CN104119425B - The antigenic epitope of simulation norfloxacin and application thereof - Google Patents

Abstract

The invention belongs to biological technical field, relate to the antigenic epitope of norfloxacin, its aminoacid sequence is DHAYWPKFWGKA.Norfloxacin antigenic epitope of the present invention can replace the expensive and norfloxacin standard substance of strong toxicity, and the immunology detection of norfloxacin it is applied to as competition antigen or solid-phase coating antigen, this antigenic epitope has and the immunoreation characteristic of natural norfloxacin molecular mimicry, and effect is the best.Decrease the norfloxacin harm to health, saved cost, there is the highest using value.

Description

Antigen mimotope that mimics norfloxacin and its application

technical field

The invention belongs to the field of biotechnology, and in particular relates to norfloxacin antigen mimic epitopes and applications thereof.

Background technique

Norfloxacin (Norfloxacin, NOR) is a broad-spectrum antibiotic, which can absorb water in the air, and its color will gradually become darker when exposed to light. It is slightly soluble in dimethylformamide, very slightly soluble in water or ethanol, and is Soluble in hydrochloric acid or sodium hydroxide solution, it has high antibacterial activity against aerobic gram-negative bacilli, and has good antibacterial effect on most bacteria in vitro, such as most bacteria of Enterobacteriaceae, including Citrobacter, cloacae Enterobacter, Enterobacter aerogenes, Escherichia coli, Klebsiella, Proteus, Salmonella, Shigella, Vibrio, Yersinia, etc. Norfloxacin also has antibacterial activity against multidrug-resistant bacteria in vitro. It also has good antibacterial effect on penicillin-resistant Neisseria gonorrhoeae, Haemophilus influenzae and Moraxella catarrhalis. Norfloxacin is a fungicide that inhibits DNA synthesis and replication by acting on the A subunit of bacterial DNA helicase, leading to bacterial death. However, due to the abuse of antibiotics, the detection of norfloxacin residues is particularly important.

At present, the methods for detecting norfloxacin in food mainly include high performance liquid chromatography, gas chromatography, thin layer chromatography and immunological detection. It has been widely used in the detection of sand stars. However, in the process of establishing an immunological detection method, standard norfloxacin must be used as a raw material to prepare a competing antigen or a solid-phase coated antigen. Norfloxacin is not only expensive but also highly carcinogenic. It poses a great threat to the health of personnel and the environment, thus restricting the application and promotion of immunological detection methods to a certain extent. 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 the phage display peptide library technology, the polypeptide (antigen mimic epitope) that can specifically bind to the target molecule (anti-norfloxacin monoclonal antibody) is screened out from the peptide library. The similar immune response characteristics of norfloxacin molecules, through the obtained norfloxacin antigen mimetic epitope, can replace the expensive and highly toxic norfloxacin standard substance, and can be used as a competitive antigen or solid-phase coating antigen Immunological detection of norfloxacin.

Contents of the invention

In the present invention, the anti-norfloxacin monoclonal antibody is used as the target molecule, the target molecule is solid-phase-coated on the microtiter plate, put into the phage random display loop seven and twelve peptide libraries, respectively, and affinity panning is carried out to obtain Four antigenic mimotopes of norfloxacin (one each for cyclic heptadeptide and dodecapeptide). Their amino acid sequences are as follows:

Norfloxacin antigen mimic epitope 1: Norfloxacin antigen mimic epitope (polypeptide) screened from a random cyclic heptapeptide library of bacteriophage, its amino acid sequence is: CKFGFEPFC.

Norfloxacin antigen mimic epitope 2: Norfloxacin antigen mimic epitope (polypeptide) screened from phage random cyclic heptapeptide library,

Its amino acid sequence is: CNTARWPFC.

Norfloxacin antigen mimic epitope 3: Norfloxacin antigen mimic epitope (polypeptide) screened from phage random dodecapeptide library,

Its amino acid sequence is: DHAYWPKFWGKA.

Norfloxacin antigen mimic epitope 4: Norfloxacin antigen mimic epitope (polypeptide) screened from phage random dodecapeptide library,

Its amino acid sequence is: SRMGPENWDKWY.

The present invention also relates to the nucleotide sequence encoding the above antigen mimotope amino acid sequence, preferably AAG TTTGGG TTT GAG CCG TTT; GTT AAT ACG GCG AGG TGG CCG TTT; GAT CAT GCG TAT TGG CCGAAG TTT TGG GGT AAG GCT; AGT CGG ATG GGT CCG GAG AAT TGG GAT AAG TGG TAT.

In the above-mentioned antigen mimotope (polypeptide) structure, the uppercase English letters represent one of the 21 known natural L-type amino acid residues or their D-type isomers, that is, C represents cysteine residues , D stands for aspartic acid residues, P stands for proline residues, R stands for arginine residues, K stands for lysine residues, H stands for histidine residues, I stands for isoleucine residues , V represents a valine residue, Y represents a tyrosine residue, S represents a serine residue, F represents a phenylalanine residue, E represents a glutamic acid residue, and M represents a methionine residue. The two cysteine residues at the N-terminus and C-terminus of mimotope 1 form an intramolecular disulfide bond, which is a ring structure.

The mimotope (polypeptide) of the norfloxacin antigen mentioned in the present invention can be produced in large quantities by phage amplification, chemical synthesis or genetic engineering recombinant expression. Phage amplification means that the phage displaying the norfloxacin antigen mimic epitope (polypeptide) will be reproduced in large quantities through biological amplification to produce phage particles displaying the norfloxacin antigen mimic epitope (polypeptide). Chemical synthesis refers to the synthesis of polypeptides by chemically synthesizing them according to the published amino acid sequences of mimotope 1, mimotope 2, mimotope 3, and mimotope 4. The way of genetic engineering recombinant expression refers to the gene encoding mimotope 1, mimotope 2, mimotope 3 or mimotope 4, by cloning into the expression vector, and carrying out norfloxacin in the form of polypeptide-fusion protein. Large-scale preparation of antigen mimotopes.

The present invention also relates to the application of the mimetic epitope of the norfloxacin antigen 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 applying the norfloxacin antigen mimic epitope (CKFGFEPFC, CNTARWPFC, DHAYWPKFWGKA, SRMGPENWDKWY) of the present invention, the synthetic mimic epitope can be used for immunological detection and analysis, and the norfloxacin antigen displayed by phage amplification The phage particles of the antigen mimic epitope (polypeptide) are directly used for analysis and detection. Of course, the norfloxacin antigen mimic epitope can also be excised from the phage instead of the norfloxacin standard for immunological detection and analysis.

It also relates to the application of norfloxacin antigen mimetic epitope as solid phase antigen or competing antigen in immunological detection and analysis.

It also relates to the application of norfloxacin antigen mimic epitope as solid-phase antigen in colloidal gold immunochromatographic detection and analysis.

The aforementioned norfloxacin antigen mimic epitope can replace the expensive and highly toxic norfloxacin standard substance, and can be used as a competitive antigen or solid-phase coating antigen in the immunological detection of norfloxacin. The antigen mimic epitope Has similar immune response properties to the natural norfloxacin molecule and works very well.

The beneficial effect of the present invention is: the norfloxacin antigen mimetic epitope of the present invention can replace the expensive and highly toxic norfloxacin standard substance, and be used as the competition antigen or solid-phase coated antigen for the immunization of norfloxacin According to scientific testing, the antigen mimic epitope has similar immune response characteristics to natural norfloxacin molecules, and the effect is very good. The harm of norfloxacin to human health is reduced, the cost is saved, and the invention has high application value.

Description of drawings

Figure 1. Indirect competition ELISA standard curve established with norfloxacin antigen mimotope. (The numbers c7-13, c7-15, 12-24, and 12-29 represent four antigen mimotopes of norfloxacin respectively, and their amino acid sequences are CKFGFEPFC, CNTARWPFC, DHAYWPKFWGKA, SRMGPENWDKWY respectively)

detailed description

Example 1. Affinity panning and identification of norfloxacin antigen mimotope

1) Affinity panning of norfloxacin antigen mimotope: the specific method is: dilute anti-norfloxacin monoclonal antibody with 10 mM PBS (pH 7.4), and coat 96 wells with a final concentration of 100 μg/mL ELISA plates were incubated 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, discard the blocking solution, wash 5 times with TBST, add 100 μl phage peptide library (phage display ring heptapeptide library or dodecapeptide library, purchased from NEB Company, dilute the phage stock solution 10 times with TBS, about 1.0× 10 ¹¹ pfu), shake and react for 1 hour at 22-26°C. 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.

During the second and third rounds of panning, the coated anti-norfloxacin 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 remaining steps Ditto.

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-norfloxacin monoclonal antibody with 10 mM PBS (pH 7.4), coat 96-well microtiter plate with 10 μg/mL, and incubate at 4 °C overnight. 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 The substrate solution was developed in the dark for 5 minutes, and the absorbance at 450 nm was read with a microplate reader (ThermoScientific Multiskan FC). The phage clones with OD ₄₅₀ greater than 2 times of the negative control were selected as positive clones.

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

Example 2. The sequencing of the mimotope encoding gene of norfloxacin antigen and the determination of its amino acid sequence

The phages identified by indirect competition ELISA displaying mimotopes of the norfloxacin antigen 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 sequences of the mimotopes of the norfloxacin antigen can be obtained: CKFGFEPFC, CNTARWPFC, DHAYWPKFWGKA and SRMGPENWDKWY.

Example 3. The application of mimotopes of norfloxacin antigens as competitive antigens 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, the sample extract is ready for use.

(2) Coating and sealing

Dilute anti-norfloxacin monoclonal antibody with 10 mM PBS (pH 7.4), coat the 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, then wash the plate 6 times with PBST stand-by.

(3) Establishment of standard curve

Take out the strips treated in step (2), and put 50 µl of phage displaying norfloxacin antigen mimic epitope (1.0×10 ¹¹ pfu) and a series of 50 µl of norfloxacin standards with different concentrations into each well products and incubated 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. The color was then developed with TMB substrate and the OD ₄₅₀ was read. Take the logarithm of norfloxacin concentration as the abscissa, and the binding rate (OD ₄₅₀ of the wells with norfloxacin added/OD ₄₅₀ × 100% of the wells without norfloxacin) as the ordinate to establish an indirect competition standard curve (figure 1).

(4) Detection of samples

Take out the strips treated in step (2), add 50 µl of phage displaying norfloxacin antigen mimic epitope (1.0×10 ¹¹ pfu) and test 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 norfloxacin in the sample according to the standard curve.

Embodiment 4. Norfloxacin antigen mimotope is used 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, the sample extract is ready for use.

(2) Coating and sealing

Dilute the phage displaying the mimotope of norfloxacin antigen (2.0×10 ¹¹ pfu) with 10 mM PBS (pH 7.4), coat 100 microliters on the 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-norfloxacin monoclonal antibody (0.5ng/ml) and a series of 50 μl norfloxacin standards of different concentrations to each well, and incubate at 37°C 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. Take the logarithm of norfloxacin concentration as the abscissa, and the binding rate (OD ₄₅₀ of the wells with norfloxacin added/OD ₄₅₀ × 100% of the wells without norfloxacin) as the ordinate to establish an indirect competition standard curve .

(4) Detection of samples

Take out the strips treated in step (2), add 50 μl anti-norfloxacin monoclonal antibody (0.5ng/ml) and a series of 50 μl norfloxacin standards of different concentrations to each well, and incubate at 37°C 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. Take the logarithm of norfloxacin concentration as the abscissa, and the binding rate (OD ₄₅₀ of the wells with norfloxacin added/OD ₄₅₀ × 100% of the wells without norfloxacin) as the ordinate to establish an indirect competition standard curve

Example 5. Application of Norfloxacin 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, the sample extract is ready for use.

(2) Lattice of phage and control lines

Dilute the phage (2.0×10 ¹¹ pfu) displaying the mimotope of norfloxacin antigen with 10 mM PBS (pH 7.4), and streak the phage on the nitrocellulose membrane (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 norfloxacin antibody

Add the norfloxacin antibody dropwise into 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 one-tenth of the volume of 10 % BSA solution, after stirring for 15 minutes, let it stand for 30 minutes, remove the supernatant after centrifugation, and obtain the colloidal gold-labeled norfloxacin antibody solution.

(4) Assembly of colloidal gold detection card

Spray the colloidal gold-labeled norfloxacin antibody on the colloidal gold pad (1.0 μg/ml), and assemble the sample pad, colloidal gold pad, nitrocellulose membrane and absorbent paper dotted with detection lines and control lines, Cut into test strips and put them into test cards for later use.

(5) Detection of samples

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

Example 5 Mass preparation of norfloxacin antigen mimotope

(1) By means of phage amplification

The phage displaying the mimotope of the norfloxacin antigen was added to 20 ml of culture inoculated with ER 2738, and 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 by way of norfloxacin antigen mimic epitope-fusion protein

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

PCR reaction system: (50 µL)

10 × Pyrobest Buffer (Mg2+ plus) 5 µL

dNTP Mixture (each for 2.5 mM) 4µL

M13KE insert extension primer (10 mM) 1µL

-96 gIII sequencing primer (10 mM) 1 µL

Phage DNA Template 1µL

Pyrobest DNA Polymerase 0.5 µL

Sterile ddH2O 37.5 µL

PCR reaction conditions:

95°C for 5 minutes

Then 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 sequence of norfloxacin antigen mimotope 1 is AAG TTT GGG TTT GAG CCG TTT; the coding gene sequence of norfloxacin antigen mimotope 2 is: GTT AAT ACG GCG AGG TGG CCG TTT; The coding gene sequence of antigen mimotope 3 is GAT CAT GCG TAT TGG CCG AAG TTT TGG GGT AAG GCT; the coding gene sequence of norfloxacin antigen mimotope 4 is: AGT CGG ATG GGT CCG GAG AAT TGG GAT AAG TGG TAT .

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.

Expression of mimotope-MBP fusion protein of norfloxacin antigen

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 of 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 of 1 M Tris-HCl to the supernatant, pH 7.4, to obtain norfloxacin antigen mimic epitope-MBP fusion protein.

SEQUENCE LISTING

<110> Nanchang University

<120> Antigen Mimotope Mimicking Norfloxacin and Its Application

<130> 1

<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 9

<212> PRT

<213> Artificial sequence

<400> 1

Cys Lys Phe Gly Phe Glu Pro Phe Cys

1 5

<210> 2

<211> 9

<212> PRT

<213> Artificial sequence

<400> 2

Cys Asn Thr Ala Arg Trp Pro Phe Cys

1 5

<210> 3

<211> 12

<212> PRT

<213> Artificial sequence

<400> 3

Asp His Ala Tyr Trp Pro Lys Phe Trp Gly Lys Ala

1 5 10

<210> 4

<211> 12

<212> PRT

<213> Artificial sequence

<400> 4

Ser Arg Met Gly Pro Glu Asn Trp Asp Lys Trp Tyr

1 5 10

<210> 5

<211> 21

<212>DNA

<213> Artificial sequence

<400> 5

aagtttgggt ttgagccgtt t 21

<210> 6

<211> 24

<212>DNA

<213> Artificial sequence

<400> 6

gttaatacgg cgaggtggcc gttt 24

<210> 7

<211> 36

<212>DNA

<213> Artificial sequence

<400> 7

gatcatgcgt attggccgaa gttttggggt aaggct 36

<210> 8

<211> 36

<212>DNA

<213> Artificial sequence

<400> 8

agtcggatgg gtccggagaa ttgggataag tggtat 36

Claims (6)

1. An antigen mimotope that mimics norfloxacin, characterized in that the amino acid sequence is SRMGPENWDKWY. 2. The nucleotide encoding the amino acid sequence of the antigen mimotope described in claim 1. 3. The nucleotide according to claim 2, the sequence is AGT CGG ATG GGT CCG GAG AAT TGG GAT AAGTGG TAT. 4. The application of the antigen mimotope described in claim 1 in immunological detection and analysis. 5. The application according to claim 4, characterized in that the mimotope of the norfloxacin antigen is used in immunological detection and analysis with solid-phase antigen or competition antigen. 6. The application according to claim 4, characterized in that the mimotope of the norfloxacin antigen is used as a solid-phase antigen in colloidal gold immunochromatography detection and analysis.