Peptide specifically binding to tetrodotoxin and uses thereof

Abstract

The present invention relates to a novel peptide discovered using bacteriophage display screening. According to the present invention, it has been confirmed that the peptide specifically binds to tetrodotoxin and can accurately detect tetrodotoxin. By utilizing this, it is possible to determine whether various targets, such as food and raw materials thereof, possess tetrodotoxin, thereby contributing to the supply of safe food.

Description

Technology Field

[0001] The present invention relates to a peptide that specifically binds to tetrodotoxin and its uses, and more specifically, to a peptide that specifically binds to tetrodotoxin, a type of potent neurotoxin, and a method for detecting tetrodotoxin using the same. Background Technology

[0003] As domestic sea surface temperatures rise due to global warming, the catch of various tropical fish species is increasing domestically. In particular, a large number of highly toxic species have been discovered recently, posing a serious threat to fisheries resources, the marine environment, and public health, thus requiring countermeasures.

[0004] Among the various toxins found in the aforementioned tropical fish, tetrodotoxin (TTX) is a low-molecular-weight marine toxin associated with fatal food poisoning, known to be approximately 1,200 times more toxic than cyanide. Although initially thought to occur only in pufferfish, it has now been confirmed to occur in various species, including shrimp, crabs, marine gastropods, and gobies; the blue-ringed octopus, which has recently been frequently observed along the Korean coast, also falls into this category. When tetrodotoxin enters the body, it blocks sodium channels in the sodium-potassium pumps of motor neurons, thereby disrupting the formation of action potentials and halting the electrochemical activity of nerve cells, which paralyzes voluntary muscles. This can cause severe illness generally characterized by tongue paralysis, nausea, vomiting, respiratory paralysis, diarrhea, and abdominal pain, and in extreme cases, leads to suffocation due to respiratory failure. Furthermore, tetrodotoxin is heat-stable and is not inactivated during traditional cooking processes. Therefore, some countries and organizations have enacted relevant regulations in consideration of the severity of tetrodotoxin poisoning. For example, the Japanese government has set a regulatory limit of 2 mg / kg (2 ppm) for the tetrodotoxin equivalent in food, and the U.S. Food and Drug Administration (FDA) restricts the import of pufferfish products into the United States.

[0005] Generally, liquid chromatography (LC) is mainly used as a quantitative detection method for tetrodotoxin, and enzyme-linked immunosorbent assay (ELISA) is used as a rapid detection method.

[0006] However, although antibodies possess high specificity and sensitivity, their production costs are very high, they are complex, and they have low stability. This poses a significant cost-consuming problem, given the nature of seafood monitoring, which requires the periodic testing of numerous samples.

[0007] Therefore, there is a need to establish a system that is fast and cost-effective.

[0008] Accordingly, while researching various techniques for rapidly and accurately detecting tetrodotoxin, the inventors discovered a novel peptide that specifically binds to tetrodotoxin and confirmed that the peptide is specific to tetrodotoxin, thereby arriving at the present invention. Prior art literature

[0010] Japanese Published Patent Application No. 2008-00241423 The problem to be solved

[0011] The present invention aims to solve the problems of the prior art by providing a novel peptide that specifically binds to tetrodotoxin, a composition for detecting tetrodotoxin using the same, a kit, a biochip, etc., and a method for detecting tetrodotoxin and a screening method.

[0012] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below. means of solving the problem

[0014] According to one embodiment of the present invention, a peptide for detecting tetrodotoxin is provided, comprising a part or the whole sequence of an amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NOs 1 to 9.

[0015] In the present invention, the peptide may be characterized by comprising a part or the whole sequence of the amino acid sequence represented by SEQ ID NO. 5 or 9.

[0016] In the present invention, the peptide may be characterized as being a dimer, trimer, or multimer.

[0017] In the present invention, the peptide may be characterized by being labeled with any one selected from the group consisting of chromogenic enzymes, radioisotopes, chromophores, luminescent substances, and fluorescent substances.

[0018] In another embodiment of the present invention, a composition for detecting tetrodotoxin is provided, comprising one or more peptides selected from the group consisting of amino acid sequences represented by SEQ ID NOs 1 to 9.

[0019] In another embodiment of the present invention, a kit for detecting tetrodotoxin is provided, comprising one or more peptides selected from the group consisting of amino acid sequences represented by SEQ ID NOs 1 to 9.

[0020] In another embodiment of the present invention, a biochip for detecting tetrodotoxin is provided, comprising one or more peptides selected from the group consisting of amino acid sequences represented by SEQ ID NOs 1 to 9.

[0021] In another embodiment of the present invention, a method for detecting tetrodotoxin is provided, comprising: a step of treating a sample with one or more peptides selected from the group consisting of amino acid sequences represented by SEQ ID NOs 1 to 9; and a step of detecting a reaction between the peptide and the sample.

[0022] In the present invention, the detection of the reaction may be characterized by being performed by 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.

[0023] In another embodiment of the present invention, a screening method for peptides that specifically bind to tetrodotoxin is provided, comprising the steps of: preparing a complex in which tetrodotoxin and a carrier protein are combined; and selecting a peptide that specifically binds to the complex.

[0024] In the present invention, the carrier protein may be characterized as being albumin or ovalbumin. Effects of the invention

[0026] The novel peptide for detecting tetrodotoxin according to the present invention exhibits high accuracy and sensitivity to tetrodotoxin, and a detection kit or biochip containing the same can also rapidly and accurately detect tetrodotoxin.

[0027] Therefore, by utilizing this, it is possible to verify the presence of tetrodotoxin in various substances such as food and raw materials, thereby contributing to the safe supply of food, management of fisheries resources, the marine environment, and public health. Brief explanation of the drawing

[0029] Figure 1 is according to Example 1 and shows the results of confirming the binding status and extent of tetrodotoxin and the carrier protein ovalbumin (OVA) by MALDI-TOF analysis. Figure 2 is according to Example 2 and illustrates the process of discovering a peptide receptor that specifically binds to tetrodotoxin. Figure 3 is according to Example 3 and shows the results of confirming the binding power of a bacteriophage candidate group containing a peptide according to the present invention to tetrodotoxin. Figure 4 is according to Example 3 and shows the results of confirming the binding power to tetrodotoxin according to the concentration of phage particles of a bacteriophage candidate group containing a peptide according to the present invention. Figure 5 is according to Example 3 and shows the results of confirming the binding ability of a bacteriophage candidate group containing a peptide according to the present invention to tetrodotoxin according to the concentration of tetrodotoxin-OVA. Figure 6 is according to Example 3 and shows the results of comparing the binding strength of a bacteriophage candidate group containing a peptide selected according to the present invention with control groups containing tetrodotoxin and other toxins. Figure 7 is according to Example 4 and shows the results of a molecular docking simulation for predicting the binding site between the amino acid sequence of a peptide selected according to the present invention and tetrodotoxin. Specific details for implementing the invention

[0030] Hereinafter, to explain more specifically, examples will be provided for detailed explanation. However, the following examples are illustrative and the scope of the present invention is not limited thereto.

[0031] According to one embodiment of the present invention, a peptide for detecting tetrodotoxin is provided, comprising a part or the whole sequence of an amino acid sequence selected from the group consisting of amino acid sequences represented by SEQ ID NOs 1 to 9.

[0032] APVRTHF (Sequence No. 1).

[0033] SAPRHLP (Sequence No. 2).

[0034] HSLPAFL (Sequence No. 3).

[0035] DYTDVSH (Sequence No. 4).

[0036] LAPVYLGSRHIE (Sequence No. 5).

[0037] VGSANRTSPHEA (Sequence No. 6).

[0038] SSADGLPRWNHY (Sequence No. 7).

[0039] GSTFSVGPHVLD (Sequence No. 8)

[0040] EPPSYIHSTGTS (Sequence No. 9).

[0041] In addition, the above sequence number 1 may represent a group of candidate names for TTX-7-R2-1, sequence number 2 for TTX-7-R3-4, sequence number 3 for TTX-7-R3-5, sequence number 4 for TTX-7-R3-8, sequence number 5 for TTX-12-R2-1, sequence number 6 for TTX-12-R2-5, sequence number 7 for TTX-12-R3-9, sequence number 8 for TTX-12-R3-11, and sequence number 9 for TTX-12-R3-13.

[0042] Specifically, in the present invention, the peptide may be characterized by comprising a part or the whole sequence of the amino acid sequence represented by SEQ ID NO. 5 or 9, and in terms of selecting a peptide having an excellent detection effect of tetrodotoxin and a docking simulation binding effect with an increased binding strength in proportion to the concentration of the bacteriophage candidate or the concentration of the target toxicity, it may most suitably comprise a part or the whole sequence of the amino acid sequence represented by SEQ ID NO. 5.

[0043] The amino acid sequence of the peptide according to the present invention may be obtained by finding a sequence that binds to tetrodotoxin using a bacteriophage display peptide library Ph.D.-7 or Ph.D.-12 (New England BioLab), which is designed to express hundreds of millions of different peptides obtained by artificially inserting a gene sequence to express a linear peptide having a sequence of 12 random amino acids at the end of a gene producing p², a type of coat protein, in the genome of M13 bacteriophage, and then infecting E. coli. Suitablely, the sequence that binds to tetrodotoxin may be obtained by using Ph.D.-12.

[0044] The above M13 bacteriophage is a nano-sized material that has the property of infecting only specific strains of E. coli, and there have been no reports to date regarding the possibility of it infecting the human body through mutation. In particular, it obtained FDA approval in 2006 and is being used as an additive to prevent bacterial contamination in instant foods. It is receiving attention not only as an alternative that can solve the problem of antibiotic resistance but also as a bio-evolved nanomaterial that is harmless to the human body. Furthermore, using the above bacteriophage has the advantages of a simple manufacturing process, improved productivity and stability, and the ability to utilize various genetic and chemical modifications.

[0045] The peptide according to the present invention is a small molecule peptide that has the advantage of being three-dimensionally stabilized due to its small size, easily penetrating mucous membranes, and recognizing molecular targets even deep within tissues. Furthermore, the small molecule peptide according to the present invention is relatively simple to mass-produce compared to antibodies and has almost no toxicity. In addition, the small molecule peptide according to the present invention has the advantage of high binding affinity to target substances and does not undergo denaturation even during heat or chemical treatment. Moreover, due to its small molecular size, it can be attached to other proteins and utilized as a fusion protein.

[0046] The peptide according to the present invention may be prepared by chemical synthesis known in the art. Representative methods include, but are not limited to, liquid or solid-phase synthesis, fragment condensation, and F-MOC (9-fluorenylmethoxycarbonyl) or T-BOC (tert-butyloxycarbonyl) chemical methods. Additionally, the peptide of the present invention may be prepared by genetic engineering methods. First, a DNA sequence encoding the peptide is constructed according to conventional methods. The DNA sequence may be constructed by PCR amplification using appropriate primers. Alternatively, the DNA sequence may be synthesized by standard methods known in the art, for example, using an automated DNA synthesizer (e.g., Biosearch or those sold by Applied Biosystems). The constructed DNA sequence is inserted into a vector containing one or more expression control sequences (e.g., promoters, enhancers, etc.) that are operatively linked to the DNA sequence and regulate the expression of the DNA sequence, and host cells are transformed with the recombinant expression vector formed therefrom. The generated transformant is cultured under appropriate medium and conditions to express the DNA sequence, and a substantially pure peptide encoded by the DNA sequence is recovered from the culture. The recovery may be performed using methods known in the art (e.g., chromatography). The term "substantially pure peptide" means that the peptide according to the present invention does not substantially contain any other proteins derived from the host. Furthermore, the peptide according to the present invention may be in a form in which the N-terminus or C-terminus is modified or protected by various organic groups to protect against protein-cleaving enzymes in vivo and to increase stability.That is, the C-terminus of the above peptide may be modified into a form that can be modified to increase stability, without any particular limitations, but preferably, it may be modified into a hydroxyl group (-OH) or an amino group (-NH2).

[0047] In addition, the N-terminus of the above peptide may be modified to a form that can be modified to increase stability, without any particular limitations, but preferably, it may be modified to a group selected from the group consisting of acetyl, fluorenyl methoxycarbonyl (Fmoc) group, formyl group, palmitoyl group, myristyl group, stearyl group, and polyethylene glycol (PEG).

[0048] In addition, the peptide according to the present invention is characterized by having any one of the following amino acids: a D-type, an L-type, a peptide mimic including a peptoid monomer, or a non-natural amino acid.

[0049] In the present invention, the peptide may be characterized as being a dimer, trimer, or multimer, and may include various functional groups at the N-terminus or C-terminus, but is not limited thereto.

[0050] As examples of the various functional groups mentioned above, the N-terminal functional group may be any one selected from the group consisting of free amines, acetylation, biotin, and fluorophore, and the C-terminal functional group may be any one selected from the group consisting of free acid, amidation, biotin, and fluorophore, but is not limited thereto.

[0051] In addition, the peptide in the present invention may be characterized by being labeled with any one selected from the group consisting of chromogenic enzymes, radioisotopes, chromophores, luminescent substances, and fluorescent substances, but is not limited thereto.

[0052] For example, the above-mentioned chromogenic enzyme may be peroxidase or alkaline phosphatase, the above-mentioned radioisotope may be 124I, 125I, 111In, 99mTc, 32P, 35S, and the above-mentioned luminescent material or fluorescent material may be FITC, RITC, rhodamine, Texas Red, fluorescein, phycoerythrin, quantum dots, etc.

[0053] Similarly, the detectable label may be an antibody epitope, substrate, cofactor, inhibitor, or affinity ligand.

[0054] These markings may be performed during the process of synthesizing the peptide of the present invention, or may be additionally performed on a peptide that has already been synthesized.

[0055] If a fluorescent substance is used as a detectable label, the fluorescence pattern of the peptide bound to tetrodotoxin can be observed by fluorescence mediated tomography (FMT), and tetrodotoxin can be detected according to the observed fluorescence pattern.

[0056] It is clear to those skilled in the art that biological functional equivalents that may be included in the range of peptides for detecting tetrodotoxin of the present invention are limited to those comprising variations in amino acid sequences that exhibit biological activity equivalent to that of the peptides of the present invention.

[0057] These amino acid variations are based on the relative similarities of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be considered biologically functional equivalents.

[0058] In introducing mutations, the hydropathic index of amino acids may be considered. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0059] The hydrophobic amino acid index is very important in conferring interactive biological functions of peptides. It is a known fact that similar biological activity can be achieved by substituting with amino acids having similar hydrophobic indices. When introducing a variation based on the hydrophobic index, the substitution is preferably made between amino acids exhibiting a difference in hydrophobic index within ±2, more preferably within ±1, and even more preferably within ±0.5.

[0060] Meanwhile, it is also well known that substitution between amino acids having similar hydrophilicity values ​​results in peptides having uniform biological activity. As disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​are assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); aspalate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); Phenylalanine (-2.5); tryptophan (-3.4).

[0061] When introducing a variation based on hydrophilicity values, substitutions are made between amino acids that exhibit a difference in hydrophilicity values ​​within preferably ±2, more preferably ±1, and even more preferably ±0.5.

[0062] Amino acid exchanges in peptides that do not change the overall activity of the molecule are known in the art (H. Neurath, RLHill, The Proteins, Academic Press, New York, 1979). The most common exchanges are exchanges between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0063] Considering the variant having the aforementioned biologically equivalent activity, the peptide for tetrodotoxin detection of the present invention is interpreted to include a sequence that exhibits substantial identity with the sequence listed in the sequence list. The above substantial identity refers to a sequence that exhibits at least 80% homology, more preferably 90% homology, when the peptide sequence of the present invention is aligned with any other sequence to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art. Any alignment method known in the art may be used without limitation for sequence comparison.

[0064] In another embodiment of the present invention, a composition for detecting tetrodotoxin is provided, comprising one or more peptides selected from the group consisting of amino acid sequences represented by SEQ ID NOs 1 to 9, which are peptides according to the present invention.

[0065] For example, any one or more peptides selected from the group consisting of amino acid sequences represented by SEQ ID NOs. 1 to 9 may also be interpreted to include sequences exhibiting substantial identity with the sequences listed in the sequence list, as previously described. The above substantial identity refers to a sequence that exhibits at least 80% homology, more preferably 90% homology, when the peptide sequence of the present invention is aligned with any other sequence to the greatest extent possible and the arranged sequence is analyzed using an algorithm commonly used in the art. Any alignment method known in the art may be used without limitation for sequence comparison. The composition for detecting tetrodotoxin according to the present invention may be used to confirm or diagnose whether tetrodotoxin is present in various subjects suspected of having tetrodotoxin.

[0066] In another embodiment of the present invention, a kit for detecting tetrodotoxin is provided, comprising one or more peptides selected from the group consisting of amino acid sequences represented by SEQ ID NOs. 1 to 9, which are peptides according to the present invention.

[0067] The tetrodotoxin detection kit of the present invention may include one or more other component compositions or devices suitable for the method of analyzing peptides for tetrodotoxin detection, and may additionally include a buffer or reaction solution that maintains the structure or physiological activity of the peptides stably. In addition, to maintain stability, it may be provided in a state maintained at 4°C.

[0068] In addition, to facilitate the identification, detection, and quantification of the peptide according to the present invention bound to tetrodotoxin, the peptide included in the kit of the present invention may be provided in a labeled state as described above. Meanwhile, if the peptide of the present invention is provided unlabeled, the kit of the present invention may additionally include a component for exploring the degree of binding or the location thereof with tetrodotoxin of the peptide of the present invention in vitro or in vivo. The above component may be a known compound for labeling the peptide of the present invention, or an antibody against a specific receptor that binds to the peptide of the present invention or a secondary antibody against these, and a reagent for detection thereof, for exploration through an antigen-antibody reaction.

[0069] For example, the kit of the present invention can be used to determine whether various objects, agricultural products, or fishery products possess tetrodotoxin, and can be used to determine whether a substance possessing tetrodotoxin has been consumed. The above determination can be performed by modifying a conventional immunoassay method or protocol.

[0070] In addition, diagnosis can be performed by using the peptide of the present invention instead of the antibody in conventional immunoassay, and by using the same process.

[0071] In another embodiment of the present invention, a biochip for detecting tetrodotoxin is provided, comprising one or more peptides selected from the group consisting of amino acid sequences represented by SEQ ID NOs. 1 to 9, which are peptides according to the present invention.

[0072] The peptide according to the present invention can be specifically immobilized on various chips (gold, silver, magnetic beads, silica, graphene, carbon nanotubes, etc.) that can be used as biochips. However, the features of the present invention are related to the features of the peptide described above, and manufacturing the peptide into a biochip in this manner is a part that can be done according to known technology.

[0073] Using the biochip for detecting tetrodotoxin according to the present invention, reactions between proteins, reactions between proteins and reaction ligands, reactions between target ligands and reaction proteins, or reactions between target ligands and reaction ligands can be detected.

[0074] The above reaction ligand may be a low molecular weight biomaterial, peptide, carbohydrate, aptamer, fatty acid, or lipid that reacts with a protein or peptide, but is not limited thereto. In addition, the above reaction protein may be an enzyme or antibody, but is not limited thereto. Furthermore, the above target ligand may be a low molecular weight compound, nucleic acid, peptide, carbohydrate, aptamer, fatty acid, or lipid that reacts with a protein or peptide, but is not limited thereto. In addition, the above reaction detection may be performed using a labeled or unlabeled material.

[0075] In another embodiment of the present invention, a method for detecting tetrodotoxin is provided, comprising: a step of treating a sample with one or more peptides selected from the group consisting of amino acid sequences represented by SEQ ID NOs. 1 to 9, which are peptides according to the present invention; and a step of detecting a reaction between the peptide and the sample.

[0076] Through this, it is possible to detect the presence or absence of tetrodotoxin in a sample, quantify the amount of tetrodotoxin, and confirm whether a substance containing tetrodotoxin has been ingested.

[0077] In the present invention, the term "sample" is used in the broadest sense. On the one hand, it includes both biological and environmental samples. Furthermore, the sample may include specimens of synthetic origin. On the other hand, it may include specimens or cultures (e.g., microbial cultures). The sample may be a sample derived from agricultural or aquatic products. In the case of the aquatic product sample, it may preferably be fish or shellfish. In addition, the above biological samples may be used to confirm whether a substance containing tetrodotoxin has been ingested using the peptide according to the present invention, and include whole blood, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, leukocytes, peripheral blood mononuclear cells, buffy coat, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, and bronchial aspirate. It may be one or more selected from the group consisting of aspirate, synovial fluid, joint aspirate, organ secretions, cell, cell extract, and cerebrospinal fluid, but is not limited thereto.

[0078] In the method of the present invention, the detection of the reaction may be performed by 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, but is not limited thereto.

[0079] In another embodiment of the present invention, a screening method for peptides that bind to tetrodotoxin is provided.

[0080] Specifically, a screening method for peptides that specifically bind to tetrodotoxin can be provided, comprising the steps of: preparing a complex in which tetrodotoxin and a carrier protein are combined; and selecting peptides that specifically bind to said complex.

[0081] In the screening method of the present invention, the carrier protein may be characterized as being albumin or ovalbumin.

[0082] In addition, in the screening method of the present invention, the peptide that specifically binds to the complex may be characterized by comprising a part or the whole sequence of one or more amino acid sequences selected from the group consisting of amino acid sequences represented by SEQ ID NOs 1 to 9.

[0084] The contents of the present invention described above shall apply equally unless they are mutually contradictory, and implementations made by a person skilled in the art with appropriate modifications are also included within the scope of the present invention.

[0085] Redundant content is omitted out of consideration for the complexity of this specification, and terms not otherwise defined in this specification have the meanings commonly used in the technical field to which this invention belongs.

[0087] The present invention will be described in more detail below through examples. However, it will be obvious to those skilled in the art that the following examples are provided merely to facilitate a better understanding of the present invention and that the scope of the present invention is not to be interpreted as being limited by the examples.

[0089] Examples

[0090] Example 1. Formation of tetrodotoxin haptens and tetrodotoxin carriers

[0091] A specific binding bioreceptor for tetrodotoxin was discovered through a phage display technique in which a tetrodotoxin carrier (tetrodotoxin-OVA) was synthesized using formaldehyde as a medium and then immobilized on a plate for use in phage display.

[0092] When small molecules such as tetrodotoxin are immobilized on a carrier protein and used for phage display rather than being directly immobilized on a plate, they can be discovered more efficiently. Representative proteins used as carrier proteins include bovine serum albumin (BSA) or ovalbumin (OVA), both of which have carboxylic acid and amine functional groups.

[0093] In addition, the binding of the tetrodotoxin carrier (tetrodotoxin-OVA) was carried out in the following manner.

[0094] First, 1.4 mg of tetrodotoxin is prepared by dissolving it in 1.4 mL of phosphate buffered saline (50 mM, pH 7.4). 50 mg of ovalbumin is dissolved in 1 mL of phosphate buffered saline (10 mM, pH 7.5). 40 μL of the prepared ovalbumin solution is taken, and 60 μL of acetate buffer (1 M, pH 7.4) and 140 μL of tetrodotoxin solution are added and mixed evenly. 8.2 μL of formaldehyde solution is added to the mixture, and the mixture is stirred at room temperature for 72 hours to proceed with tetrodotoxin carrier (tetrodotoxin-OVA) conversion.

[0095] Haptened tetrodotoxin was obtained by removing unreacted substances through a dialysis process, and the binding status and extent of tetrodotoxin and the carrier protein ovalbumin (OVA) were confirmed by MALDI-TOF analysis and are shown as the results in Figure 1.

[0096] Referring to Figure 1, it can be seen that the ovalbumin peak at 44.7 kDa binds to tetrodotoxin to form a hapten, and then shifts to a broad peak around 48.5 kDa, thus confirming that a tetrodotoxin carrier with approximately 12 tetrodotoxins immobilized per ovalbumin was successfully obtained.

[0098] Example 2: Discovery of peptides capable of specific and selective binding to tetrodotoxin

[0099] In order to discover peptides that specifically bind to tetrodotoxin, peptides that specifically bind to tetrodotoxin were discovered using the tetrodotoxin carrier prepared in Example 1 (described as tetrodotoxin-OVA complex) and a bacteriophage display peptide library.

[0100] Specifically, the Ph.D.-12 and Ph.D.-7 (New England BioLab) M13 bacteriophage random peptide library (Phage display peptide library) was used, which consists of recombinant M13 bacteriophages expressing hundreds of millions of different linear or circular peptide receptors obtained by infecting E. coli after artificially inserting a gene sequence to express a peptide receptor with a random sequence of 12 or 7 amino acids at the end of the gene sequence producing pIII, a type of coat protein, in the M13 bacteriophage genome.

[0101] To discover tetrodotoxin high-specificity peptide receptors, a total of three pannings were performed. The panning process was carried out using streptavidin-coated plates (Streptavidin High Binding Capacity Coated 96-Well Plates (Thermo Scientific, Cat. No. 15501)) after labeling the tetrodotoxin-OVA complex prepared in Example 1 with biotin. This is schematically illustrated in Figure 2.

[0103] 1) Ph.D.-7 M13 Bacteriophage Library

[0104] In the first experiment, 100 μL of biotinylated tetrodotoxin-OVA complex at a concentration of 1 μM and 1X10 Ph.D.-7 M13 bacteriophage library 131 μL of a PFU / mL concentration was mixed into a 1.5 mL Eppendorf tube and reacted at room temperature for 1 hour to form a pre-complex. Before the reaction was completed, a plate coated with streptavidin was prepared by washing it three times repeatedly for 30 seconds with 0.1 M PBS (phosphate-buffered saline). Next, 100 μL of the pre-complex was added to the well and stirred at 150 rpm for 10 minutes at room temperature; then, to remove non-specific binding, 1 μL of 0.1 mM biotin was added to the solution and stirred at 150 rpm for 5 minutes at room temperature. After this process, the agenetic M13 bacteriophage was washed 10 times repeatedly with 200 μL of PBST (0.1% Tween 20 added to 0.1 M PBS). Finally, the phage that specifically binds to tetrodotoxin was eluted in 100 μL of 0.2 M Glycin-HCl (pH 2.2) solution, and 15 μL of Tris-HCl (pH 9.0) was added for pH neutralization.

[0105] During the three repeated experiments performed using the above process, it was observed that the concentration of phages eluted from each library tended to increase with each repeated panning, as shown in Table 1 below.

[0106]

[0107] In addition, the amino acid sequences of M13 bacteriophages that bind to tetrodotoxin were identified through DNA sequencing, and four candidate bacteriophages that showed high frequency or had similar sequences were selected and are shown in Table 2 below.

[0108]

[0110] 2) Ph.D.-12 M13 Bacteriophage Library

[0111] Next, a total of three pannings were performed for the discovery of peptide receptors using the Ph.D.-12 M13 bacteriophage library, and the process and concentration throughout the experiment were carried out under the same conditions as the receptor discovery using the Ph.D.-7 M13 bacteriophage library.

[0112] In this experiment, the concentration of eluted phages showed the trend shown in Table 3 below, and five candidate phages with sequence similarity or repetitive binding were selected and shown in Table 4 below.

[0114]

[0115]

[0117] Example 3: Confirmation of the binding affinity of bacteriophage candidates to tetrodotoxin

[0118] 1) Amplification of M13 bacteriophage candidates

[0119] According to Example 2 above, a total of 9 M13 bacteriophage candidates selected through a total of 3 phage displays for tetrodotoxin were amplified for each phage in order to be used in an ELISA experiment.

[0120] Specifically, colonies of the E. coli strain (ER2738) were placed in 20 mL of LB medium and cultured overnight at 37°C while stirring at 210 rpm. Phage stock was added to 20 mL of LB medium (OD: 0.04) along with pre-cultured E. coli, and cultured for 4.5 hours at 37°C and 210 rpm. After the culture was finished, the culture medium was transferred to a centrifuge tube and centrifuged at 4,500 g for 10 minutes at 4°C to remove E. coli. Next, the supernatant was transferred to a new centrifuge tube, and the above process was repeated twice to completely remove any remaining E. coli. 16 mL of the supernatant from the culture medium from which E. coli had been removed was combined with 4 mL of 20% PEG / 2.5 mol NaCl, and the mixture was stored at 4°C for 12 hours. The next day, the reaction solution was centrifuged at 12,000 g for 15 minutes at 4°C, and the supernatant was removed. The remaining pellet was then dissolved in 1 mL of 0.1 M PBS, transferred to a microtube, and centrifuged at 14,000 rpm for 5 minutes at 4°C to remove remaining impurities. After centrifugation, the supernatant was transferred to a microtube, 200 μL of 20% PEG / 2.5 mol NaCl was added, and the mixture was placed on ice and stored at 4°C for 1 hour. After the reaction time was complete, the solution was centrifuged at 14,000 rpm for 10 minutes, and the supernatant was removed. The precipitate containing M13 bacteriophage was then dissolved in 200 μL of 0.1 M PBS to obtain amplified M13 bacteriophage. Subsequently, phage titration was performed to calculate the phage concentration (PFU / mL: Plaque forming units / mL), and the concentrations of the 9 candidate phages were 5×10 11 I adjusted it to PFU / mL.

[0121] 2) Perform enzyme immunoassay (ELISA) using M13 bacteriophage candidates

[0122] Enzyme-linked immunoassay (ELISA) was performed using the above-mentioned amplified M13 bacteriophage candidates.

[0123] Specifically, streptavidin-coated plates (Streptavidin High Binding Capacity Coated 96-Well Plates (Thermo Scientific, Cat. No. 15501)) were prepared, and the process of adding and removing 200 μL of 0.1 M PBS was repeated three times. Subsequently, 100 μL of a 1 μM biotinylated tetrodotoxin-OVA complex and 100 μL of a 1 μM biotinylated OVA (to be used as a control) were added to each plate well, sealed, and incubated overnight at 4°C. The next day, the cultured plates were removed and preheated at room temperature for 15 minutes. Next, a blocking solution was prepared by adding 0.2 g of BSA to 40 mL of NaHCO3 (pH 9.4); after removing the solution from the plates, 200 μL of this solution was added to each well, sealed, and reacted at 37°C for 1 hour. After the reaction was complete, the plates were placed at room temperature. After cooling, 1 μL of 20 mM biotin was added and the reaction was carried out for an additional 15 minutes. Subsequently, after removing all the solution, the sample was washed 6 times with 0.5% Tween-20 in 0.1 M PBS, and 100 μL of phage candidates (concentration: 5 × 10¹¹ PFU / mL) were added to a plate immobilized with 0VA-TTX and reacted at room temperature for 1 hour. After the reaction was complete, the sample was washed 6 times with 0.5% Tween-20 in 0.1 M PBS, 100 μL of anti-M13-HRP antibody:blocking solution diluted 0.5:5000 was added, and the sample was reacted at room temperature with stirring at 150 rpm for 1 hour. Next, the sample was washed 6 times repeatedly using 200 μL of PBST (0.1% Tween 20 added to 0.1 M PBS), and then 100 μL of substrate (TMB) was added and reacted for 10 minutes.Finally, 100 μL of 2 M sulfuric acid (H2SO4) was added to terminate the reaction, and the absorbance (OD) value was measured at 450 nm and the results are shown in Figures 3 to 5.

[0124] Figure 3 shows the experimental results of confirming the binding affinity of peptides composed of amino acid sequences of SEQ ID NOs 1 to 9 to a control group and tetrodotoxin using an enzyme immunoassay. It was confirmed that peptides composed of amino acid sequences of SEQ ID NOs 5 and 9 showed a higher binding affinity to tetrodotoxin on plates coated with tetrodotoxin-OVA compared to OVA used as a control group.

[0125] Accordingly, peptides composed of the amino acid sequences of SEQ ID NOs. 5 and 9, which exhibited higher binding affinity to tetrodotoxin, were primarily selected.

[0126] FIG. 4 shows phages labeled with peptides consisting of the amino acid sequences of SEQ ID NOs. 5 and 9 selected according to the results of FIG. 2 at different concentrations (1×10 10 ~1X10 12 By comparing the binding affinity after treatment with PFU / mL, it was confirmed that the binding affinity to tetrodotoxin increased as the concentration of the bacteriophage candidate increased.

[0127] Figure 5 compares the binding affinity to tetrodotoxin according to tetrodotoxin-OVA concentrations (0, 0.0625, 0.125, 0.25, 0.5, and 1 μM) of peptides composed of amino acid sequences of SEQ ID NOs. 5 and 9 selected according to the results of Figure 3, and it was confirmed that the binding affinity increased in proportion to the increase in the concentration of the target toxin.

[0128] 3) Selection of optimal peptides for tetrodotoxin detection

[0129] After constructing a standard curve using the change in binding strength of peptides composed of the amino acid sequences of SEQ No. 5 (TTX-12-R2-1) and 9 (TTX-12-R3-13), the limit of detection (LOD) and limit of quantification (LOQ) of each candidate group were calculated and compared and are shown in Table 5 below.

[0130]

[0132] Referring to Table 5 above, it was confirmed that the peptide consisting of the amino acid sequence of Sequence No. 5 (TTX-12-R2-1) exhibited superior detection and quantification limits compared to other candidates.

[0133] In addition, Figure 6 shows the results of comparing the binding affinity of a peptide composed of the amino acid sequence of SEQ ID NO. 5 (TTX-12-R2-1) to tetrodotoxin and its binding affinity to various control groups, including saxitoxin, another type of marine toxin, using the same ELISA method as above. It was confirmed that the peptide composed of the amino acid sequence of SEQ ID NO. 5 (TTX-12-R2-1) exhibited a higher binding affinity to tetrodotoxin compared to the control groups.

[0134] Referring to Examples 1 to 3 and Figures 5 to 6 above, it was found that the peptide composed of the amino acid sequence of SEQ ID NO. 5 (TTX-12-R2-1) is the optimal peptide for tetrodotoxin detection, and therefore, the peptide composed of the amino acid sequence of SEQ ID NO. 5 (TTX-12-R2-1) was selected as the peptide for tetrodotoxin detection.

[0136] Example 4: Prediction of binding sites between tetrodotoxin and selected tetrodotoxin-specific peptides via molecular docking simulation

[0138] According to the above example, the free DockThor web server (www.dockthor.lncc.br) was used to predict the binding site between the amino acid sequence of a peptide consisting of the amino acid sequence of selected sequence number 5 (TTX-12-R2-1) and tetrodotoxin.

[0139] Specifically, the structure of the amino acid sequence (LAPVYLGSRHIE) of the peptide composed of the amino acid sequence of SEQ ID NO. 5 (TTX-12-R2-1) was obtained using the Pep-Fold 3.0 program (https: / / bioserv.rpbs.univ-paris-diderot.fr / services / PEP-FOLD3 / ), and the structure of tetrodotoxin was used in the docking process by utilizing the structure registered in PubChem (PubChem CID: 11174599) (https: / / pubchem.ncbi.nlm.nih.gov). The docking simulation was conducted by selecting 750 models for the binding process and setting the simulation process to repeat 1,000,000 times for each model 24 times, and the results are shown in Figure 7.

[0140] Referring to Figure 6, the amino acid sequence (LAPVYLGSRHIE) of the peptide composed of the amino acid sequence of SEQ ID NO. 5 (TTX-12-R2-1) showed a binding energy (binding strength) of -6.198 Kcal / mol for tetrodotoxin, and in particular, it was confirmed that arginine (Arg) at position 9 and glutamic acid (Glu) at position 12 of the peptide's amino acid sequence showed high binding strength to the C3 (RMSD: 1.388), C8 (RMSD: 1.320), and C10 (RMSD: 2.234) positions of tetrodotoxin, respectively.

[0141] In other words, this means that a peptide consisting of the amino acid sequence of sequence number 5 (TTX-12-R2-1) can bind to tetrodotoxin.

[0143] In summary, the peptide of the present invention, discovered using a bacteriophage display technique, can specifically bind to tetrodotoxin and can be usefully utilized for the detection of tetrodotoxin. In particular, in the case of the peptide composed of the amino acid sequences of SEQ ID NOs 5 (TTX-12-R2-1) and 9 (TTX-12-R3-13), the binding strength increases in proportion to the concentration of the bacteriophage candidate group or the concentration of the target toxin, thereby providing a superior tetrodotoxin detection effect.

[0144] In addition, the peptide composed of the amino acid sequence of SEQ ID NO. 5 (TTX-12-R2-1) exhibits excellent binding effects in docking simulations, and has the highest accuracy and sensitivity against tetrodotoxin.

[0146] Accordingly, the peptide according to the present invention can rapidly and accurately detect tetrodotoxin, and by using this, it is possible to confirm whether tetrodotoxin is present in various objects such as food and raw materials, thereby contributing to the safe supply of food, management of fishery resources, marine environment, and public health.

[0148] As specific parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention.

[0149] Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims

Claim 1 Sequence number 5 A peptide for detecting tetrodotoxin consisting of an amino acid sequence represented by Claim 2 delete Claim 3 delete Claim 4 A peptide for detecting tetrodotoxin according to claim 1, characterized in that the peptide is labeled with any one selected from the group consisting of a chromogenic enzyme, a radioisotope, a chromopore, a luminescent substance, and a fluorescent substance. Claim 5 A composition for detecting tetrodotoxin comprising a peptide having an amino acid sequence represented by SEQ ID NO.

5. Claim 6 A kit for detecting tetrodotoxin comprising a peptide consisting of an amino acid sequence represented by SEQ ID NO.

5. Claim 7 A biochip for detecting tetrodotoxin comprising a peptide consisting of an amino acid sequence represented by SEQ ID NO.

5. Claim 8 A method for detecting tetrodotoxin, comprising: a step of treating a sample with a peptide consisting of an amino acid sequence represented by SEQ ID NO. 5; and a step of detecting the reaction between the peptide and the sample. Claim 9 A method for detecting tetrodotoxin according to claim 8, wherein the detection of the reaction is performed by 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. Claim 10 delete Claim 11 delete Claim 12 delete

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