KR-20260064760-A - Domoic acid-specific binding peptides and uses thereof

Abstract

The present invention relates to a peptide that specifically binds to domoic acid and its use. By using bacteriophage display screening, a peptide that binds to domoic acid, a representative toxin of amnesic shellfish toxins, with very high specificity and affinity was discovered. This allows for the rapid and accurate detection of domoic acid, thereby enabling the rapid and accurate determination of whether seafood is contaminated and contributing to the supply of safe food.

Inventors

• 박종필
• 박태정
• 조채환
• 박찬영

Assignees

• 중앙대학교 산학협력단

Dates

Publication Date

20260508

Application Date

20241028

Claims (12)

1. A peptide consisting of any one amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 6 that specifically binds to domoic acid.
2. In paragraph 1, The above peptide is characterized by being composed of SEQ ID NO. 5 or 6.
3. In paragraph 1, A peptide characterized in that the above peptide is a dimer, a trimer, or a polymer.
4. A polynucleotide encoding the amino acid sequence of the peptide of claim 1.
5. A recombinant expression vector comprising the polynucleotide of claim 4.
6. Transformed bodies other than humans transformed with the recombinant expression vector of paragraph 5.
7. A composition for detecting domoic acid comprising the peptide of claim 1.
8. In Paragraph 7, A composition characterized in that the above-mentioned peptide is labeled with one selected from the group consisting of chromogenic enzymes, radioactive isotopes, chromophores, luminescent substances, and fluorescent substances.
9. A kit for detecting domoic acid containing the peptide of claim 1.
10. A domoic acid detection chip comprising the peptide of claim 1.
11. (a) a step of treating a sample with a peptide that specifically binds to the domoic acid of claim 1; and (b) a step of detecting the reaction between the above peptide and the sample A method for detecting domoic acid, comprising
12. In Paragraph 11, A detection method characterized by performing the detection of the above reaction using one or more methods selected from the group consisting of cyclic voltammetry, square wave voltammetry, electrochemical impedance spectroscopy, quartz crystal microbalance, surface plasmon resonance, lateral flow assay (LFA)/variable flip angle (VFA), enzyme-linked immunoassay, fluorescence resonance energy transfer, and surface-enhanced Raman spectroscopy.

Description

Domoic acid-specific binding peptides and uses thereof The present invention relates to a peptide that specifically binds to domoic acid and its uses. Domoic acid, a representative toxin of amnesic shellfish poisoning (ASP), is produced by toxic diatoms such as Pseudonitzschia spp. , including Pseudonitzschia multiseries (P. multiseries), P. australis, and P. delicatissima . Domoic acid has a structure similar to excitatory neurotransmitters of the central nervous system, such as glutamate and kainic acid. When domoic acid administered from outside the body reaches the brain, it acts as an agonist for L-glutamate, a central neurotransmitter, binding to glutamate receptors—which are ion channel types of nerve cells—and causing necrosis of the hippocampus, thalamus, and synovial cells in the brain, thereby inducing amnesia. Domoic acid accumulates in the bodies of bivalves and crustaceans, such as crabs, that feed primarily on toxic diatoms ( P. SPP ). When humans consume these organisms, the substance enters the body; in small amounts, symptoms such as vomiting, diarrhea, abdominal pain, headache, and dizziness appear within 30 minutes to 24 hours and persist for several days. In high doses, it causes serious illnesses, including gastroenteritis, renal failure, arrhythmia, and neurological disorders such as memory loss. The most representative example of domoic acid infection is the 1987 case in Canada involving 107 infections resulting from the consumption of mussels contaminated with domoic acid; in this case, 4 people died and 12 suffered from memory impairment. Consequently, the United States regulates the substance by setting an allowable limit of 20 mg/kg for fish and shellfish, Canada for seafood, and the EU and Australia for bivalves. Antibody-based ELISA (Enzyme-linked immunosorbent assay) is commonly used for the detection of domoic acid, and EuroProxima’s "EuroProxima Domoic acid ELISA kit" and PerkinElmer’s "MaxSignal® Domoic Acid ELISA kit" are available on the market to a limited extent. While antibody-based detection methods have the advantage of high specificity and sensitivity, they have the disadvantages of high production costs, complex production procedures, and low stability. Using the aforementioned ELISA kits for monitoring seafood for domoic acid, the causative agent of amnesic shellfish poisoning, incurs high costs; since this is unsuitable given the nature of seafood monitoring, which requires periodic testing of an unspecified number of samples, the establishment of a new detection system is necessary. Accordingly, the inventors conducted research to develop a method to detect domoic acid, which is present in trace amounts in shellfish, cheaply, quickly, and accurately. As a result, they discovered a novel peptide receptor that binds to domoic acid with high sensitivity and specificity, and developed a technology to quantitatively and qualitatively analyze domoic acid using this. Figure 1 shows the manufacturing process of magnetic beads with immobilized domosan used in the present invention and the results of measuring whether domosan is immobilized on the surface of the magnetic beads through a FITC labeling test using a fluorescence measurement method. Figure 2 is a schematic diagram showing a method for performing bacteriophage display to discover novel peptide receptors capable of highly specific and highly selective binding to domoic acid. Figure 3 shows the experimental results confirming the binding affinity of peptide candidate groups to domoic acid using an enzyme immunoassay. Figure 4 shows the results of enzyme immunoassay to test the binding strength of selected Da-12-4R#3 (Sequence No. 2), Da-7-2R#9 (Sequence No. 5), and Da-7-4R#5 (Sequence No. 6) peptides to a control group and domoic acid. Figure 5 shows the results of enzyme immunoassay to test the binding strength of additionally selected Da-7-2R#9 (sequence number 5) and Da-7-4R#5 (sequence number 6) peptides according to differences in domoic acid concentration. Figure 6 shows the results of an enzyme immunoassay to calculate the limit of detection (LOD) and limit of quantification (LOQ) values for the Da-7-2R#9 (sequence number 5) and Da-7-4R#5 (sequence number 6) peptides. Figure 7 shows the results of confirming domoic acid immobilized on the surface of HRP through MALDI/TOF/MS analysis. Figure 8 is a schematic diagram of a Direct-competitive ELISA (dc-ELISA) for detecting domoic acid. Figure 9 shows the results of enzyme immunoassay to test the binding strength of DA BP1 (sequence number 7) and DA BP2 (sequence number 8) to domoic acid concentrations. Figure 10 shows the results of an enzyme immunoassay to test the non-specific binding affinity of DA BP1 (sequence number 7) and DA BP2 (sequence number 8) to HRP. Figure 11 is a figure showing the experimental results confirming the structural change resulting from the binding of the DA BP1 (sequence number 7) peptide with domoic acid. Figure 11 is a figure sh