CN104356207B - Fumonisins B1Dodecapeptide antigenic epitope and its application - Google Patents

Abstract

The invention belongs to biological technical field, it is related to fumonisins B₁Dodecapeptide antigenic epitope, its amino acid sequence be FYTSPGRTSHYM.FB of the present invention₁Antigenic epitope can replace expensive and strong toxicity FB₁Standard items, and it is applied to FB as competition antigen or solid-phase coating antigen₁Immunology detection, the antigenic epitope have and natural FB₁The similar immune response characteristic of molecule, effect is very good.Reduce FB₁Harm to health, has saved cost, with very high application value.

Description

Dodecapeptide Antigen Mimic Epitope of Fumonisin B1 and Its Application

technical field

The invention belongs to the field of biotechnology, and in particular relates to _a fumonisin B1 antigen mimic epitope and an application thereof.

Background technique

Fumonisins B ₁ (Fumonisins B ₁ , FB ₁ ) is a common mycotoxin, mainly produced by Fusarium moniliforme. Studies have shown that FB ₁ has strong toxic effects on humans and animals, including nerve Toxicity, reproductive toxicity, embryotoxicity and teratogenic carcinogenicity, etc., the International Center for Cancer Research has divided FB ₁ into Group 2B, which is a possible carcinogen to humans. At present, most countries have set limits on the residual content of FB ₁ in grains, grains and foods. For example, the Food and Agriculture Organization of the United Nations (FAO) stipulates that the maximum daily intake of FB ₁ , FB ₂ , and FB ₃ is 2 µg. /kg body weight; Switzerland stipulates that the total residue limit of FB ₁ and FB ₂ in grain is 1000 µg/L.

At present, the methods for detecting FB ₁ in food mainly include high performance liquid chromatography, gas chromatography, thin _- layer chromatography and immunological detection. has been widely applied. However, in the process of establishing an immunological detection method, the FB ₁ standard must be used as a raw material to prepare a competing antigen or a solid-phase coated antigen. FB ₁ is not only expensive but also highly carcinogenic, which is harmful to the health and The environment poses a great threat, which to a certain extent restricts the application and promotion of immunological detection methods. In view of this, people began to use anti-idiotypic antibody and antigen mimotope technology to replace harmful small molecule standard products, and some progress has been made. The main feature of phage display peptide library technology is that it can effectively screen out phage display peptides that specifically bind to the target. Molecules, exploration of unknown protein spatial structure epitopes, and development of new vaccines are widely used.

In the present invention, by using phage display peptide library technology, a polypeptide (antigen mimic epitope) capable of specifically binding to _a target molecule (anti-FB ₁ monoclonal antibody) is screened out from the peptide library, and the antigen mimic epitope has the same Molecularly similar immune response characteristics, the obtained FB ₁ antigen mimic epitope can replace the expensive and highly toxic FB ₁ standard, and it can be used as a competitive antigen or solid-phase coating antigen for the immunological detection of FB ₁ .

Contents of the invention

In the present invention, the anti-FB ₁ monoclonal antibody is used as the target molecule, the target molecule is solid-phase-coated on a microtiter plate, put into a phage random display dodecapeptide library, and affinity panned to obtain seven kinds of FB ₁ antigen mimics gauge. Their amino acid sequences are as follows:

NNAAMYSEMATD, FYTSPGRTSHYM, IHQELRYTKDSP, GDGVHKSHDIRG, TTLQMRSEMADD, SMLNDYRDYTTH, TRDKSSMLERWP.

The present invention also relates to the nucleotide sequence encoding the amino acid sequence of the above-mentioned antigen mimotope, corresponding to:

AATAATGCGGCGATGTATTCGGAGATGGCTACTGA、TTTTATACTAGTCCGGGTCGGACGAGTCATTATATG 、ATTCATCAGGAGTTGCGTTATACTAAGGATTCTCCG、GGGGATGGGGTGCATAAGTCGCATGATATCCGTGGG、ACTACGCTTCAGATGCGTAGTGAGATGGCTGATGAT、TCGATGCTTAATGATTATCGTGATTATACTACTCAT、ACTCGGGATAAGTCGTCGATGTTGGAGCGTTGGCCG

In the structure of the above-mentioned antigen mimotope (polypeptide), the uppercase English letters represent one of the 21 known natural L-type amino acid residues or their D-type isomers, C represents the cysteine residue, D represents aspartic acid residues, P represents proline residues, R represents arginine residues, K represents lysine residues, H represents histidine residues, I represents isoleucine residues, V stands for valine residues, Y stands for tyrosine residues, S stands for serine residues, F stands for phenylalanine residues, E stands for glutamic acid residues, M stands for methionine residues, G stands for Glycine residues, L for leucine residues, Q for glutamine residues, W for tryptophan residues, N for asparagine residues, A for alanine residues, T for threonine residues base.

The FB1 antigen mimic epitope (polypeptide) mentioned in the present invention can be produced in large quantities through phage amplification, chemical synthesis or genetic engineering recombinant expression. Phage amplification refers to the production of phage particles displaying FB1 antigen mimic epitopes (polypeptides) in large numbers through biological amplification of phage displaying FB1 antigen mimic epitopes (polypeptides). Chemical synthesis refers to the synthesis of polypeptides by chemically synthesizing them according to the amino acid sequence of the published FB1 antigen mimic epitope. The way of genetic engineering recombinant expression refers to the gene encoding the FB1 antigen mimic epitope is cloned into the expression vector, and a large amount of FB1 antigen mimic epitope is prepared in the form of polypeptide-fusion protein.

The present invention also relates to the application of the fumonisin B ₁ antigen mimic epitope in immunological detection and analysis. The types of immunological detection include enzyme-linked immunosorbent assay, colloidal gold immunochromatography, immunospot hybridization and other immunological analysis detection types based on antigen-antibody specific reaction.

When the fumonisin B ₁ antigen mimic epitope (NNAAMYSEMATD, FYTSPGRTSHYM, IHQELRYTKDSP, GDGVHKSHDIRG, TTLQMRSEMADD, SMLNDYRDYTTH, TRDKSSMLERWP) of the present invention is applied, the synthesized mimic epitope can be used for immunological detection and analysis, and will be amplified by phage The obtained phage particles displaying the FB ₁ antigen mimic epitope (polypeptide) are directly used for analysis and detection. Of course, the FB ₁ antigen mimic epitope can also be excised from the phage instead of the FB ₁ standard for immunological detection and analysis.

It also relates to the application _of fumonisin B1 antigen mimic epitope as solid phase antigen or competition antigen in immunological detection and analysis.

It also relates to the application of fumonisin B1 antigen mimic epitope as _a solid-phase antigen in the detection and analysis of colloidal gold immunochromatography.

The aforementioned fumonisin B ₁ antigen mimic epitope can replace the expensive and highly toxic FB ₁ standard product, and is used as a competitive antigen or solid-phase coating antigen for the immunological detection of FB _1. The antigen mimic epitope has the same The natural FB ₁ molecule has similar immune response properties and works very well.

The beneficial effects of the present invention are: the fumonisin B ₁ antigen mimic epitope of the present invention can replace the expensive and highly toxic FB ₁ standard product, and be applied to the immunological detection of FB ₁ as a competing antigen or a solid-phase coated antigen , the antigen mimic epitope has similar immune response properties to the natural FB ₁ molecule, and the effect is very good. The harm of FB ₁ to human health is reduced, the cost is saved, and the invention has high application value.

Description of drawings

Figure 1 is an indirect competition ELISA standard curve established with FB1 antigen mimotope. The linear regression equation of the standard curve is y = -144.5ln(x) + 196.18, R² = 0.9609, the IC ₅₀ value is 0.562ng/mL, the linear detection range is 0.171~2.420 ng/mL, and the lowest detection limit is 0.121 ng/mL .

detailed description

Example 1. Affinity panning and identification of FB ₁ antigen mimotope

1) Affinity panning of FB ₁ antigen mimic epitope: the specific method is: dilute anti-FB ₁ monoclonal antibody with 10 mM PBS (pH 7.4), and coat a 96-well microtiter plate with a final concentration of 100 μg/mL, Incubate overnight at 4°C. The next day, after washing 10 times with TBST (50 mM NaCl, pH 7.5 containing 0.1% Tween-20 (v/v) ), add 300 μl of blocking solution (3% BSA-PBS) and incubate at 4°C for 2 hours. After 2 hours, the blocking solution was discarded, washed 5 times with TBST, and 100 μl of phage peptide library (phage display dodecapeptide library, purchased from NEB Company, 10-fold diluted phage stock solution with TBS, about 1.0×10 ¹¹ pfu) was added to each well. The reaction was shaken at 22-26°C for 1 hour. Unbound phages were discarded, washed 10 times with TBST, bound phages were eluted with 0.2 M Glycine-HCl (pH 2.2), and immediately neutralized with 15 μl 1 M Tris-HCl (pH 9.1). Take 10 μl of the eluted phage to measure the titer, and the rest was used to infect 20 mL of the E. coli ER2738 strain that had grown to the pre-logarithmic stage for amplification. On the third day, the phage was purified by PEG/NaCl precipitation, and the titer of the amplified phage was determined.

In the second and third rounds of panning, the coated anti-FB ₁ monoclonal antibody concentrations were 75 μg/mL and 50 μg/mL, respectively, and the TBST concentrations used were 0.25% and 0.5%, and the rest of the steps were the same as above.

2) Identification of positive phage clones: 20 phage plaques were randomly selected from the plate for measuring phage titer after the third round of panning, and the phages were amplified. I-ELISA) to identify positive phage clones, the specific method is as follows: first, dilute anti-FB ₁ monoclonal antibody with 10 mM PBS (pH 7.4), coat 96-well microtiter plate with 10 μg/mL, and incubate overnight at 4°C. The next day, after washing 3 times with PBST (10 mM PBS, 0.05% Tween-20 (v/v) ), block with PBS containing 3% skimmed milk powder, incubate at 37°C for 1 hour; add 100 μl of phage plaque amplification solution (1.0×10 ¹¹ pfu), using the original phage peptide library as a negative control, incubate at 37 °C for 1 hour; add 100 µl of HRP-labeled anti-M13 phage secondary antibody diluted 1:5000 times, and incubate at 37 °C for 1 hour; add 100 µl TMB bottom The solution was developed in the dark for 5 minutes, and the absorbance at 450 nm was read with a microplate reader (Thermo Scientific Multiskan FC). The phage clones with OD ₄₅₀ greater than 2 times of the negative control were selected as positive clones.

3) Identification of FB ₁ antigen mimic epitope: The identification of FB ₁ antigen mimic epitope was carried out by indirect competition ELISA, the specific method was: dilute anti-FB ₁ monoclonal antibody with 10 mM PBS (pH 7.4), 10 µg/ mL coated ELISA plate, incubated overnight at 4°C; the next day, after washing 3 times with PBST (10 mM PBS, 0.05% Tween-20 (v/v)), blocked with PBS containing 3% skimmed milk powder, 37 Incubate at ℃ for 1 hour; add 50 µl of phage clones (1.0×10 ¹¹ pfu) identified as positive by indirect ELISA and 50 µl of FB ₁ standard (concentration range 0-20 ng/ml), and incubate at 37°C for 1 hour; add Dilute HRP-labeled anti-M13 phage secondary antibody 100 μl at 1:5000, incubate at 37°C for 1 hour; add 100 μl TMB substrate solution, develop color in the dark for 5 minutes, read OD ₄₅₀ , it can bind to anti-FB ₁ monoclonal antibody, and can Phage blocked by FB ₁ standard, identified as FB ₁ mimotope.

Example 2. Sequencing of the gene encoding the FB ₁ antigen mimotope and determination of its amino acid sequence

The phages identified by indirect competition ELISA displaying FB1 antigen mimic epitopes were amplified, and the DNA sequencing templates of the phages were extracted. The brief process is as follows: For phage amplification, after the first step of centrifugation, transfer 800 µl of the phage-containing supernatant to a new centrifuge tube. Add 200 µl PEG/NaCl to precipitate the phage. After centrifugation, resuspend the pellet in 100 µl iodide buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 4 M NaI), add 250 µl absolute ethanol to precipitate DNA, centrifuge and wash with 70% ethanol Precipitation (DNA sequencing template). The pellet was finally resuspended in 20 µl of sterilized water, and 2 µl was taken for agarose gel electrophoresis analysis; 5 µL of phage template was used for DNA sequencing, and the -96 gIII sequencing primers were: 5'- ^HO CCC TCA TAG TTA GCG TAA CG -3'. According to the DNA sequencing results and the codon table, the amino acid sequence of the FB ₁ antigen mimotope can be obtained. Their amino acid sequences are as follows:

NNAAMYSEMATD, FYTSPGRTSHYM, IHQELRYTKDSP, GDGVHKSHDIRG, TTLQMRSEMADD, SMLNDYRDYTTH, TRDKSSMLERWP.

Example 3. Application of FB ₁ antigen mimotope as a competitive antigen in ELISA

(1) Sample extraction

Weigh 5g of the sample (cereals and related foods), add 25ml of 60% methanol-PBS solution, shake at 200 rpm for 5 minutes; filter the extract with No. 1 filter paper, take 1ml of the filtrate and add 4ml of PBS ( Phosphate buffer, pH = 7.2) After mixing, it is the sample extract, ready for use.

(2) Coating and sealing

Dilute anti-FB ₁ monoclonal antibody with 10 mM PBS (pH 7.4), coat the plate with 10 µg/mL, and incubate overnight at 4°C. The next day, after washing 3 times with PBST (10 mM PBS, 0.05% Tween-20 (v/v)), block with PBS containing 3% skimmed milk powder, incubate at 37°C for 1 hour, then wash the plate 6 times with PBST stand-by.

(3) Establishment of standard curve

Take out the strips treated in step (2), put 50 µl of phage displaying FB ₁ antigen mimic epitope (1.0×10 ¹¹ pfu) and a series of 50 µl of FB ₁ standard with different concentrations into each well, 37°C Incubate for 1 hour. Add 1:5000 dilution of HRP-labeled anti-M13 phage secondary antibody and incubate at 37°C for 1 hour. The color was then developed with TMB substrate and the OD ₄₅₀ was read. Taking the logarithm of FB ₁ concentration as the abscissa and the binding rate (OD ₄₅₀ of wells with FB ₁ added/OD ₄₅₀ of wells without FB ₁ × 100%) as ordinate, an indirect competition standard curve was established. The results showed that the standard curve was S-shaped, and the linear correlation was good (Figure 1). As shown in Figure 1, the indirect competition ELISA standard curve was established with the FB1 antigen mimotope (amino acid sequence NNAAMYSEMATD). The linear regression equation of the standard curve was y = -144.5ln(x) + 196.18, R² = 0.9609, the IC50 value was 0.562ng/mL, the linear detection range was 0.171~2.420 ng/mL, and the lowest detection limit was 0.121 ng/mL.

(4) Detection of samples

Take out the strips treated in step (2), add 50 µl of phage displaying FB ₁ antigen mimic epitope (1.0×10 ¹¹ pfu) and sample extract to each well, and incubate at 37°C for 1 hour. Add 1:5000 dilution of HRP-labeled anti-M13 phage secondary antibody and incubate at 37°C for 1 hour. Then use TMB substrate to develop color, read OD ₄₅₀ , calculate the binding rate, and deduce the content of FB ₁ in the sample according to the standard curve.

Example 4. Application of FB ₁ antigen mimotope as solid-phase antigen in ELISA

(1) Sample extraction

Weigh 5g of the sample (cereals and related foods), add 25ml of 60% methanol-PBS solution, shake at 200 rpm for 5 minutes; filter the extract with No. 1 filter paper, take 1ml of the filtrate and add 4ml of PBS ( Phosphate buffer, pH = 7.2) After mixing, it is the sample extract, ready for use.

(2) Coating and sealing

Dilute phage displaying FB ₁ antigen mimotope (2.0×10 ¹¹ pfu) with 10 mM PBS (pH 7.4), coat 100 microliters on a microtiter plate, and incubate overnight at 4°C. The next day, after washing 3 times with PBST (10 mM PBS, 0.05% Tween-20 (v/v)), block with PBS containing 3% skimmed milk powder, incubate at 37°C for 1 hour, then wash the plate 6 times with PBST stand-by.

(3) Establishment of standard curve

Take out the strips treated in step (2), add 50 µl anti-FB ₁ monoclonal antibody (0.5 ng/ml) and a series of 50 µl FB ₁ standards of different concentrations into each well, and incubate at 37°C for 1 hour. Add 1:2000 dilution of HRP-labeled goat anti-mouse IgG secondary antibody and incubate at 37°C for 1 hour. The color was then developed with TMB substrate and the OD ₄₅₀ was read. Taking the logarithm of FB ₁ concentration as the abscissa and the binding rate (OD ₄₅₀ of wells with FB ₁ added/OD ₄₅₀ of wells without FB ₁ × 100%) as ordinate, an indirect competition standard curve was established.

(4) Detection of samples

Take out the strips treated in step (2), add 50 µl anti-FB ₁ monoclonal antibody (0.5 ng/ml) and a series of 50 µl FB ₁ standards of different concentrations into each well, and incubate at 37°C for 1 hour. Add 1:2000 dilution of HRP-labeled goat anti-mouse IgG secondary antibody and incubate at 37°C for 1 hour. The color was then developed with TMB substrate and the OD ₄₅₀ was read. Taking the logarithm of FB ₁ concentration as the abscissa and the binding rate (OD ₄₅₀ of wells with FB ₁ added/OD ₄₅₀ of wells without FB ₁ × 100%) as ordinate, an indirect competition standard curve was established.

Example 5. Application of FB ₁ antigen mimotope as solid-phase antigen in colloidal gold immunochromatographic analysis

(1) Sample extraction

Weigh 5g of the sample (cereals and related foods), add 25ml of 60% methanol-PBS solution, shake at 200 rpm for 5 minutes; filter the extract with No. 1 filter paper, take 1ml of the filtrate and add 4ml of PBS ( Phosphate buffer, pH = 7.2) After mixing, it is the sample extract, ready for use.

(2) Lattice of phage and control lines

Dilute the phage (2.0×10 ¹¹ pfu) displaying the FB ₁ antigen mimotope of the present invention with 10 mM PBS (pH 7.4), and streak the phage on the nitrocellulose membrane with a dot array instrument or a micropipette (pore size 0.2-0.45 microns), as the detection line; 0.5 mg/ml HRP-labeled goat anti-mouse IgG secondary antibody was streaked on the same nitrocellulose membrane (located above the detection line) , the distance is greater than 5 mm), as a control line.

(3) Colloidal gold-labeled FB ₁ antibody

Add the FB ₁ antibody dropwise to the colloidal gold solution (pH=8.2), and stir while dripping. After 30 minutes, take 1% PEG and add it to the above solution, continue stirring for 15 minutes, and then add 1/10 volume of 10% BSA After stirring the solution for 15 minutes, let it stand for 30 minutes, remove the supernatant after centrifugation, and obtain a colloidal gold-labeled FB ₁ antibody solution.

(4) Assembly of colloidal gold detection card

Spray the colloidal gold-labeled FB ₁ antibody on the colloidal gold pad (1.0 μg/ml), assemble the sample pad, colloidal gold pad, nitrocellulose membrane and absorbent paper dotted with detection lines and control lines, cut into Test strips, loaded into the test card for use.

(5) Detection of samples

Add the sample extract to the sample pad and let it stand for 10 minutes. If the sample contains FB ₁ and exceeds the detection threshold of the colloidal gold detection test paper, the detection line area will not develop color, but the control line area will develop color; if the sample does not contain FB ₁ and lower than the detection threshold of the colloidal gold detection test paper, the detection line area is colored, and the control line area is also colored. If no color develops in the area of the control line, the test strip is invalid.

Example 5 Mass preparation of FB ₁ antigen mimotope

(1) By means of phage amplification

The phage displaying the mimotope of FB1 antigen was added to 20 ml of the culture inoculated with ER 2738, and the culture was shaken at 37 degrees and 220 rpm for 4.5 h. Transfer the culture to another centrifuge tube, centrifuge at 10,000 rpm 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 stand at 4°C for 120 min . Centrifuge the PEG/NaCL solution at 10,000 rpm at 4°C for 15 min. Discard the supernatant, centrifuge briefly and aspirate the residual supernatant. Add 1mL TBS for resuspension, which is the phage amplification solution.

(2) Preparation in the form of FB ₁ antigen mimic epitope-fusion protein

A. PCR amplification of exogenous coding gene of FB1 antigen mimotope

PCR reaction system: (50 µL)

10 × Pyrobest Buffer (Mg2+ plus) 5 µL

dNTP Mixture (each for 2.5 mM) 4 µL

M13KE insert extension primer (10 mM) 1 µL

-96 gIII sequencing primer (10 mM) 1 µL

Phage DNA Template 1 µL

Pyrobest DNA Polymerase 0.5 µL

Sterile ddH2O 37.5 µL

PCR reaction conditions: 95°C for 5 min, followed by 95°C for 30 sec, 55°C for 30 sec, 72°C for 40 sec, and 72°C for 10 min for a total of 30 cycles.

PCR products were purified using a PCR product recovery kit and quantified by a micronucleic acid quantifier. The coding gene sequences of FB1 antigen mimotope respectively correspond to:

AATAATGCGGCGATGTATTCGGAGATGGCTACTGA、TTTTATACTAGTCCGGGTCGGACGAGTCATTATATG 、ATTCATCAGGAGTTGCGTTATACTAAGGATTCTCCG、GGGGATGGGGTGCATAAGTCGCATGATATCCGTGGG、ACTACGCTTCAGATGCGTAGTGAGATGGCTGATGAT、TCGATGCTTAATGATTATCGTGATTATACTACTCAT、ACTCGGGATAAGTCGTCGATGTTGGAGCGTTGGCCG

B. Double enzyme digestion of exogenous coding gene and expression vector

The exogenous coding gene and expression vector (pMAl-pIII, NEB company, which can express MBP fusion protein) were double-digested with ACC65I and Eag I enzymes, respectively.

C. Ligation and transformation of digested products

The plasmid pMal-PIII and the target fragment were mixed at a ratio of 1:10 (molar ratio), ligated in a water bath at 16 °C for 12 h, and 10 μL of the ligated product was added to 100 μL of competent cell TB1, and mixed well. After ice-bathing for 30 min, heat shock in a water bath at 42°C for 90 s, immediately ice-bath for 5 min, add 600 μL LB liquid culture medium, incubate at 37°C, 200 rpm for 1 h, centrifuge at 10,000 rpm for 2 min, remove the supernatant and take About 200 μL was spread on LB-A solid (Ampr) medium, cultured overnight at 37°C, and positive clones were obtained.

D. Expression of FB ₁ antigen mimotope-MBP fusion protein

Pick a single colony of the positive clone obtained above and inoculate it in 5 mL LB-A, 0.2% sucrose, 37°C, 220 r/min, and culture overnight with shaking. v/v) Inoculate in 50 mL of LB-A, 0.2% sucrose medium, inoculate 3 bottles respectively, shake culture at 37°C, 220 r/min, when the bacterial concentration OD600 of the culture reaches 0.6, add to the three bottles of culture Add IPTG to a final concentration of 0.2 mmol/L, culture with shaking at 220 r/min, centrifuge the inducer (PEG solution) at 4 °C, 4000 g for 20 min to collect the bacterial pellet, and discard the supernatant. Resuspend cells in 400 mL 30 mM Tris-HCl, 20% sucrose, pH 8.0 (80 mL/g cell wet weight), add EDTA to 1 mM, shake at room temperature for 5-10 min, 8000g, 4°C, centrifuge for 20 min , discard the supernatant, resuspend the pellet in 400 ml pre-cooled 5 mM MgSO4, shake on ice for 10 min, centrifuge at 8000g, 4°C for 20 min, keep the supernatant, add 8 mL 1 M Tris-HCl to the supernatant, pH 7.4, FB1 antigen mimotope-MBP fusion protein was obtained.

SEQUENCE LISTING

<110> Nanchang University

<120> Dodecapeptide Antigen Mimotope of Fumonisin B ₁ and Its Application

<130> 1

<160> 14

<170> PatentIn version 3.3

<210> 1

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<213> Artificial sequence

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Asn Asn Ala Ala Met Tyr Ser Glu Met Ala Thr Asp

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Phe Tyr Thr Ser Pro Gly Arg Thr Ser His Tyr Met

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Ile His Gln Glu Leu Arg Tyr Thr Lys Asp Ser Pro

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Gly Asp Gly Val His Lys Ser His Asp Ile Arg Gly

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Thr Thr Leu Gln Met Arg Ser Glu Met Ala Asp Asp

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Ser Met Leu Asn Asp Tyr Arg Asp Tyr Thr Thr His

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Thr Arg Asp Lys Ser Ser Met Leu Glu Arg Trp Pro

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aataatgcgg cgatgtattc ggagatggct actgat 36

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ttttatacta gtccgggtcg gacgagtcat tatatg 36

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attcatcagg agttgcgtta tactaaggat tctccg 36

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ggggatgggg tgcataagtc gcatgatatc cgtggg 36

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actacgcttc agatgcgtag tgagatggct gatgat 36

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tcgatgctta atgattatcg tgattatact actcat 36

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<212>DNA

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actcgggata agtcgtcgat gttggagcgt tggccg 36

Claims (2)

1. The dodecapeptide mimotope of fumonisin B ₁ , characterized in that the amino acid sequence is: FYTSPGRTSHYM, IHQELRYTKDSP. 2. The nucleotide encoding the mimotope amino acid sequence of the dodecapeptide antigen described in claim 1.