A method for screening β-lactoglobulin-specific binding polypeptides using a phage display library

Abstract

The present invention relates to a method for screening polypeptides specifically binding to β-lactoglobulin using a phage display library, and the method comprises the following steps: (1) after activating and culturing ER2738 strain, adding phages with gradient dilution to the host bacteria for expanded culture to obtain a recombinant phage library; (2) performing three rounds of panning on a random 12-mer peptide library using β-lactoglobulin as a bait, amplifying the target phages that bind, and sequencing with -96gIII sequencing primer, and obtaining polypeptides specifically binding to β-lactoglobulin after translation; (3) synthesizing the polypeptides obtained in the above steps, and verifying the affinity and specificity through indirect competitive ELISA. The polypeptides screened in the present invention can achieve specific recognition of β-lactoglobulin and have extremely high application value in the detection of β-lactoglobulin.

Description

Technical Field

[0001] The present invention relates to the field of food detection, and particularly to a method for screening β-lactoglobulin-specific binding polypeptides. Background Art

[0002] Cow's milk is one of the eight major food allergens determined by the Codex Alimentarius Commission (CAC). Allergic reactions caused by it are common in infants and young children. When the disease occurs, it can affect multiple systems throughout the body. In severe cases, it can lead to anaphylactic shock and even endanger life. Among cow's milk allergic people, 82% of allergic patients are allergic to cow's milk β-lactoglobulin.

[0003] For many years, β-lactoglobulin, as the main allergenic milk protein, has been the focus of researchers. Its molecule consists of 162 amino acid residues, with a molecular weight of 18.4 kDa, mainly existing in the form of a dimer. Its highly structured nature contributes to the characteristics of acid resistance and protease hydrolysis resistance. After being digested by the human body, intact β-lactoglobulin and peptide fragments still exist, triggering allergic reactions. Therefore, β-lactoglobulin can be used as an effective marker for detecting whether cow's milk protein is contained in food. In order to enable cow's milk allergic patients to avoid contact with cow's milk allergens as much as possible, the detection of β-lactoglobulin is particularly crucial. Screening β-lactoglobulin-specific binding polypeptides is of great significance for its rapid and accurate detection.

[0004] Phage display library technology has currently developed into a powerful tool for discovering new properties and changing the properties of existing polypeptides. It is simple to operate, relatively low in cost, has an ultra-large library capacity of 10 12-13 and is more likely to screen out polypeptide sequences that match the target substance. So far, there has been no report on screening polypeptides that specifically bind to β-lactoglobulin using phage display library technology. Summary of the Invention

[0005] The purpose of the present invention is to provide a peptide segment that specifically binds to β-lactoglobulin and a screening method therefor.

[0006] For the above invention purpose, the present invention provides the following technical solutions:

[0007] The first aspect of the present invention provides a method for screening polypeptides that specifically bind to β-lactoglobulin using a phage display library, and the method includes the following steps:

[0008] (1) After activating and culturing the ER2738 strain, adding gradient-diluted phages to the host bacteria in the enlarged culture to obtain a recombinant phage library;

[0009] (2) Using β-lactoglobulin as a bait, perform three rounds of panning on a random 12-mer peptide library, amplify the bound target phages, and sequence them with the -96gIII sequencing primer. After translation, obtain the polypeptides that specifically bind to β-lactoglobulin;

[0010] (3) Synthesize the polypeptides obtained in step (2), verify the affinity of the polypeptides for β-lactoglobulin and the binding specificity of the polypeptides to different proteins by indirect competitive ELISA, and screen out the polypeptides that specifically recognize β-lactoglobulin.

[0011] Preferably, step (1) includes the following steps:

[0012] Inoculate the ER2738 strain into LB-Tet medium and culture it overnight for activation; inoculate a single colony into LB-Tet medium and culture it overnight until the OD 600 is 0.4 - 0.6; according to the 10-fold serial dilution method, dilute the phages with LB medium. After equally dividing the host bacteria for large-scale culture as required, add the diluted gradient phages to each tube, mix and incubate; spread the infected host bacteria on a plate and culture it overnight; pick independent plaques, inoculate them into an agar plate and culture it overnight; add the overnight culture to LB liquid medium and culture it with shaking; centrifuge the above culture at high speed, collect the supernatant, add PEG / NaCl solution, mix well and let it stand overnight; centrifuge the above solution at high speed and collect the precipitate, resuspend it with sterile PBS solution to obtain the amplified phage culture.

[0013] More preferably, step (1) includes the following steps:

[0014] (1) Inoculate the ER2738 strain into LB-Tet medium and culture it overnight for activation; inoculate a single colony into LB-Tet medium and culture it overnight until the OD 600 is 0.4 - 0.6;

[0015] (2) Dissolve IPTG and Xgal in DMF for use; add agar to LB medium, and at the same time add the IPTG / Xgal stock solution to prepare a plate, and set it aside after cooling and solidifying;

[0016] (3) According to the 10-fold serial dilution method, dilute the phages with LB medium. After equally dividing the host bacteria for large-scale culture as required, add the diluted gradient phages to each tube, mix and incubate;

[0017] (4) Spread the infected host bacteria in step (3) on the plate prepared in step (2); culture it overnight, count the plaques the next day, and calculate the titer of M13K07 phage;

[0018] (5) Pick independent plaques and inoculate them into an agar plate for overnight culture;

[0019] (6) Add the overnight culture to LB liquid medium and shake culture;

[0020] (7) Centrifuge the above culture at high speed, collect the supernatant, add a PEG / NaCl solution with a volume of 1 / 5 of the supernatant volume, mix well and let it stand overnight;

[0021] (8) After centrifuging the above solution at high speed, collect the precipitate, resuspend it with sterile PBS solution, which is the amplified phage culture;

[0022] (9) Take the amplified phage culture to measure the titer, and save the remaining solution for future use.

[0023] Further preferably, the step (1) includes the following steps:

[0024] (1) Inoculate the ER2738 strain into LB-Tet medium and activate it by overnight culture at 37°C; inoculate single colonies into 5 mL of LB-Tet medium at a ratio of 1:100 and culture overnight at 37°C until the OD 600 reaches 0.4 - 0.6;

[0025] (2) Prepare an agar plate: Weigh 1.25 g of IPTG and 1 g of Xgal, dissolve them in 25 mL of DMF, store at -20°C for later use; add 15 g of agar to 1 L of LB medium, and at the same time add 1 mL of the IPTG / Xgal stock solution to prepare the plate. After cooling to room temperature and solidifying, store it at 4°C in the dark for later use;

[0026] (3) According to the 10-fold serial dilution method, dilute the phage with LB medium. After equally dividing the host bacteria for large-scale culture as required, add 10 μL of the serially diluted phage to each tube, mix gently and incubate at room temperature for 30 min;

[0027] (4) Spread the infected host bacteria in step (3) on the plate prepared in step (2); culture overnight at 37°C, count the plaques the next day, and calculate the titer of M13K07 phage; the titer measurement method: Dilute 1 μL of phage in 100 μL of LB liquid medium at a dilution ratio of 1:100, serially dilute it to 6 concentration gradients, then add 900 μL of ER2738 bacterial liquid in the logarithmic phase respectively, incubate at 37°C for 30 min, spread it on a preheated LB-Tet plate, and culture it inverted overnight at 37°C; count the blue plaques and calculate the phage titer;

[0028] (5) Pick independent plaques and inoculate them into an agar plate, and culture overnight at 37°C;

[0029] (6) Add 500 μL of the overnight culture to 50 mL of LB liquid medium, place it at 37 °C, and shake it at 200 rpm for 3 - 6 h;

[0030] (7) Centrifuge the above culture at high speed at 8000 rpm for 20 min, collect the supernatant, add the PEG / NaCl solution with a volume 1 / 5 of the supernatant volume, mix well by inverting up and down several times, and then let it stand overnight at 4 °C;

[0031] (8) Centrifuge the above solution at 4 °C at high speed at 8000 rpm for 20 min, collect the precipitate, and resuspend it with 200 μL of sterile PBS solution to obtain the amplified phage culture;

[0032] (9) Take 10 μL of the amplified phage culture to measure the titer, and store the remaining solution at 4 °C for standby.

[0033] Preferably, step (ii) includes the following steps:

[0034] Dilute the β - lactoglobulin standard with PBS buffer, take a 96 - well ELISA plate and coat it overnight; discard the coating solution, add gelatin solution for blocking; take the phage solution prepared in step (i), dilute it with gelatin solution, add it to the ELISA plate for incubation; discard the phage sample, add triethylamine and let it stand, quickly neutralize the eluate with Tris - HCl; measure the titer of the recombinant phage in the eluate; concentrate and purify the phage particles for the next round of screening; repeat the above steps twice to complete three rounds of panning; sequence the positive phage clones and translate the sequencing results into amino acid sequences.

[0035] More preferably, step (ii) includes the following steps:

[0036] (1) Dilute the β - lactoglobulin standard with PBS buffer, take a 96 - well ELISA plate and coat it overnight;

[0037] (2) Discard the coating solution, add 1% gelatin solution to each well for blocking;

[0038] (3) Take the phage solution prepared in step (i), dilute it with a 1% gelatin solution, and add it to the ELISA plate for incubation;

[0039] (4) Discard the phage sample, wash it with PBST and PBS, add triethylamine and let it stand, quickly neutralize the eluate with Tris - HCl;

[0040] (5) According to the operation method of step (i), measure the titer of the recombinant phage in the eluate;

[0041] (6) Concentration and purification of phage particles: Concentrate and purify phage particles according to the operation methods in steps (3)-(8) of step (1) for the next round of screening;

[0042] (7) Repeat steps (1)-(6) twice to complete the second and third rounds of panning;

[0043] (8) Sequence the positive phage clones with a positive to negative ratio greater than 2.1. The sequencing primer is -96gIII, and the sequencing direction is reverse sequencing;

[0044] (9) Find the 36 gene sequences between the two restriction enzyme sites and translate them into amino acid sequences on the website https: / / web.expasy.org / translate / .

[0045] Further preferably, step (2) includes the following steps:

[0046] (1) Antigen coating: Dilute β-lactoglobulin standard with PBS buffer to 4 μg / mL. Take a 96-well enzyme-linked immunosorbent assay (ELISA) plate, select 3 replicates, add 100 μL to each well, and coat overnight at 4°C. Use PBS as a negative control;

[0047] (2) Blocking: Discard the coating solution, add 150 μL of 1% gelatin solution to each well, and block at room temperature for 1 h;

[0048] (3) Incubating phages: Wash 4 times with PBST. Take the phage solution prepared in the first step, dilute it with 1% gelatin solution to 5×10 11 pfu / mL, add it to the ELISA plate, 100 μL / well, and incubate at room temperature for 2 h;

[0049] (4) Elution: Discard the phage sample, wash 10 times with PBST, then wash 5 times with PBS. Add 100 μL of freshly prepared 0.1 M triethylamine to each well, let it stand at room temperature for 10 min, and quickly aspirate the eluate and neutralize it with an equal volume of 1 M Tris-HCl at pH 7.4;

[0050] (5) Determination of phage titer in the eluate: Determine the titer of recombinant phages in the eluate according to the operation method in the first step;

[0051] (6) Concentration and purification of phage particles: Concentrate and purify phage particles according to the operation methods in steps (3)-(8) of step (1) for the next round of screening;

[0052] (7) Repeat steps (1)-(6) twice to complete the second and third rounds of panning;

[0053] (8) The positive phage clones with a positive to negative ratio greater than 2.1 were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing primer was -96gIII (5’---CCCTCATAGTTAGCGTAACG---3’), and the sequencing direction was reverse sequencing;

[0054] (9) The 36 gene sequences between the two restriction enzyme sites were found and translated into amino acid sequences on the website https: / / web.expasy.org / translate / . The translation result from 3’-5’ is the polypeptide sequence displayed by the phage.

[0055] Preferably, the polypeptides obtained in the step (2) include FNSDSRSTHQED, NTACDMSGRHCH, EIRLSDSPTSGL, SDAWKISTITTM, EVEASSQSPEFY, GFPVPTKEGNRH, QSMAYYVWYADF, YPTGIVSYDDTM, ATTSAELSNEKL, MNTEIETMNSRK, and SMPRLWDNTSNT.

[0056] More preferably, the polypeptides obtained in the step (2) are as follows:

[0057]

[0058] Preferably, the method for verifying the affinity between the polypeptide and β-lactoglobulin by indirect competitive ELISA in the step (3) includes: coupling the polypeptide with β-lactoglobulin, coating the peptide-protein complex on a 96-well plate with PBS coating solution, blocking with 1% gelatin solution, adding rabbit anti-β-lactoglobulin polyclonal antibody for incubation, discarding the primary antibody, adding goat anti-rabbit enzyme-labeled secondary antibody for incubation, developing color and measuring the OD 450nm value, and calculating the competitive inhibition rate of the polypeptide against β-lactoglobulin.

[0059] More preferably, the method for verifying the affinity between the polypeptide and β-lactoglobulin by indirect competitive ELISA in the step (3) includes the following steps:

[0060] (1) Coupling the polypeptide with β-lactoglobulin: Dissolve β-lactoglobulin in MES, dissolve the polypeptide screened in the step (2) in DMSO, add EDC solution and mix, then add NHS solution and mix, and dialyze the reaction solution in deionized water;

[0061] (2) Antigen coating: Coat the peptide-protein complex on a 96-well plate with PBS coating solution and shake;

[0062] (3) Blocking: Pour out the coating solution, wash with PBST, add 1% gelatin solution for blocking and incubate;

[0063] (4) Primary antibody: Discard the blocking solution, wash with PBST, add rabbit anti-β-lactoglobulin polyclonal antibody, and incubate.

[0064] (5) Secondary antibody: Discard the primary antibody, wash with PBST, add goat anti-rabbit enzyme-labeled secondary antibody, and incubate.

[0065] (6) Color development: Discard the secondary antibody, wash with PBST, add TMB substrate solution, terminate the reaction with H2SO4 solution, and measure the OD 450nm value, and calculate the competitive inhibition rate of the polypeptide against β-lactoglobulin.

[0066] Further preferably, the method for verifying the affinity between the polypeptide and β-lactoglobulin by indirect competitive ELISA in step (3) includes:

[0067] (1) Peptide-protein conjugation: Dissolve 8 mg of β-lactoglobulin in 0.8 mL of 0.1 M MES, and dissolve 4 mg of the small peptide in 0.4 mL of DMSO; mix the protein solution and the peptide solution evenly at a volume ratio of 1:2, add 0.1 mL of 1 M EDC solution drop by drop, mix for 30 min, then add 3.7 mL of 7 mM NHS, and mix for 2 hours; finally, dialyze the solution against deionized water overnight.

[0068] (2) Antigen coating: Coat 100 μL of 2 μg / mL peptide-protein complex on each well of a 96-well plate with PBS coating solution, shake at 37 °C for 1 hour, use β-lactoglobulin at the same concentration as the positive control and PBS as the negative control.

[0069] (3) Blocking: Pour out the coating solution, wash repeatedly with PBST 3 times, place on a shaker and gently shake for 10 min, add 300 μL of 1% gelatin solution for blocking, and incubate at 37 °C for 1 hour.

[0070] (4) Primary antibody: Discard the blocking solution, wash with PBST, add 100 μL of rabbit anti-β-lactoglobulin polyclonal antibody diluted at a ratio of 1:4000, and incubate at 37 °C for 1 hour.

[0071] (5) Secondary antibody: Discard the primary antibody, wash with PBST, add 100 μL of goat anti-rabbit enzyme-labeled secondary antibody diluted at a ratio of 1:100000, and incubate at 37 °C for 1 hour.

[0072] (6) Color development: Discard the secondary antibody, wash with PBST, add 100 μL of TMB substrate solution, keep in the dark for 5 minutes; terminate the reaction with 50 μL of 2 mol / L H2SO4 solution, measure the OD 450nm value, and calculate the competitive inhibition rate of the polypeptide against β-lactoglobulin.

[0073] Preferably, the binding specificity between the polypeptide and different proteins in step (iii) is the binding specificity between the polypeptide and β-lactoglobulin, α-lactalbumin, bovine serum albumin, and casein.

[0074] Preferably, the polypeptide used for verification by indirect competitive ELISA in step (iii) is selected from one or more of the following peptide segments: FNSDSRSTHQED, NTACDMSGRHCH, EIRLSDSPTSGL, SDAWKISTITTM, EVEASSQSPEFY, GFPVPTKEGNRH, QSMAYYVWYADF, YPTGIVSYDDTM, ATTSAELSNEKL, MNTEIETMNSRK, and SMPRLWDNTSNT.

[0075] More preferably, the polypeptide used for verification by indirect competitive ELISA in step (iii) includes FNSDSRSTHQED, NTACDMSGRHCH, EIRLSDSPTSGL, SDAWKISTITTM, EVEASSQSPEFY, GFPVPTKEGNRH, QSMAYYVWYADF, YPTGIVSYDDTM, ATTSAELSNEKL, MNTEIETMNSRK, and SMPRLWDNTSNT.

[0076] Preferably, the polypeptide screened out in step (iii) is selected from one or more of the following peptide segments: EVEASSQSPEFY, QSMAYYVWYADF, and YPTGIVSYDDTM.

[0077] More preferably, the polypeptide screened out in step (iii) is QSMAYYVWYADF.

[0078] The second aspect of the present invention provides a polypeptide specifically binding to β-lactoglobulin screened by the above method, and the polypeptide is selected from one or more of the following peptide segments: FNSDSRSTHQED, NTACDMSGRHCH, EIRLSDSPTSGL, SDAWKISTITTM, EVEASSQSPEFY, GFPVPTKEGNRH, QSMAYYVWYADF, YPTGIVSYDDTM, ATTSAELSNEKL, MNTEIETMNSRK, and SMPRLWDNTSNT.

[0079] Preferably, the polypeptide is selected from one or more of the following peptide segments: SDAWKISTITTM, EVEASSQSPEFY, QSMAYYVWYADF, and YPTGIVSYDDTM.

[0080] More preferably, the polypeptide is selected from one or more of the following peptide segments: EVEASSQSPEFY, QSMAYYVWYADF, and YPTGIVSYDDTM.

[0081] Even more preferably, the polypeptide is QSMAYYVWYADF.

[0082] The third aspect of the present invention provides the application of a polypeptide that specifically binds to β-lactoglobulin in the detection of β-lactoglobulin. The polypeptide is selected from one or more of the following peptide segments: FNSDSRSTHQED, NTACDMSGRHCH, EIRLSDSPTSGL, SDAWKISTITTM, EVEASSQSPEFY, GFPVPTKEGNRH, QSMAYYVWYADF, YPTGIVSYDDTM, ATTSAELSNEKL, MNTEIETMNSRK, and SMPRLWDNTSNT.

[0083] Preferably, the polypeptide is selected from one or more of the following peptide segments: SDAWKISTITTM, EVEASSQSPEFY, QSMAYYVWYADF, and YPTGIVSYDDTM.

[0084] More preferably, the polypeptide is selected from one or more of the following peptide segments: EVEASSQSPEFY, QSMAYYVWYADF, and YPTGIVSYDDTM.

[0085] Even more preferably, the polypeptide is QSMAYYVWYADF.

[0086] Preferably, the detection of β-lactoglobulin is for non-diagnostic purposes.

[0087] The present invention has the following beneficial effects:

[0088] In the present invention, Escherichia coli ER2738 is infected with phages to form recombinant phages containing polypeptide library information. After three rounds of panning of this polypeptide library with β-lactoglobulin, the target phages are amplified and sequenced. After translation, the polypeptide sequences that specifically bind to β-lactoglobulin are obtained. After synthesizing the polypeptide sequences, the affinity and specificity are verified by indirect competitive ELISA. The polypeptides screened in the present invention can specifically recognize β-lactoglobulin and have extremely high application value in the detection of β-lactoglobulin. Description of the Drawings

[0089] Figure 1 It is a schematic diagram of the screening process of the phage display library in Example 1 of the present invention;

[0090] Figure 2It is the result graph of the phage recovery rate of the three-round biological screening in Example 1 of the present invention;

[0091] Figure 3 It is the result graph of the inhibition rate of various peptide segments on β-lactoglobulin in Example 1 of the present invention;

[0092] Figure 4 It is the result graph of the inhibition rate of various peptide segments on α-lactalbumin (A), bovine serum albumin (B) and casein (C) in Example 1 of the present invention. Detailed implementation manners

[0093] The present invention is further illustrated by way of examples, but the present invention is not limited to the following examples only.

[0094] Example 1: Screening of polypeptides specifically binding to β-lactoglobulin using a phage display library

[0095] The first step: Phage amplification and titer determination

[0096] (1) Inoculate the ER2738 strain into LB-Tet medium and culture it overnight at 37°C for activation; inoculate a single colony into 5 mL of LB-Tet medium at a ratio of 1:100 and culture it overnight at 37°C until the OD 600 reaches 0.4 - 0.6;

[0097] (2) Prepare an agar plate: Weigh 1.25 g of IPTG and 1 g of Xgal, dissolve them in 25 mL of DMF, and store them at -20°C for later use; add 15 g of agar to 1 L of LB medium, and at the same time add 1 mL of IPTG / Xgal stock solution to prepare the plate. After cooling at room temperature until it solidifies, store it at 4°C in the dark for later use;

[0098] (3) According to the 10-fold serial dilution method, dilute the phage with LB medium. After equally dividing the host bacteria cultured on a large scale as required, add 10 μL of the diluted gradient phage to each tube respectively, mix gently, and incubate at room temperature for 30 min;

[0099] (4) Spread the host bacteria infected in step (3) on the plate prepared in step (2); culture it overnight at 37°C. Count the phage plaques the next day and calculate the titer of M13K07 phage; The titer determination method: Dilute 1 μl of phage in 100 μl of LB liquid medium at a dilution ratio of 1:100, continuously dilute it into 6 concentration gradients, then add 900 μl of ER2738 bacterial liquid in the logarithmic phase respectively, incubate at 37°C for 30 min, spread it on a preheated LB-Tet plate, and culture it upside down overnight at 37°C; Count the blue plaques and calculate the phage titer;

[0100] (5) Pick independent plaques and inoculate them into an agar plate, and incubate overnight at 37°C;

[0101] (6) Add 500 μL of the overnight culture to 50 mL of LB liquid medium, place it at 37°C, and shake culture at 200 rpm for 3 - 6 h;

[0102] (7) Centrifuge the above culture at high speed at 8000 rpm for 20 min, collect the supernatant, add a PEG / NaCl solution with a volume 1 / 5 of the supernatant volume, mix well by inverting up and down multiple times, and then let it stand overnight at 4°C;

[0103] (8) Centrifuge the above solution at 4°C at high speed of 8000 rpm for 20 min, collect the precipitate, and resuspend it with 200 μL of sterile PBS solution to obtain the amplified phage culture;

[0104] (9) Take 10 μL of the amplified phage culture to measure the titer, and store the remaining solution at 4°C for standby.

[0105] The second step: Panning of the 12 - peptide phage display library

[0106] (1) Antigen coating: Dilute the β - lactoglobulin standard with PBS buffer to 4 μg / mL, take a 96 - well ELISA plate, select 3 replicate wells, add 100 μL to each well, and coat overnight at 4°C. Use PBS as the negative control;

[0107] (2) Blocking: Discard the coating solution, add 150 μL of 1% gelatin solution to each well, and block at room temperature for 1 h;

[0108] (3) Incubate phages: Wash 4 times with PBST, take the phage solution prepared in the first step, dilute it with 1% gelatin solution to 5×10 11 pfu / mL, add it to the ELISA plate, 100 μL / well, and incubate at room temperature for 2 h;

[0109] (4) Elution: Discard the phage sample, wash 10 times with PBST, then wash 5 times with PBS, add 100 μL of freshly prepared 0.1 M triethylamine to each well, let it stand at room temperature for 10 min, and quickly aspirate the eluate and neutralize it with an equal volume of 1 M Tris - HCl at pH 7.4;

[0110] (5) Determination of the phage titer in the eluate: According to the operation method of the first step, determine the titer of the recombinant phages in the eluate;

[0111] (6) Concentration and purification of phage particles: According to the operation methods of (3) - (8) in the first step, concentrate and purify the phage particles for the next round of screening;

[0112] (7) Repeat steps (1)-(6) twice to complete the second and third rounds of panning;

[0113] Keep the input amount of phage at 1×10 11 pfu all the time. 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 8.3×10 3 pfu, 3.2×10 5 pfu, and 2.7×10 7 pfu respectively. The output of phage 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 biological screening, the recovery rate of phage gradually increases, and the enrichment efficiency increases from 8.3×10 -8 to 2.7×10 -4 , indicating that the phage peptide library has specifically bound to β-lactoglobulin and has been effectively enriched.

[0114] Table 1 Results of three rounds of screening of phage peptide library against β-lactoglobulin

[0115] Round Input (pfu / ml) Output (pfu / ml) Recovery rate (%) 1 <![CDATA[1×10 11 > <![CDATA[8.3×10 3 > <![CDATA[8.3×10 -8 <!-- 7 -->]]> 2 <![CDATA[1×10 11 > <![CDATA[3.2×10 5 > <![CDATA[3.2×10 -6 > 3 <![CDATA[1×10 11 > <![CDATA[2.7×10 7 > <![CDATA[2.7×10 -4 >

[0116] (8) Send the positive phage clones with a positive-to-negative ratio greater than 2.1 to Shanghai Sangon Biological Engineering Co., Ltd. for sequencing. The sequencing primer is -96gIII (5’---CCCTCATAGTTAGCGTAACG---3’, SEQ ID NO.12), and the sequencing direction is reverse sequencing;

[0117] (9) Find the 36-gene sequence between the 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).

[0118] Table 2 Candidate peptide sequences

[0119]

[0120] Step 3: Analysis of the inhibitory effect of peptides on the immune binding activity of β-lg

[0121] (1) Peptide - protein conjugation: The 11 polypeptides obtained in the second step were entrusted to Anhui Guoping Pharmaceutical Co., Ltd. for solid - phase synthesis. Dissolve 8 mg of β - lactoglobulin in 0.8 mL of 0.1 M (N - morpholino) ethanesulfonic acid (MES), and dissolve 4 mg of the polypeptide in 0.4 mL of DMSO; mix the protein solution and the peptide solution evenly at a volume ratio of 1:2, drop - wise add 0.1 mL of 1 M EDC solution, mix for 30 min, then add 3.7 mL of 7 mM NHS, and mix for 2 hours; finally, dialyze the solution against deionized water overnight;

[0122] (2) Antigen coating: Coat 100 μL of 2 μg / mL peptide - protein complex on each well of a 96 - well plate with PBS coating solution, shake at 37 °C for 1 hour, use β - lactoglobulin at the same concentration as the positive control and PBS as the negative control;

[0123] (3) Blocking: Pour out the coating solution, wash it repeatedly 3 times with PBST, place it on a shaker and gently shake for 10 min, add 300 μL of 1% gelatin solution for blocking, and incubate at 37 °C for 1 hour;

[0124] (4) Primary antibody: Discard the blocking solution, wash with PBST, add 100 μL of rabbit anti - β - lactoglobulin polyclonal antibody diluted at a ratio of 1:4000, and incubate at 37 °C for 1 hour;

[0125] (5) Secondary antibody: Discard the primary antibody, wash with PBST, add 100 μL of goat anti - rabbit enzyme - labeled secondary antibody diluted at a ratio of 1:100000, and incubate at 37 °C for 1 hour;

[0126] (6) Color development: Discard the secondary antibody, wash with PBST, add 100 μL of TMB substrate solution, and keep it in the dark for 5 minutes; terminate the reaction with 50 μL of 2 mol / L H2SO4 solution and measure the OD 450nm value.

[0127] Compared with the control group, the IgG - binding ability of the complexes of P4, P5, P7, and P8 with β - lactoglobulin decreased, while that of the other polypeptide - protein complexes increased. Whether it increased or decreased, it indicated that the peptide segments affected the structure of β - lactoglobulin to a certain extent due to their interaction with β - lactoglobulin, resulting in changes in IgG - binding ability. Among Figure 3 them, among the polypeptide - protein complexes with a positive inhibition rate, the inhibition rates of polypeptides P4, P5, P7, and P8 on β - lactoglobulin were 13.06%, 60.86%, 87.99%, and 75.03% respectively, and the inhibition rate of P7 was the highest. Among the polypeptides with a negative inhibition rate, the inhibition rate of P9 was the highest, at - 19.67%. Among the 11 peptide segments, there were extremely significant differences between P5, P7, P8 and the blank group, indicating that these 3 peptide segments had obvious affinity for β - lactoglobulin.

[0128] Step 4: Peptide specificity verification

[0129] According to the method in Step 3, couple the peptides with the best affinity (P5, P7, and P8) screened out with α-lactalbumin, casein, and bovine serum albumin, and conduct an indirect competitive ELISA experiment to measure the OD 450nm value and calculate the competitive inhibition rate.

[0130] P5, P7, and P8 have strong affinities for β-lactoglobulin, and α-lactalbumin, bovine serum albumin, and casein, which are abundant in cow's milk, are likely to interfere with the detection of β-lactoglobulin. Therefore, select these 3 peptides and 3 cow's milk proteins to complete the specificity experiment. The inhibition rates of 11 peptides against α-lactalbumin (A), bovine serum albumin (B), and casein (C) are as Figure 4 shown. The competitive inhibition rates of P5 against the three proteins are 56.63%, 77.61%, and 73.90% respectively. The competitive inhibition rates of P7 against the three proteins are 45.77%, 31.7%, and 1.28% respectively. The competitive inhibition rates of P8 against the three proteins are 142.4%, 78.1%, and 78.7% respectively. By comparing the results of the indirect competitive ELISA inhibition rates of P5, P7, P8, and the three candidate proteins, P5 has a low binding ability to α-lactalbumin and strong binding abilities to bovine serum albumin and casein; P7 has the lowest inhibition rate against the three proteins, indicating that its binding ability to the three proteins is very weak; while P8 has strong binding abilities to α-lactalbumin, bovine serum albumin, and casein. The results show that the P7 polypeptide has a low cross-reactivity with α-lactalbumin, bovine serum albumin, and casein, showing good specificity.

[0131] Although the specific implementation of the present invention has been described, those skilled in the art should recognize that various changes and modifications can be made to the present invention without departing from the scope or spirit of the present invention. Therefore, the present invention is intended to cover all such changes and modifications that fall within the scope of the appended claims and their equivalents.

Claims

1. A polypeptide that specifically binds to β-lactoglobulin, characterized in that, The polypeptide is QSMAYYVWYADF.

2. Application of a polypeptide specifically binding to β-lactoglobulin in the detection of β-lactoglobulin, characterized in that, The polypeptide is QSMAYYVWYADF, and the application is for non-diagnostic and non-therapeutic purposes.

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