Sesame allergen Ses i 2-specific binding polypeptide and screening method thereof

Abstract

The present invention relates to a Ses i 2-specific binding polypeptide of sesame allergen and a screening method thereof. The specific binding polypeptide includes the polypeptides shown in SEQ ID NO.1-5. The method includes the following steps: (I) Activate and culture Escherichia coli strain 2738, add phages with gradient dilution to obtain a recombinant phage library; (II) Perform three rounds of panning with Ses i 2 protein as the target, amplify the bound target phages, sequence them with 96gIII sequencing primers, and obtain a polypeptide specifically binding to Ses i 2 protein after translation; (III) Synthesize the polypeptide obtained in the above steps and verify the affinity by bio-layer interferometry. The screening method uses a phage library to screen for peptide aptamers of sesame protein Sesi 2, which have high affinity and specificity and can be used for the detection of Ses i 2 protein.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a specific binding polypeptide, i.e., a peptide aptamer, that specifically recognizes the allergen protein Ses i 2, and in particular, a method for screening a peptide aptamer of the allergen protein Ses i 2 based on phage display technology. Background Art

[0002] Sesame (Sesamum indicum L.) is a global agricultural crop with high oil, protein, and bioactive ingredient contents, and has effects such as regulating the stomach and intestines, moisturizing the skin, and anti-aging.

[0003] In the Uniprot protein database, Ses i 2 is named as a 2S seed storage protein, also known as 2S storage albumin and β-globulin, belonging to the prolamin superfamily, containing 148 amino acids, and having a relative molecular mass of 7×103. Related research indicates that Ses i 2 can be recognized by the sera of 80% of sesame-allergic patients and is one of the main allergens of sesame. Ses i 2 has the highest homology with cashew Ana o 3, hazelnut Cor a 14.0101, and Jug r 1, followed by peanut Ara h 2, Ara h 6, and Ara h7, making it possible for Sesi 2 to have clinical cross-allergic reactions with other nuts. Sesi 2 has strong resistance to digestion by pepsin, trypsin, and chymotrypsin in its natural state, and there are no glycosylation modification sites in its amino acid sequence. Due to the strong anti-digestibility of Sesi 2 and the high possibility of clinical cross-allergic reactions, Ses i 2 has a strong sensitization risk.

[0004] Peptide aptamers are extremely small and simple molecules composed of 5-20 amino acid residues. They are small in size and are usually embedded as a loop in a stable protein scaffold. They have good stability and affinity, low production costs, and can be obtained in large quantities through chemical synthesis or bacterial expression. Due to these advantages, peptide aptamers have received extensive attention in the past decade and have been applied to the fields of immunoassay, theranostics, and biosensors. In this application, peptide aptamers are also referred to as specific binding polypeptides.

[0005] The peptide phage library of phage display screening technology has a large capacity, containing billions of variants, and can interact with target molecules such as proteins, cells, toxins, and metals. The polypeptides or proteins displayed can maintain their relative spatial structures and biological activities, and it is an effective technology for screening, identifying, and isolating affinity peptides. After 3-5 rounds of biological screening, peptides with the highest affinity for the target molecule are enriched.

[0006] Although peptide aptamers have been obtained through phage display technology in the prior art, there is no report on screening peptide aptamers against sesame allergen proteins. It is of great significance to screen Sesi 2 peptide aptamers with high affinity and specificity through phage display technology and apply them to the detection of allergen proteins. Summary of the Invention

[0007] In view of the deficiencies of the prior art, the present invention provides a peptide aptamer that specifically recognizes the allergen protein Ses i 2, and the peptide aptamer has high affinity and high specificity.

[0008] The present invention provides a method for screening a peptide aptamer that specifically recognizes the allergen protein Ses i 2. The method uses the sesame allergen Ses i 2 protein as the target protein and a 12-mer phage display library for multiple rounds of screening. The enrichment effect is evaluated by detecting the titer of phages in the eluate of each round until obvious enrichment occurs. The advantages of this method are that the peptide phage library has a large capacity, containing billions of variants, high screening efficiency, and the displayed polypeptides can maintain their relative spatial structures and biological activities.

[0009] The object of the present invention is to provide a peptide segment that specifically binds to the sesame allergen protein Ses i 2 and a method for screening the same.

[0010] In view of the above object of the invention, the present invention provides the following technical solutions:

[0011] The first aspect of the present invention provides a method for screening polypeptides that specifically bind to Ses i 2 protein using a phage display library, and the method includes the following steps:

[0012] (1) Synthesize recombinant sesame allergen protein Ses i 2;

[0013] (2) Culture host bacteria 2738;

[0014] (3) Screen the peptide phage display library;

[0015] (4) Detect the phage titer;

[0016] (5) Enrich and purify phage particles;

[0017] (6) Further enrich phage particles and determine the titer;

[0018] (7) Determine the surface peptide sequence of positive phages and synthesize peptide aptamers;

[0019] (8) Determine the affinity of peptide aptamers.

[0020] The peptide phage library is: a prefabricated 12-mer phage display library, which is purchased from Kamede Biotech Company.

[0021] Specifically, the method includes the following steps:

[0022] (1) Express the Ses i 2 protein through genetic recombination technology;

[0023] (2) Activate the 2738 host bacteria, and culture a small amount of the activated 2738 host bacteria overnight;

[0024] (3) Peptide phage display library screening: Coat the Ses i 2 protein antigen on an ELISA plate, block, wash, coat the mother liquor of the prefabricated peptide phage display library, wash, and finally elute the product to obtain the phages that bind to the Ses i 2 protein;

[0025] (4) Phage titer detection: Culture the 2738 host bacteria monoclonal to the logarithmic phase, dilute the phages obtained in step (3) and mix them with the 2738 host bacteria, let them stand for infection, pour the plate and culture overnight, count the plaques and calculate the titer;

[0026] (5) Phage particle enrichment and purification: Activate the 2738 host bacteria monoclonal, prepare the bacterial solution infected with the phages in step (4), add LB liquid medium, culture in a shaker, and separate by centrifugation and precipitation to obtain the amplified first-round enrichment product;

[0027] (6) Further enrichment and titer determination of phage particles: After diluting the first-round enrichment product, add the 2738 bacterial solution with OD600 = 0.3 - 0.4, infect in a shaker, then pour the plate in the dark, observe the next day to see if blue plaques grow, repeat the above steps twice to complete three rounds of panning, and effectively enrich the phages that specifically bind to the Ses i 2 protein;

[0028] (7) Determine the surface peptide sequence of the positive phage and synthesize the peptide aptamer: Sequence the positive phage clone, translate the sequencing result into an amino acid sequence, and synthesize the peptide aptamer with the corresponding polypeptide sequence;

[0029] (8) Determine the affinity of the peptide aptamer, and verify the affinity between the polypeptide and the Ses i 2 protein through the biofilm interference technology: Specifically capture the Ses i 2 protein through the NTA chip, dilute the polypeptide obtained in step (7) with PBST + 5% DMSO buffer, add it to a 96-well plate, and perform the affinity test after setting the program;

[0030] The peptide phage library is: a prefabricated 12-peptide phage display library purchased from KMD Biotech Co., Ltd. For the method as described above, preferably, the expression of the Ses i 2 protein in step (1) includes the following steps:

[0031] Insert the gene of Ses i 2 into the expression vector pET28a through the cloning sites BamHI (GGATCC)-XhoI (CTCGAG), and introduce it into the Rosetta strain;

[0032] Inoculate the preserved bacteria and culture until the OD600 of the bacteria is 0.6 - 0.8. Add isopropyl-β-D-1-thiogalactoside to the culture and then culture to induce the expression of the fusion protein. Harvest the bacteria and prepare samples for SDS-PAGE analysis;

[0033] The strain is preferably Rosetta(DE3) E.coli Strain.

[0034] Preferably, the activation of the 2738 host bacteria in step (2) includes the following steps:

[0035] Take two portions of LB liquid medium, both add tetracycline. Add 2738 glycerol bacteria to one portion, and use the other portion as a blank control. Culture in a shaker at 37°C and 210 rpm for 4 h;

[0036] Take a tetracycline plate, dip a small amount of activated 2738 glycerol bacteria, streak on the tetracycline plate, and place it in an incubator at 37°C to culture overnight;

[0037] The concentration of tetracycline is preferably 500 mg / mL.

[0038] Preferably, the screening of the peptide phage display library in step (3) includes the following steps:

[0039] Dilute the Ses i 2 protein with PBS. Take 4 wells and coat 5 μg / well on the ELISA plate. Coat overnight at 4°C or for 2 h at 37°C, and discard the residual solution;

[0040] Add the blocking solution, place at room temperature for 1 h, and wash the plate quickly;

[0041] Take 9 μL of 0.01 M PBS buffer and mix it with 1 μL of the peptide phage display library mother liquor, and add it to the above-mentioned washed wells. Shake at room temperature for 1 h;

[0042] Transfer the liquid in the wells of the ELISA plate coated with the peptide phage display library mother liquor to a centrifuge tube, and wash the plate;

[0043] Add the elution buffer to the above wells, shake at room temperature for 10 min, transfer the elution buffer to the centrifuge tube, and add Tris-Hcl with a pH of 8.0 to neutralize the eluate, which is the first-round elution product, that is, the phage that binds to the Ses i 2 protein, and store it at 4°C; The elution buffer is preferably Tris-Hcl with a pH of 3.0.

[0044] Preferably, the phage titer detection in step (4) includes the following steps:

[0045] Inoculate monoclonal 2738 host bacteria into low-salt LB medium, and culture at 37 °C and 250 rpm until the logarithmic phase, i.e., OD600 = 0.4 - 0.6;

[0046] Take the phage liquid to be detected, i.e., the first-round eluate obtained in step (3), dilute it 10-fold with LB medium, take a sample of an appropriate dilution, add an equal amount of 2738 host bacteria in the logarithmic growth phase, and mix well. Incubate statically at 37 °C for 1.5 h;

[0047] Add low-salt LB upper gel containing IPTG and X-gal to the infected bacterial liquid, mix well, pour it onto the plate of LB lower gel, and place it in an incubator at 37 °C to culture overnight;

[0048] Finally, count the plaques and calculate the titer.

[0049] Preferably, the enrichment and purification of phage particles in step (5) include the following steps:

[0050] Prepare LB liquid medium, add 100 mg / mL tetracycline with a volume of one-thousandth of the liquid medium and a single colony on the 2738 host bacteria plate, and culture in a shaker at 37 °C and 210 rpm for 4 h for activation;

[0051] Mix 1 μL of the first-round eluate with 10 μL of the activated 2738 host bacteria, and place at 37 °C for 1.5 h;

[0052] Add the infected bacterial liquid into LB liquid medium, and culture in a shaker at 37 °C and 250 rpm for 4.5 h;

[0053] Take out the medium, collect the amplified phages, i.e., the first-round enrichment product, and store at 4 °C.

[0054] Preferably, the further enrichment and titer determination of phage particles in step (6) include the following steps:

[0055] Prepare low-salt LB liquid medium, add 3 μL of 50 mg / mL tetracycline and a single colony picked from the streaked plate of 2738 host bacteria, and culture in a shaker at 37 °C and 250 rpm until OD600 = 0.3 - 0.4;

[0056] Add 1 μL of the first-round enrichment product to 9 μL of low-salt LB liquid medium for dilution;

[0057] The lower layer of glue is placed in the dark and preheated at 37°C. The 2738 bacterial solution with OD600 = 0.3 - 0.4 is mixed with an equal volume of diluted enriched product, and they are infected in a shaker at 37°C and 150 rpm for 1.5 h. Then, the plate is poured, placed upside down in an incubator at 37°C, and cultured in the dark overnight. Observe the next day to see if blue spots appear;

[0058] Repeat the above steps to complete three rounds of screening, and perform titer determination to enrich phages that specifically bind to the Ses i 2 protein.

[0059] Preferably, the determination of the surface peptide sequence of the positive phage and the synthesis of the peptide aptamer in step (7) include the following steps:

[0060] Perform nucleotide sequencing on positive phage clones with a positive-to-negative ratio greater than 2.1, translate the nucleotide sequence into a polypeptide sequence, and synthesize corresponding polypeptide fragments according to the polypeptide sequence.

[0061] Preferably, the determination of the specificity of the peptide aptamer in step (9) includes the following steps:

[0062] Select 5 peptides with the highest affinity for Ses i 2, dilute them with PBST + 5% DMSO buffer to 1 mM, specifically capture the sesame allergens Ses i 1, Ses i 2, Ses i 3, Ses i 4, Ses i 5, Ses i 6, Ses i 7 and the cross-allergens peanut and Brazil nut proteins through an NTA chip. Dilute the proteins to 20 μg / ml, add the samples to a 96-well plate, and perform an affinity test after setting the program to screen for polypeptides that specifically bind to Ses i 2.

[0063] Preferably, the polypeptide used for verifying the biolayer interferometry technique in step (8) is selected from one or more of the following peptide segments: KLAGPDVTAKYV, DGRHDPTLFMTS, QSVFQQQHPQLT, QSTDHARLFRTG, and LVRYSIFPGGTD.

[0064] More preferably, the polypeptide screened in step (8) is selected from one or more of the following peptide segments: KLAGPDVTAKYV, QSVFQQQHPQLT, QSTDHARLFRTG.

[0065] Further preferably, the polypeptide screened in step (8) is KLAGPDVTAKYV.

[0066] The second aspect of the present invention provides the application of a polypeptide that specifically binds to the Ses i 2 protein in the detection of the Ses i 2 protein. The polypeptide is selected from one or more of the following peptide segments: KLAGPDVTAKYV, DGRHDPTLFMTS, QSVFQQQHPQLT, QSTDHARLFRTG, and LVRYSIFPGGTD. Preferably, the polypeptide is selected from one or more of the following peptide segments: KLAGPDVTAKYV, DGRHDPTLFMTS, QSVFQQQHPQLT, QSTDHARLFRTG.

[0067] More preferably, the polypeptide is selected from one or more of the following peptide segments: KLAGPDVTAKYV, QSVFQQQHPQLT, QSTDHARLFRTG.

[0068] Even more preferably, the polypeptide is KLAGPDVTAKYV.

[0069] Preferably, the detection of the Ses i 2 protein is for non-diagnostic purposes.

[0070] The present invention has the following beneficial effects:

[0071] In the present invention, phages are used to infect Escherichia coli 2738 to form recombinant phages containing polypeptide library information. After three rounds of panning of this polypeptide library with the Ses i 2 protein, and amplification and sequencing of the target phages, polypeptide sequences that specifically bind to the Sesi 2 protein are obtained after translation. After synthesizing the polypeptide sequences, the affinity is verified by the biofilm interference technique. The polypeptides screened in the present invention can specifically recognize the Ses i 2 protein and have extremely high application value in the detection of the Ses i 2 protein.

[0072] The present invention screens allergen protein Ses i 2 peptide aptamers through phage display technology. As the number of panning rounds increases, peptide aptamers with high affinity are enriched. After sequencing and translation, the amino acid sequences of peptide aptamers with high affinity and high specificity for the target protein can be obtained. It can be used for detecting the allergen protein Ses i 2 and for the purpose of preventing sesame allergy.

[0073] The present invention also synthesizes the sesame protein Ses i 2 with immunological activity by means of prokaryotic expression, and characterizes its molecular weight by SDS-PAGE. Description of the Drawings

[0074] Figure 1 It is the titer determination of the three-round phage display library screening process in Example 1 of the present invention;

[0075] Figure 2This is for measuring the affinity between the peptide segment and Ses i 2 protein in Example 1 of the present invention by BLI; Detailed implementation mode

[0076] Key biological materials:

[0077] Host strain 2738 (Tianjin Carmed Biotech Co., Ltd.).

[0078] Pre-prepared 12-mer phage display library (Tianjin Carmed Biotech Co., Ltd.).

[0079] The present invention will be further described by way of examples, but the present invention is not limited to the following examples.

[0080] Example 1 Screening for polypeptides specifically binding to sesame allergen Ses i 2 using a phage display library (I) Expression of Sesi 2 protein

[0081] Insert the gene of Ses i 2 into the expression vector pET28a through the cloning sites BamHI (GGATCC)-XhoI (CTCGAG), and introduce it into the Rosetta (DE3) E. coli Strain; inoculate the preserved bacteria and culture until the OD600 of the bacteria is 0.6 - 0.8, add isopropyl-β-D-1-thiogalactoside (IPTG) to the culture, and then culture to induce the expression of the fusion protein. Harvest the bacteria and prepare samples for SDS-PAGE analysis to identify the obtained Ses i 2 protein.

[0082] (II) Culturing and activating host strain 2738

[0083] (1) Activation of host strain 2738: Take two centrifuge tubes, pour 3 mL of LB liquid medium, add 3 μL of 500 mg / mL tetracycline. Add 20 μL of 2738 glycerol bacteria to one tube, and use the other tube as a blank control. Culture in a shaker at 37 °C and 210 rpm for 4 h;

[0084] (2) Take a tetracycline plate, dip a small amount of the activated 2738, streak it on the tetracycline plate, and place it in an incubator at 37 °C to culture overnight.

[0085] (III) Screening of the peptide phage display library

[0086] (1) Antigen coating: Use PBS as the coating buffer, dilute the Ses i 2 protein, and coat 5 μg / well in 4 wells on the enzyme-linked immunosorbent assay (ELISA) plate, coat overnight at 4 °C or for 2 h at 37 °C;

[0087] (2) Blocking: Invert the ELISA plate containing the protein on a clean paper towel, tap it forcefully to remove the residual solution, add 400 μL of blocking solution (such as 2% skim milk powder), and let it stand at room temperature for 1 h;

[0088] (3) Washing: Invert the ELISA plate filled with the blocking solution on a clean paper towel, flick it forcefully to remove the residual solution, then quickly wash the plate with 200 μL of 0.01 M PBST buffer, invert it on a clean paper towel, and flick it forcefully to remove the residual solution. Repeat this process 3 times.

[0089] (4) Coating the mother liquor of the prefabricated peptide phage display library: Mix 90 μL of 0.01 M PBS buffer and 10 μL of the mother liquor of the peptide phage display library, add the mixture to the washed wells, and shake it on an ELISA plate shaker at room temperature for 1 h.

[0090] (5) Washing: Use a pipette to aspirate the liquid in the wells of the ELISA plate coated with the mother liquor of the peptide phage display library, transfer it to a centrifuge tube, invert the ELISA plate on a clean paper towel, flick it forcefully to remove the residual solution, then quickly wash the plate with 200 μL of 0.01 M PBST buffer, and flick it forcefully to remove the residual solution. Repeat this process 5 times.

[0091] (6) Eluting the product: Add 200 μL of elution buffer (Tris-Hcl with pH 3.0) to the wells of the eluted ELISA plate, shake it at room temperature on an ELISA plate shaker for 10 min, transfer the elution buffer to a centrifuge tube, and add 15 μL of Tris-Hcl with pH 8.0 to neutralize the eluate. This is the first-round elution product, namely the phage that binds to the Ses i 2 protein, and store it at 4°C.

[0092] (IV) Phage titer detection

[0093] (1) Take two centrifuge tubes, pour 3 mL of low-salt LB medium into each tube. Inoculate one tube with the 2738 monoclonal and use the other tube as a blank control. Culture at 37°C and 250 rpm until the logarithmic phase (OD600 = 0.4 - 0.6).

[0094] (2) Phage dilution: Take 10 μL of the phage liquid to be detected, i.e., the first-round elution product obtained in step (III), dilute it in a 10-fold gradient with LB medium, take 200 μL of an appropriate dilution of the sample, add it to 200 μL of 2738 cells in the logarithmic growth phase, and mix well. Incubate statically at 37°C for 1.5 h.

[0095] (3) Pouring the plate: Add 3 - 4 mL of low-salt LB upper gel containing IPTG and X-gal to the infected bacterial liquid, mix well, pour it onto the LB lower gel plate, and place it in a 37°C incubator to culture overnight.

[0096] (5) Count the plaques and calculate the titer.

[0097] (V) Enrichment and purification of phage particles

[0098] (1) Activation of 2738: Take two centrifuge tubes, pour 3 mL of LB liquid medium into them, add 3 μL of 100 mg / mL tetracycline. Add a single colony picked from the 2738 plate to one of the tubes, and use the other tube as a blank control. Incubate them in a shaker at 37 °C and 210 rpm for 4 h;

[0099] (2) Infection: Take a centrifuge tube, add 200 μL of the activated 2738 host bacteria and 20 μL of the eluate from the first round, and place it at 37 °C for 1.5 h;

[0100] (3) Add the infected bacterial liquid into a 200 mL conical flask containing about 30 mL of LB liquid medium, and incubate it in a shaker at 37 °C and 250 rpm for 4.5 h (foam appears and does not dissipate in a short time);

[0101] (4) Take out the medium, transfer it to a centrifuge tube, and centrifuge at 6000 rpm for 10 min; Transfer the supernatant to another centrifuge tube, add 1 / 5 volume of PEG6000 + NaCl, seal it in a bag, and place it at 4 °C for precipitation overnight;

[0102] (5) Take out the centrifuge tube from 4 °C, centrifuge at 6000 rpm for 20 min; Discard the supernatant, and dissolve the precipitate with 1 mL of PBS;

[0103] (6) Centrifuge at 12000 rpm for 5 min, transfer the supernatant to another centrifuge tube, add 1 / 5 volume of PEG6000 + NaCl, seal it in a bag, and precipitate at 4 °C for 1 h;

[0104] (7) Centrifuge at 12000 rpm for 10 min, discard the supernatant, and dissolve the precipitate with 200 μL of PBS;

[0105] (8) Centrifuge at 12000 rpm for 10 min, transfer the supernatant to another centrifuge tube, which is the enriched product of the first round, and store it at 4 °C.

[0106] (6) Further enrichment of phage particles and titer determination

[0107] (1) Activation of 2738: Take two centrifuge tubes, pour 3 mL of LB liquid medium into them, add 3 μL of 50 mg / mL tetracycline. Add a single colony picked from the 2738 streaked plate to one of the tubes, and use the other tube as a blank control. Incubate them in a shaker at 37 °C and 250 rpm until OD600 = 0.3 - 0.4;

[0108] (2) Enrichment product dilution: Take sterile centrifuge tubes and add 180 μL of low-salt LB liquid medium to each tube. Add 20 μL of the enrichment product to the first tube and mix well. Take 20 μL from the first tube and add it to the second tube, mix well. Then take 20 μL from the second tube and add it to the third tube, mix well, and so on.

[0109] (3) Infection: Place the lower gel in a 37 °C dark place for preheating. Take sterile centrifuge tubes and add 200 μL of 2738 bacterial solution with OD600 = 0.3 - 0.4 and 200 μL of the diluted elution product respectively. Incubate in a shaker at 37 °C and 150 rpm for 1.5 h.

[0110] (4) Pouring the plate (operating in the dark): According to the preparation method of the upper gel, dissolve the low-salt LB solid medium (prepared with agarose), cool it to 60 °C, add X-gal with a final concentration of 40 μg / mL and IPTG with a final concentration of 1 mM, and mix well. At the same time, take a beaker or glass cylinder and add an appropriate amount of hot water. When the upper gel cools to about 37 °C and the hot water cools to about 45 °C, pour the bacterial solution in the 10 mL tube into 3 - 4 mL of the upper gel, quickly tap the bottom of the tube on the table to mix well, insert it into the 45 °C warm water, and mix at most 6 tubes each time. Then tap the bottom of the tube on the table again to mix well, quickly pour it onto the lower gel plate, place it upright in the dark for 10 min, then invert it and place it in a 37 °C incubator for overnight culture in the dark.

[0111] (5) Observe the next day to see if blue plaques appear.

[0112] (6) Repeat steps (1) - (5) to complete the subsequent experiments: the second round of elution → the second round of elution product titration → the second round of enrichment → the second round of enrichment product titration → the third round of elution → the third round of elution product titration. The titration of the elution products in the three rounds is as Figure 1 .

[0113] The results of the three rounds of biopanning are shown in Table 1. The phage elution titers in the first, second, and third rounds are 1.68×10 -3 , 8.8×10 -3 , 4×10 -2 respectively. The output of phages is relatively low, which is due to the elution of a large number of non-specific and relatively weak specific phages. With the progress of the biological screening, the recovery rate of phages gradually increases, and the enrichment efficiency increases from 1.68×10 -3 to 4×10 -2 , indicating that the peptide phage display library specifically binds to the Ses i 2 protein and is effectively enriched, and phages specifically binding to the Ses i2 protein are enriched.

[0114] Table 1 Results of three rounds of screening of the peptide phage display library against the Ses i 2 protein

[0115]

[0116] (VII) Sequence determination and peptide aptamer synthesis

[0117] (1) Pick multiple monoclonal phages from the plates after three rounds of screening and send them to Qingke Biotechnology for sequencing. The sequencing primer is the 96Ⅲ sequencing primer (5’---CCCTCATAGTTAGCGTAACG---3’), and the sequencing direction is reverse sequencing;

[0118] (2) Find the 36-gene sequence between two restriction enzyme sites and translate it into an amino acid sequence on the website https: / / web.expasy.org / translate / . The translation result from 3’-5’ is the candidate polypeptide sequence displayed by the phage (Table 2). Synthesize the polypeptide fragment of the corresponding peptide aptamer according to the polypeptide sequence.

[0119] Table 2 Candidate peptide sequences

[0120]

[0121] (VIII) Peptide aptamer affinity determination

[0122] Specifically capture Ses i 2 protein through an NTA chip. After the signal reaches 5.5 nm, bind it to Ses i 2-1, Ses i2-2, Ses i 2-3, Ses i 2-4, and Ses i 2-5 respectively.

[0123] (1) Dilute Ses i 2 protein and immobilize it on the NTA chip.

[0124] (2) Dilute the peptide segments Ses i 2-1, Ses i 2-2, Ses i 2-3, Ses i 2-4, and Ses i 2-5 with PBST + 5% DMSO buffer and add them to a 96-well plate.

[0125] (3) Program setting: baseline detection, binding, dissociation.

[0126] (4) Chip position: Place the chip in columns 8 and 9 and wet the chip.

[0127] (5) Run the program.

[0128] Preferably, it further includes step (IX) verifying the affinity between the polypeptide and Ses i 2 protein by bio-layer interferometry

[0129] Because the Ses i 2 protein is tagged with His, the Ses i 2 protein is specifically captured by the NTA chip. After the signal reaches 5.5 nm, it binds to Ses i 2-1, Ses i 2-2, Ses i 2-3, Ses i 2-4, and Ses i 2-5 respectively.

[0130] The method includes:

[0131] (1) Dilute the Ses i 2 protein to 20 μg / ml and solidify it for 600 s.

[0132] (2) Dilute the peptide segments Ses i 2-1, Ses i 2-2, Ses i 2-3, Ses i 2-4, and Ses i 2-5 with PBST + 5% DMSO buffer to 1 mM and add them to a 96-well plate. Among them: add PBST + 5% DMSO to columns 3 and 11; add the diluted Ses i 2-1, Ses i 2-2, and Ses i 2-3 to A4, B4, and C4 in sequence; add the diluted Ses i 2-4 and Ses i 2-5 to B5 and C5 in sequence; add PBST + 5% DMSO to D4 and D5 for 0-concentration deduction; add nickel chloride and glycine·HCl to columns 10 and 12 respectively for chip regeneration.

[0133] (3) Program setting

[0134]

[0135]

[0136] (4) Chip position: A total of 6 chips are used in this experiment and placed in columns 8 and 9. The chips are wetted 10 min before the start of the experiment. The wetting buffer is 1×PBS.

[0137] (5) Run the program.

[0138] After baseline acquisition, protein immobilization, binding, and dissociation, the binding data of the protein to 5 peptide segments are as Figure 2 . It can be seen from the dissociation binding curve that the binding signal of the Ses i 2-1 peptide segment is relatively strong and the dissociation is relatively slow, and the binding effect is good. The binding signal of the Ses i2-2 peptide segment is weak and the dissociation is fast, and the affinity is poor. The affinity constant KD value, association constant Kon, and dissociation constant Kdis of each peptide are shown in Table 3. Among them, the affinity of the Ses i 2-1 peptide segment reaches 5.01x10 -6 M, which is 2-5 orders of magnitude higher than the other 4 peptide segments, and the Ses i 2-1 peptide segment has the strongest affinity. Finally, the Ses i 2-1 peptide segment is selected as the aptamer of the sesame allergen Sesi 2 peptide.

[0139] Table 3 Affinity constant, binding constant and dissociation constant of peptide segments and Ses i 2 protein

[0140]

[0141] The above is only one of the embodiments of the present invention, and it is not intended to limit the present invention in other forms. Any person skilled in the art can use the disclosed technical content to make changes or modifications into equivalent embodiments with equivalent changes. However, any simple modification, equivalent change and improvement made to the above embodiments based on the technical essence of the present invention without departing from the technical solution content of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A polypeptide that specifically binds to the sesame allergen Ses i 2, characterized in that: The specifically binding polypeptide is: Ses i 2-1, with the sequence: KLAGPDVTAKYV.

2. A kit comprising the polypeptide according to claim 1.

3. Use of the kit according to claim 2 in the preparation of a product for detecting sesame allergen Ses i 2 protein.

4. Use of the polypeptide according to claim 1 in the preparation of a product for detecting sesame allergen Ses i 2 protein.

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