Peanut allergen polypeptide as well as screening method and application thereof

Abstract

The invention provides a peanut allergen polypeptide as well as a screening method and application thereof. And screening conformational epitopes of Ara h 5 and Ara h 8 proteins by using a phage display technology. The Ara h 5 protein and the Ara h 8 protein are used as antigens to immunize BALB / c mice to prepare polyclonal IgE and IgG antibodies of anti-target protein. ELISA (enzyme-linked immuno sorbent assay) and dot immunoblotting detection show that the titers of anti-Ara h 5 and anti-Ara h 8 protein polyclone IgG and anti-Ara h 8 protein polyclone IgE are 1: 64000 and 1: 320 respectively, and the antibody has specificity. The antibody is taken as a target spot, and the conformational epitope sequences of the Ara h 5 protein, which are screened by a phage display technology, are WETIYSR and FHWWYLK. And the conformational epitopes of the Ara h 8 protein are FPYMKFV, FPYMKFR, SMFARID and SFHWWLF (Small Form of Horse and White White Link Factor).

Description

TECHNICAL FIELD

[0001] The application belongs to the field of anti-allergy biotechnology, and particularly relates to a peanut allergen polypeptide and a screening method and application thereof. BACKGROUND

[0002] An allergic reaction refers to a reaction of tissue damage or functional disorder of an immunized body when the same antigen is received again. In recent years, the incidence of allergic diseases has shown a continuous upward trend. It is reported that the prevalence of allergy is as high as 22% globally, but the public's awareness of the disease is still relatively lagging. Allergic diseases can cause respiratory obstruction, central nervous system disorder, skin redness and itching, and other symptoms, which seriously affect the work and life of patients.

[0003] As one of the nine major allergens, peanut allergy is an IgE-mediated hypersensitivity reaction to specific peanut proteins. Because the results can be fatal, and the prevalence is increasing, peanut allergy has become a serious social problem. Peanuts have the characteristics of high thermal stability, acid resistance and enzyme resistance. General processing methods cannot remove the allergens of peanut food. Moreover, unlike the allergic reactions caused by milk, eggs and other foods, peanut allergy often has a lifelong nature, and only 10% of patients develop immune tolerance to peanuts as they age. Although the allergens of peanuts belong to only a few protein families, the structures and contents of these allergen proteins are different, and the reasons for the severe allergic reactions caused by peanuts cannot be explained at present.

[0004] In recent years, research on allergen epitopes has mainly focused on linear epitopes of allergens. A large number of studies have identified linear epitopes of different allergens, but there are still few studies on the identification and role of conformational epitopes. Moreover, conformational epitopes are only found in B cell epitopes, which are the core part of antigens. Conformational epitopes usually account for 90% of total antibodies that bind to antigens. Most food allergens have the characteristics of thermal stability and resistance to protease hydrolysis, and can maintain their original structure after entering the human body, or still have allergenic activity after being absorbed and transported to various parts of the body. Therefore, conformational epitopes play a key role in allergenicity. Previous research results have found that the conformational epitopes of peanut protein Ara h 5 and Ara h 8 allergens also play a key role in allergenicity, and the conformational epitopes may be the cause of allergic asthma. Therefore, it is very meaningful to characterize the conformational epitopes of peanut protein Ara h 5 and Ara h 8 allergens.

[0005] Current methods for characterizing conformational epitopes include large-scale mutagenesis, phage display technology, hydrogen / deuterium exchange, X-ray crystallography and nuclear magnetic resonance (NMR). Theoretically, the best way to determine the conformational epitope is the X-ray co-crystal structure of the antigen-antibody complex. At present, due to technical limitations, it is difficult to obtain highly pure monoclonal antibodies and proteins for crystallization. The difficulty of the application of hydrogen / deuterium exchange and nuclear magnetic resonance spectroscopy lies in the difficulty of the experiment. Hydrogen / deuterium exchange measurement requires highly pure protein-ligand and protein samples. Nuclear magnetic resonance spectroscopy requires determination of the nuclear magnetic resonance signal of each amino acid residue in the antigen, which is a great challenge for large molecule antigens. A widely used research method is to determine the key sites in protein interaction by site-directed mutagenesis. Site-directed mutagenesis can change or reduce the interaction between proteins to expose the key sites. However, this method is expensive and time-consuming. When the loss of affinity is due to changes in the structure outside the epitope, false positive results are sometimes observed. In the study of antigen epitopes, phage display peptide library as an important technology has been successfully applied.

[0006] To solve the above technical problems, the present application screens polyclonal antibodies targeting Ara h 5 protein and Ara h 8 protein by phage display peptide library, and locates the spatial epitope of Ara h 5 protein and Ara h 8 protein as allergen by using phage display technology to screen sequences combined with polyclonal antibodies. SUMMARY

[0007] The purpose of the present application is to overcome the shortcomings of insufficient research on peanut protein sensitization and lack of research on spatial epitopes of allergens, and to provide the application of phage display technology in the research of peanut product allergy mechanism. Based on phage display technology, the conformational epitopes of peanut food allergens are screened.

[0008] Peanut allergy shows strong respiratory allergic reactivity. The important proteins Ara h 5 and Ara h 8 in peanuts that cross-react with respiratory allergens can also cause respiratory allergic reactivity, and conformational epitopes play a key role in sensitization.

[0009] The present application obtains the conformational epitopes of Ara h 5 and Ara h 8 proteins by phage display technology, and screens the dominant epitopes that are beneficial to antibody binding, which perfects the sensitization mechanism of peanuts, provides an important theoretical basis for immunotherapy of food allergy, can expand new treatment approaches for peanut allergy, and provides new coping strategies for allergic patients.

[0010] To achieve the above-mentioned purposes, the present application provides the following technical solutions: The present application provides a peanut allergen polypeptide, which is derived from Ara h 5 protein and has the sequence of WETIYSR (SEQ ID NO: 1) or FHWWYLK (SEQ ID NO: 2). The present application also provides a peanut allergen polypeptide, which is characterized in that the polypeptide is derived from Ara h 8 protein and has the sequence of FPYMKFR (SEQ ID NO: 3), SFHWWLF (SEQ ID NO: 4), FPYMKFV (SEQ ID NO: 5) or SMFARID (SEQ ID NO: 6).

[0011] The present application provides a nucleic acid encoding the above-mentioned allergen polypeptide.

[0012] The present application provides an expression cassette, a recombinant vector, a recombinant protein, a recombinant cell or a recombinant bacterium, which can express the above-mentioned peanut allergen polypeptide. Meanwhile, the present application also provides an expression cassette, a recombinant vector, a recombinant protein, a recombinant cell or a recombinant bacterium, which has the above-mentioned nucleic acid encoding the allergen polypeptide.

[0013] The present application also provides a prophylactic or therapeutic agent containing the above-mentioned allergen polypeptide as an effective ingredient.

[0014] The present application also provides the use of the above-mentioned allergen polypeptide as an effective ingredient of a prophylactic or therapeutic agent. Further, the allergen protein can be used as at least one of an effective ingredient of a prophylactic or therapeutic agent, which is a T-cell reactive polypeptide fragment, a B-cell reactive polypeptide fragment, a protein constructed by fusing a polypeptide fragment with a carrier protein, or a polynucleotide. The present application provides the use of the above-mentioned allergen polypeptide as an antigen, a hapten, an immunogen or an immunomodulator.

[0015] The present application also provides an antibody, which can bind to Ara h 5 protein and / or Ara h 8 protein. Preferably, the antibody can be one of a polyclonal antibody, a monoclonal antibody, a chimeric antibody or a humanized modified antibody.

[0016] The present application also provides a medicament for preventing or treating allergic diseases, which is a single or complex preparation containing the above-mentioned allergen polypeptide or the above-mentioned nucleic acid or gene encoding the allergen polypeptide.

[0017] Preferably, the medicament is an allergen vaccine.

[0018] The present application also provides a kit for allergen diagnosis, which contains the above-mentioned allergen polypeptide or the nucleic acid or gene encoding the allergen polypeptide.

[0019] The application also provides the use of the allergen polypeptide, nucleic acid or gene, expression cassette, recombinant vector, recombinant protein, recombinant cell or recombinant bacteria in the production process of a kit for diagnosing peanut protein allergy or a medicine or a pharmaceutical composition thereof for preventing or treating peanut allergy, content calibration or quality control of a finished product.

[0020] In another aspect, the application also provides the use of the above-mentioned peanut allergen polypeptide as an antigen to prepare an antibody.

[0021] The application 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 medicine. Preferably, the anti-peanut allergy product is a skin microneedle. Preferably, the above-mentioned product contains an adjuvant in the form of a liposome.

[0022] In another aspect, the application also provides the use of the above-mentioned peanut allergen polypeptide as an anti-peanut allergy product, including improving the tolerance of the body to a specific allergen. Preferably, the specific allergen is peanut or a peanut product, and preferably the peanut product is a food.

[0023] The application also provides the use of an anti-peanut allergy product containing the above-mentioned peanut allergen polypeptide in the preparation of a medicine for reducing the sensitivity of the body to an allergen, and the allergen is peanut protein.

[0024] The antibodies prepared by the method can reduce the sensitization of peanut protein, can be applied to the development of low-sensitization peanut protein products, the preparation of allergy-preventing and / or treating medicines based on epitopes, have high titers, can reduce production costs, and are suitable for industrial production.

[0025] Further, the antibodies against Ara h 5 and Ara h 8 proteins prepared by the method can be used in anti-allergy medicines, and the anti-allergy medicines include but are not limited to loratadine, cetirizine and astemizole.

[0026] Further, the antibodies against Ara h 5 and Ara h 8 proteins prepared by the method can be used in anti-allergy foods, and the anti-allergy foods include but are not limited to peanut oil, peanut drinks and peanut snacks.

[0027] The application obtains cloned antibodies by immunizing animals, purifies serum antibodies by using a HiTrap™ Protein G HP column, and determines the titer of the antibodies by using ELISA and dot immunoblotting. The results show that a clear characteristic peak appears at the specific elution position, and the titer is high, proving that the cloned antibodies prepared and purified by the method have the required functionality and specificity.

[0028] Further, the present application can obtain positive monoclonal phages capable of specifically binding to IgE antibodies and IgG antibodies against Ara h 5 and Ara h 8 proteins by biopanning of phage random peptide library, and the results show that, after three rounds of screening, the positive monoclonal phages can be enriched in large quantities, and the ELISA detection results show that the ability of the screened positive monoclonal phages to specifically bind to IgE antibodies and IgG antibodies against Ara h 5 and Ara h 8 proteins is significantly enhanced, indicating that the method effectively screens the required specific antibodies. The results of Pepitope three-dimensional structure comparison also show that the screened sequences have a high match with Ara h 5 or Ara h 8.

[0029] The present application finally confirms the sequences of the anti-Ara h 5 and Ara h 8 proteins prepared by the method through the sequences screened in the spatial structure. The results show that the WETIYSR and FHWWYLK sequences are conformational epitopes of Ara h 5 protein, and the FPYMKFR, SFHWWLF and FPYMKFV sequences are conformational epitopes of Ara h 8 protein. The results can be used for the determination of key amino acids of epitopes and the development of subsequent immunotherapy, in addition, the modification of the simulated epitopes can help to develop blocking antibodies or epitope-specific immunotherapy for peanut allergy.

[0030] The present application performs phage display of peanut allergen proteins Ara h 5 and Ara h 8 through phage display technology, screens corresponding phage antibodies and optimizes. Through multiple rounds of screening, allergen polypeptides with high efficient binding to antibodies are obtained, the binding activity of the screened sequences to antibodies is verified by ELISA, and the three-dimensional structure of the screened sequences and antibodies is compared by Pepitope, indicating that the screened allergen polypeptide sequences are Ara h 5 and Ara h 8 protein antibody epitopes, and can be used for preparing or screening anti-allergy products, and establishing body tolerance products and applications to specific allergens.

[0031] The present application has the following advantages relative to the prior art: 1. The application of phage display technology to anti-allergy substances opens up a path for the application of phage display technology and promotes the further development of the method.

[0032] 2. Sequences with good titer and strong binding ability to IgE and IgG antibodies are successfully screened.

[0033] 3. It is clear that the conformational epitopes on Ara h 5 protein that can be allergens are WETIYSR and FHWWYLK, and the conformational epitopes on Ara h 8 protein that can be allergens are FPYMKFR, SFHWWLF, FPYMKFV and SMFARID.

[0034] 4. The determination of Ara h 5 and Ara h 8 protein conformational epitopes provides a theoretical basis for the development of low-sensitization peanut proteins and immunotherapy based on epitopes. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 is the purification profile of serum antibodies and SDS-PAGE verification. Figure 1 A is Ara h 5 antibody, Figure 1 B is Ara h 8 antibody, Figure 1 C is SDS-PAGE verification.

[0036] Figure 2 is the comparison of the titers of polyclonal IgE antibody and IgG antibody binding phage after three rounds of screening. Figure 2 A is IgE antibody, Figure 2 B is IgG antibody.

[0037] Figure 3 is the binding ability of the target sequence of the screening to IgE antibody. Figure 3 A is Ara h 5 peptide segment, Figure 3 B is Ara h 8 peptide segment.

[0038] Figure 4 is the binding ability of the target sequence of the screening to IgG antibody. Figure 4 A is Ara h 5 peptide segment, Figure 4 B is Ara h 8 peptide segment.

[0039] Figure 5 is the positioning of the polypeptide sequence screened in the Ara h 5 protein structure (IgE).

[0040] Figure 6 is the positioning of the polypeptide sequence screened in the Ara h 8 protein structure (IgE).

[0041] Figure 7 is the positioning of the polypeptide sequence screened in the Ara h 5 protein structure (IgG).

[0042] Figure 8 is the positioning of the polypeptide sequence screened in the Ara h 8 protein structure (IgG). DETAILED DESCRIPTION

[0043] Objects, advantages, and features of the present application will become apparent to those skilled in the art from the following detailed description in conjunction with the exemplary embodiments. It is to be understood, however, that the description is carried out only by way of example and not limitation. The essentials of the specification are merely to help the skilled in the art to understand the specific details of the present application.

[0044] Example 1 Preparation of cloned antibodies 1. Immunization of animals (1) The amino acid sequences of peanut Ara h 5 and Ara h 8 proteins were obtained from the NCBI database, and the codons were optimized to the dominant codons of E. coli. The gene sequence was synthesized and expressed in E. coli. The purified recombinant protein was used as the immunogen of recombinant allergen Ara h 5 and Ara h 8 protein. The 6-8-week-old BALB / c mice were immunized with the recombinant protein dissolved in PBS and mixed with an equal volume of complete Freund's adjuvant (the final protein concentration was 100 μg / mL). 50 μL of the immunogen was injected subcutaneously on the back of the neck of female BALB / c mice.

[0045] (2) After the first injection, 3 booster immunizations were performed every other week with an equal amount of immunogen mixed with incomplete Freund's adjuvant. Both the first immunization and the booster immunization were performed by subcutaneous injection, and attention was paid to evenly distribute the injection on the back of the neck of the mice to avoid skin ulceration caused by concentrated immunization.

[0046] (3) Three days after the fourth immunization, the blood was collected from the orbit for ELISA determination. If the antibody titer was qualified, the mice were sacrificed, and the serum was collected for antibody purification.

[0047] Note: The experiment was approved by the China Agricultural University Experimental Animal Welfare and Animal Experiment Ethics Review Committee.

[0048] 2. Antibody purification experiment (1) The serum antibodies were purified by HiTrap™ Protein G HP column. Before loading the protein on the column, the column was washed with 10 column volumes of binding buffer (20 mM sodium phosphate, pH 7.0) at a speed of 1 mL / min.

[0049] (2) The collected serum was diluted 1:5 (diluent 20 mM sodium phosphate, pH 7.0) and loaded on the column. The serum protein was loaded on the column at a speed of 1 mL / min, and 5 column volumes of binding buffer were used for washing until the baseline was stable.

[0050] (3) Elution was performed with 5 times the column volume of elution buffer (0.1 M glycine-HCl, pH 2.7) until the baseline was stable. The eluted collection tube was added with 1 M Tris-HCl, pH 9.0 neutralization liquid (added according to 10:1).

[0051] (4) The specific elution liquid was replaced with a dialysis bag to change the buffer to 0.01 mol / L PBS, and SDS-PAGE was performed to identify the purified antibody. Figure 1 )。

[0052] 3. ELISA assay of antibody titer (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.

[0053] (2) After blocking and washing, IgG antibodies were diluted according to 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, IgE antibodies were diluted according to 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, and the control group normal serum was diluted at the same ratio, and was added to the 96-well plate and incubated at 37°C for 2 h.

[0054] (3) After washing, the corresponding secondary antibody was added and incubated for 1 h, 100 μL of HRP-labeled rabbit anti-mouse IgG (1:10000) was added for IgG antibodies, and 100 μL of HRP-labeled rat anti-mouse IgE (1:2000) was added for IgE antibodies.

[0055] (4) After washing, TMB was added for color development, and the absorbance value of each well at 450 nm was determined using a multifunctional enzyme marker.

[0056] 4. Dot immunoblotting method for determining titer (1) 1.5 μl of 2 μg / mL Ara h 5 and Ara h 8 proteins were taken on an NC membrane and blocked for 1 h.

[0057] (2) IgG antibodies were diluted at 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000 and incubated for 2 h.

[0058] (3) Incubate IgE antibody diluted 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280 at 4°C overnight.

[0059] (4) After washing, incubate corresponding secondary antibody rabbit anti-mouse IgG (1:10000) or rat anti-mouse IgE (1:2000) at room temperature for 1 h.

[0060] (5) After washing, add ECL chemiluminescence reagent for development and exposure for analysis.

[0061] Example 2 Screening of allergen epitopes by phage display technology targeting antibodies 1. Resuscitation and culture of phage host bacteria ER2738 (1) Use a sterile inoculation loop to take a small amount of E. coli ER2738 glycerol stock, streak it on LB Tet+ plates, and incubate at 37°C overnight. Tet is tetracycline.

[0062] (2) Pick a single colony in LB Tet+ medium, and incubate at 37°C overnight with shaking to keep the bacteria. LB Tet+ plates and bacterial liquid are stored at 4°C for no more than 3 weeks.

[0063] 2. Biological panning of phage peptide library (1) Coating: Dilute the IgG and IgE polyclonal antibodies (100 µg / mL) obtained in Example 1 with coating solution (0.1 M NaHCO3 (pH 8.6)) to coat the enzyme- labeled plates, 100 µL / well, numbered A for positive selection, and incubate at 4°C overnight. In addition, dilute normal mouse serum 5 times with coating solution to coat the enzyme-labeled plates, 100 µL / well, numbered B for negative selection, and incubate at 4°C overnight.

[0064] (2) Blocking: Discard the coating solution on each plate, then tap the front of each plate firmly on a clean paper towel to remove residual solution. Fill each plate or well completely with blocking buffer. Incubate at 4°C for 2 hours.

[0065] (3) Binding: Aspirate the blocking solution from the B-panelling wells, wash thoroughly 6 times with TBST (TBS + 0.1% [v / v] Tween-20), and tap out the wash solution. Add 100 μL of the random 7-mer phage library solution (1011phage, original phage library 1013phage / mL, Ph.D.-7 Phage Display Peptide Library Kit, New England Biolabs, Cat#E8100S) diluted with TBST to each well of the B-panelling, and slowly shake at room temperature for 1 hour. Aspirate the blocking solution from the A-panelling wells, wash thoroughly 6 times with TBST (TBS + 0.1% [v / v] Tween-20), and tap out the wash solution. Add the phage solution from the B-panelling to the A-panelling, and slowly shake at room temperature for 1 hour.

[0066] (4) Elution: Aspirate the phage solution from the A-panelling, wash thoroughly 6 times with TBST (TBS + 0.1% [v / v] Tween-20), and tap out the wash solution each time using a clean paper towel to prevent cross-contamination. This is to remove the non-specifically bound phage.

[0067] (5) Extraction: Add 100 μL of the extraction solution (0.2 mol / L glycine- hydrochloric acid (pH 2.2), 1 mg / mL BSA filtered and sterilized) to each well, and slowly shake at room temperature for 15 minutes.

[0068] (6) Neutralization: Collect the solution into a small tube previously added with 15 μL of the stop solution (1 mol / L Tris-HCl, pH 9.1), and mix well to neutralize.

[0069] (7) Titering: Dilute the phage with sterilized TBST solution, and add 10 μL to 200 μL of fresh ER2738 bacterial solution cultured to the logarithmic phase (OD600 value of 0.5), mix well, and then incubate at room temperature for 5 minutes. Add 3 mL of preheated LB-TOP top agar (preheated in a 45°C water bath) to the mixture, mix well, and then pour onto an LB solid plate containing X-Gal / IPTG. Incubate at 37°C overnight, and count the plaques the next day.

[0070] Titering formula: pfu (plague forming unit) / mL = plaque number x dilution factor x total volume (mL).

[0071] (8) Amplification: The remaining eluted phage solution is amplified the next day, and the overnight culture in LB medium is used to inoculate 100 mL of fresh LB medium, and incubate at 37°C until the OD600 value reaches 0.5. Add 100 μL of the eluted phage solution to the culture, and incubate at 37°C for 4 hours. Tet+The ER2738 bacterial broth grown in culture medium was diluted 1:100 into 20 mL of LB medium and the remaining eluate was added to a 250 mL Erlenmeyer flask. The flask was incubated at 37°C with 220 rpm 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 re-spun (pellet discarded). The top 80% of the supernatant was transferred to a sterile new test tube and 1 / 6 volume of 20% PEG / 2.5 M NaCl was added. The phage was allowed to precipitate overnight at 4°C. The next day the phage was pelleted by centrifugation at 12,000 g for 15 minutes at 4°C, the supernatant was discarded, the tube was briefly re-spun, and the remaining supernatant was removed with a pipette. The pellet was suspended with 1 mL of TBS. The suspension was transferred to a sterile microfuge tube and centrifuged at 14,000 rpm for 5 minutes at 4°C to pellet the remaining cells. The supernatant was transferred to a new microfuge tube and re-precipitated by adding 1 / 6 volume of 20% PEG / 2.5 M NaCl. The mixture was incubated on ice for 60 minutes. The mixture was centrifuged at 14,000 rpm for 10 minutes at 4°C, the supernatant was discarded, the tube was briefly re-spun, and the remaining supernatant was removed with a micropipette. The pellet was resuspended with 200 μL of TBS solution. The amplified phage library was ready for use.

[0072] (9) Repeat selection: The second and third rounds of selection were repeated, except that the concentration of TBST used for washing was increased (second round: 0.5% TBST; third round: 1% TBST) and the number of washes was increased (second round: 10 times; third round: 15 times). The remaining steps were the same as the first round of selection. The eluate was tested for phage titer, and the plaques from the last round of selection were retained.

[0073] After three rounds of biopanning (as shown in Tables 1 and 2 below), phage that bound to IgE antibodies and IgG antibodies against Ara h 5 and Ara h 8 proteins were obtained, respectively, as shown in Table 3. Figure 2

[0074] Table 1. Conditions and results of biopanning of phage display peptide library with IgE antibodies as target

[0075] Table 2. Conditions and results of biopanning of phage display peptide library with IgG antibodies as target

[0076] Example 3. Amplification and sequencing of monoclonal phage and verification of binding properties ​1. Amplification of monoclonal phage and DNA sequencing (1) Using a pipette tip, pick isolated blue patches from the titration plate, ensuring that each patch contains one DNA sequence. Then transfer to the ER2738 bacterial liquid which is diluted according to 1:100 of the overnight culture. Incubate the test tube at 37°C for 4.5 hours with shaking.

[0077] (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 re-spin. Using a pipette, transfer the upper 80% of the supernatant to a new tube. This is the amplified phage stock.

[0078] (3) Transfer 500 μL of supernatant containing phage to a new microcentrifuge tube. Add 200 μL of 20% PEG / 2.5 M NaCl, mix by inverting several times, and let stand at room temperature for 20 minutes. Centrifuge at 14,000 rpm for 10 minutes at 4°C, discard the supernatant. Briefly re-spin. Carefully aspirate and discard any remaining supernatant.

[0079] (4) Knock the tube to completely suspend the pellet in 100 μL of iodide buffer. Add 250 μL of ethanol and incubate at room temperature for 15 minutes. Centrifuge at 14,000 rpm for 10 minutes at 4°C in a centrifuge, discard the supernatant.

[0080] (5) Wash the pellet with 0.5 mL of 70% ethanol (stored at -20°C), re-centrifuge, discard the supernatant, and briefly dry the pellet under vacuum. Suspend the pellet in 30 μL of sterile water. Send to Suzhou Jinweizhi Biotechnology Co., Ltd., and the sequencing primer is -96 gIII universal primer.

[0081] Through biotagging of the phage random peptide library, positive monoclonal phage capable of specifically binding to IgE antibodies and IgG antibodies against Ara h 5 and Ara h 8 proteins were screened, and 20 positive monoclonal phage were picked for DNA sequencing. As a result, 16 polypeptide sequences capable of binding to IgE antibodies against Ara h 5 protein, 15 polypeptide sequences capable of binding to IgE antibodies against Ara h 8 protein, and 20 polypeptide sequences capable of binding to IgG antibodies against Ara h 5 protein and Ara h 8 protein were obtained.

[0082] For the IgE-binding positive monoclonal phage-displayed polypeptide sequences against Ara h 5 protein, a total of 3 binding peptides were screened. The most frequent one was FHWWYLK with a frequency of 11 / 16. For the IgE-binding positive monoclonal phage-displayed polypeptide sequences against Ara h 8 protein, a total of 5 binding peptides were screened. The most frequent ones were FPYMKFR and FPYMKFV with a frequency of 6 / 15 and 5 / 15, respectively.

[0083] In addition, for the IgG-binding positive monoclonal phage-displayed polypeptide sequences against Ara h 5 protein, a total of 5 binding peptides were screened. The most frequent ones were FLHRDND and WETIYSR with a frequency of 6 / 20 and 5 / 20, respectively. And WETIYSR was the same as the sequence screened by IgE. For the IgG-binding positive monoclonal phage-displayed polypeptide sequences against Ara h 8 protein, a total of 6 binding peptides were screened. The most frequent ones were FPYMKFV and FPYMKFR with a frequency of 7 / 20 and 6 / 20, respectively. The same as the polypeptide sequences displayed by the IgE-binding positive monoclonal phage against Ara h 8 protein.

[0084] 2. ELISA verification of the binding activity of the screened sequences to antibodies (1) Add 5 μL of amplified phage stock to 20 mL of culture and incubate at 37°C with vigorous shaking for 4.5 hours. Transfer the culture to a centrifuge tube and centrifuge at 12,000 g for 10 minutes at 4°C. Transfer the supernatant to a new tube and re-spin, discarding the pellet. Transfer 80% of the supernatant to a new tube, add 1 / 6 volume of 20% PEG / 2.5 M NaCl, and precipitate overnight at 4°C. Centrifuge the precipitate at 12,000 g for 15 minutes at 4°C. Discard the supernatant, re-spin briefly, and then remove the remaining supernatant with a pipette. Resuspend the pellet in 1 mL of TBS. Transfer the suspension to a microcentrifuge tube and spin at 14,000 rpm for 5 minutes at 4°C to pellet the remaining cells. Transfer the supernatant to a new microcentrifuge tube and re-precipitate with 1 / 6 volume of 20% PEG / 2.5 M NaCl. Incubate on ice for 6 minutes. Microcentrifuge at 14,000 rpm for 10 minutes at 4°C. Discard the supernatant, re-spin briefly, and then remove the remaining supernatant with a micropipette. Resuspend the pellet in 50 μL of TBS and determine the titer.

[0085] (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 to each well. 5 Bacteriophages. Incubated at room temperature for 2 hours. Unselected bacteriophages were used as a control group.

[0086] (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 1 M hydrochloric acid to each well and measure the absorbance at 450 nm using a microplate reader.

[0087] 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. 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.

[0088] Example 4: Conformational Epitope Localization and Analysis The amino acid sequences bound to IgE antibodies and IgG antibodies against Ara h 5 protein and Ara h 8 protein screened by phage random peptide library were respectively aligned with the three-dimensional structure of Ara h 5 protein and Ara h 8 protein by Pepitope method.

[0089] The results are shown in Table 1. Figures 5-8 As shown in Table 1, for screening the IgE conformational epitope sequence of potential Ara h 5, the matching degree of sequence WETIYSR and sequence FHWWYLK in the spatial structure is the highest, and they are in a cluster of aggregation in the spatial structure. For screening the IgE conformational epitope sequence of potential Ara h 8, the sequences with the highest matching degree are FPYMKFV and FPYMKFR, while sequence HTYSSTT does not correspond in the protein spatial structure, and there is only one positive clone, which may be a false positive sequence. For screening the IgG conformational epitope sequence of potential Ara h 5, the matching degree of sequences FLHRDND, WETIYSR, YSFTLHA and FHWWYLK in the spatial structure is the highest. For screening the IgG conformational epitope sequence of potential Ara h 8, the sequences with the highest matching degree are FPYMKFV and FPYMKFR.

[0090] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the technical principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A peanut allergen polypeptide, characterized in that, The polypeptides are derived from the Ara h 8 protein and have the sequences FPYMKFR, SFHWWLF, FPYMKFV and / or SMFARID.

2. Use of a peanut allergen polypeptide according to claim 1 as an antigen, hapten, immunogen or immunomodulator.

3. Anti-peanut allergy product prepared from a peanut allergen polypeptide according to claim 1.

4. The anti-peanut allergy product as described in claim 3, characterized in that, The anti-peanut allergy product is a vaccine and / or a medicament.

5. The anti-peanut allergy product as described in claim 4, characterized in that, The product comprises an adjuvant in the form of a liposome.

6. An antibody, characterized in that, The antibodies are obtained by target screening using a peanut allergen polypeptide according to claim 1.

7. The antibody of claim 6, wherein The antibodies are one of a polyclonal antibody, a monoclonal antibody or a chimeric antibody or a humanized modified antibody.

8. Use of an anti-peanut allergy product according to any one of claims 3-5, characterized in that, The degree of tolerance of the body to a specific allergen is increased, preferably the specific allergen is peanut or a peanut product, preferably the peanut product is a food product.

9. Use of an anti-arachis allergen product according to any one of claims 3 to 5 for the manufacture of a medicament for reducing the sensitivity of the body to allergens, characterised in that, The allergen is a peanut protein.

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