Enrofloxacin-mimic antigen epitope peptide, and preparation method and application thereof

[0038] Example 1. Panning and identification of ENR antigen mimotope

[0039] (1) Affinity panning of ENR antigen mimotope: the specific method is: 0.1M NaHCO 3 (pH 8.6) Dilute the anti-ENR monoclonal antibody, and add the final concentration of 80μg/ml to a 96-well microtiter single well, and coat overnight at 4°C. The next day, it was quickly washed 6 times with TBST [50mM Tris-HCl (pH7.5), 150mM NaCl, 0.1% Tween-20 (v/v)], then 350μl of 1% OVA blocking solution was added and blocked at 4°C for 2h. Discard the blocking solution, wash with TBST 6 times, add 120μl of phage peptide library (phage display hepeptide library, purchased from NEB, dilute the phage with TBST, add 2.0×10 11 pfu), react at 25°C for 50 min. The phage in the well was discarded, washed 10 times with TBST, patted dry, eluted with 0.2M Glycine-HCl (pH 2.2) for 8 min, and then neutralized with 15 μl of 1M Tris-HCl (pH 9.1). Take 5μl of the eluted phage to measure the titer, and use the rest for E.coli ER2738 strain for amplification. The phage was purified with PEG/NaCl the next day, and the titer of the amplified phage was determined. In the second and third rounds of panning, the coated anti-ENR monoclonal antibody concentrations were 40 μg/ml and 20 μg/ml, respectively, the blocking solution was 1% BSA and 1% OVA, and the TBST concentration used was 0.3 % And 0.5%. In the elution mode, 0.2MGlycine-HCl (pH 2.2) was still used in the second round, and ENR standard (10ng/ml) competitive elution was used in the third round, and the remaining steps were the same as above.

[0040] (2) Identification of positive phage clones: 24 phage plaques were randomly selected from the plate where the phage titer was measured after the third round of panning for phage amplification and purification, and the purified product was purified by indirect enzyme-linked immunosorbent method. For positive identification, the specific operation is as follows: Use 0.1M NaHCO 3 (pH 8.6) Dilute the anti-ENR monoclonal antibody, coat a 96-well ELISA plate with 1μg/ml, and incubate overnight at 4°C. After washing 3 times with PBST [10mM PBS, pH7.4, 0.05% Tween-20 (v/v)] the next day, blocking with 1% BSA, blocking for 2 hours at 4°C; discarding the blocking solution, and washing 3 times with PBST; 100μl plaque purification solution (about 1.0×10 10 pfu), with the original phage peptide library as a negative control, incubate at 37°C for 45min, wash 6 times with PBST; add 100μl of HRP-labeled anti-M13 phage secondary antibody diluted 1:5000, incubate at 37°C for 45min, wash 6 times with PBST; add 100μl TMB substrate solution, protect from light for 10 minutes, read the absorbance value at 450nm with a microplate reader. Select OD 450nm Phage clones that are 2 times larger than the negative control are considered positive clones.

[0041] (3) Identification of ENR antigen mimic epitope: use indirect competitive enzyme-linked immunosorbent assay to determine the epitope of all positive phage clones, the specific operation is as follows: use 0.1M NaHCO 3 (pH 8.6) Dilute the anti-ENR monoclonal antibody, coat a 96-well ELISA plate with 1μg/ml, and incubate overnight at 4°C. After washing 3 times with PBST [10mM PBS, pH7.4, 0.05% Tween-20 (v/v)] the next day, block with 1% BSA and block for 2h at 4℃; discard the blocking solution, wash 3 times with PBST, add 50μl of positive phage clones (about 1.0×10 10 pfu) and 50μl ENR standard (10ng/ml), incubate at 37℃ for 45min, wash 6 times with PBST; add 100μl of 1:5000 diluted HRP-labeled anti-M13 phage secondary antibody, incubate at 37℃ for 45min, wash 6 times with PBST; add 100μl TMB Substrate solution, protected from light for 10 minutes, read the absorbance value at 450nm with a microplate reader. Phages that can be inhibited by ENR standard products are identified as ENR antigen mimotopes.

[0042] Example 2. Sequencing of ENR antigen mimotope coding gene and determination of its amino acid sequence

[0043] The phage identified as the mimic epitope of the ENR antigen by indirect competitive enzyme-linked immunosorbent assay was amplified, and the phage DNA was extracted. The operation process is as follows: amplify the target phage, after the amplified product is centrifuged, transfer 500μl of phage supernatant to a new centrifuge tube; add 200μl of PEG/NaCl to precipitate the phage, stand for 20min, and centrifuge at 14000rpm for 10min. The supernatant was discarded, the pellet was resuspended in 100μl of iodide buffer (10mM Tris-HCl (pH 8.0), 1mM EDTA, 4M NaI), 250μl of absolute ethanol was added for precipitation, and the pellet was allowed to stand for 15min and centrifuged at 14000rpm for 10min. After discarding the supernatant, wash the precipitate (DNA sequencing template) with 70% ethanol, centrifuge at 14000 rpm for 10 min, discard the supernatant, and briefly vacuum dry. Resuspend the pellet in 30μl TE buffer (10mM Tris-HCl (pH 8.0), 1mM EDTA), take 4μl for agarose gel electrophoresis analysis; take 10μL phage template for DNA sequencing, the -96gIII sequencing primer is: 5'- HOCCC TCA TAG TTA GCG TAA CG-3'. According to the DNA sequencing results and the codon table, the amino acid sequence of the ENR antigen mimotope can be obtained. The amino acid sequence of all plaques is GKPYITW.

[0044] Example 3. Application of ENR antigen mimotope as a competitive antigen in enzyme-linked immunoassay

[0045] (1) Sample extraction

[0046] Remove the fat and connective tissue of the pork, break it up with a blender, weigh 2g into a 15ml centrifuge tube, add 4ml of 50% methanol-PBS solution, shake on a shaker for 3min; centrifuge at 4000rpm for 10min. Repeat the above shaking and centrifugation steps for the precipitation, combine the two supernatants, centrifuge again at 10000rpm, 4℃ for 10min to collect the supernatant, filter with a microporous membrane (pore size 0.22μm), dilute the filtrate with PBS in equal volume, and mix well spare. Take 50μl of filtrate directly into the micropores for enzyme-linked immunoassay determination.

[0047] (2) Antibody coating

[0048] Use 0.1M NaHCO 3 (pH 8.6) Dilute the anti-ENR monoclonal antibody, coat the microplate with 1μg/ml, and incubate overnight at 4°C. After washing 3 times with PBST [10mM PBS, 0.05% Tween-20(v/v)] the next day, blocking with 1% BSA, blocking at 4℃ for 2h, washing the plate 3 times with PBST, pat dry, and placing it at 4℃ for use .

[0049] (3) Establishment of standard curve

[0050] Take out the slats processed in step (2), add 50μl ENR antigen mimotope phage (about 1.0×10 10 pfu) and a series of 50μl ENR standards of different concentrations, incubated at 37°C for 45 min, washed with PBST 6 times; 100μl 1:5000 diluted HRP-labeled anti-M13 phage secondary antibody was added, incubated at 37°C for 45 min, washed with PBST 6 times. Add 100μl TMB substrate for color development, read the OD with a microplate reader 450nm Absorbance value. Taking the logarithm of ENR concentration as the abscissa, the binding rate (OD 450nm / OD of holes without ENR 450nm ×100%) is the ordinate, the ENR indirect competition ELISA standard curve was established, and the results showed a good linear correlation ( figure 1 ). Such as figure 1 , Standard curve of indirect competitive enzyme-linked immunosorbent assay method established by ENR antigen mimic epitope. ENR antigen mimotope (GKPYITW) and IC of anti-ENR antibody 50 The value is 89.5 pg/ml.

[0051] (4) Testing of samples

[0052] Take out the slats processed in step (2), add 50μl ENR antigen mimotope phage (about 1.0×10 10 pfu) and the sample extract to be tested, incubated at 37°C for 45 min. Discard the reaction solution, wash with PBST 6 times; add 1:5000 diluted HRP-labeled anti-M13 phage secondary antibody, incubate at 37°C for 45 min, discard the reaction solution, wash with PBST 6 times; then use TMB substrate to develop color and read the OD 450nm Value, calculate the binding rate, and calculate the ENR content in the sample according to the standard curve.