A peanut allergen polypeptide, its screening method and application

Abstract

This invention provides a peanut allergen peptide, its screening method, and its application. Phage display technology is used to screen for conformational epitopes of Ara h5 and Ara h8 proteins. Polyclonal IgE and IgG antibodies against the target proteins are prepared by immunizing BALB / c mice with Ara h5 and Ara h8 proteins as antigens. ELISA and dotted immunoblotting detection revealed that the titers of polyclonal IgG and IgE against Ara h5 and Ara h8 proteins were 1:64000 and 1:320, respectively, and the antibodies exhibited specificity. Using the antibodies as targets, phage display technology screened the conformational epitope sequences of Ara h5 protein as WETIYSR and FHWWYLK. The conformational epitopes of Ara h8 protein were FPYMKFV, FPYMKFR, SMFARID, and SFHWWLF.

Description

Technical Field

[0001] This invention belongs to the field of anti-allergy biotechnology, specifically relating to a peanut allergen polypeptide, its screening method, and its application. Background Technology

[0002] An allergic reaction is a response of an already immune system to the same antigen, resulting in tissue damage or functional disorder. In recent years, the incidence of allergic diseases has been steadily increasing, with reports indicating a global prevalence as high as 22%. However, public awareness of these diseases remains relatively low. Allergic diseases can cause symptoms such as respiratory obstruction, central nervous system disorders, and itchy, swollen skin, severely impacting patients' work and daily lives.

[0003] Peanuts are among the nine major allergens. Peanut allergy is an IgE-mediated hypersensitivity reaction to specific peanut proteins. Because the consequences can be fatal and its prevalence is increasing, peanut allergy is becoming a growing social problem. Peanuts are highly heat-stable, acid-resistant, and enzyme-resistant, meaning that conventional processing methods cannot remove the allergenic properties of peanut foods. Furthermore, unlike allergic reactions to milk or eggs, peanut allergy is often lifelong, with only 10% of patients developing immune tolerance to peanuts as they age. Although peanut allergens belong to only a few protein families, the structures and amounts of these allergenic proteins vary greatly, and the reasons for severe peanut allergies remain unclear.

[0004] Recent research on allergen epitopes has primarily focused on linear epitopes of allergens, with numerous studies identifying linear epitopes for different allergens. However, research on the identification and function of conformational epitopes remains relatively limited. Furthermore, conformational epitopes are only found in B-cell epitopes and are the core component of antigens. Conformational epitopes typically account for 90% of the total antibodies binding to antigens. Most food allergens possess thermostability and resistance to protease hydrolysis, maintaining their original structure after entering the body or retaining sensitizing activity after absorption and transport throughout the body. Therefore, conformational epitopes play a crucial role in sensitization. Previous research has found that the conformational epitopes of peanut protein Arah 5 and Arah 8 allergens also play a key role in sensitization, and that conformational epitopes may be a cause of allergic asthma. Therefore, characterizing the conformational epitopes of peanut protein Arah 5 and Arah 8 allergens is highly significant.

[0005] Current methods for characterizing conformational epitopes include large-scale mutagenesis, phage display, hydrogen / deuterium exchange, X-ray crystallography, and nuclear magnetic resonance (NMR). Theoretically, the best method for determining conformational epitopes is the X-ray co-crystal structure of antigen-antibody complexes. However, current technological limitations prevent the acquisition of highly pure monoclonal antibodies and proteins for crystallization. The application of hydrogen / deuterium exchange and NMR spectroscopy is challenging due to their experimental complexity. Hydrogen / deuterium exchange measurements require highly pure protein-ligand and protein samples. NMR spectroscopy requires determining the NMR signal of each amino acid residue in the antigen, which is a significant challenge for large molecular antigens. A widely used research method is site-directed mutagenesis to identify key sites in protein-protein interactions. Site-directed mutagenesis can alter or reduce protein-protein interactions to expose these key sites. However, this method is expensive and time-consuming. False positives are sometimes observed when the loss of affinity is due to structural changes outside the epitope. In antigenic epitope research, phage display peptide libraries have been successfully applied as an important technique.

[0006] To address the aforementioned technical problems, this invention uses phage display peptide libraries to screen for polyclonal antibodies targeting Ara h5 and Ara h8 proteins, and utilizes phage display technology to screen sequences that bind to the polyclonal antibodies, thereby locating Ara h5 and Ara h8 proteins as spatial epitopes of allergens. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of insufficient research on the allergenicity of peanut protein and the lack of research on spatial epitopes of allergens by providing an application of phage display technology in the study of peanut product allergy mechanisms. Based on phage display technology, conceptual epitopes of peanut food allergens are screened.

[0008] Peanut allergy manifests as a strong respiratory allergic reaction. Important peanut proteins, Ara h5 and Ara h8, which cross-react with respiratory allergens, can also induce respiratory allergic reactions, and conformational epitopes play a crucial role in sensitization.

[0009] This invention obtains the conformational epitopes of Ara h5 and Ara h8 proteins through phage display technology and screens out the dominant epitopes that are conducive to antibody binding, thus improving the sensitization mechanism of peanuts. This provides an important theoretical basis for the immunotherapy of food allergies, expands new treatment pathways for peanut allergies, and provides new coping strategies for allergy patients.

[0010] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0011] This invention provides a peanut allergen polypeptide derived from Ara h5 protein, with the sequences WETIYSR (SEQ ID NO:1) and FHWWYLK (SEQ ID NO:2). It also provides a peanut allergen polypeptide, characterized in that the polypeptide is derived from Ara h8 protein, with the sequences FPYMKFR (SEQ ID NO:3), SFHWWLF (SEQ ID NO:4), FPYMKFV (SEQ ID NO:5), or SMFARID (SEQ ID NO:6).

[0012] The present invention provides a nucleic acid encoding the above-mentioned allergen polypeptide.

[0013] This invention provides an expression cassette, recombinant vector, recombinant protein, recombinant cell, or recombinant bacterium capable of expressing the aforementioned peanut allergen polypeptide. Furthermore, this invention also provides an expression cassette, recombinant vector, recombinant protein, recombinant cell, or recombinant bacterium containing the nucleic acid encoding the aforementioned allergen polypeptide.

[0014] The present invention also provides a preventive or therapeutic agent in which the above-mentioned allergen polypeptide is used as an active ingredient.

[0015] The present invention also provides an application of the above-mentioned allergen polypeptide as an active ingredient in a preventive or therapeutic agent. Furthermore, the allergen protein can be at least one of the following as an active ingredient in a preventive or therapeutic agent: an immunomodulatory T-cell reactive polypeptide fragment, a B-cell reactive polypeptide fragment, a protein or polynucleotide fused with a carrier protein;

[0016] This invention provides an application of the above-mentioned allergen polypeptide as an antigen, hapten, immunogen, or immunomodulator.

[0017] The present invention also provides an antibody that can bind to Ara h5 and / or Ara h8 proteins. Preferably, the antibody can be one of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, or a humanized modified antibody.

[0018] The present invention also provides a medicament for the prevention or treatment of allergic diseases, the medicament being a single-ingredient or compound preparation comprising the above-mentioned allergen polypeptide or the nucleic acid or gene encoding the above-mentioned allergen polypeptide.

[0019] Preferably, the drug is an allergen vaccine.

[0020] The present invention also provides a kit for allergen diagnosis, comprising the above-mentioned allergen polypeptide or nucleic acid or gene encoding the allergen polypeptide.

[0021] The present invention also provides the use of allergen peptides, nucleic acids or genes, expression cassettes, recombinant vectors, recombinant proteins, recombinant cells or recombinant bacteria in the production process or finished products for the preparation of reagent kits for diagnosing peanut protein allergy or drugs for the prevention or treatment of peanut allergy.

[0022] On the other hand, the present invention also provides the application of the above-mentioned peanut allergen polypeptide as an antigen in the preparation of antibodies.

[0023] This invention also provides an anti-peanut allergy product prepared from the above-mentioned peanut allergen polypeptide. Preferably, the anti-peanut allergy product is a vaccine and / or a drug. Preferably, the anti-peanut allergy product is a skin microneedle. Preferably, the above product contains an adjuvant in liposome form.

[0024] On the other hand, the present invention also provides the application of the above-mentioned peanut allergen polypeptide as an anti-peanut allergy product, including improving the body's tolerance to specific allergens. Preferably, the specific allergen is peanuts or peanut products, and preferably the peanut products are food products.

[0025] The present invention also provides the application of the anti-peanut allergy product containing the above-mentioned peanut allergen polypeptide in the preparation of a drug to reduce the body's sensitivity to allergens, wherein the allergen is peanut protein.

[0026] The antibody prepared by this method can reduce the allergenicity of peanut protein and can be applied to the development of low-allergenic peanut protein products, the preparation of drugs for the prevention and / or treatment of allergies based on epitopes, and has high potency, which can reduce production costs and is suitable for industrial production.

[0027] Furthermore, the antibodies against Ara h5 and Ara h8 proteins prepared by this method can be used in anti-allergy drugs, including but not limited to loratadine, cetirizine, and astemizole.

[0028] Furthermore, the antibodies against Ara h5 and Ara h8 proteins prepared by this method can be used in anti-allergy foods, including but not limited to peanut oil, peanut drinks, and peanut snacks.

[0029] This invention obtains clonal antibodies through animal immunization using HiTrap. TM Serum antibodies were purified using a Protein G HP column, and antibody titers were determined by ELISA and Western blotting. The results showed a distinct characteristic peak at the specific elution site, and the titer was high, demonstrating that the clonal antibodies prepared and purified using this method possess the required functionality and specificity.

[0030] Furthermore, this invention utilizes a phage random peptide library for bioselection to obtain positive monoclonal phages that specifically bind to IgE and IgG antibodies against Ara h5 and Ara h8 proteins. Results show that after three rounds of screening, a large number of positive monoclonal phages are enriched. ELISA results also demonstrate that the selected positive monoclonal phages exhibit significantly enhanced specific binding ability to IgE and IgG antibodies against Ara h5 and Ara h8 proteins, indicating that this method effectively screens for the desired specific antibodies. Pepitope three-dimensional structure alignment also shows a high degree of matching between the screened sequences and Ara h5 or Ara h8.

[0031] Finally, through sequence screening based on spatial structural localization, this invention confirmed the sequences of the anti-Ara h5 and Ara h8 proteins prepared by this method. The results showed that the WETIYSR and FHWWYLK sequences are conformational epitopes of the Ara h5 protein, while FPYMKFR, SFHWWLF, and FPYMKFV are conformational epitopes of the Ara h8 protein. These results can be used to determine key amino acids of the epitopes and for subsequent immunotherapy. Furthermore, modification with mimicking epitopes may contribute to the development of blocking antibodies or epitope-specific immunotherapies against peanut allergy.

[0032] This application utilizes phage display technology to display peanut allergen proteins Ara h5 and Ara h8, screening for and optimizing corresponding phage antibodies. Through multiple rounds of screening, allergen peptides with high antibody binding efficiency were obtained. ELISA verified the binding activity between the screened sequences and antibodies, and Pepitope alignment of the three-dimensional structures of the screened sequences and antibodies showed that the screened allergen peptide sequences are readily binding antibody epitopes for Ara h5 and Arah 8 proteins. These sequences can be used to prepare or screen anti-allergy products, as well as to establish tolerance to specific allergens and related applications.

[0033] The present invention has the following advantages over the prior art:

[0034] 1. Applying phage display technology to the research of anti-allergy substances has paved the way for the application of phage display technology and promoted the further development of this method.

[0035] 2. Sequences with good titers and strong binding ability to IgE and IgG antibodies were successfully screened.

[0036] 3. It was identified that the conformational epitopes on the Ara h5 protein that can act as allergens are WETIYSR and FHWWYLK, and the conformational epitopes on the Ara h8 protein that can act as allergens are FPYMKFR, SFHWWLF, FPYMKFV and SMFARID.

[0037] 4. The identification of conformational epitopes of Ara h5 and Ara h8 proteins provides a theoretical basis for the development of hypoallergenic peanut proteins and epitope-based immunotherapy. Attached Figure Description

[0038] Figure 1 This is a purification map of serum antibodies and SDS-PAGE verification. Figure 1 A represents the Arah5 antibody. Figure 1 B is an Arah8 antibody. Figure 1 C is for SDS-PAGE verification.

[0039] Figure 2 This is a comparison of the titers of phages bound to polyclonal IgE and IgG antibodies after three rounds of screening. Figure 2 A is an IgE antibody. Figure 2 B represents IgG antibodies.

[0040] Figure 3 It refers to the binding ability of the target sequence to the IgE antibody. Figure 3 A represents the Ara h5 peptide. Figure 3 B represents the Ara h8 peptide.

[0041] Figure 4 It refers to the binding ability of the target sequence to the IgG antibody. Figure 4 A represents the Ara h5 peptide. Figure 4 B represents the Ara h8 peptide.

[0042] Figure 5 This refers to the localization (IgE) of the selected peptide sequence within the Ara h 5 protein structure.

[0043] Figure 6 This indicates the location (IgE) of the selected peptide sequence within the Ara h 8 protein structure.

[0044] Figure 7 This indicates the location of the selected peptide sequence within the Ara h 5 protein structure (IgG).

[0045] Figure 8 This indicates the location of the selected peptide sequence within the Ara h8 protein structure (IgG). Detailed Implementation

[0046] The objects and functions of the present invention, as well as the methods for achieving these objects and functions, will be explained below with reference to exemplary embodiments. However, the present invention is not limited to the exemplary embodiments disclosed below; it can be implemented in various forms. This specification is merely intended to help those skilled in the art to comprehensively understand the specific details of the invention.

[0047] Example 1: Preparation of Clonal Antibodies

[0048] 1. Animal immunization

[0049] (1) The full amino acid sequences of peanut Ara h5 and Ara h8 proteins were obtained from the NCBI database, and their codons were optimized to conform to the dominant codons of E. coli. The gene sequences were synthesized and expressed in E. coli. The purified recombinant proteins were used as recombinant allergens Ara h5 and Ara h8 proteins as immunogens. The recombinant proteins dissolved in PBS were premixed with an equal volume of complete Freund's adjuvant and used to immunize 6- to 8-week-old BALB / c mice (final protein concentration was 100 μg / mL). 50 μL of immunogen was injected subcutaneously into the neck and back of female BALB / c mice at multiple sites.

[0050] (2) After the first injection, three booster immunizations were performed every week, using an equal amount of immunogen mixed with incomplete Freund's adjuvant. Both the first and booster immunizations were administered subcutaneously, ensuring even distribution of the injections in the subcutaneous tissue of the neck and back of the mice to avoid concentrated immunization that could cause skin ulceration.

[0051] (3) Blood was collected from the orbital cavity three days after the fourth immunization for ELISA testing. If the antibody titer was qualified, the mice were sacrificed, the serum was collected, and the antibodies were purified.

[0052] Note: The experiment was approved by the Laboratory Animal Welfare and Animal Experimentation Ethics Review Committee of China Agricultural University.

[0053] 2. Antibody purification experiment

[0054] (1) Using HiTrap TM Protein G HP column purification of serum antibodies. Before loading the protein onto the column, the column was washed with 10 column volumes of binding buffer (20 mM sodium phosphate, pH 7.0) at a rate of 1 mL / min.

[0055] (2) The collected serum was diluted 1:5 (diluent: 20 mM sodium phosphate, pH 7.0) and then loaded onto the column. Serum proteins were loaded onto the column at a rate of 1 mL / min and washed with 5 column volumes of binding buffer until the baseline stabilized.

[0056] (3) Elute with 5 column volumes of elution buffer (0.1 M glycine-HCl, pH 2.7) until the baseline is stable. Add 1 M Tris-HCl, pH 9.0 neutralizing solution (add neutralizing solution at a ratio of 10:1) to the elution collection tube.

[0057] (4) Replace the specific eluent with 0.01 mol / L PBS using a dialysis bag, and perform SDS-PAGE to identify the purified antibody. Figure 1 ).

[0058] 3. ELISA to determine antibody titer

[0059] (1) Ara h 5 and Ara h 8 proteins (10 μg / mL) were coated on a 96-well plate at 4°C using carbonate coating buffer.

[0060] (2) After blocking and washing, IgG antibody was diluted with 0.1% BSA antibody dilution buffer at 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, and 1:256000, and IgE antibody was diluted at 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, and 1:1280. Normal serum from the control group was diluted in the same proportion. The solutions were added to 96-well plates and incubated at 37°C for 2 hours.

[0061] (3) After washing, add the corresponding secondary antibody and incubate for 1 hour. Add 100 μL of HRP-labeled rabbit anti-mouse IgG (1:10000) to the IgG antibody and 100 μL of HRP-labeled rat anti-mouse IgE (1:2000) to the IgE antibody.

[0062] (4) After washing, add TMB for color development and use a multi-functional microplate reader to measure the absorbance of each well at 450 nm.

[0063] 4. Dot immunoblotting method for determining potency

[0064] (1) Take 1.5 μl of 2 μg / mL Ara h 5 and Ara h 8 protein and place it on an NC membrane and block for 1 h.

[0065] (2) Incubate IgG antibodies diluted at 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, and 1:256000 for 2 hours.

[0066] (3) Incubate IgE antibodies diluted at 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640 and 1:1280 at 4°C overnight.

[0067] (4) After washing, incubate at room temperature with the corresponding secondary antibody rabbit anti-mouse IgG (1:10000) or rat anti-mouse IgE (1:2000) for 1 hour.

[0068] (5) After washing, ECL chemiluminescent agent was added for development and exposure before analysis.

[0069] Example 2: Screening allergen epitopes using antibody-targeted phage display technology

[0070] 1. Resuscitation and culture of bacteriophage host bacterium ER2738

[0071] (1) Using a sterile inoculation loop, take a small amount of the E. coli ER2738 glycerol cryopreservation and streak it onto LB cells. Tet+ Incubate overnight at 37°C inverted on a petri dish. Tet is tetracycline.

[0072] (2) Pick a single colony in LB Tet+ In the culture medium, incubate overnight at 37°C with shaking to preserve the bacteria. LB Tet+ Petri dishes and bacterial cultures should be stored at 4°C for up to 3 weeks.

[0073] 2. Bioselection of phage peptide libraries

[0074] (1) Coating: The IgG and IgE polyclonal antibodies (100 μg / mL) obtained in Example 1 were diluted and purified with coating buffer (0.1 M NaHCO3 (pH 8.6)), coated with ELISA plates, 100 μL / well, labeled A for positive selection, and incubated overnight at 4°C; In addition, normal mouse serum was diluted 5 times with coating buffer and coated with ELISA plates, 100 μL / well, labeled B for negative selection, and incubated overnight at 4°C.

[0075] (2) Blocking: Discard the coating solution from each plate, then firmly tap it face down onto a clean paper towel to remove any residual solution. Completely fill each plate or well with blocking buffer. Incubate at 4°C for 2 hours.

[0076] (3) Binding: Aspirate the blocking solution from the B negative selection, wash thoroughly 6 times with TBST (TBS + 0.1% [v / v] Tween-20), and blot away the washing solution. Add 100 μL of random 7-peptide library liquid diluted with TBST (10¹¹ phages, original peptide library 10¹³ phages / mL, Ph.D.-7 Phage Display Peptide Library Kit, New England Biolabs, catalog number #E8100S) to each well of the B negative selection and gently shake at room temperature for 1 hour. Aspirate the blocking solution from the A positive selection, wash thoroughly 6 times with TBST (TBS + 0.1% [v / v] Tween-20), and blot away the washing solution. Add the phage solution from the B negative selection to the A positive selection, and gently shake at room temperature for 1 hour.

[0077] (4) Elution: Aspirate the phage fluid selected by A-positive bacteriophage and wash thoroughly 6 times with TBST (TBS + 0.1% [v / v] Tween-20), patting off the washing solution each time, using a clean paper towel after each wash to prevent cross-contamination. This removes non-specifically bound phages.

[0078] (5) Extraction: Add 100 μL of extraction buffer (0.2 mol / L glycine-hydrochloric acid (pH 2.2), 1 mg / mL BSA filtered and sterilized) to each well and shake slowly at room temperature for 15 minutes.

[0079] (6) Neutralization: The liquid was collected into a small tube containing 15 μL of stop solution (1 mol / L pH 9.1 Tris-HCl) and mixed to neutralize.

[0080] (7) Titration: Dilute the phages serially with sterile TBST solution, adding 10 μL of each dilution to 200 μL of fresh ER2738 bacterial culture that has reached the logarithmic growth phase (OD600 value of 0.5), mixing well, and then incubating at room temperature for 5 minutes. Add 3 mL of preheated LB-TOP top agar (preheated in a 45℃ water bath), mix well, and pour onto LB solid plates containing X-Gal / IPTG. Incubate overnight at 37℃, and count plaques the next day.

[0081] Titer calculation formula: pfu (plague forming unit) / mL = number of plaques × dilution factor × total volume (mL).

[0082] (8) Amplification: The remaining eluted phage fluid was amplified on the second day and incubated overnight in LB filtrate. Tet+The ER2738 bacterial culture in the medium was diluted 1:100 to 20 mL of LB medium, and the remaining elution buffer was added to a 250 mL Erlenmeyer flask. The culture was incubated at 37°C and 220 rpm with shaking for 4.5 hours. The culture was transferred to a sterile centrifuge tube and centrifuged at 12,000 g for 10 minutes at 4°C. The supernatant was transferred to a new tube and rotated again (discarding the precipitate). The upper 80% of the supernatant was transferred to a sterile new test tube, and 1 / 6 volume of 20% PEG / 2.5M NaCl was added. The phage was allowed to precipitate overnight at 4°C. The next day, the phage was precipitated by centrifugation at 12,000 g at 4°C for 15 minutes, the supernatant was discarded, the tube was briefly rotated again, and any remaining supernatant was removed with a pipette. The precipitate was resuspended in 1 mL of TBS. The suspension was transferred to a sterile microcentrifuge tube and centrifuged at 14,000 rpm for 5 minutes at 4°C to precipitate any remaining cells. Transfer the supernatant to a new microcentrifuge tube and add 1 / 6 volume of 20% PEG / 2.5M NaCl for reprecipitation. Incubate on ice for 60 minutes. Centrifuge at 14,000 rpm for 10 minutes at 4°C, discard the supernatant, briefly centrifuge again, and remove any remaining supernatant using a micropipette. Resuspend the particles in 200 μL of TBS solution. The amplified phage library is now obtained.

[0083] (9) Repeat screening: Repeat the second and third rounds of screening, except that the concentration of TBST used during washing is increased (round 2: 0.5% TBST; round 3: 1% TBST) and the number of elutions is increased (round 2: 10 times; round 3: 15 times), the other steps are the same as the first round of screening. The elution buffer is tested for phage titer, and the plaques obtained in the last round of screening are retained.

[0084] After three rounds of biological selection (as shown in Tables 1 and 2 below), phages capable of binding to IgE and IgG antibodies against Ara h5 and Ara h8 proteins were obtained, respectively. Figure 2 As shown.

[0085] Table 1. Selection conditions and results of phage display peptide library bioselection using IgE antibodies as targets.

[0086]

[0087]

[0088] Table 2. Selection conditions and results of phage display peptide libraries targeting IgG antibodies.

[0089]

[0090] Example 3: Monoclonal phage amplification, sequencing, and binding performance verification

[0091] 1. Amplification and DNA sequencing of monoclonal bacteriophages

[0092] (1) Using a pipette tip, pick up well-separated blue patches from the titration plate, ensuring that each patch contains a DNA sequence. Then transfer the patches to an overnight culture of ER2738 diluted 1:100. Incubate the tubes at 37°C with shaking for 4.5 hours.

[0093] (2) Transfer the culture to a microcentrifuge tube and centrifuge at 14,000 rpm for 30 seconds. Transfer the supernatant to a new tube and centrifuge again. Use a pipette to transfer the top 80% of the supernatant to the new tube. This is the amplified phage stock solution.

[0094] (3) Transfer 500 μL of the phage-containing supernatant to a new microcentrifuge tube. Add 200 μL of 20% PEG / 2.5 M NaCl, invert several times to mix, and let stand at room temperature for 20 minutes. Centrifuge at 14,000 rpm for 10 minutes at 4°C, and discard the supernatant. Briefly rotate again. Carefully pipette and discard any remaining supernatant.

[0095] (4) Tap the test tube firmly to completely suspend the precipitate in 100 μL of iodide buffer. Add 250 μL of ethanol and incubate at room temperature for 15 minutes. Centrifuge at 14,000 rpm at 4°C for 10 minutes and discard the supernatant.

[0096] (5) Wash the precipitate with 0.5 mL of 70% ethanol (stored at –20°C), centrifuge again, discard the supernatant, and briefly dry the precipitate under vacuum. Resuspend the precipitate in 30 μL of sterile water. Send to Suzhou Genewiz Biotechnology Co., Ltd., using -96gIII universal primers for sequencing.

[0097] Bioselection using a random phage peptide library identified positive monoclonal phages that specifically bind to IgE and IgG antibodies against Ara h5 and Ara h8 proteins. Twenty positive monoclonal phages from each phage were then selected for DNA sequencing. The results yielded 16 peptide sequences binding to IgE antibodies against Ara h5, 15 peptide sequences binding to IgE antibodies against Ara h8, and 20 peptide sequences binding to IgG antibodies against both Ara h5 and Ara h8 proteins.

[0098] For the polypeptide sequences displayed by IgE-positive monoclonal phages exhibiting anti-Ara h 5 protein binding, a total of 3 binding peptides were screened. The most frequent was FHWWYLK, with a frequency of 11 / 16. For the polypeptide sequences displayed by IgE-positive monoclonal phages exhibiting anti-Ara h 8 protein binding, a total of 5 binding peptides were screened. The most frequent were FPYMKFR and FPYMKFV, with frequencies of 6 / 15 and 5 / 15, respectively.

[0099] In addition, for the polypeptide sequences displayed by positive monoclonal phages binding to anti-Ara h 5 protein IgG, a total of 5 binding peptides were screened. The most frequent were FLHRDND and WETIYSR, with frequencies of 6 / 20 and 5 / 20 respectively. Furthermore, WETIYSR was identical to the sequence screened for IgE. For the polypeptide sequences displayed by positive monoclonal phages binding to anti-Ara h 8 protein IgG, a total of 6 binding peptides were screened. The most frequent were FPYMKFV and FPYMKFR, with frequencies of 7 / 20 and 6 / 20 respectively. These were identical to the polypeptide sequences displayed by positive monoclonal phages binding to anti-Ara h 8 protein IgE.

[0100] 2. ELISA verification of the binding activity between the screening sequence and the antibody.

[0101] (1) Add 5 μL of amplified phage stock solution to 20 mL of culture and incubate with vigorous shaking at 37 °C for 4.5 h. Transfer the culture to a centrifuge tube and centrifuge at 12,000 g for 10 min at 4 °C. Transfer the supernatant to a new tube and re-rotate, discarding the precipitate. Transfer the upper 80% of the supernatant to a new tube, add 1 / 6 volume of 20% PEG / 2.5M NaCl, and precipitate overnight at 4 °C. Centrifuge at 12,000 g for 15 min at 4 °C. Discard the supernatant, briefly re-rotate, and then remove the residual supernatant with a pipette. Resuspend the precipitate in 1 mL TBS. Transfer the suspension to a microcentrifuge tube and rotate at 14,000 rpm for 5 min at 4 °C to precipitate the remaining cells. Transfer the supernatant to a new microcentrifuge tube and re-precipitate with 1 / 6 volume of 20% PEG / 2.5M NaCl. Incubate on ice for 6 min. Centrifuge at 14,000 rpm for 10 minutes at 4°C. Discard the supernatant, briefly centrifuge again, and then remove any remaining supernatant using a micropipette. Resuspend the precipitate in 50 μL of TBS and determine the titer.

[0102] (2) Then, place 100 μL of 100 μg / mL antibody in 0.1 M NaHCO3, pH 8.6, and coating buffer, and incubate overnight at 4°C. Discard the excess target solution and tap the plate face down onto a paper towel. Completely fill each well with 150 μL of blocking buffer. Incubate at 4°C for 2 hours. Discard the blocking buffer and wash 6 times with TBST (0.5% Tween-20), tapping the plate face down onto a clean paper towel each time. Add 100 μL of TBST diluted with 10% Tween-20 solution to each well. 5 Bacteriophages. Incubated at room temperature for 2 hours. Unselected bacteriophages were used as a control group.

[0103] (3) Wash 6 times with TBST (0.5% Tween-20), tapping the plate face down onto a clean paper towel each time. Add 100 μL of HRP-labeled anti-M13 antibody (1:2500) diluted with blocking buffer to each well and incubate at room temperature with shaking for 2 hours. Discard the unbound antibody and wash 6 times with TBST (0.5% Tween-20). Add 100 μL of TMB to each well and incubate at 37°C in the dark for 15 minutes. Stop the reaction by adding 50 μL of 1M hydrochloric acid to each well. Measure the absorbance at 450 nm using a microplate reader.

[0104] The results are as follows Figure 3 , 4 As shown, the selected sequences exhibited significantly higher binding affinity to IgE antibodies compared to the control group. The sequence FHWWYLK showed the strongest binding affinity to IgE antibodies against Ara h5 protein, while sequences FPYMKFR and SMFARID showed the strongest binding affinity to IgE antibodies against Ara h8 protein, significantly higher than other binding sequences. The OD value of sequence YSFTLHA, which bound to IgG antibodies against Ara h5 protein, was not significantly different from the OD value of the unscreened phage peptide library, suggesting that sequence YSFTLHA was a false positive clone. However, the OD values ​​of sequences FHWWYLK and FLFRDND were significantly higher than the OD values ​​of other sequences binding to IgG antibodies against Ara h5 protein. Sequences FPYMKFV and FPYMKFR, which bound to IgG antibodies against Ara h8 protein, also showed significantly higher binding affinity than other sequences.

[0105] Example 4: Conformational Epitope Localization and Analysis

[0106] The amino acid sequences of IgE and IgG antibodies that bind to anti-Ara h5 and anti-Ara h8 proteins, respectively, selected from a phage random peptide library and screened using the Pepitope method, were compared with the three-dimensional structures of Ara h5 and Ara h8 proteins.

[0107] The results are as follows Figure 5-8 As shown, for screening potential Ara h 5 IgE conformational epitopes, sequences WETIYSR and FHWWYLK showed the highest spatial matching and were clustered together. For screening potential Ara h 8 IgE conformational epitopes, the sequences with the highest matching were FPYMKFV and FPYMKFR, while sequence HTYSSTT did not correspond to any protein in the spatial structure and had only one positive clone, possibly indicating a false positive. For screening potential Ara h 5 IgG conformational epitopes, sequences FLRRD, WETIYSR, YSFTLHA, and FHWWYLK showed the highest spatial matching. For screening potential Ara h 8 IgG conformational epitopes, the sequences with the highest matching were FPYMKFV and FPYMKFR.

[0108] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A peanut allergen polypeptide, characterized in that, The polypeptide is derived from the Ara h5 protein, with the sequence WETIYSR and / or FHWWYLK.

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