQ ID NO:12) as template DNA, Ara h 3 (using Ara h3-1-UP and Ara h 3-4-DOWN amplification), Ara h 3-1, Ara h 3-2, Ara h 3-3, and Ara h 3-4 were amplified. The PCR reaction system is shown in Table 3. The reaction conditions were: preheating at 94℃ for 5 min; denaturation at 94℃ for 60 s, annealing at 56℃ for 60 s, extension at 72℃ for 90 s, for 32 cycles; and final extension at 72℃ for 10 min. The products were stored at 4℃ and detected by 2% agarose gel electrophoresis.

[0067] Table 3. PCR Reaction System

[0068] Element Volume (μL) Template DNA 1 upstream primer 1 Downstream primer 1 sterile water 22 Ex-Taq premix 25 Total volume 50

[0069] According to the electrophoresis results ( Figure 2 The molecular weights of the bands Ara h 3, Ara h 3-1, Ara h 3-2, Ara h 3-3 and Ara h 3-4 were 1521bp, 510bp, 495bp, 483bp and 483bp, respectively, which can be observed to be consistent with the size of the target gene. The gel recovery product was stored at -20℃ and used for subsequent construction of recombinant vectors.

[0070] After PCR identification, the target gene was recovered by gel extraction.

[0071] 2. Construction of recombinant vectors for Ara h3 and its overlapping segment genes

[0072] Ligation and transformation of Ara h 3 and its overlapping segments: 4 μL of the target gene (Ara h 3, Ara h 3-1, Ara h 3-2, Ara h 3-3, and Ara h 3-4) was mixed with 1 μL of pMD TM Ligation was performed using 18-T Vector and 5 μL of Ligation buffer. After incubation overnight at 16°C, 50 μL of E. coli JM109 competent cells (purchased from TaKaRa) were added, followed by heat shock on ice, and then 500 μL of SOC medium was added. The cells were incubated at 37°C for 1 h, centrifuged at 4000 rpm for 4 min to remove the supernatant, and the remaining bacterial culture was spread on LB agar plates and incubated at 37°C for 16 h until blue-white spots formed.

[0073] Blue-white screening results are as follows Figure 3 As shown, the white spots represent positive single clones, indicating that the target gene has been successfully cloned and transformed into E. coli JM109 competent cells. To further confirm the correctness of the cloning vector, plasmids were extracted from the white spots and subjected to PCR identification. The plasmid PCR identification results are shown below. Figure 4 As shown in (a), the PCR amplification product is consistent with the size of the target gene. Double enzyme digestion of the plasmid yielded the following results: Figure 4 As shown in (b), after digestion with EcoRI and HindIII, the length of the remaining fragment matched the length of the pMD18-T vector (2692 bp), and the length of the cleaved gene was the same as the length of the target gene. These results indicate that the recombinant vectors of Ara h3, Ara h3-1, Ara h3-2, Ara h3-3, and Ara h3-4 were successfully constructed.

[0074] Example 3: Phage display technology for expressing Ara h3 and its overlapping segment genes

[0075] 1. Enzyme digestion and recovery of Ara h3 and its gene fragments

[0076] The double enzyme digestion products from Example 2 were separated using a 2% agarose gel. After cutting the target DNA gel block, the target gene fragment was recovered using a gel recovery kit.

[0077] 2. T7 phage packaging

[0078] Table 4. Phage Packaging System

[0079] Packaging System Volume (μL) Target gene 0.5 T7 vector arms 0.2 T4 DNA ligase 0.2 10× ligation buffer 0.1 Total volume 1

[0080] The mixture of the above packaging system was incubated at 16°C for 14 h, then 5 μL of phage packaging extract (T7 SelectCloning Kit, purchased from Novagen) was added, and after incubation at 22°C for 2 h, 54 μL of LB liquid medium was added to terminate the reaction, yielding recombinant phage.

[0081] 3. PCR identification of recombinant bacteriophages

[0082] E. coli JM109 was inoculated into 20 mL of solution containing carb. + In LB broth containing IPTG, revive overnight at 37°C until the broth becomes turbid. Take 5 μL of recombinant phage, 700 μL of E. coli JM109 bacterial suspension, and 5 mL of top agar, mix well, and then plate onto LB agar plates. + ), incubate for 4 hours until plaques appear, such as Figure 5 As shown in (a). Randomly selected plaques were dissolved in TE buffer, lysed at 65°C for 10 min, centrifuged at 12000 rpm for 2 min, and the DNA in the supernatant was used as a template for PCR identification. The PCR system was: 80°C pre-denaturation for 5 min; 94°C denaturation for 50 s, 55°C annealing for 1 min, 72°C extension for 1 min, repeated for 35 cycles; followed by a 6 min extension; and stored at 4°C. Results are shown below. Figure 5 As shown in (b), electrophoresis showed that the band size was consistent with the expected target band, indicating that the recombinant phage of Ara h3 and its segmented genes was successfully constructed.

[0083] 4. Large-scale culture of recombinant phages and purification of expressed proteins

[0084] Add 5 μL of recombinant phage to 50 mL of E. coli JM109 bacterial culture for infection. After clarification, centrifuge at 8000 rpm for 10 min. Add PEG 8000 and NaCl to the supernatant, gently shake to dissolve, and incubate overnight at 4°C to precipitate. The next day, centrifuge the sample at 10000 rpm for 10 min to collect phage protein particles. Resuspend in 200 μL of sterile SM solution. After 1 h, add 500 μL of chloroform, centrifuge at 5000 rpm for 10 min, collect the supernatant purified and enriched phage protein, and store at -80°C.

[0085] Example 4: Screening of epitope regions that enhance Ara h3 sensitization through baking

[0086] The phage protein prepared in Example 3 was diluted to 10 μg / mL using CBS buffer (carbonate buffer), and 100 μL was added to each well of an ELISA plate for coating. Three replicates were set up, along with negative and blank controls. The plates were incubated overnight at 4°C. The next day, the liquid in the wells was discarded, the plates were blotted dry, and 200 μL of PBST buffer was added to each well for washing five times, 5 min each time. 200 μL of blocking buffer (5% skim milk powder) was added to each well and incubated at 37°C for 2 h. After washing, 50 μL of primary antibody (baked Ara h 3 rabbit polyclonal antibody prepared in Example 1 (1:204800 dilution) or natural Ara h 3 rabbit polyclonal antibody (1:51200 dilution)) was added to each well and incubated at 37°C for 1 h. After washing, 50 μL of goat anti-rabbit IgG antibody diluted 5000 times was added to each well and incubated at 37°C for 1 h. After washing, TMB was added for color development for 5 min, and the OD was measured. 450 Value. Based on the relative changes in the reaction between bacteriophage proteins and baked Ara h3 rabbit polyclonal antibody (hereinafter referred to as baked antibody) or natural Ara h3 rabbit polyclonal antibody (hereinafter referred to as natural antibody), the epitope region where baking significantly enhances the antigenicity of Ara h3 was located. The natural Ara h3 rabbit polyclonal antibody is prepared by referring to "Preparation of Baked Ara h3 Rabbit Polyclonal Antibody", which is obtained by replacing "baked Ara h3" with "untreated natural Ara h3".

[0087] The stronger the binding affinity of phage proteins to antibodies, the higher their antigenicity. Indirect ELISA results are as follows: Figure 6 As shown in the figure. The results indicate that the full-length Ara h3 and the Ara h3-2 fragment exhibit the strongest binding affinity to the rabbit polyclonal antibody derived from natural Ara h3, suggesting that Ara h3-2 possesses strong antigenicity. The relative change in antibody binding affinity for the Ara h3-3 fragment reached 37.93% (relative change = (OD of baked antibody group)). 450 -Natural Antibody Group OD 450 ) / Natural Antibody Group OD 450The percentage (×100%) was significantly higher than other fragments, indicating that Ara h 3-3 contains more epitopes similar to the baked Ara h 3 protein. These epitopes may be more significantly exposed in the baked Ara h 3 protein, making them easier for the baking antibody to recognize and bind, resulting in a significant enhancement of its antigenicity. Therefore, it is speculated that Ara h 3-3 may have the most newly generated or exposed epitopes during the baking process, and its antigenicity enhancement is the most significant. Ara h 3-3 was selected for the next step of overlapping segmentation and localization.

[0088] Example 5: Precisely positioned baking of epitope regions that enhance Ara h3 sensitization

[0089] Referring to the method in Example 2, the screened Ara h 3-3 was further fragmented to precisely locate the dominant antigen region. The second round of fragmented amino acid sequences are as follows:

[0090] Ara h 3-3-A, 1~63aa (its encoding gene is 189bp):

[0091] GGNIFSGFTPEFLEQAFQVDDRQIVQNLRGETESEEEGAIVTVRGGLRILSP DRKRRAD EEEE ;

[0092] Ara h 3-3-B, 53–116aa (its encoding gene is 192 bp):

[0093] DRKRRADEEEE YDEDEYEYDEEDRRRGRGSRGRGNGIEETICTASAKKN IGRNRSPDI YNPQAG ;

[0094] Ara h 3-3-C, 102~161aa (its encoding gene is 180bp):

[0095] IGRNRSPDIYNPQAG SLKTANDLNLLILRWLGPSAEYGNLYRNALFVAHYNTNAHSIIY R.

[0096] Table 5. Second round of segmented primer design for Ara h 3

[0097]

[0098] Using the Ara h 3-3 coding gene as template DNA, the overlapping segments Ara h 3-3-A, Ara h 3-3-B, and Ara h 3-3-C were amplified. The PCR amplification results of the overlapping segments A, B, and C are shown below. Figure 7 As shown; the PCR identification results of the recombinant plasmids of Ara h 3-3 overlapping segments A, B, and C are as follows. Figure 8 As shown in (a); the enzyme digestion identification results of the recombinant plasmids of Ara h 3-3 overlapping segments A, B, and C are as follows. Figure 8 (b) shows the PCR identification results of the recombinant phages from the overlapping segments A, B, and C of Ara h 3-3. Figure 9 As shown, the phage proteins were then enriched and purified.

[0099] Indirect ELISA was used to screen for the epitope regions that most significantly enhanced the antigenicity of Ara h 3-3 during baking. The results are as follows: Figure 10 As shown in the figure. The results indicate that Ara h 3-3-B has the lowest antigenicity and the greatest relative variation. Ara h 3-3-B may have the most newly generated or exposed epitopes, and the region with the most significant antigenic enhancement may be Ara h 3-3-B. Therefore, Ara h 3-3-B was selected for the next step of overlapping segmentation and localization.

[0100] Third round of segmented amino acid sequences:

[0101] Ara h 3-3-B-Ⅰ, 1~25aa (its encoding gene is 75bp):

[0102] DRKRRADEEEEYDEDEY EYDEEDRR ;

[0103] Ara h 3-3-B-II, 18–43aa (its encoding gene is 78 bp):

[0104] EYDEEDRR RGRGSRGRGN GIEETICT ;

[0105] Ara h 3-B-Ⅲ, 36–64aa (encoding gene 87bp):

[0106] GIEETICT ASAKKNIGRNRSPDIYNPQAG.

[0107] Table 6. Primer design for Ara h 3-3-B

[0108]

[0109] Using the Ara h 3-3-B encoding gene as template DNA, PCR amplification was performed on the overlapping segments Ara h 3-3-B: Ara h3-3-B-Ⅰ, Ara h 3-3-B-Ⅱ, and Ara h 3-3-B-Ⅲ. The PCR amplification results are shown below. Figure 11As shown; the PCR identification results of the recombinant plasmids of Ara h 3-3-B overlapping segments I, II, and III are as follows. Figure 12 As shown in (a); the enzyme digestion identification results of the recombinant plasmids of Ara h 3-3-B overlapping segments I, II, and III are as follows. Figure 12 (b) shows the PCR identification results of the recombinant phages from the overlapping segments I, II, and III of Ara h 3-3-B. Figure 13 As shown, the phage proteins were then enriched and purified.

[0110] Indirect ELISA was used to screen for the epitope regions that most significantly enhanced the antigenicity of Ara h 3-3-B during baking. The results are as follows: Figure 14 As shown. This result indicates that Ara h 3-3-B-II has low antigenicity and the greatest relative variation, suggesting that this fragment may have the most newly generated or exposed epitopes, and the region with the most significant antigenic enhancement is likely Ara h 3-3-B-II (amino acid sequence: 305 EYDEEDRRRGRGSRGRG NGIEETICT 330 ).

[0111] Example 6: Dot-Blot and ELISA detection of synthetic peptides

[0112] 1. Peptide synthesis and purity identification

[0113] Three polypeptides, Ara h 3-3-B-II-P1, Ara h 3-3-B-II-P2, and Ara h 3-3-B-II-P3 (abbreviated as P1, P2, and P3, with sequences detailed in Table 7), were synthesized using Fmoc solid-phase technology based on the dominant region Ara h 3-3-B-II. The purity and molecular weight of the three polypeptides were then identified using high-performance liquid chromatography (HPLC) and mass spectrometry (MS).

[0114] Table 7. Analysis of Synthetic Peptides

[0115] polypeptide sequence Molecular weight (Da) purity(%) P1 (C)EYDEEDRRRGRGSR 1780.84 95.42 P2 (C)RRGRGSRGRGNGI 1398.55 97.24 P3 (C)GRGNGIEETICT 1249.37 96.52

[0116] The detection results of the three peptides by MS and HPLC are shown in Table 7 and Figure 15 As shown, the purity is all above 95%.

[0117] 2. SDS-PAGE identification of coupled peptides

[0118] SDS-PAGE was used to verify the coupling effect of the peptides. This method achieves electrophoretic separation based on the difference in protein molecular weight. The successful coupling of the peptides with BSA can be determined by comparing the migration positions of the coupled products with those of standard molecular weight proteins. The coupled peptide solution was adjusted to 2 mg / mL with PB buffer. 5 mL of 12% separating gel and 2 mL of 5% stacking gel were prepared. 12 μL of the coupled peptide was mixed with 4× loading buffer, and electrophoresis was performed at 120 V until the staining agent reached the bottom of the gel. Staining was continued for 1.5 h, followed by destaining until the bands were clear. The identification results are as follows: Figure 16 As shown, the molecular weights of the three peptides P1, P2, and P3 are 1780.84 Da, 1398.55 Da, and 1249.37 Da, respectively. The molecular weight of BSA is 66.43 kDa. The molecular weight of the conjugated peptide-BSA complex is significantly increased compared to the BSA monomer, and is about 30 kDa higher than the molecular weight of the BSA carrier protein alone. This indicates that each conjugate contains about 15 peptides, confirming successful conjugation of the peptides to BSA, and can be used for subsequent experiments.

[0119] 3. Dot-Blot and ELISA detection of synthetic peptides

[0120] Dot-Blot Procedure: The nitrocellulose membrane was soaked in PBST solution for 1 hour and then air-dried. 2 μL each of 2 mg / mL Ara h 3, phage expression protein Ara h 3-3-B-II, peptides (P1, P2, and P3), BSA, and PBS were spotted onto the membrane, and the membrane was dried for 1 hour. The membrane was washed 5 times with PBST and incubated in 5% skim milk powder for 2 hours. After washing, the membrane was immersed in 20 mL of diluted natural Ara h 3 rabbit polyclonal antibody and the baked Ara h 3 rabbit polyclonal antibody obtained in Example 1, respectively, and reacted at 37°C for 1 hour. After washing, the membrane was immersed in 20 mL of diluted goat anti-rabbit enzyme-labeled secondary antibody and reacted at 37°C for 1 hour. The membrane was then stained with an ACE kit for 10 minutes. Dot-Blot results are shown below. Figure 17 As shown, Ara h3, phage-expressed proteins Ara h3-3-B-II, and P1 showed colorimetric results in the chromogenic solution, while the other samples did not show colorimetric results. Furthermore, the natural Ara h3 rabbit polyclonal antibody and the baked Ara h3 rabbit polyclonal antibody reacted in the same way as the samples. This indicates that P2 and P3 did not bind to rabbit anti-Ara h3 serum, and P1 contains a linear epitope that specifically binds to rabbit anti-Ara h3 serum.

[0121] ELISA identification of synthetic peptides, such as Figure 18As shown, peptide P1 exhibited specific binding reactions with both baked and natural Ara h3 rabbit polyclonal antibodies (P / N > 2.1), indicating that peptide P1 contains a linear antigenic epitope. Furthermore, the OD of the reaction between peptide P1 and baked and natural Ara h3 rabbit polyclonal antibodies was [not specified]. 450 The values ​​showed no significant difference, and the synthetic peptide lacked spatial conformation, indicating that the antigenicity of the linear epitope in peptide P1 remained stable during baking. Comparison of the antigenicity of the phage protein Arah3-3-B-II revealed that the antigenicity of Arah3-3-B-II was higher than that of the linear peptide P1. This difference suggests that the Arah3-3-B-II region may contain both linear and conformational epitopes. Therefore, baking may primarily enhance the antigenicity of Arah3 by altering its conformational epitope, while the linear epitope remains stable and undamaged.

[0122] 4. Sensitization detection of peptides and phage-expressed proteins

[0123] To detect the sensitizing effects of phage-expressed proteins (Ara h 3, Ara h 3-3, Ara h 3-3-B, Ara h 3-3-B-II) and peptides (P1, P2, P3), they were reacted with serum from peanut-allergic patients (from Henan Provincial People's Hospital). The specific steps were as follows: phage-expressed proteins were diluted to 10 μg / mL with CBS buffer, and 100 μL was added to each well of an ELISA plate for coating. After incubation overnight at 4°C, the liquid in the wells was discarded. The plate was then washed 5 times with 200 μL of PBST buffer (PBS buffer containing 0.05% Tween 20) per well, blotted dry, and allowed to stand for 5 min. 200 μL of blocking buffer (5% skim milk powder) was added to each well and incubated at 37°C for 2 h. After washing, 50 μL of serum from peanut-allergic patients (1:400 dilution) was added to each well and incubated at 37°C for 1 h. After washing, goat anti-human enzyme-labeled secondary antibody (1:2000 dilution) was added and incubated at 37°C for 1 h. After washing, TMB was added for color development for 5 min, and OD was measured. 450 value.

[0124] Table 8 shows that the three-round phage expressed proteins Ara h 3-3, Ara h 3-3-B, and Ara h 3-3-B-II all exhibit sensitizing activity, with Ara h 3-3-B-II being the dominant sensitizing epitope region. The OD of peptide P1... 450 A positive result indicates that it can specifically bind to serum IgE in patients with peanut allergy, suggesting that the linear epitope contained in peptide P1 has sensitizing properties. The sequence of peptide P1 is as follows: 305 EYDEEDRRRGRGSR 318 It overlaps with the fourth known sensitizing epitope of Ara h3, DEDEYEYDEEDRG, by 6 amino acids but is unrelated. The overlapping region between the two is...305 EYDEEDR 311 .

[0125] Table 8. Sensitization assay of phage proteins and peptides

[0126] protein <![CDATA[OD 450 ]]> degree of reaction Ara h 3 <![CDATA[0.328±0.013 a ]]> +++++ Ara h 3-3 <![CDATA[0.256±0.0146 b ]]> ++++ Ara h 3-3-B <![CDATA[0.219±0.0124 c ]]> +++ Ara h 3-3-B-Ⅱ <![CDATA[0.191±0.0026 d ]]> ++ polypeptide P1 <![CDATA[0.169±0.0074 e ]]> + polypeptide P2 <![CDATA[0.147±0.0026 f ]]> - Peptide P3 <![CDATA[0.131±0.0051 f ]]> - BSA <![CDATA[0.071±0.0054 g ]]> -

[0127] Example 7: Locating key amino acids in the dominant region of Ara h3

[0128] 1. Conjugation and SDS-PAGE identification of mutant peptides

[0129] Based on the results of peptide Dot-Blot and ELISA experiments, the positive peptide P1 was selected for an alanine substitution experiment to synthesize a mutant peptide and identify key amino acids. Alanine is one of the most common amino acids in proteins, widely distributed in protein structures. It can eliminate the interference of side chains on antigen-antibody binding while maintaining the conformation and properties of the main chain. Based on these characteristics, alanine is often selected as a substitute amino acid in mutation experiments to study the function and role of other amino acids. Alanine was substituted in the positive peptide P1 to synthesize a mutant peptide, and its purity and molecular weight were identified. The results are shown in [Figure number missing]. Figure 19 The structures of all 14 peptides were accurate and their purity was above 95%. The results of the mutant peptide conjugation identification are as follows: Figure 20 As shown, the molecular weight of the conjugates (mutant peptide and BSA) is more than 30 kDa larger than that of BSA, indicating that the mutant peptide and BSA are successfully conjugated.

[0130] Table 9. Mutant Peptides

[0131] name sequence mutation site purity(%) M1 <![CDATA[(C) A YDEEDRRRGRGSR]]> <![CDATA[E 1 ]]> 95.21 M2 <![CDATA[(C)E A DEEDRRRGRGSR]]> <![CDATA[Y 2 ]]> 96.28 M3 <![CDATA[(C)EY A EEDRRRGRGSR]]> <![CDATA[D 3 ]]> 97.36 M4 <![CDATA[(C)EYD A EDRRRGRGSR]]> <![CDATA[E 4 ]]> 96.08 M5 <![CDATA[(C)EYDE A DRRRGRGSR]]> <![CDATA[E 5 ]]> 98.32 M6 <![CDATA[(C)EYDEE A RRRGRGSR]]> <![CDATA[D 6 ]]> 96.08 M7 <![CDATA[(C)EYDEED A RRGRGSR]]> <![CDATA[R 7 ]]> 97.48 M8 <![CDATA[(C)EYDEEDR A RGRGSR]]> <![CDATA[R 8 ]]> 97.12 M9 <![CDATA[(C)EYDEEDRR A GRGSR]]> <![CDATA[R 9 ]]> 97.98 M10 <![CDATA[(C)EYDEEDRRR A RGSR]]> <![CDATA[G 10 ]]> 97.49 M11 <![CDATA[(C)EYDEEDRRRG A GSR]]> <![CDATA[R 11 ]]> 96.44 M12 <![CDATA[(C)EYDEEDRRRGR A SR]]> <![CDATA[G 12 ]]> 95.29 M13 <![CDATA[(C)EYDEEDRRRGRG A R]]> <![CDATA[S 13 ]]> 96.25 M14 <![CDATA[(C)EYDEEDRRRGRGS A ]]> <![CDATA[R 14 ]]> 96.70

[0132] Note: (C): Cyscysteine ​​is used as a protecting group in polypeptide synthesis; A Ala alanine replaces the original amino acid residue;

[0133] 2. Identification of key amino acids

[0134] The antigenicity and sensitization of the mutant peptide were detected by Dot-Blot and ELISA methods, respectively. If the binding ability of the mutant peptide to serum was significantly reduced after a specific amino acid mutation, it indicates that the amino acid plays a key role in antigenicity and sensitization. The Dot-Blot results are as follows: Figure 21 As shown, the six mutant peptides 1, 3, 5, 7, 9, and 13 did not specifically bind to rabbit polyantiserum, indicating that the amino acids replaced by these peptides are key amino acids affecting their antigenicity, namely E305, D307, E309, R311, R313, and S317. Figure 22As shown, after reacting the mutant peptides with the serum of peanut-allergic patients, seven mutant peptides, namely mutant peptides 1, 2, 3, 5, 7, 9, and 13, did not specifically bind to the serum of peanut-allergic patients. This indicates that the amino acids replaced by these peptides are key amino acids affecting their sensitization, namely E305, Y306, D307, E309, R311, R313, and S317. Among them, the five amino acid sites E305, Y306, D307, E309, and R311 have been confirmed as key sensitizing amino acids, which are significantly correlated with the key amino acid sites discovered in this invention.

[0135] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An epitope polypeptide derived from the peanut allergen Ara h3, characterized in that, The epitope polypeptide contains the sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:

8.

2. The epitope polypeptide according to claim 1, characterized in that, The amino acid sequences of the epitope polypeptides are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:

11.

3. The use of the epitope polypeptide according to claim 1 or 2 in the preparation of a kit for detecting peanut allergen proteins.

4. A kit for detecting peanut allergen proteins, characterized in that, The kit contains reagents for detecting the epitope peptides of claim 1 or 2.

5. The reagent kit according to claim 4, characterized in that, The reagent contains an antibody against the epitope polypeptide of claim 1 or 2.

6. An anti-peanut allergy product, characterized in that, The product contains antibodies, which are obtained by screening epitope peptides as targets as described in claim 1 or 2.

7. The anti-peanut allergy product according to claim 6, characterized in that, The antibody is a polyclonal antibody, a monoclonal antibody, a chimeric antibody, and / or a humanized modified antibody.

8. The anti-peanut allergy product according to claim 6, characterized in that, The product targets peanuts or peanut products as the allergen.

9. The anti-peanut allergy product according to claim 6, characterized in that, The peanuts mentioned are roasted peanuts.

10. The method for locating epitope peptides according to claim 1 or 2, characterized in that, The epitope polypeptide contains EYDEEDRRRGRGSR, and the epitope polypeptide is an epitope region that enhances the sensitization of Ara h3 during baking. The positioning method includes the following steps: (1) Baked Ara h3 rabbit-derived polyclonal antibodies were prepared by immunizing white rabbits with baked Ara h3; (2) The amino acid sequence of Ara h 3 is segmented by overlap; (3) The protein Ara h3 and its overlapping segments are presented on the surface of the phage; (4) Using indirect enzyme-linked immunosorbent assay (ELISA), the epitope regions that enhance the sensitization of Ara h3 by baking were precisely located, and the amino acid sequence of the fragment that most significantly enhances the sensitization of Ara h3 by baking was obtained.

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