CN116640183A - Antigen mimic epitope of zearalenone toxin, application and detection kit - Google Patents
Antigen mimic epitope of zearalenone toxin, application and detection kit Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses an antigen mimic epitope of zearalenone toxin, application and a detection kit, and belongs to the technical field of biology. The invention provides an antigen mimic epitope of zearalenone toxin, wherein a polypeptide capable of being specifically combined with an anti-ZEN monoclonal antibody is screened from a peptide library by using phage display peptide library technology, namely the antigen mimic epitope has immune response characteristics similar to those of a natural ZEN molecule, and the obtained ZEN antigen mimic epitope replaces a ZEN standard product with high price and strong toxicity and is used as a competitive antigen or a solid-phase coating antigen for immunological detection of ZEN. The antigen mimotope disclosed by the invention is used as a standard product for immunological detection, so that the harm of ZEN to human health is reduced, the cost is saved, and the antigen mimotope has high application value.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an antigen mimic epitope of zearalenone toxin, and application and a detection kit thereof.
Background
Zearalenone (ZEN) is a mycotoxin produced by fusarium fungi and mainly contaminates crops such as corn, barley, wheat, oat, sorghum and the like. ZEN has strong estrogenic effect and can produce reproductive toxicity, and also has immune toxicity, liver toxicity, genetic toxicity and potential carcinogenicity. The toxins can enter animals and humans through ZEN contaminated grains and products thereof or ZEN-residue meat, milk, etc., and pose a threat to animal husbandry and human health.
Currently, methods for detecting ZEN include high performance liquid chromatography, gas chromatography, thin layer chromatography, high performance liquid chromatography-mass spectrometry, and immunological detection methods. The immunological detection method has the advantages of simple pretreatment, easy grasp in operation, strong specificity, high sensitivity and the like, so that the immunological detection method becomes a detection method which is widely applied. However, in the process of establishing an immunological detection method, a ZEN standard substance is necessary to be used as a raw material for preparing a competitive antigen or a solid-phase coated antigen, so that ZEN is expensive and has extremely strong carcinogenicity, and causes extremely serious threat to the health and environment of detection personnel, thereby restricting the application and popularization of the immunological detection method to a certain extent.
Disclosure of Invention
The invention aims to provide an antigen mimic epitope of zearalenone toxin, application and a detection kit, wherein the antigen mimic polypeptide can be specifically combined with an anti-ZEN monoclonal antibody, has immune response characteristics similar to those of a natural ZEN molecule, and can replace a ZEN standard product with high price and strong toxicity.
The invention provides an antigen mimic epitope of zearalenone toxin, and the amino acid sequence of the antigen mimic epitope is shown as SEQ ID No.1 or SEQ ID No. 2.
The invention also provides a nucleotide sequence for encoding the antigen mimotope.
Preferably, the nucleotide sequence is shown as SEQ ID No.3 or SEQ ID No. 4.
The invention also provides application of the antigen mimotope or phage containing the antigen mimotope or fusion protein of the antigen mimotope and a label in preparation of a zearalenone toxin detection reagent.
Preferably, the zearalenone toxin detection reagent comprises an immunological detection reagent for zearalenone toxin.
Preferably, the antigen mimotope is used as a solid phase antigen or as a competitor antigen when detected by immunological means.
The invention also provides an immunological detection kit of the zearalenone toxin, which takes the antigen mimic epitope as an antigen standard.
The beneficial effects are that: the invention provides an antigen mimic epitope of zearalenone toxin, which is characterized in that a polypeptide capable of specifically binding with a target molecule (anti-ZEN monoclonal antibody) is screened from a peptide library by using phage display peptide library technology, namely the antigen mimic epitope has immune response characteristics similar to those of a natural ZEN molecule, and the obtained ZEN antigen mimic epitope is used for replacing a ZEN standard substance with high price and strong toxicity and is used as a competitive antigen or a solid-phase coating antigen for immunological detection of ZEN. The antigen mimotope disclosed by the invention is used as a standard product for immunological detection, so that the harm of ZEN to human health is reduced, the cost is saved, and the antigen mimotope has high application value.
Drawings
FIG. 1 is a graph showing the results of indirect ELISA for identifying phage positive clones; in the figures, 1-24 represent 24 phage clones, and Blank represents a negative control;
FIG. 2 is a graph showing the results of indirect competition ELISA for identifying ZEN phage display mimotopes; 1-24 in the figure represent 24 phage clones;
FIG. 3 is a standard ELISA curve constructed using ZEN antigen mimotope 1 as a competitor antigen;
FIG. 4 is a standard ELISA curve constructed using ZEN antigen mimotope 2 as a competitor antigen.
Detailed Description
The invention provides an antigen mimic epitope of zearalenone toxin, wherein the amino acid sequence of the antigen mimic epitope is shown as SEQ ID No.1 (TPWTTBL) or SEQ ID No.2 (TPWTESE).
The antigen mimotopes of the present invention are preferably selected from peptide libraries by phage display peptide library technology, and the antigen mimotopes are cyclic heptapeptide antigen mimotopes. And the antigen mimotope preferably further comprises adding a cysteine residue (C) at each of N-terminal and C-terminal, respectively, to form a cyclic structure by intramolecular disulfide bond. The antigen mimotopes of the invention, capital English letters respectively represent twenty-one known natural L-type amino acid residue or one of D-type isomers thereof, namely, C represents cysteine residue, T represents threonine residue, P represents proline residue, W represents tryptophan residue, H represents histidine residue, L represents leucine residue, E represents glutamic acid residue and S represents serine residue.
The invention also provides a nucleotide sequence for encoding the antigen mimotope.
The nucleotide sequence of the invention is shown as SEQ ID No.3 (ACT CCG TGGACT CCG CAT CTG) or SEQ ID No.4 (ACT CCT TGGACT GAG TCT GAG).
The preparation method of the ZEN antigen mimotope is not particularly limited, and the ZEN antigen mimotope can be prepared by a conventional method in the field, such as a large number of preparation methods by phage amplification, chemical synthesis or genetic engineering recombinant expression. Phage amplification refers to the mass propagation of phage displaying ZEN antigen mimotopes in a bioamplification mode to produce phage particles displaying ZEN antigen mimotopes. Chemical synthesis refers to the synthesis of polypeptides by means of chemical synthesis of the polypeptides according to the amino acid sequence of the published mimotope. The recombinant expression mode of gene engineering refers to cloning the gene encoding the mimotope into an expression vector and performing mass preparation of the ZEN antigen mimotope in the form of polypeptide-fusion protein.
The invention also provides application of the antigen mimotope or phage containing the antigen mimotope or fusion protein of the antigen mimotope and a label in preparation of a zearalenone toxin detection reagent.
In the detection of ZEN, particularly immunological detection, using the above antigen mimotopes, the present invention preferably exists in the form of a cyclic heptapeptide, and more preferably comprises using synthetic mimotopes for immunological detection analysis, or using phage particles displaying ZEN antigen mimotopes obtained by phage amplification directly for analysis detection, or cleaving ZEN antigen mimotopes from phage to replace ZEN standard substances for immunological detection analysis. When detected by immunological methods, the present invention preferably uses the antigen mimotope as a solid phase antigen or as a competing antigen.
The invention also provides an immunological detection kit of the zearalenone toxin, which takes the antigen mimic epitope as an antigen standard.
In order to further illustrate the present invention, the following descriptions of the antigen mimotopes of zearalenone toxin, and the application and detection kit provided by the present invention are provided in detail with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Affinity panning and identification of ZEN antigen mimotopes
(1) Affinity panning of ZEN antigen mimotopes: the specific method comprises the following steps: the anti-ZEN monoclonal antibody was diluted with 10mM TBS (pH 7.4) and coated with 96-well ELISA plates at a final concentration of 100. Mu.g/mL and incubated overnight at 4 ℃. The next day after washing 10 times with TBST (50 mM NaCl, pH7.4 containing 0.1% Tween-20 (v/v)), 300. Mu.L of blocking solution (3% BSA-TBS) was added and incubated at 4℃for 2 hours. After 2 hours, the blocking solution was discarded, washed 5 times with TBST, and 100. Mu.L of phage peptide library (phage display Cycloheptapeptide library, available from NEB Co., about 1.0X10) was added to each well, and phage stock was diluted 10-fold with TBS 11 pfu), and carrying out oscillation reaction for 1 hour at 22-26 ℃. Unbound phage were discarded, washed 10 times with TBST, bound phage eluted with 0.2M Glycine-HCl (pH 2.2), and immediately neutralized with 15. Mu.L of 1M Tris-HCl (pH 9.1). Titer was measured on 10. Mu.L of eluted phage, the remainder used to infect 20mLE.coli ER2738 strain grown to log phase was amplified. The third day the phage were purified by PEG/NaCl precipitation and the titer of the amplified phage was determined.
In the panning of the second, third and fourth rounds, the coated anti-ZEN monoclonal antibodies had concentrations of 75. Mu.g/mL, 50. Mu.g/mL and 25. Mu.g/mL, respectively, and TBST concentrations of 0.25%, 0.1% and 0.5% were used, the remaining steps of the second and third rounds were the same, no further amplification was necessary after the fourth round was completed, and the post-elution titers were measured directly.
TABLE 1 enrichment of phages in three rounds of affinity panning
(2) Identification of positive phage clones: 24 phage plaques were randomly picked from the plates after the fourth round of panning to perform phage amplification, and positive phage clones were identified by indirect enzyme-linked immunosorbent assay (Enzyme Linked Immunosorbent Assay, ELISA) using the following specific methods: first, the anti-ZEN monoclonal antibody was diluted with 10mM TBS (pH 7.4), 2. Mu.g/mL coated 96-well ELISA plates, and incubated overnight at 4 ℃. The following day, after 3 washes with TBST (10mM TBS,0.05%Tween-20 (v/v)), blocking with TBS containing 3% skimmed milk powder, and incubation at 37deg.C for 1 hr; 100. Mu.L of phage plaque amplification liquid (1.0X10) 11 pfu), with the original phage peptide library as a negative control, incubated for 1 hour at 37 ℃; HRP-labeled anti-M13 phage secondary antibody 100. Mu.L was added at 1:30000 (original concentration 3.29. Mu.g/mL) and incubated at 37℃for 1 hour; adding 100 mu LTMB substrate solution, developing in dark for 10min, adding 50 mu LH 2 SO 4 (2M) stop solution, and the absorbance at 450nm was read by an enzyme-labeled instrument (SP-Max 3500 FL). Selecting 0D 450 Phage clones 2-fold larger than negative control were positive clones, and the results are shown in FIG. 1.
(3) Identification of ZEN antigen mimotopes: the method for identifying the ZEN antigen mimotope by adopting an indirect competition ELISA method comprises the following specific steps: anti-ZEN monoclonal antibodies were diluted with 10mM TBS (pH 7.4), coated with 2. Mu.g/mL ELISA plates and incubated overnight at 4 ℃; the next dayAfter washing 3 times with TBST (10mM TBS,0.05%Tween-20 (v/v)), blocking with TBS containing 3% skimmed milk powder, and incubating at 37deg.C for 1 hr; 50. Mu.L of phage clones (1.0X10) positive by indirect ELISA were added 11 pfu) and 50 μl zen standard (concentration range 0-100 ng/mL), 37 ℃ for 1 hour; HRP-labeled anti-M13 phage secondary antibody 100. Mu.L was diluted 1:30000 (original concentration 3.29. Mu.g/mL) and incubated at 37℃for 1 hour; adding 100 μl of TMB substrate solution, developing for 10min in dark, adding 50 μ L H 2 SO 4 (2M) stop solution, read the absorption value at 450nm, can bind to the anti-ZEN monoclonal antibody, and can be blocked by ZEN standard, identify as ZEN antigen mimotope, the results are shown in figure 2.
Example 2
Sequencing of ZEN antigen mimotope coding gene and determination of amino acid sequence thereof
And (3) amplifying the phages No. 10 and No. 16 which are identified and displayed with the ZEN antigen mimotopes through indirect competition ELISA, and extracting the DNA sequencing templates of the phages. The brief procedure is as follows:
phage amplification was performed and after the first centrifugation step, 500. Mu.L of phage-containing supernatant was transferred to a new centrifuge tube. Phage was precipitated by adding 200. Mu.L PEG/NaC1 and left at room temperature for 10min. After centrifugation, the pellet was resuspended in 100. Mu.L of iodide buffer (10 mM Tris-HCl (pH 8.0), 1mM EDTA,4M NaI), 250. Mu.L of absolute ethanol was added to pellet DNA, incubated at room temperature for 10min, and after centrifugation the pellet was washed with 70% ethanol as a template for DNA sequencing. The pellet was finally resuspended in 30. Mu.L of Tris-HCl (pH 8.0), 1mM EDTA and analysed by agarose gel electrophoresis; DNA sequencing was performed on 10. Mu.L of phage template, which was-96 gIII sequencing primer SEQ ID No.5:5' - HO CCC TCA TAG TTA GCG TAA CG-3'. According to the DNA sequencing result and codon table, the amino acid sequences of ZEN antigen mimotopes, CTPWTPHLC (mimotope 1) and CTPWTESEC (mimotope 2) can be obtained.
Example 3
Application of ZEN antigen mimic epitope as competitive antigen in ELISA
(1) Coating and sealing
The anti-ZEN monoclonal antibody was diluted with 10mM TBS (pH 7.4), coated with 2. Mu.g/mL, and incubated overnight at 4 ℃. The following day was washed 3 times with TBST (10mM TBS,0.05%Tween-20 (v/v)) and blocked with TBS containing 3% nonfat dry milk, incubated at 37℃for 1 hour, and plates were washed 6 times with PBST for use.
(2) Establishment of a Standard Curve
The strip treated in step (2) was removed and 50. Mu.L of phage displaying the ZEN antigen mimotopes was added to each well (1.0X10) 11 pfu) and a series of 50 μl zen standards at different concentrations, incubated at 37 ℃ for 1 hour. HRP-labeled anti-M13 phage secondary antibody was added at 1:30000 and incubated for 1h at 37 ℃. Then developed with TMB substrate and OD read 450 Binding rate (OD of wells with ZEN added) on the abscissa of log of ZEN concentration 450 OD of wells without ZEN added 450 X 100%) is on the ordinate, and an indirect competition standard curve is established.
An indirect competition ELISA standard curve is established by using the mimotope 1, and the result shows that the standard curve is S-shaped, has better linear correlation, and has a detection range of 2.00-9.40 ng/mL and half inhibition concentration (Halfmaximal inhibitory concentration, IC) 50 ) 5.18ng/mL (FIG. 3). Similarly, an indirect competition ELISA standard curve is established by using the mimotope 2, the detection range is 2.14-10.50 ng/mL, and the IC 50 5.43ng/mL (FIG. 4).
Example 4
Large scale preparation of ZEN antigen mimotopes
(1) In phage amplification mode
Phage displaying the ZEN antigen mimotope was added to 20mL of the culture inoculated with ER2738 and cultured with shaking at 37 ℃ at 220rpm for 4.5 hours. The culture was transferred to another centrifuge tube, centrifuged at 10000rpm at 4℃for 10min, 80% of the upper supernatant was transferred to a fresh tube, 1/6 volume of PEG/NaCl was added, and the mixture was allowed to stand at 4℃for 120min. PEG/NaCl stationary solution was centrifuged at 10000rpm at 4℃for 15min. The supernatant was discarded and the residual supernatant was aspirated after a short centrifugation. Adding 1mLTBS for resuspension, thus obtaining phage amplification liquid.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (7)
1. An antigen mimotope of zearalenone toxin is characterized in that the amino acid sequence of the antigen mimotope is shown as SEQ ID No.1 or SEQ ID No. 2.
2. A nucleotide sequence encoding the antigen mimotope of claim 1.
3. The nucleotide sequence according to claim 2, wherein the nucleotide sequence is shown as SEQ ID No.3 or SEQ ID No. 4.
4. Use of an antigen mimotope according to claim 1 or a phage comprising an antigen mimotope according to claim 1 or a fusion protein of an antigen mimotope and a tag according to claim 1 for the preparation of a zearalenone toxin detection reagent.
5. The use according to claim 4, wherein the zearalenone toxin detection reagent comprises an immunological detection reagent for zearalenone toxin.
6. The use according to claim 4 or 5, wherein the antigen mimotope is used as a solid phase antigen or as a competing antigen when detected by immunological means.
7. An immunological detection kit of zearalenone toxin, characterized in that the antigen mimotope of claim 1 is used as an antigen standard.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310602033.2A CN116640183A (en) | 2023-05-26 | 2023-05-26 | Antigen mimic epitope of zearalenone toxin, application and detection kit |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202310602033.2A CN116640183A (en) | 2023-05-26 | 2023-05-26 | Antigen mimic epitope of zearalenone toxin, application and detection kit |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017184590A1 (en) * | 2016-04-18 | 2017-10-26 | The Broad Institute Inc. | Improved hla epitope prediction |
| BR102017020980A2 (en) * | 2017-09-29 | 2019-04-16 | Universidade Federal Do Parana | AFLATOXINE MIMETIC PEPTIDIC SEQUENCE APPLIED AS IMMUNOGEN |
| CN113173973A (en) * | 2021-05-24 | 2021-07-27 | 河南中泽生物工程有限公司 | Zearalenone mimic epitope and application thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017184590A1 (en) * | 2016-04-18 | 2017-10-26 | The Broad Institute Inc. | Improved hla epitope prediction |
| BR102017020980A2 (en) * | 2017-09-29 | 2019-04-16 | Universidade Federal Do Parana | AFLATOXINE MIMETIC PEPTIDIC SEQUENCE APPLIED AS IMMUNOGEN |
| CN113173973A (en) * | 2021-05-24 | 2021-07-27 | 河南中泽生物工程有限公司 | Zearalenone mimic epitope and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| JINGMING ZHOU等: "Induction of anti-Zearalenone immune response with mimotopes identified from a phage display peptide library", TOXICON, vol. 199, pages 1 - 6 * |
| 徐富勇等: "噬菌体展示系统表达玉米赤霉烯酮模拟表位肽", 生物技术通报, no. 1, pages 144 - 150 * |
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