A mycophenolic acid mimotope peptide and application thereof

Abstract

The application discloses a fengycin mimotope peptide which can be specifically combined with fengycin antibody, and a sensitive and rapid competitive enzyme-linked immunoassay method can be established by using the phage displaying the mimotope peptide or the mimotope peptide. 50 The IC50 is 0.30 ng / mL, and the LOD is 0.02 ng / mL, and the sensitivity of the method is improved by 60 times compared with the sensitivity of a traditional enzyme-linked immunoassay method based on a hapten; the competitive immunoassay method based on the mimotope peptide has good cross reactivity to other common structural analogues.

Description

Technical Field

[0001] This invention relates to the detection of food, and more specifically, to a bongkrekic acid mimic epitope peptide and its application. Background Technology

[0002] Bongkrekic acid (BA) is a biotoxin secreted by Burkholderia gladioli pv. Cocovenenans (BGC), which easily contaminates cereal products (fermented cornmeal, glutinous corn dumplings, etc.) and spoiled white fungus. Its primary target organs are the liver, brain, and kidneys; poisoning can lead to death from respiratory failure.

[0003] For the detection of bongkrekic acid, instrumental analysis methods require expensive equipment, professional personnel for operation and maintenance, complex pretreatment, long chromatographic times, and cannot achieve rapid on-site detection. Immunoassay, on the other hand, is an analytical technique based on the specific binding reaction of antigen and antibody, suitable for on-site screening, simple, rapid, and low-cost, making it a commonly used rapid food safety detection technology. Chinese patent CN 113156125 A discloses a test strip and method for detecting bongkrekic acid; Chinese patent CN110058008 A discloses a method for detecting bongkrekic acid in food and its application; and Chinese patent CN 113511992 A discloses a bongkrekic acid hapten, its preparation method, and its application. However, bongkrekic acid is highly toxic, and direct coating may pose potential hazards to operators.

[0004] Phage-display peptide library screening yields mimicry epitope peptides (green and non-toxic), which can successfully replace complete antigens in immunoassays. The resulting heterologous immunoassay method significantly improves upon traditional chemical antigen immunoassays. This method is convenient, time-saving, labor-saving, low-cost, and stable. Most importantly, it reduces the risk of laboratory personnel exposure to toxic environments. Mimicry epitope peptides have broad application prospects in the field of immune system analysis and detection; however, no mimicry epitope peptides have been reported for use in BA (bioimmunoassay) detection, either domestically or internationally. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a bongkrekic acid mimic epitope peptide and its application.

[0006] The first objective of this invention is to provide a bongkrekic acid mimic epitope peptide.

[0007] A second objective of this invention is to provide a gene encoding a bongkrekic acid mimic epitope peptide.

[0008] A third objective of this invention is to provide biomaterials.

[0009] The fourth objective of this invention is to provide a bacteriophage.

[0010] The fifth object of the present invention is to provide an immunogenic substance.

[0011] The sixth objective of this invention is to provide a non-diagnostic detection method for bongkrekic acid.

[0012] A seventh object of the present invention is to provide the use of one or more of the bongkrekic acid mimic epitope peptide, the gene, the biomaterial, the bacteriophage, or the substance in non-diagnostic detection of bongkrekic acid and / or in a bongkrekic acid detection kit.

[0013] The eighth object of the present invention is to provide a detection kit for bongkrekic acid.

[0014] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0015] This invention claims protection for a bongkrekic acid mimic epitope peptide, the amino acid sequence of which is shown in SEQ ID NO:1 or 3.

[0016] All bongkrekic acid mimic epitope peptides obtained through biological or chemical synthesis fall within the scope of protection of this invention.

[0017] The present invention also claims protection for a gene encoding a bongkrekic acid mimic epitope peptide, which encodes a peptide with an amino acid sequence as shown in SEQ ID NO:1 or 3, or its reverse complementary sequence encoding a peptide with an amino acid sequence as shown in SEQ ID NO:1 or 3.

[0018] Preferably, the nucleotide sequence is as shown in SEQ ID NO:2 or 4, or the reverse complementary sequence of the nucleotide sequence shown in SEQ ID NO:2 or 4.

[0019] This invention also claims protection for biological materials, which are any of the following:

[0020] (1) An expression cassette containing the gene;

[0021] (2) A recombinant vector containing the gene;

[0022] (3) Recombinant microorganisms containing the aforementioned gene;

[0023] (4) A cell line containing the aforementioned gene;

[0024] (5) A recombinant vector containing the expression cassette described in (1);

[0025] (6) Recombinant microorganisms containing the recombinant vector described in (2) or (5);

[0026] (7) A cell line containing the recombinant vector described in (2) or (5).

[0027] The present invention also claims protection for a bacteriophage having the bongkrekic acid mimic epitope peptide displayed on its surface.

[0028] Specifically, the bongkrekic acid mimic epitope peptide is displayed at the N-terminus of the phage PⅢ capsid protein.

[0029] Specifically, the phage uses the M13 phage plasmid as a vector, and the gene is inserted into the gⅢ gene of the membrane protein encoded by the phage.

[0030] The present invention also claims protection for an immunogenic substance, namely, the bongkrekic acid mimic epitope peptide coupled with a carrier protein.

[0031] Preferably, the carrier protein is OVA or BSA.

[0032] Most preferably, the carrier protein is OVA.

[0033] This invention also claims a non-diagnostic detection method for bongkrekic acid, using an antibody against bongkrekic acid as a coating antibody and the bongkrekic acid mimic epitope peptide, the bacteriophage, and / or the substance as competitive antigens for enzyme-linked immunosorbent assay (ELISA).

[0034] Preferably, the antibody against bongkrekic acid is a monoclonal antibody.

[0035] More preferably, the monoclonal antibody is secreted by a hybridoma cell line that was deposited at the China Center for Type Culture Collection on May 12, 2021, with accession number CCTCCNO:C2021127 and classified as hybridoma cell line A5.

[0036] As a specific embodiment, the enzyme-labeled plate was coated with the bongkrekic acid antibody and then blocked;

[0037] The phage and the sample to be tested were added together to the microwells of the antibody-coated ELISA plate, incubated, washed, and then HRP-labeled anti-M13 phage HRP secondary antibody were added. After washing again, TMB chromogenic solution was added, and the reaction was carried out in the dark. The reaction was terminated with 10% H2SO4, and the absorbance was read (450 nm).

[0038] After establishing a standard curve for a series of concentrations of bongkrekic acid, the OD value of the test sample is used to interpret the test sample.

[0039] The application of one or more of the bongkrekic acid mimic epitope peptide, the gene, the biomaterial, the bacteriophage, or the substance in non-diagnostic detection of bongkrekic acid and / or in bongkrekic acid detection kits is also within the scope of protection of this invention.

[0040] The bongkrekic acid mimic epitope peptide obtained by biological or chemical synthesis can be coupled or uncoupled with a carrier protein to serve as a complete bongkrekic acid antigen, thereby establishing an immunological detection method.

[0041] The present invention also claims a bongkrekic acid detection kit, wherein the bongkrekic acid mimic epitope peptide, the gene, the biomaterial, the bacteriophage, or the substance are one or more of these.

[0042] Preferably, it contains the bacteriophage and an antibody against bongkrekic acid.

[0043] More preferably, the antibody against bongkrekic acid is a monoclonal antibody.

[0044] More preferably, the monoclonal antibody is secreted by a hybridoma cell line that was deposited at the China Center for Type Culture Collection on May 12, 2021, with accession number CCTCCNO:C2021127 and classified as hybridoma cell line A5.

[0045] Compared with the prior art, the present invention has the following beneficial effects:

[0046] This invention provides the first-ever acquisition of a bongkrekic acid mimic epitope peptide, which can specifically bind to bongkrekic acid antibodies. A sensitive and rapid competitive enzyme-linked immunosorbent assay (ELISA) was established using the phage displaying the mimic epitope peptide described in this invention. The competitive immunosorbent assay method constructed using the mimic epitope peptide provided in this invention achieves an IC50 score of [missing value]. 50 The sensitivity of this method is 0.30 ng / mL and the LOD is 0.02 ng / mL. Compared with the sensitivity of the traditional hapten-based enzyme-linked immunosorbent assay (CN114045266A), the sensitivity of this method is improved by 60 times. The competitive immunoassay method based on the mimic epitope peptide provided by this invention has good cross-reactivity with other common structural analogs. Attached Figure Description

[0047] Figure 1 A schematic diagram illustrating simulated peptide screening for bacteriophages.

[0048] Figure 2 Phage-ELISA screening results for phage-mimicked epitope peptides.

[0049] Figure 3 To establish a standard curve for the detection of bongkrekic acid based on phage display of mimic epitope peptides. Detailed Implementation

[0050] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0051] 1. Main reagents

[0052] Peptone, yeast extract, agar, IPTG, Xgal, PEG8000, horseradish peroxidase-labeled anti-M13 monoclonal antibody (Sino Biological).

[0053] 2. Main reagent formulation

[0054] LB liquid medium: 1g peptone, 0.5g yeast extract, 1g NaCl, add 100mL of tertiary water, autoclave, and store at room temperature.

[0055] Top medium: 1g peptone, 0.5g yeast extract, 0.5g NaCl, 0.7g agar, add 100mL of tertiary water, autoclave, and store at room temperature.

[0056] IPTG / Xgal mixture: 1.25g IPTG, 1g Xgal, dissolved in 25ml LDM and sterilized through an organic membrane.

[0057] LB / IPTG / Xgal plates: Sterilize 1L LB medium with 15g / L agar, cool to below 70°C, add 1ml IPTG / Xgal mixture, mix well, and pour into plates. This plate medium should be stored in the dark at 4°C.

[0058] Tet: 20 mg / mL, dissolved in anhydrous ethanol:water = 1:1.

[0059] 20% PEG8000 / NaCl: 80g PEG8000, 58.44g NaCl, diluted to 400mL with grade III water, autoclaved, and stored at room temperature.

[0060] TBS: First, prepare Tris-HCl (pH = 7.5): Add 15.764g Tris to 200mL of grade III water, and adjust the pH to 7.5 with HCl; dissolve 17.532g NaCl in 150mL of Tris-HCl, and then bring the volume to 200mL. Autoclave and store at room temperature.

[0061] 3. Anti-bongkrekic acid antibody: Prepared by the Guangdong Provincial Key Laboratory of Food Safety, South China Agricultural University, it is a monoclonal antibody against bongkrekic acid secreted by a hybridoma cell line. The hybridoma cell line was deposited at the China Center for Type Culture Collection on May 12, 2021, with accession number CCTCC NO:C2021127 and classified as hybridoma cell line A5. The bongkrekic acid monoclonal antibody was purified by protein G column chromatography and used in subsequent examples.

[0062] Example 1: Screening of mimic epitope peptides that specifically bind to bongkrekic acid antibodies

[0063] I. Experimental Methods

[0064] 1. Phage screening and amplification (technical routes such as...) Figure 1 (As shown)

[0065] (1) Panning: Dilute the purified anti-bongkrekic acid antibody (bongkrekic acid monoclonal antibody secreted by hybridoma cell line, which was deposited at China Center for Type Culture Collection on May 12, 2021, with accession number: CCTCC No. C2021127, and classified as hybridoma cell line A5) with 0.01 mol / L PBS, take 100 μL, 100 μg / mL into a high-adsorption ELISA plate (3 replicates), and coat overnight at 4℃.

[0066] (2) Wash the microplate from step (1) twice with PBST (300 μL / well), and incubate with blocking buffer (3% (w / v) skim milk powder) at 37°C for 1 hour.

[0067] (3) After drying the blocking solution of the ELISA plate in step (2), add 100 μL of 1011 pfu / mL phage display heptapeptide library (diluted with 5% skim milk powder) to each well of the ELISA plate, i.e., one round of input, and incubate at 4°C for 2 h.

[0068] (4) Wash 10 times with pre-cooled PBST, then wash 10 times with cold PBS, and then add 1 μg / mL of bongkrekic acid and react at 4℃ for 4h.

[0069] (5) Collect the supernatant from step (4), and add the supernatant (take a small amount of phage to measure the titer, i.e., one round of output) to a container containing 20 mL of E. coli ER2738 (OD2738). 600 In a 250 mL Erlenmeyer flask containing 0.5 g of phage, the phage was amplified and cultured at 37 °C and 250 rpm for 4.5 h on a shaker; at the same time, a small amount of supernatant was taken to measure the titer of the phage.

[0070] (6) Transfer the amplified phage into a 50 mL centrifuge tube, centrifuge at 12000 rpm for 10 min at 4 °C, and collect the supernatant.

[0071] (7) Add 1 / 6 volume of 20% PEG8000 / NaCl to the supernatant of (6), mix thoroughly, and incubate overnight in an ice bath at 4°C.

[0072] (8) Centrifuge the solution from step (7) again at 4°C and 12,000 rpm for 10 min, remove the supernatant, add 1 mL LTBS to resuspend the precipitate, and centrifuge again under the same conditions.

[0073] (9) Take 350 μL of TBS to resuspend the precipitate from step (8) (i.e., the second round of input) for the next round of screening.

[0074] (10) Steps (1) to (9) constitute one round of screening and amplification. Repeat steps (1) to (9) twice as the second and third rounds of screening and amplification, respectively. The antibody concentrations used in step (1) in the second and third rounds of screening and amplification are 50 μg / mL and 10 μg / mL, respectively. The bongkrekic acid used in step (4) in the second and third rounds of screening and amplification is 1 μg / mL and 500 ng / mL, respectively.

[0075] 2. Determination of bacteriophage titer

[0076] (1) Take 10 mL of LB liquid medium, add 0.1% tetracycline (w / w), inoculate with Escherichia coli ER2738, and incubate at 37°C and 250 rpm until OD500. 600 It is 0.5;

[0077] (2) Place LB / IPTG / Xgal plates in a 37°C oven for at least 1 hour; preheat Top medium to maintain a temperature of around 45°C.

[0078] (3) Dilute the supernatant or amplified phage: Dilute the supernatant by 10-10%. 3 The amplified phage was diluted 10 times. 8 ~10 10 times.

[0079] (4) Take 10 μL of diluted phage and add it to 200 μL of prepared E. coli ER2738 with an OD600 of about 0.5, and mix well. Add it to 3 mL of prepared Top medium, mix well, spread it evenly on the LB / IPTG / Xgal plate prepared in step (2), cool for 10 min, and incubate upside down in a 37°C incubator overnight.

[0080] (5) Record the blue phage spots on the plate to calculate the titer of the phage.

[0081] II. Experimental Results

[0082] The results are shown in Table 1. The results show that enrichment begins from the second round of screening, and the output is highest in the third round. According to the instructions of the phage display library, screening should only be performed in three rounds. Based on this result, a number of clones were picked from the titer assay plate of the third round output for screening positive clones.

[0083] Table 1. Phage titers

[0084]

[0085]

[0086] Example 2: Identification of mimic epitope peptides that specifically bind to bongkrekic acid antibodies

[0087] I. Experimental Methods

[0088] 1. After the third round of screening in Example 1, the titer of the supernatant was determined. A culture plate with fewer than 100 blue phages was selected, and 40 blue phage plaques were randomly selected for amplification and identification.

[0089] 2. Blue phage plaque amplification: A single phage plaque was inoculated into 1 mL of ER2738 (OD) solution. 600 In a 4 mL centrifuge tube containing 0.01 to 0.05 g of the sample, incubate at 37°C and 250 rpm for 4.5 h.

[0090] 3. Centrifuge the culture medium at 12,000 rpm for 10 min at 4℃. The supernatant can be used for subsequent identification and sequencing of positive clones based on phage enzyme-linked immunosorbent assay (ELISA).

[0091] (1) The specific method of identification is as follows:

[0092] The purified anti-bongkrekic acid antibody (10 μg / mL) was diluted with PBS, and 100 μL was placed in a high-adsorption ELISA plate and coated overnight at 4°C.

[0093] The next day, the ELISA plate was washed twice with PBST (300 μL / well), incubated with 3% (w / v) skim milk powder at 37°C for 1 hour.

[0094] Take 50 μL of the obtained supernatant and mix it with an equal volume of 1 μg / mL bongkrekic acid or PBS and add it to the enzyme label well. At the same time, use 1 μg / mL OVA / BSA / LF as a control experiment to detect non-specific binding ability.

[0095] After incubating at room temperature for 1 hour and washing 7 times with PBST, 100 μL of anti-M13 phage antibody-HRP (horseradish peroxidase-labeled anti-M13 monoclonal antibody) was diluted 5000 times (v / v) with PBST and added to the wells. The mixture was then incubated at 37°C for 30 minutes.

[0096] Wash 5 times again, add 100 μL of TMB liquid substrate buffer to each well, and incubate at 37°C for 10 minutes.

[0097] Finally, the absorbance was read (450 nm) after terminating the reaction with 50 μL of 10% (v / v) H2SO4.

[0098] The criteria for selecting positive clones are: having an antibody that can bind (absorbance value higher than 1.5), being competitive with bongkrekic acid (absorbance value lower than 0.5), and having weak binding ability to OVA / BSA / LF (absorbance value lower than 0.2).

[0099] (2) Sequencing

[0100] The identified positive clones were sequenced using sequencing primer 96gIII to obtain the gene sequence. The sequence of sequencing primer 96gIII is: TTTTGAAATCTAGCAATGCGATTGATACTCCCG.

[0101] II. Experimental Results

[0102] like Figure 2 As shown, 37 of the 40 selected clones were positive and subsequently sequenced. Sequencing identification revealed two different mimic epitope peptide sequences.

[0103] The sequencing results are shown in Table 2.

[0104] Table 2. Phage display mimic epitope peptide sequencing results

[0105]

[0106] Specifically, the obtained zymogenic epitope peptide is displayed at the N-terminus of the phage PⅢ capsid protein, that is, the phage uses the M13 phage plasmid as a vector, and the gene sequence is inserted into the gⅢ gene of the membrane protein encoded by the phage.

[0107] Although both phages screened could mimic the binding of bongkrekic acid's mimic epitope peptide to anti-bongkrekic acid antibodies, and their sensitivity was not significantly different (inhibition rate of 50% ± 1% for 1 ng / mL bongkrekic acid), the titer determination results for phages with absorbance values ​​ranging from 1 to 1.2 showed some differences in affinity. N.10 (SEQ ID NO: 4) had a stronger affinity (10¹¹ pfu / mL), while N.1 had a weaker affinity (10¹³ pfu / mL), with a titer of 10¹² pfu / mL. Therefore, the phage with the highest affinity (N.10) was selected to construct the standard curve.

[0108] Example 3: Application of mimic epitope peptides that specifically bind to bongkrekic acid antibodies as competitive antigens in enzyme-linked immunosorbent assays (ELISA).

[0109] I. Experimental Methods

[0110] 1. Antibody coating

[0111] Dilute the anti-bongkrekic acid antibody with PBS, coat the ELISA plate with 10 μg / mL, and incubate overnight at 4°C. The next day, wash twice with PBST, and block with 3% (w / v) skim milk powder at 37°C for 1 hour. After drying, it can be stored at 4°C for subsequent experiments.

[0112] 2. Establishment of the standard curve

[0113] Phage ELISA: 50 μL of N.10 phage clone (10 12 Add bongkrekic acid (pfu / mL) and 50 μL of PBS or a series of concentrations (12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.195, 0.0976, 0.0488, 0.0244, 0.0122 and 0.0061 ng / mL) to the microwells coated with the antibody. Incubate at 37°C for 45 min. After washing 7 times with PBST, add 100 μL of 5000-fold (v / v) diluted HRP-labeled anti-M13 phage HRP (secondary antibody). Wash 5 times again with PBST. Add 100 μL of LMB chromogenic solution and develop in the dark for 10 min. Add 50 μL of 10% (v / v) H2SO4.

[0114] The logarithm of the concentration of each bongkrekic acid standard is used as the x-axis, and the corresponding B / B0 is used as the y-axis (B0 is the absorbance value measured in the well with a bongkrekic acid concentration of 0, and B is the absorbance value of the well with bongkrekic acid concentration in other series).

[0115] II. Experimental Results

[0116] Standard curve such as Figure 3 The method for detecting the LOD (IC50) of bongkrekic acid 10 The concentration was 0.02 ng / mL, and the IC50 concentration was [missing information]. 50 The concentration was 0.30 ng / mL, and the quantitative range was 0.06–1.52 ng / mL (y = -0.45lgX + 0.28).

[0117] Example 4: Antigen epitope peptides that specifically bind to bongkrekic acid antibodies.

[0118] I. Experimental Methods

[0119] The cross-reactivity (CR) of five common toxins—toxin flavin, aflatoxin, ochratoxin, zearalenone, and fumonisin—was determined with bongkrekic acid as the cross-reactivity (CR) factor of 100%.

[0120] The detection method is as follows:

[0121] 50 μL of N.10 phage clone and 50 μL of the derivative of the test compound were added to the microwells coated with antibody and incubated at 37 °C for 45 min. After washing 7 times with PBST, 100 μL of HRP-labeled anti-M13 phage HRP secondary antibody diluted 5000-fold (v / v) was added, followed by washing 5 times with PBST again. 100 μL of TMB chromogenic solution was added, and the mixture was incubated in the dark for 10 min. 50 μL of 10% H2SO4 was added. The absorbance was read (450 nm).

[0122] II. Experimental Results

[0123] The results are shown in Table 3. The cross-reactivity rates of other common toxins were all less than 0.1%, indicating that the antigenic epitope peptides that specifically bind to bongkrekic acid antibodies obtained by screening have good specificity and are expected to enable rapid detection of bongkrekic acid without interference.

[0124] Table 3 shows the cross-reactions of phage-derived mimicry epitope peptides with common toxins.

[0125]

[0126]

[0127] Example 5: A method for detecting bongkrekic acid

[0128] 1. Antibody coating

[0129] Dilute the anti-bongkrekic acid antibody with PBS, coat the ELISA plate with a concentration of 10 μg / mL, and incubate overnight at 4°C. The next day, wash twice with PBST, and block with 3% (w / v) skim milk powder at 37°C for 1 hour. After drying, store at 4°C for subsequent experiments.

[0130] 2. Processing of the sample to be tested

[0131] The samples to be tested (such as fermented rice noodle products, white fungus, or black fungus samples) are pulverized separately. 1g of each sample is weighed and added to a clean centrifuge tube, followed by 3mL of extraction solvent (ethyl acetate). After shaking well, the tube is centrifuged at 3000 rpm for 3–5 minutes. The supernatant is collected and dried. Finally, the sample is reconstituted with 200μL of reconstitution solution (0.1M PB solution), and shaken well again to obtain the sample solution to be tested.

[0132] 3. Phage ELISA

[0133] Add 50 μL of N.10 phage clone and 50 μL of the test sample solution to the wells coated with antibody. Incubate at 37°C for 45 min. After washing 7 times with PBST, add 100 μL of HRP-labeled anti-M13 phage HRP secondary antibody diluted 5000-fold (v / v). Wash 5 times again with PBST. Add 100 μL of TMB chromogenic solution and develop in the dark for 10 min. Add 50 μL of 10% H2SO4. Read the absorbance (450 nm).

[0134] 4. Result Interpretation

[0135] Substitute the absorbance value Y obtained from the sample to be tested into the standard curve established above (Y = -0.45lgX + 0.28) to calculate the content of bongkrekic acid X in the sample to be tested.

[0136] Example 6: A kit for detecting bongkrekic acid

[0137] 1. Composition

[0138] Anti-bongkrekic acid antibody (monoclonal antibody against bongkrekic acid secreted by a hybridoma cell line, which was deposited at the China Center for Type Culture Collection on May 12, 2021, with accession number CCTCC No. C2021127 and classified as hybridoma cell line A5), N.10 phage clone obtained from Example 2, bongkrekic acid, PBS, PBST, HRP-labeled anti-M13 phage HRP secondary antibody, TMB chromogenic solution, and 10% (v / v) H2SO4.

[0139] 2. Instructions for use

[0140] Same as Example 5.

[0141] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A bongkrekic acid mimic epitope peptide, characterized in that, Its amino acid sequence is shown in SEQ ID NO:1 or 3.

2. A gene encoding a bongkrekic acid mimic epitope peptide, characterized in that, It encodes a peptide with an amino acid sequence as shown in SEQ ID NO: 1 or 3, or its reverse complementary sequence encodes a peptide with an amino acid sequence as shown in SEQ ID NO: 1 or 3.

3. A biomaterial, characterized in that, It is any one of the following: (1) An expression cassette containing the gene of claim 2; (2) A recombinant vector containing the gene of claim 2; (3) A recombinant microorganism containing the gene described in claim 2; (4) A cell line containing the gene of claim 2; (5) A recombinant vector containing the expression cassette described in (1); (6) Recombinant microorganisms containing the recombinant vector described in (2) or (5); (7) A cell line containing the recombinant vector described in (2) or (5).

4. A bacteriophage, characterized in that, The surface displays the bongkrekic acid mimic epitope peptide as described in claim 1.

5. An immunogenic substance, characterized in that, The bongkrekic acid mimic epitope peptide of claim 1 is coupled with a carrier protein.

6. A non-diagnostic detection method for bongkrekic acid, characterized in that, An antibody against bongkrekic acid was used as the coating antibody, and the bongkrekic acid mimic epitope peptide of claim 1, the bacteriophage of claim 4, and / or the substance of claim 5 were used as competitive antigens for enzyme-linked immunosorbent assay (ELISA).

7. The detection method according to claim 6, characterized in that, The antibody against bongkrekic acid is a monoclonal antibody.

8. The use of one or more of the following in the non-diagnostic detection of bongkrekic acid and / or the preparation of a bongkrekic acid detection kit: the bongkrekic acid mimic epitope peptide of claim 1, the gene of claim 2, the biomaterial of claim 3, the bacteriophage of claim 4, or the substance of claim 5.

9. A kit for detecting bongkrekic acid, characterized in that, The bongkrekic acid mimic epitope peptide of claim 1, the gene of claim 2, the biomaterial of claim 3, the bacteriophage of claim 4, or the substance of claim 5, or more of these.

10. The detection kit according to claim 9, characterized in that, It contains the bacteriophage as described in claim 4 and an antibody against bongkrekic acid.

Images