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CN-122011113-A - Okadaic acid mimotope peptide and application thereof

CN122011113ACN 122011113 ACN122011113 ACN 122011113ACN-122011113-A

Abstract

The application relates to okadaic acid mimotope peptide and application thereof, wherein the amino acid sequence of the okadaic acid mimotope peptide is shown as SEQ ID NO. 1,3, 5, 7, 9, 11, 13 or 15. The method can be specifically combined with okadaic acid antibody, and a sensitive and rapid competitive enzyme-linked immunoassay method is established by utilizing the phage displaying the mimotope peptide, wherein the IC 50 is 0.83 ng/mL, the LOD is 0.21 ng/mL, the detection range is 0.35-1.98 ng/mL, and the competitive immunoassay method established based on the mimotope peptide is more sensitive than the traditional immunoassay method, has good cross reaction to other marine toxins, avoids the use of toxin antigens, and is green and environment-friendly.

Inventors

  • XU ZHENLIN
  • HUANG QIDAN
  • Ao Riqileng
  • LUO LIN
  • LEI HONGTAO
  • SHEN YUDONG
  • WANG HONG

Assignees

  • 华南农业大学

Dates

Publication Date
20260512
Application Date
20251223

Claims (10)

  1. 1. The okadaic acid mimotope peptide is characterized in that the amino acid sequence is shown as SEQ ID NO. 1,3, 5, 7, 9, 11, 13 or 15.
  2. 2. A biomaterial characterized by being one or more of the following: (1) A gene encoding the okadaic acid mimotope peptide of claim 1; (2) An expression cassette comprising the gene of (1); (3) A recombinant vector comprising the gene of (1) or the expression cassette of (2); (4) A recombinant microorganism comprising the gene described in (1), the expression cassette described in (2) or the recombinant vector described in (3); (5) A cell line comprising the gene described in (1), the expression cassette described in (2) or the recombinant vector described in (3).
  3. 3. The biomaterial according to claim 2, wherein the recombinant microorganism is a phage having the okadaic acid mimotope peptide of claim 1 expressed on the surface.
  4. 4. A conjugate, characterized in that it is the okadaic acid mimotope peptide of claim 1 conjugated with a carrier protein.
  5. 5. A method for detecting okadaic acid for non-diagnostic purposes, comprising the steps of: An enzyme-linked immunosorbent assay is carried out by using an anti-okadaic acid antibody as a coating antibody and using the okadaic acid mimotope peptide of claim 1, the phage of claim 3 or the conjugate of claim 4 as a competitive antigen.
  6. 6. The method of claim 5, wherein the anti-okadaic acid antibody is a monoclonal antibody obtained by immunizing a mouse with okadaic acid-conjugated bovine serum albumin as an immunogen and then screening the immunized mouse.
  7. 7. A kit for detecting okadaic acid, comprising any one or more of the okadaic acid mimotope peptide of claim 1, the phage of claim 3 or the conjugate of claim 4.
  8. 8. The kit of claim 7, further comprising an antibody against okadaic acid.
  9. 9. Use of the okadaic acid mimotope peptide of claim 1, the biomaterial of claim 2 or the conjugate of claim 4 for detecting okadaic acid.
  10. 10. Use of the okadaic acid mimotope peptide of claim 1, the biomaterial of claim 2 or the conjugate of claim 4 for the preparation of a kit for detecting okadaic acid.

Description

Okadaic acid mimotope peptide and application thereof Technical Field The application relates to the technical field of immunodetection, in particular to okadaic acid mimotope peptide and application thereof. Background Okadaic acid, also known as okadaic acid, is a small-molecule lipophilic marine toxin produced by algae such as red tide algae, fusarium and Protopanax, belongs to polyether marine biotoxin, and is a main component in diarrhea shellfish toxins, and is named after being separated from okadaic Tian Ruan sponge for the first time. Protein hyperphosphorylation is induced by inhibiting threonine and serine phosphatase activities, so that the medicine has low toxicity and no specific medicine treatment. As a cancerogenic factor with great harm, the said cancerogenic factor has destruction to liver cell and nerve cell of human body, long-term toxic effect and long-term accumulation of cancerogenic factor. Okadaic acid does not destroy toxic effects due to high temperature conditions, and symptoms such as diarrhea, nausea and vomiting can appear after people eat okadaic acid polluted seafood by mistake, so no specific medicine exists at present. Therefore, it is indispensable to monitor okadaic acid in foods. The traditional okadaic acid detection method mainly comprises chromatographic detection, biological detection and immunological detection. The chromatographic detection needs expensive instruments, the pretreatment of the sample is complex, the biological detection needs longer detection time, the accuracy is not enough, and the requirements of on-site rapid detection cannot be met. The immunological technology has the advantages of strong specificity, high sensitivity, no need of complex instruments and the like, so the immunological technology is a common technology for rapidly detecting toxins at present. However, in the detection process, the okadaic acid standard substance is used as a raw material for artificial antigen chemical synthesis, the reaction steps in the synthesis process are complicated, the synthesis raw material is expensive, and a large amount of toxic standard substances and organic solvents are required in the preparation process, so that the physical health of operators is greatly harmed. Therefore, the research on the green and nontoxic small molecule detection holoantigen substitute is an important development direction for establishing environment-friendly immunoassay. In recent years, researchers successfully replace small molecule antigens for immunological detection by utilizing phage display random peptide library technology to panning with strong specificity and high specificity mimotopes. Compared with the competitor synthesized by traditional chemistry, the mimotope protein has the advantages of quick and simple acquisition, stable property and low cost, and most importantly, the mimotope protein can reduce the risk of exposure of laboratory personnel to toxic environment. In the field of immunodetection of toxic and harmful substances, the mimotope has wide application prospect in replacing antigen for immunoassay. Disclosure of Invention The invention aims to overcome the defects of the prior art and provides okadaic acid mimotope peptide and application thereof. The first object of the present invention is to provide a okadaic acid mimotope peptide. A second object of the present invention is to provide a biomaterial. A third object of the present invention is to provide a conjugate. The fourth object of the present invention is to provide a method for detecting okadaic acid. A fifth object of the present invention is to provide a kit for detecting okadaic acid. A sixth object of the present invention is to provide the use of the okadaic acid mimotope peptide, the biological material or the conjugate for detecting okadaic acid. The seventh object of the present invention is to provide an application of the okadaic acid mimotope peptide, the biological material or the conjugate in preparing a kit for detecting okadaic acid. In order to achieve the above object, the present invention is realized by the following technical scheme: (1) Purified okadaic Tian Suanshan clone antibody was coated on a high-adsorption ELISA plate, the ELISA plate was blocked with 3% nonfat milk powder, and then a random linear dodecapeptide phage display library was added to the ELISA plate for panning, according to a bind-elute-amplify panning protocol, through 3 rounds of enrichment panning. Wherein, the dosage of the antibody coated by the three rounds of elutriation screens and the dosage of okadaic acid standard used for competitive elution of phage are sequentially reduced; (2) After 3 rounds of panning, 95 phage monoclonal were randomly picked for preliminary identification of phage ELISA, and the 24 positive clones obtained were amplified and sequenced to find 8 sequences in total. According to the invention, a competition type mode panning screen is utilized to display phage