A diethylstilbestrol mimotope peptide, its preparation method and application

Abstract

The present invention belongs to the technical field of phage display peptide libraries, and specifically relates to a diethylstilbestrol mimotope peptide, a preparation method thereof and an application. The amino acid sequence of the antigen mimotope peptide of diethylstilbestrol is YVYPQMP. When applied, the antigen mimotope can be used for immunological detection by biologically synthesizing the antigen mimotope peptide, or the phage displaying the diethylstilbestrol mimotope can be directly obtained through phage amplification for immunoassay, or the diethylstilbestrol mimotope can be separated from the phage to replace the diethylstilbestrol standard for immunological detection and analysis. The detection process is simple and convenient, the result is accurate, and the sensitivity is high. The present invention effectively improves the detection sensitivity of diethylstilbestrol in food, and at the same time reduces the harm to the human body and the ecological environment during the detection process of diethylstilbestrol, and has outstanding application value.

Description

Technical Field

[0001] The invention belongs to the technical field of phage display peptide library, and particularly relates to a diethylstilbestrol mimetic epitope peptide and a preparation method and application thereof. Background Art

[0002] Diethylstilbestrol is a synthetic non-steroidal estrogen that can promote animal growth and is widely used in animal husbandry and fisheries. However, its lower concentration can significantly affect the development of the human reproductive system and produce side effects such as teratogenicity and carcinogenicity. Therefore, most countries have completely banned the use of diethylstilbestrol as an additive. The 235 Announcement of the Ministry of Agriculture of my country pointed out that diethylstilbestrol should not be detected in animal food. However, the abuse of diethylstilbestrol still exists. Timely, efficient and sensitive detection of diethylstilbestrol residues in edible agricultural products is an important means to control its hazards and ensure food safety.

[0003] At present, the detection methods of diethylstilbestrol are mainly gas chromatography, high performance liquid chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, enzyme-linked immunosorbent assay, etc. Chromatographic detection methods are highly sensitive, but the equipment is expensive, the sample pretreatment is cumbersome, and it is difficult to use for large-scale screening. Although the immunological detection method is simple, convenient, sensitive, and low-cost, the implementation process must use diethylstilbestrol standard as a raw material for artificial antigen chemical synthesis. Diethylstilbestrol is not only highly teratogenic and carcinogenic, but also expensive, and causes great harm to the health of operators and the ecological environment. In recent years, researchers have used phage display random peptide library technology to select highly specific and specific analog epitopes to successfully replace small molecule antigens for immunological detection. However, since these antigen analog epitopes are obtained from random natural peptide libraries, they are limited by the capacity and diversity of natural peptide libraries, and there will be a large amount of missing information on specific target antigen analog epitope peptides, resulting in poor detection performance of the selected antigen epitope peptides to replace small molecule antigens for immunological detection. Therefore, it is very necessary to select diethylstilbestrol mimetic epitope peptides with better detection performance, so as to not only avoid the use of diethylstilbestrol standards and reduce detection costs in establishing immunoassay methods for diethylstilbestrol, but also improve the detection sensitivity of diethylstilbestrol immunological methods and ensure the safety of animal-derived food. Summary of the invention

[0004] The purpose of the present invention is to solve the deficiencies of the prior art and provide a diethylstilbestrol mimetic epitope peptide and a preparation method and application thereof, specifically adopting the following technical solutions:

[0005] In a first aspect of the present invention, there is provided an antigen mimetic epitope peptide of diethylstilbestrol, the amino acid sequence of which is YVYPQMP.

[0006] In a second aspect of the present invention, there is provided a gene encoding the above antigen mimotope peptide, and its nucleotide sequence is: TATGTGTATCCTCAGATGCCT.

[0007] In a third aspect of the present invention, there is also provided a method for preparing the above antigen mimotope peptide, comprising the following steps:

[0008] Adding the phage with the diethylstilbestrol antigen mimotope to the culture solution of Escherichia coli ER2738 in the early logarithmic phase, culturing with shaking for 4.5 h, centrifuging for 10 min after the culture is completed, taking the supernatant and adding PEG / NaCl, standing overnight at 4 °C, then centrifuging again, resuspending the precipitate in TBS, centrifuging for 5 min again to precipitate the residual cells, obtaining the supernatant, adding PEG / NaCl, incubating on ice for 60 min, centrifuging for 10 min, discarding the supernatant, and finally resuspending the precipitate in TBS, 0.02% NaN 3 3.

[0009] The method for preparing the above antigen mimotope peptide specifically comprises the following steps:

[0010] (1) Adding the phage with the diethylstilbestrol mimotope to 20 mL of the culture solution of Escherichia coli ER2738 in the early logarithmic phase, culturing with shaking at 37 °C and 150 rpm for 4.5 h;

[0011] (2) Transferring the culture to another centrifuge tube, centrifuging at 4 °C and 7000 g for 10 min, transferring the upper 80% of the supernatant to a fresh tube, adding 1 / 6 volume of PEG / NaCl, and standing at 4 °C overnight;

[0012] (3) Centrifuging at 4 °C and 7000 g for 15 min, pouring off the supernatant, resuspending the precipitate in 1 mL of TBS, and centrifuging at 4 °C and 7000 g for 5 min to precipitate the residual cells.

[0013] (4) Transferring the supernatant to another fresh centrifuge tube, adding 1 / 6 volume of PEG / NaCl, and incubating on ice for 60 min. Centrifuging at 4 °C and 7000 g for 10 min, discarding the supernatant, and resuspending the precipitate in 200 µL of TBS, 0.02% NaN 3 3.

[0014] In the present invention, since the acquisition of these antigenic mimotopes is derived from a random natural peptide library, restricted by the capacity and diversity of the natural peptide library, there will be a large amount of missing information on specific target antigenic mimotope peptides. Therefore, the inventor targeted to construct a phage display diethylstilbestrol mimotope peptide library and screened out diethylstilbestrol mimotopes with better detection performance from it. This method can not only avoid the use of diethylstilbestrol standards and reduce the detection cost, but also improve the detection sensitivity of the diethylstilbestrol immunological method and ensure the food safety of animal-derived products.

[0015] As a further preferred embodiment, the phage with diethylstilbestrol antigenic mimotope is obtained by the following process:

[0016] (1) Dissolve the anti-diethylstilbestrol monoclonal antibody in 1×PBS and coat it overnight at 4 °C; after removing the liquid, wash it 3 times with TBST, add 1% OVA blocking solution, and react at 4 °C for 2 h;

[0017] (2) After the reaction, remove the liquid, wash it 3 times with TBST, then add the phage display diethylstilbestrol mimotope peptide library, and gently shake and react at 16 °C - 22 °C for 1 h. Discard the liquid and wash it 10 times with TBST to remove unbound phages;

[0018] (3) Add the eluent, gently shake for 8 min, take out the eluent and add the neutralization buffer to obtain the first-round eluted phages. Amplify and purify the obtained first-round eluted phages;

[0019] (4) Perform the second-round and third-round competitive affinity panning in sequence according to the steps of the first round; finally obtain the phage with diethylstilbestrol antigenic mimotope;

[0020] Among them, in step (3), the first round adopts acid elution, and the eluent is Glycine-HCl with pH = 2.2 and a concentration of 0.2 M;

[0021] In step (4), during the second-round and third-round competitive affinity panning, competitive elution is adopted, and the eluent is a diethylstilbestrol standard with a concentration of 1 ng / mL. And the amount of phages amplified and purified in the previous round added each time is 1×10 10 pfu; the antibody coating concentrations for the second and third-round competitive affinity panning are 5 µg / mL and 1 µg / mL respectively, and the blocking solutions are 1% BSA and 1% OVA in sequence; the elution times for phages and diethylstilbestrol standards are 45 min and 30 min respectively.

[0022] As a further preferred embodiment, the construction method of the phage display diethylstilbestrol mimotope peptide library includes the following steps:

[0023] S1: First, obtain the core mimotope of diethylstilbestrol. Targetedly construct a phage display diethylstilbestrol mimotope peptide library using genetic recombination technology, and adopt a 6-encoding method to design and synthesize random heptapeptide nucleotides, extension primers, and sequencing primers.

[0024] S2: Place the random heptapeptide nucleotides and 3-fold molar amount of extension primers in a PCR instrument to form an annealing product and further extend it to form an extended product. Use KpnI-HF enzyme and EagI-HF enzyme to perform double digestion on the formed extended product and M13KE vector respectively. After double digestion of the extended product, perform non-denaturing polyacrylamide gel electrophoresis, stain it, cut the gel to recover the DNA band, add elution buffer, and shake it overnight at 37 °C to elute the DNA. The next day, use the phenol-chloroform method to recover the double-digested product of the random heptapeptide nucleotide double strand to obtain the inserted sequence. After double digestion of the M13KE vector, separate the target fragment by agarose gel electrophoresis, and recover and purify it with an agarose gel recovery kit to obtain the double-digested M13KE product.

[0025] S3: Connect the double-digested M13KE product and the inserted sequence in a molar ratio of 1:10 overnight. Purify the ligation product using the phenol-chloroform method, and dissolve the precipitate with sterile TE buffer. The ligation product is electrotransformed into ER2738 competent cells in batches, add SOC culture medium, and the culture after shaking is the electrotransformation culture. Expand the electrotransformation culture in a ratio of 1:50, add it to the ER2738 bacterial solution in the early logarithmic growth phase, shake and incubate for 4.5 h, and centrifuge for 20 min after the incubation ends. Transfer the supernatant and discard the cells, add 1 / 6 volume of PEG / NaCl, incubate overnight at 4 °C, centrifuge for 20 min after the incubation ends, discard the supernatant, completely resuspend the phage precipitate in TBS, and gently shake it overnight at 4 °C. Then centrifuge for 10 min, take the supernatant, add 1 / 6 volume of PEG / NaCl to precipitate the phage, and incubate at 4 °C for 1 h. After the incubation ends, centrifuge for 20 min and discard the supernatant. Resuspend the final precipitate in 40 mL of TBS, gently shake it at 4 °C for 24 - 48 h, add an equal amount of sterile glycerol, and mix well to obtain the phage display diethylstilbestrol mimotope peptide library.

[0026] As a further preferred embodiment, the sequences of the heptapeptide nucleotide, extension primer, and sequencing primer are as follows:

[0027] Random heptapeptide nucleotide L7: 5’-CATGTTTCGGCCGAACCTCCACCMNNMNNMNNAGGATACACATAAGAGTGAGAATAGAAAGGTACCCGGG-3’;

[0028] Extension primer EP: 5’-CATGCCCGGGTACCTTTCTATTCTC-3’;

[0029] Sequencing primer - 96 gIII: 5'-CCCTCATAGTTAGCGTAACG-3'.

[0030] As a further preferred embodiment, the above-mentioned TBST comprises the following components:

[0031] Tris-HCl buffer with a pH of 7.5 and a concentration of 50 mM, NaCl solution with a concentration of 150 mM, and Tween-20 with a concentration of 0.1% - 0.5%. More preferably, the concentration of Tween-20 is 0.1% during the first round of washing, 0.3% during the second round of washing, and 0.5% during the third round of washing. Among them, the concentration of the detergent Tween-20 has a greater impact on the results of panning. Adding a certain concentration of Tween-20 to TBS as the washing solution aims to reduce the non-specific interaction between phages and antibodies and blocking proteins. Through three rounds of affinity panning, the use concentrations of Tween-20 in the washing solution are 0.1%, 0.3%, and 0.5% respectively, which can effectively avoid non-specific phage adsorption and pan for phages with higher affinity.

[0032] The present invention also provides a method for preparing an antigen mimotope peptide - fusion protein similar in function to the above-mentioned antigen mimotope peptide, comprising the following steps:

[0033] (1) Using the phage replicative form DNA with a diethylstilbestrol mimotope as a template, EP and - 96 gIII as primers, amplifying the gene encoding the diethylstilbestrol mimotope, and recovering and purifying the PCR product using a common DNA product purification kit. Double-digesting the PCR product and the pMAL-pIII vector with KpnI and EagI enzymes; ligating the digested target fragment with the vector overnight. Removing the salt ions in the reaction system by the phenol-chloroform method and purifying the ligation product. Taking 5 μL of the ligation product and adding it to 100 μL of TB1 competent cells, mixing well, and electrotransforming the ligation product into TB1 competent cells using a gene introduction instrument, immediately adding 1 mL of SOC culture medium, and culturing at 37 °C with shaking at 150 rpm for 40 min to obtain the electrotransformation culture. Coating the electrotransformation culture on an LB-Amp solid medium and culturing overnight at 37 °C to obtain positive clones.

[0034] (2) Picking a single colony from the above-mentioned obtained positive clones and inoculating it into 5 mL of LB-Amp, 0.2% glucose, and culturing overnight at 37 °C with shaking at 220 rpm.

[0035] (3) Inoculate the overnight culture into 50 mL of LB-Amp, 0.2% glucose medium at an inoculation amount of 1% (v / v), and culture with shaking at 37 °C and 220 rpm; when the OD 600 of the cultured bacteria reaches 0.6, add 0.1 mol / L IPTG to the culture medium to a final concentration of 0.3 mmol / L, and culture with shaking at 15 °C and 220 rpm for 16 h.

[0036] (4) Centrifuge the inducer at 4 °C and 4000 g for 20 min, collect the cell precipitate, discard the supernatant, resuspend the cells in 15 mL of a solution containing 30 mM Tris-HCl, 20% glucose and pH 8.0, add EDTA to a final concentration of 1 mmol / L, and shake at room temperature for 10 min. Centrifuge at 4 °C and 8000 g for 20 min, discard the supernatant, and resuspend the precipitate in 15 mL of pre-cooled 5 mM MgSO 4 , and shake on ice for 10 min.

[0037] (5) Centrifuge at 4 °C and 8000 g for 20 min, retain the supernatant, and add 300 μL of 1 mol / L Tris-HCl pH 7.4 to the supernatant to obtain the diethylstilbestrol mimotope peptide-MBP fusion protein.

[0038] In addition, the above antigenic epitope peptide can also be obtained by the method of polypeptide chemical synthesis based on its amino acid sequence.

[0039] In the fourth aspect of the present invention, the application of the above antigen mimotope peptide in immunological detection and analysis is also provided.

[0040] In the fifth aspect of the present invention, the application of the above antigen mimotope peptide as a competitive antigen in an enzyme-linked immunosorbent assay for detecting diethylstilbestrol in food is also provided. Preferably, the above food includes chicken, fish and shrimp meat.

[0041] The beneficial effects of the present invention are:

[0042] The hexestrol mimotope peptide obtained by targeting in the present invention has more immune response characteristics similar to those of natural hexestrol molecules, and can be used to replace the carcinogenic, teratogenic and expensive hexestrol standard. When the antigen mimotope is applied, the mimotope peptide can be synthesized by biological means for immunological detection, or the phage displaying the hexestrol antigen mimotope can be directly obtained by phage amplification for immunoassay, or the hexestrol antigen mimotope can be separated from the phage to replace the hexestrol standard for immunological detection and analysis. The detection process is simple, convenient, accurate and highly sensitive. The present invention effectively improves the detection sensitivity of hexestrol in food, and at the same time reduces the harm to the human body and the ecological environment during the detection process of hexestrol, and has outstanding application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The indirect competitive ELISA standard curve established by using the hexestrol mimotope peptide provided by the present invention as a competitive antigen is shown;

[0044] Figure 2 The comparison of the detection sensitivities between the hexestrol mimotope peptide provided by the present invention as a competitive antigen and other hexestrol mimotope peptides is shown. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0045] The following will clearly and completely describe the concept, specific structure and technical effects of the present invention in combination with the embodiments and drawings, so as to fully understand the purpose, scheme and effects of the present invention. It should be noted that, without conflict, the embodiments in the present application and the features in the embodiments can be combined with each other.

[0046] In the technical solution of the present invention, the hexestrol mimotope peptide can also be called the hexestrol antigen mimotope.

[0047] In the amino acid sequence of the above antigen epitope, the capital English letters respectively represent one of the known natural L-type amino acid residues or their D-type isomers, where M represents methionine residue, P represents proline residue, Q represents glutamine residue, V represents valine residue, and Y represents tyrosine residue.

[0048] In the embodiments of the present scheme, unless otherwise specified, the eluent used is 0.2 M Glycine-HCl (pH 2.2); the neutralization buffer is 1 M Tris-HCl (pH 9.1); M13KE is a phage and is an expression vector; TBST includes the following components: Tris-HCl buffer with a pH of 7.5 and a concentration of 50 mM, a NaCl solution with a concentration of 150 mM and 0.1% Tween-20.

[0049] Example 1

[0050] Construction and Identification of a Phage Display Library of Diethylstilbestrol Mimotope Peptides

[0051] (1)Design of Random Heptapeptide Nucleotides and Primers: In the previous stage, the core mimotope YVYP of diethylstilbestrol antigen was obtained from a natural peptide library. Therefore, using YVYP as the backbone, a random heptapeptide nucleotide sequence was artificially synthesized, and a phage display library of diethylstilbestrol mimotope random heptapeptides was constructed by gene recombination technology. Using the (NNK)6 coding method, the designed random heptapeptide nucleotide (L7) has the sequence shown in SEQ ID No.2:

[0052] 5’-CATGTTTCGGCCGAACCTCCACCMNNMNNMNNAGGATACACATAAGAGTGAGAATAGAAAGGTACCCGGG-3’;

[0053] Extension primer (EP) has the sequence shown in SEQ ID No.3: 5’-CATGCCCGGGTACCTTTCTATTCTC-3’;

[0054] Sequencing primer (-96 gIII) has the sequence shown in SEQ ID No.4: 5’-CCCTCATAGTTAGCGTAACG-3’.

[0055] (2)Annealing, extension and double digestion of the inserted sequence: Add 5 µg of the synthesized random heptapeptide nucleotide template L7 and 3-fold molar amount of the extension primer EP to 50 µL of TE buffer containing 100 mmol / L NaCl, mix well, place in a PCR instrument, heat to 95 °C, and gradually cool to 37 °C to form the annealed product. The extension is carried out according to the following reaction system: 95 µL of sterile water, 20 µL of 10×Buffer2, 32 µL of 2.5 mmol / L dNTPs, 3 µL of Klenow enzyme, and 50 µL of the annealing system. After mixing well, incubate in a water bath at 37 °C for 1 h and at 65 °C for 15 min to form the extended product. The formed extended product is digested with KpnI-HF enzyme and EagI-HF enzyme. The double digestion system is as follows: 162 µL of sterile water, 40 µL of 10×Cutsmart Buffer, 4 µL of 20 U / µL KpnI-HF enzyme, 4 µL of 20 U / µL EagI-HF enzyme, and 190 µL of the extended product. Mix well and incubate in a water bath at 37 °C for 4 h. The double-digested product is concentrated by the phenol-chloroform method. After non-denaturing polyacrylamide gel electrophoresis and staining with Utral GelRed, the DNA band is cut out and recovered. Use 400 µL of 100 mmol / L sodium acetate (PH = 4.5), 1 mmol / L EDTA, 0.1% SDS, and shake overnight at 37 °C to elute the DNA. The next day, the double-digested product of the L7 double strand is recovered by the phenol-chloroform method, which is the inserted sequence.

[0056] (3)Double digestion and purification of the M13KE vector: Mix 3.5 µg of the M13KE vector with 5 µL of 10×Cutsmart Buffer, 2 µL of 20 U / µL KpnI-HF, and 3 µL of 20 U / µL EagI-HF enzyme. Add sterile water to 50 µL, mix well, and perform double digestion at 37 °C for 2 h. After digestion, the target fragment is separated by agarose gel electrophoresis, and the double-digested M13KE product is recovered and purified using an agarose gel recovery kit.

[0057] (4) Ligation and transformation: The ligation system consists of 20 ng of the inserted sequence, 100 ng of the double-digested product of M13KE, 2 μL of 10× ligase buffer, and 1 μL of T4 DNA ligase. Add distilled water to make the total volume of the system 20 μL. After overnight ligation at 16 °C, heat-terminate the ligation reaction at 65 °C for 15 min. Purify the ligation product by the phenol-chloroform method and dissolve the precipitate in sterile TE buffer. Divide 20 μL of the ligation product into 4 aliquots for electroporation. Each time, take 5 μL and add it to 100 μL of ER2738 competent cells for electroporation (adjust the electroporation parameters to 25 μF, 200 Ω, 2.5 kV). Immediately add 1 mL of SOC medium and incubate at 37 °C with shaking at 150 rpm for 40 min to obtain the electrotransformed culture. Expand the electrotransformed culture at a ratio of 1:50 and add it to the ER2738 bacterial solution in the early logarithmic growth phase. Incubate at 37 °C with shaking at 250 rpm for 4.5 h. Centrifuge at 5000 g at 4 °C for 20 min. Transfer the supernatant and discard the cells. Add 1 / 6 volume of PEG / NaCl and incubate overnight at 4 °C. Centrifuge at 5000 g at 4 °C for 20 min and discard the supernatant. Resuspend the phage precipitate completely in 100 mL of TBS and gently shake overnight at 4 °C. Centrifuge at 5000 g at 4 °C for 10 min to remove residual cells. Transfer the supernatant to a new tube and discard the precipitate. Add 1 / 6 volume of PEG / NaCl to precipitate the phage and incubate at 4 °C for 1 h. Centrifuge at 5000 g at 4 °C for 20 min and discard the supernatant. Resuspend the final precipitate in 40 mL of TBS and gently shake at 4 °C for 24 - 48 h. Add an equal volume of sterile glycerol and mix well to obtain the phage display diethylstilbestrol mimotope peptide library, which can be stored at -20 °C for long-term use.

[0058] (5) Identification of the phage display diethylstilbestrol mimotope peptide library: Take 100 μL of the transformed culture for dilution and dilute it 10^1 - 10^n times with LB respectively. Take 10 μL of each diluted transformed culture and add it to 200 μL of the ER2738 culture in the mid-logarithmic phase. Incubate for 5 min, mix well, and then add it to the top agar maintained at 48 °C in a water bath. Mix well and immediately spread it on the LB / IPTG / X-gal plate. Incubate the plate upside down at 37 °C overnight. Perform blue plaque counting the next day. Randomly select 20 blue plaques from the plate, extract DNA, and sequence it. Calculate the library capacity and identify the library diversity based on the number of blue plaques, the positive sequencing rate, and the sequence difference rate.

[0059] Example 2

[0060] Panning and Identification of Diethylstilbestrol Antigen Mimotopes Derived from the Phage Display Diethylstilbestrol Mimotope Peptide Library

[0061] (1) The specific steps of affinity panning of diethylstilbestrol antigen mimetic epitopes are as follows:

[0062] Dissolve anti-diethylstilbestrol monoclonal antibody in 1×PBS (20 µg / mL), 100 µL per well, and coat overnight at 4°C. The next day, discard the liquid in the wells, wash three times with TBST (50 mM Tris-HCl pH7.5, 150 mM NaCl, 0.1% Tween-20), add 300 µL / well of 1% OVA blocking solution, and block for 2 h at 4°C. Discard the liquid in the wells, wash three times with TBST, add 100 µL of phage-displayed diethylstilbestrol mimetic epitope peptide library to each well, and gently shake for 1 h at 16-22°C. Discard the liquid in the wells, wash 10 times with TBST to remove unbound phages. Add 100 µL / well of elution buffer, shake gently at room temperature for 8 min, remove the elution buffer and add 15 µL of neutralization buffer, which is the first round of elution of phages. The first round of elution was amplified and purified, and the second and third rounds of competitive affinity panning were performed in sequence. The amount of phage added each time was 1×10 10 pfu, the antibody coating concentrations for the second and third rounds of panning were 5 µg / mL and 1 µg / mL, respectively, the blocking solutions were 1% BSA and 1% OVA, respectively, the Tween-20 concentrations in the TBST used were 0.3% and 0.5%, respectively, the competitive incubation time between phage and 1 ng / mL diethylstilbestrol standard was 45 min and 30 min, respectively, and the rest of the steps were the same as above.

[0063] (2) The specific steps for identifying the mimetic epitope of diethylstilbestrol antigen are as follows:

[0064] From the plate where the phage titer was measured after the third round of panning, 50 phage blue spots were randomly selected for amplification and purification. All phage clones were positively identified by indirect ELISA. 3 Anti-diethylstilbestrol monoclonal antibody was diluted at pH 8.6 and coated on the ELISA plate at a concentration of 1 µg / mL, and incubated overnight at 4 °C. The next day, the plate was washed three times with PBST (10 mM PBS, 0.05% Tween-20), blocked with 1% BSA at 4 °C for 2 h, washed three times with PBST, and patted dry. 100 µL of phage purification solution was added to each well, and the original phage display peptide library was used as a negative control. The plate was incubated at 37 °C for 45 min, and washed three times with PBST. 100 µL / well of 1:5000 diluted horseradish peroxidase-labeled anti-M13 phage enzyme-labeled secondary antibody was added, and the plate was incubated at 37 °C for 30 min. The plate was washed three times with PBST, and 100 µL of TMB substrate was added for color development, and the OD was measured by an ELISA reader. 450 Select the absorbance value of OD 450Phages greater than 2-fold of the negative control are positive clones.

[0065] Indirect competitive ELISA was used to identify the mimotopes of diethylstilbestrol antigen for all positive phage clones. The monoclonal antibody against diethylstilbestrol was diluted with 0.1 mol / L NaHCO 3 pH 8.6 and coated on the ELISA plate at a concentration of 1 µg / mL, and incubated overnight at 4 °C. The next day, after washing 3 times with PBST (10 mM PBS, 0.05% Tween-20), the reaction was blocked with 1% BSA at 4 °C for 2 h, and then washed 3 times with PBST and patted dry. 50 µL of positive phage clones and 50 µL of diethylstilbestrol standards were added to each well, incubated at 37 °C for 45 min, and then washed 3 times with PBST. 100 µL of horseradish peroxidase-labeled anti-M13 phage enzyme-linked secondary antibody diluted 1:5000 was added to each well and incubated at 37 °C for 30 min. After washing 3 times with PBST, 100 µL of TMB substrate was added for color development, and the absorbance value of OD 450 was measured with an enzyme-linked immunosorbent assay reader. Phages that could be inhibited by diethylstilbestrol standards were identified as phages with diethylstilbestrol antigen mimotopes.

[0066] The phages identified as diethylstilbestrol mimotopes by indirect competitive ELISA were amplified, and phage DNA was extracted. After amplification of the phages, they were centrifuged at 13500 g for 10 min at 4 °C. 500 µL of the phage supernatant was transferred to a fresh tube, 400 µL of PEG / NaCl was added, and the mixture was inverted up and down to mix well, and then left standing at room temperature for 20 min. Centrifuged at 13500 g for 10 min at 4 °C, the supernatant was discarded, and the precipitate was resuspended in 100 µL of iodide buffer, 250 µL of absolute ethanol was added, and the mixture was left standing at room temperature for 20 min. Centrifuged at 13500 g for 10 min at 4 °C, the supernatant was discarded, the precipitate was washed with 0.5 mL of 70% ethanol (pre-cooled at -20 °C), centrifuged, and briefly dried under vacuum. The washed precipitate was dissolved in 40 µL of sterile water, 5 µL was taken for agarose gel electrophoresis analysis, and 10 µL was taken for DNA sequencing. The sequencing primer was (-96 gIII): 5’-CCCTCATAGTTAGCGTAACG-3’. The sequencing results showed that the DNA sequence was as shown in SEQ ID No.1: 5’-TATGTGTATCCTCAGATGCCT-3’, and according to the DNA codon table, the amino acid sequence of the diethylstilbestrol mimotope could be obtained as: YVYPQMP.

[0067] The amplification process of the above phages with diethylstilbestrol mimotopes is as follows:

[0068] (1)Add the phage with the diethylstilbestrol mimotope to 20 mL of the Escherichia coli ER2738 culture medium in the early logarithmic phase, and culture it with shaking at 37 °C and 150 rpm for 4.5 h;

[0069] (2)Transfer the culture to another centrifuge tube, centrifuge at 7000 g at 4 °C for 10 min, transfer the upper 80% of the supernatant to a fresh tube, add 1 / 6 volume of PEG / NaCl, and let it stand overnight at 4 °C;

[0070] (3)Centrifuge at 7000 g at 4 °C for 15 min, pour off the supernatant, resuspend the precipitate in 1 mL of TBS, and centrifuge at 7000 g at 4 °C for 5 min to precipitate the residual cells.

[0071] (4)Transfer the supernatant to another fresh centrifuge tube, add 1 / 6 volume of PEG / NaCl, and incubate on ice for 60 min. Centrifuge at 7000 g at 4 °C for 10 min, discard the supernatant, and resuspend the precipitate in 200 µL of TBS, 0.02% NaN 3 .

[0072] Example 3

[0073] Indirect competitive ELISA standard curve established with diethylstilbestrol mimotope peptide as the competitive antigen

[0074] (1)Dilute the anti - diethylstilbestrol monoclonal antibody with 0.1 mol / L NaHCO 3 at pH 8.6, coat the enzyme - linked immunosorbent assay (ELISA) plate at a concentration of 1 µg / mL, and incubate overnight at 4 °C.

[0075] (2)The next day, after washing 3 times with PBST (10 mM PBS, 0.05% Tween - 20), block the reaction with 1% BSA at 4 °C for 2 h, wash 3 times with PBST and pat dry.

[0076] (3)Add 50 µL of the phage with the diethylstilbestrol mimotope and 50 µL of the diethylstilbestrol standard or the extract of the sample to be tested to each well, incubate at 37 °C for 45 min, and wash 3 times with PBST.

[0077] (4)Add 100 µL / well of the horseradish peroxidase - labeled anti - M13 phage enzyme - linked secondary antibody, incubate at 37 °C for 30 min. Wash 3 times with PBST, add 100 µL of TMB substrate for color development, and measure the absorbance value of OD 450 .

[0078] (5)Using the logarithm of the diethylstilbestrol concentration as the abscissa and the binding rate (B / B 0 ×100%, where B is the OD of the added diethylstilbestrol 450, B 0 is OD without adding diethylstilbestrol 450 ), using it as the ordinate, draw the indirect competitive ELISA standard curve of diethylstilbestrol. The results are as attached Figure 1 shown. The results show good linear correlation, and for the indirect competitive ELISA standard curve established with YVYPQMP as the diethylstilbestrol mimotope peptide, its IC 50 value is 146 pg / ml. Use the same method to establish the ELISA standard curves of other diethylstilbestrol mimotopes (YVYPQQK, YVYPPGP, YVYPQSR). The results are as Figure 2 shown. The IC 50 values of the diethylstilbestrol mimotope peptides YVYPQQK, YVYPPGP, and YVYPQSR and the anti-diethylstilbestrol antibody are 185 pg / ml, 249 pg / ml, and 393 pg / ml respectively; and from Figure 2 it can be seen that the detection sensitivity of the diethylstilbestrol mimotope YVYPQMP selected from the phage display diethylstilbestrol mimotope peptide library constructed by targeting is significantly higher than that of the diethylstilbestrol mimotopes (YVYPQQK, YVYPPGP, YVYPQSR) selected from the phage display random natural peptide library.

[0079] Example 4

[0080] Use the diethylstilbestrol mimotope peptide provided by the present invention as a competitive antigen in enzyme-linked immunosorbent assay to detect diethylstilbestrol in chicken, fish, and shrimp meat

[0081] (1) Sample treatment: Break chicken meat, fish meat, and shrimp meat separately with a blender. Weigh 2 g each into 50 mL centrifuge tubes, add 6 mL of acetonitrile-acetone (84 + 16, v / v), shake at 220 rpm for 10 min, and centrifuge at 1000 g at 15 °C for 10 min. Take 3 mL of the supernatant and dry it under nitrogen at 60 °C in a water bath. Add 0.5 mL of chloroform, vortex for 20 s, add 2 mL of 2 mol / L sodium hydroxide solution, vortex for 30 s, and centrifuge at 1000 g for 5 min. Take 1 mL of the supernatant, add 200 µL of 6 mol / L phosphoric acid solution, vortex for 5 s, add 3 mL of acetonitrile for extraction, and shake for 10 min. Centrifuge at 1000 g for 10 min, take 1 mL of the upper organic phase, and dry it under nitrogen at 60 °C in a water bath. Dissolve the residue with 1 mL of buffer working solution, and take 50 µL as the test sample solution for analysis.

[0082] (2) Antibody coating and blocking: Use 0.1 mol / L NaHCO 3Dilute the anti-diethylstilbestrol monoclonal antibody at pH 8.6 and coat the enzyme-linked immunosorbent assay (ELISA) plate at a concentration of 1 µg / mL, incubating overnight at 4 °C. The next day, after washing 3 times with PBST (10 mM PBS, 0.05% Tween-20), block the reaction with 1% BSA at 4 °C for 2 h, wash 3 times with PBST and pat dry.

[0083] (3) Establishment of the standard curve: Take the ELISA plate processed in step (2), add 50 µL of phage mimicking the epitope of diethylstilbestrol antigen and 50 µL of a series of diethylstilbestrol standard products with different concentrations to each well, incubate at 37 °C for 45 min, and wash 3 times with PBST. Add 100 µL of horseradish peroxidase-labeled anti-M13 phage enzyme-linked secondary antibody per well, incubate at 37 °C for 30 min. Wash 3 times with PBST, add 100 µL of TMB substrate for color development, and measure the absorbance value of OD 450 with an enzyme-linked immunosorbent assay reader. Using the logarithm of the diethylstilbestrol concentration as the abscissa and the binding rate (B / B 0 × 100%, where B is the OD 450 after adding diethylstilbestrol, and B 0 is the OD 450 without adding diethylstilbestrol) as the ordinate, plot the indirect competitive ELISA standard curve for diethylstilbestrol.

[0084] (4) Detection of samples: Take the ELISA plate processed in step (2), add 50 µL of phage mimicking the epitope of diethylstilbestrol antigen and 50 µL of the extract of the sample to be tested to each well, incubate at 37 °C for 45 min, and wash 3 times with PBST. Add 100 µL of horseradish peroxidase-labeled anti-M13 phage enzyme-linked secondary antibody per well, incubate at 37 °C for 30 min. Wash 3 times with PBST, add 100 µL of TMB substrate for color development, and measure the absorbance value of OD 450 with an enzyme-linked immunosorbent assay reader. Using the logarithm of the diethylstilbestrol concentration as the abscissa and the binding rate (B / B 0 × 100%, where B is the OD 450 after adding diethylstilbestrol, and B 0 is the OD 450 without adding diethylstilbestrol) as the ordinate, plot the indirect competitive ELISA standard curve for diethylstilbestrol. According to the standard curve, calculate the measured values of the diethylstilbestrol standard added to the samples as shown in Table 1. According to the calculation formula of the spike recovery rate, the spike recovery rates of adding diethylstilbestrol in chicken, fish, and shrimp meat are 80.95 - 94.12%, and the relative standard deviation is less than 9%, and the test results are accurate and reliable.

[0085]

[0086] In the formula: P represents the spike recovery rate; m 0 represents the mass of the standard substance added; m1 represents the mass of the analyte in the test sample; m 2 represents the mass of the analyte in the spiked test sample.

[0087] Table 1 Recovery rates of spiked diethylstilbestrol in chicken, fish and shrimp meat

[0088]

[0089] Although the description of the present invention has been quite detailed and several of the described embodiments have been particularly described, it is not intended to be limited to any of these details or embodiments or any particular embodiment, but should be regarded as providing a broad interpretation of these claims in view of the prior art by reference to the appended claims, so as to effectively cover the intended scope of the present invention. In addition, the present invention is described above in terms of embodiments foreseeable by the inventors for the purpose of providing a useful description, and those non-substantive modifications to the present invention that are not currently foreseeable may still represent equivalent modifications of the present invention.

Claims

1. An antigen mimetic epitope peptide of diethylstilbestrol, characterized in that: Its amino acid sequence is YVYPQMP.

2. A gene encoding the antigen mimetic epitope peptide according to claim 1, characterized in that: Its nucleotide sequence is: TATGTGTATCCTCAGATGCCT.

3. A method for preparing the antigen mimetic epitope peptide according to claim 1, characterized in that: The following steps are involved: The phage with the epitope mimicking the diethylstilbestrol antigen was added to the culture medium of Escherichia coli ER2738 in the early logarithmic phase and cultured with shaking for 4.5 h. After the culture was completed, the culture was centrifuged for 10 min. The supernatant was taken and PEG / NaCl was added. The culture was allowed to stand overnight at 4°C and then centrifuged again. The precipitate was resuspended in TBS and centrifuged again for 5 min to precipitate the residual cells. The supernatant was obtained and PEG / NaCl was added. The culture was incubated on ice for 60 min and centrifuged for 10 min. The supernatant was discarded and the precipitate was finally resuspended in TBS and 0.02% NaN3.

4. The use of the antigen mimicking epitope peptide according to claim 1 in immunological detection and analysis, characterized in that: The antigen-mimicking epitope peptide is used as a competitive antigen in enzyme-linked immunosorbent assay to detect diethylstilbestrol in food.

5. The use according to claim 4, characterized in that: Food includes chicken, fish and shrimp.

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