CN115094064A - Aptamer specifically bound with domoic acid and application thereof - Google Patents

Abstract

The invention relates to the technical field of biomedical engineering, and provides an aptamer specifically combined with domoic acid and application thereof. The sequence general formula of the aptamer is as follows: 5' -ATTGGCACTCCACGCATAGG-N ₄₀ -CCTATGCGTGCTACC GTGAA-3'; wherein N is any one of A, T, G, C four deoxyribonucleotide bases, 40 represents the number of random bases, preferably the sequence shown in any one of SEQ ID NO.10, SEQ ID NO.12 and SEQ ID NO.14, and most preferably the sequence shown in any one of SEQ ID NO. 14. The aptamer can be prepared into a domoic acid separation and enrichment reagent, a purification reagent, an antagonist or a neutralizing agent, and can also be prepared into a domoic acid detection reagent, a kit or a sensorThe method is used for detecting DA in drinking water samples, and can lay a foundation for preparing drugs for preventing or treating DA poisoning and removing DA in water or aquatic products.

Description

Aptamer specifically bound with domoic acid and application thereof

Technical Field

The invention relates to the technical field of biomedical engineering, in particular to an aptamer specifically bound with domoic acid and application thereof.

Background

Domoic Acid (DA) belongs to Amnesic Shellfish Toxins (AST or ASP), is an excitatory proline derivative and a strong neurotoxic biological toxin, wherein the toxicity symptoms are mainly manifested by abdominal pain, diarrhea and vomiting, and are accompanied by memory loss, confusion, incapability of identifying families and friends and the like, and severe Poisoning patients are in a coma state or even die.

Domoic acid is produced by diatoms of the genera phaeophyta and rhombohedral, and when these diatoms proliferate in large quantities, DA can accumulate through the filter feeding enrichment and food chain of shellfish, clams. After eating aquatic products contaminated by DA by mistake, humans can suffer from amnesic shellfish poisoning, and DA can affect digestive tract, cardiovascular system and central nervous system of human and animals, has excitation effect on brain stem region related to viscera function, and has obvious neurotoxicity effect on brain region related to memory. In addition, DA binds to glutamate receptors of the human central nervous system (hippocampus cerebri), causing paralysis of the nervous system and resulting brain damage. The research shows that when the content of domoic acid in the shellfish tissue reaches 40mg/kg, poisoning of consumers can be caused, 150mg/kg has death risk, and the maximum limit which can be tolerated by human beings through eating is 20 mg/kg. In the world, the Canada firstly establishes a safety limit standard of 20 mug/g of shellfish meat, and Europe and Japan also successively list the toxin as a shellfish conventional detection item.

The chemical structure of domoic acid is very similar to Kainic Acid (KA) and glutamic acid (glumate). The domoic acid has eight isomers A-H, which are probably reaction products of DA after being irradiated by ultraviolet rays, but are not natural products of algae; the main contaminant to shellfish and crustaceans is DA, which is the most toxic of all isomers. The distribution of domoic acid is worldwide, and although the reports of domoic acid detected in China are relatively rare, in China's sea area, a plurality of pseudo-rhombohedral algae are detected and widely distributed in China's coast, wherein nine pseudo-rhombohedral algae are potential toxigenic species, and the domoic acid is reported to be generated in non-China sea areas. Domoic acid is hardly detected in China at the present stage, probably because the pollution of the rhombohedral algae is not serious, but also because the existing detection method is not sensitive enough, an effective detection technology is lacked.

Domoic acid, one of red tide algal toxins, is closely related to outbreak of red tide, and has a relationship with global ecological balance and marine environmental safety in detection and protection. In addition, domoic acid is an important neurotoxin, which can cause memory loss and even incapacity of human beings, if the domoic acid is used as a biological warfare agent in war, the consequences can not be imagined, and no effective treatment method for the poisoning caused by domoic acid exists at present, so that the research on the domoic acid can provide a theoretical basis for maintaining national defense safety, consolidating defense and clinical treatment. Furthermore, domoic acid can pollute aquatic organisms through filter feeding enrichment and food chains to further harm human health, the food safety of marine products is concerned with the health of every country as a big country for aquaculture and consumption in China, and the domoic acid has thermal stability and cannot completely lose toxicity by a common cooking means, so that the effective detection and quality control of the marine products from the source are the best means for preventing poisoning. Therefore, the method is important for detecting and protecting the marine organism toxin domoic acid.

Researchers have developed a variety of methods for detecting domoic acid, including biological, physicochemical, and immunochemical methods. Among them, Mouse Bioassay (MBA) is the earliest bioassay method for detecting domoic acid, and half of domoic acid mice have lethal amount LD50 of about 10mg/kg, but this method is inferior in reproducibility, low in sensitivity, and incapable of quantitative analysis, and has an ethical problem. The physical-chemical analysis method combining various chemical instruments has the advantage of high detection sensitivity, for example, the high performance liquid chromatography is the most widely applied method for detecting the domoic acid at present and is listed as a national standard method by various countries. The domoic acid is analyzed by high performance liquid chromatography, and the edible standard safe concentration is 2mg/100 g. The method is always a hotspot of research of people, a plurality of novel methods are derived, the accuracy is very high, but the physical and chemical analysis method usually consumes a long time, instruments and equipment are expensive, the sample pretreatment process is complicated, and professional technicians are required. In addition, immunochemical methods, such as enzyme-linked immunosorbent assay (ELISA), have simple and convenient operation, a plurality of commercialized kits have been developed based on the ELISA, and the colloidal gold test strip is more suitable for field detection. However, the preparation of antibodies is complex and time consuming, especially for small marine toxins, which are less or even non-immunogenic, whereas their toxicity is harmful to the experimental animals and cells. The prepared antibody is not easy to store for a long time, and cross reaction is also an inevitable defect. In conclusion, the conventional domoic acid detection method still has certain defects, and therefore, a new detection technology needs to be developed to avoid the defects of the method.

The aptamer is a molecule which is formed by rolling and folding single-stranded DNA or RNA to form a specific tertiary structure under the condition that a complementary strand does not exist, has a certain function and is similar to an antibody in function. Nucleic acid aptamers are generally obtained by screening through a Systematic evolution of ligands by exponential enrichment technique (SELEX), which mainly forms a complex with a relatively stable spatial structure with a target through hydrogen bonds, hydrophobic interactions, electrostatic interactions, van der waals forces, and the like; in addition, the aptamer binds to the target molecule and induces a mutual binding action, and when the single-stranded oligonucleotide approaches the target molecule indefinitely, the spatial structure of the single-stranded oligonucleotide changes, thereby forming a spatial structure that binds to the target molecule. Targets in the current field of aptamer research relate to small molecules, proteins, pathogenic bacteria, viruses, cells, tissues and even living animals, and the application range covers environmental monitoring, diagnosis, drug delivery and treatment, and has a very deep prospect. The novel molecular recognition element aptamer has the advantages of high affinity and specificity, is easy to chemically synthesize in a large amount, and is easy to modify and fix on the surface of a carrier, so that a biosensor and a research and development kit are convenient to construct, and the appearance of the aptamer brings new revelation for the detection, prevention and treatment of domoic acid.

However, there are few reports on molecular recognition probes that specifically bind domoic acid, high-affinity aptamers that specifically bind domoic acid, and the identification, optimization, and application of affinity constants for the aptamers.

Disclosure of Invention

The present invention has been made to solve the above problems, and an object of the present invention is to provide an aptamer specifically binding to domoic acid and use thereof.

The first purpose of the present invention is to provide a plurality of single-stranded DNA aptamers capable of binding specifically to DA with high affinity, and to test the affinity between the plurality of aptamers and DA to obtain an affinity constant (K) _d ) Five aptamers of smaller value, C1, C12, C58, C87, and C100.

A second object of the invention is to provide a truncated form of the affinity constant (K) for the above-mentioned aptamers _d ) The lowest C1 was optimized to provide DA aptamers C1-s, C1-b, C1-d of comparable or even superior binding capacity, but shorter length. Wherein, the affinity constant (K) of C1-d _d ) The lowest, 1.09X 10 ^-7 M, the binding force is far higher than that before truncation.

The third purpose of the invention is to provide the application of the aptamer, such as the application of the aptamer in preparing DA separation enrichment reagent, purification reagent, neutralizer or antagonist; the application in the preparation of DA detection reagent, kit or sensor; the application in preparing medicaments for preventing or treating DA poisoning; and the application in the rapid detection of DA in water bodies or aquatic products. Meanwhile, a foundation is laid for the preparation of medicaments for preventing or treating DA poisoning, the removal of DA in water or aquatic products, and the research of various applications such as DA biological functions, action mechanisms and the like.

The main technical scheme of the invention is as follows: a single-stranded DNA aptamer capable of specifically binding with DA with high affinity is screened and obtained by a Capture-SELEX (Capture-SELEX) technique (C1). And truncating the aptamer C1 according to the prediction result of the online tool, the mfold web server, on the secondary structure of the online tool, and obtaining the optimized aptamer C1-d. By combining with a biosensor platform, a DA aptamer sensor can be prepared and used for rapid detection of DA. In addition, the DA aptamer can lay a foundation for preparing medicaments for preventing or treating DA poisoning and removing DA in water bodies or aquatic products.

In a first aspect of the invention, there is provided an aptamer which binds specifically to domoic acid, the aptamer having the general sequence: 5' -ATTGGCACTCCACGCATAGG-N ₄₀ -CCTATGCGTGCTACCGTGAA-3'; wherein N represents any one of bases A, T, C and G, and N ₄₀ Representing a random sequence of 40nt in length.

Screening by a Capture-SELEX (Capture-SELEX) technology to obtain the following representative sequences:

c1: as shown in SEQ ID NO: 1 is shown in the specification;

c12: as shown in SEQ ID NO: 3 is shown in the specification;

c58: as shown in SEQ ID NO: 5 is shown in the specification;

c87: as shown in SEQ ID NO: 7 is shown in the specification;

c100: as shown in SEQ ID NO: shown in fig. 8.

In a second aspect of the invention, five DA aptamers of comparable binding capacity but shorter length, designated aptamer C1-s, aptamer C1-a, aptamer C1-b, aptamer C1-C and aptamer C1-d, have the sequences shown in SEQ ID NO. 10-SEQ ID NO.14, respectively, were optimized in a truncated manner for the most potent of the above aptamers C1.

The five aptamers can be combined with DA, wherein the affinity of the aptamer C1-s and the aptamer C1-b is equivalent to that before truncation, and the affinity between the aptamer C1-d and DA is farHigher than before truncation, reaches 1.09X 10 ^-7 And M. Therefore, the aptamer C1-d is preferable.

Preferably, the aptamer or preferably the aptamer may be chemically modified at its 3 'end or 5' end with biotin, FITC, thiol, and the like.

In a third aspect, the invention provides the use of an aptamer, for example, in the preparation of a domoic acid separation and enrichment reagent, a purification reagent, a neutralizing agent or an antagonist; the application in preparing a domoic acid detection reagent, a kit or a sensor is used for quickly detecting domoic acid in a water body or an aquatic product; the application in the preparation of medicament for treating domoic acid poisoning. Also provides the application of the domoic acid preparation in preparing and removing the domoic acid in the water body or the aquatic products.

Preferably, the therapeutic domoic acid toxic drug is an aptamer specifically binding to domoic acid as the only active ingredient or a pharmaceutical composition comprising a suitable ligand specifically binding to domoic acid. The pharmaceutical composition is also a medicine for neutralizing domoic acid, or a domoic acid antagonist and the like, and can relieve and cure symptoms such as nausea, vomit, diarrhea, bellyache, diarrhea, memory loss, confusion, coma and the like caused by domoic acid poisoning.

Preferably, the prepared preparation can completely remove the domoic acid in the water body or the aquatic product, or reduce the content of the domoic acid in the water body or the aquatic product to be below the standard regulated and recommended by the health organization and the grain and agriculture organization of the United nations.

In a fourth aspect of the present invention, there is provided a composition of an aptamer specifically binding to domoic acid, wherein the aptamer specifically binding to domoic acid is used as an active ingredient, and further comprises a pharmaceutically or detectably acceptable pharmaceutical carrier. The composition can be used for preparing anti-domoic acid drugs or detection reagents.

The composition of the invention and pharmaceutically or detectably acceptable auxiliary materials are combined into a pharmaceutical preparation composition, so that the curative effect is exerted more stably, the preparations can ensure the conformation integrity of the aptamer core sequence disclosed by the invention, and simultaneously can protect the multifunctional group of the protein and prevent the protein from degrading (including but not limited to agglomeration, deamination or oxidation).

In the fifth aspect of the invention, the application of the aptamer C1-d in rapid detection of DA in aquatic products or water bodies is particularly provided, and people are prevented from being poisoned mainly by drinking polluted water and eating polluted aquatic products by mistake.

The invention has the following beneficial guarantee and effects:

in view of the structural characteristics of small molecular weight, difficult fixation and the like of domoic acid, the invention screens and obtains the single-stranded DNA aptamer which is combined with domoic acid with high affinity and high specificity based on the Capture-SELEX (Capture-SELEX) technology. The aptamers are used as molecular recognition probes specifically bound with domoic acid, and have the advantages of high affinity, strong specificity, good stability, low immunogenicity, easiness in synthesis, modification and labeling and the like. The method can be used for separating and enriching trace domoic acid in a sample, analyzing and detecting domoic acid, preparing a medicament for neutralizing domoic acid, preparing a medicament for antagonizing domoic acid, and removing domoic acid in a water body.

Experiments prove that the aptamer can be quickly and specifically combined with domoic acid, wherein the affinity between the aptamer C1-d and DA is the highest and reaches 1.09X 10 ^-7 And M. Therefore, the aptamer obtained by screening can be prepared into an aptamer sensor or a detection reagent and applied to the detection of DA in a drinking water body sample. In addition, the aptamers can also lay a foundation for the preparation of drugs for preventing or treating DA poisoning and the removal of DA in water bodies or aquatic products.

In addition, according to the characteristics of DA molecules, the single-stranded DNA aptamer capable of being specifically combined with domoic acid with high affinity is obtained by adopting a Capture-SELEX (Capture-SELEX) method through forward screening and reverse screening, and the method has the characteristics of simplicity and convenience in operation, high repeatability and the like, greatly simplifies the construction technical route, and is low in production cost and short in purification period. The aptamer serving as a novel molecular recognition probe has the advantages of low cost, stable property, convenience in modification and the like, and is suitable for large-scale application in industrial production of biological medicines.

Drawings

Fig. 1 is a schematic diagram of DA aptamer screening based on Capture-SELEX (Capture-SELEX) technology.

FIG. 2 is a graph showing the results of the binding rate of the aptamer to the target toxin domoic acid in each round of screening.

FIG. 3 is a diagram showing the prediction of the secondary structure of the truncated sequence of aptamer C1 by mfold software.

FIG. 4 is a graph showing the results of measuring the affinity of the aptamer C1-d for domoic acid.

FIG. 5 is a graph showing the results of identifying the specificity of aptamer C1-d for domoic acid.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Example 1 construction of random ssDNA libraries and primers therefor

(1) Construction of a random ssDNA library of 80 nucleotides in length

The DA aptamer library consists of 80 bases, both ends of the DA aptamer library are fixed regions containing 20 bases, and the middle of the DA aptamer library is a random region 5' -ATTGGCACTCCACGCATAGG-N containing 40 bases ₄₀ -CCTATGCGTGCTACCGTGAA-3'; wherein N represents any one of bases A, T, C, G, N ₄₀ Representing a random sequence of length 40 nt.

(2) Construction of primers

An upstream primer: 5'-ATTGGCACTCCACGCATAGG-3' (SEQ ID NO.15),

a downstream primer 1: 5'-TTCACGGTAGCACGCATAGG-3' (SEQ ID NO.16),

a downstream primer 2: 5 '-poly (dA20) -Spacer 18-TTCACGGTAGCACGCATAGG-3' (SEQ ID NO. 17).

Example 2 Choroic acid aptamer Screen

In order to obtain aptamers that bind with high affinity and high specificity to Domoic Acid (DA), 12 rounds of screening were performed by fixing ssDNA library by Capture-SELEX technology. The screening process is shown in figure 1, the Capture-SELEX is covalently linked with a single-stranded oligonucleotide sequence, namely a Capture sequence (Capture sequence), through a solid phase carrier such as magnetic beads containing special groups, and meanwhile, the screening library is designed to contain a base sequence capable of being complementarily paired with the Capture sequence, namely a Docking sequence (Docking sequence), so that the screening library is fixed on the solid phase carrier through the base complementary pairing action of the Docking sequence and the Capture sequence.

According to the research, a Capture oligo with a Biotin label at the 3' end which is complementary with the end of a library fixed sequence is designed, the biotinylated Capture oligo and the library are paired in a buffer solution through annealing, and after the library and the SA magnetic beads are fully incubated, the library is fixed on the SA magnetic beads due to the interaction of the Biotin and the SA proteins. According to fig. 1 and 2, in the screening process, reverse screening is introduced from the 6 th round, i.e. the ssDNA library is incubated with the reverse target Kainic Acid (KA), and after washing, free target toxin Domoic Acid (DA) is added for further incubation; and finally recovering the aptamer specifically binding to the DA. The retention of aptamer binding to target DA is shown in fig. 2, and the retention of ssDNA no longer increases by 12 rounds of screening, i.e. the screening endpoint is considered to be reached.

The specific operation steps of the screening process are as follows:

(1) and (3) incubation: in each round of screening, the ssDNA library and capture sequences were dissolved and mixed in the screening buffer at a molar ratio of 1: 2. Keeping the temperature at 95 ℃ for 10min, slowly cooling to 60 ℃, keeping for 1min, then slowly cooling to 25 ℃, wherein the cooling rate is 0.1 ℃/s, and keeping the temperature at 25 ℃ for 5min for later use; then the mixed solution with good renaturation and the streptomycete are mixedAnd plain magnetic beads, before use in screening buffer washing at least five times. The DNA concentration after renaturation was used

Quantification was performed with a 2.0 fluorimeter and labeled C1. The renaturated mixture was mixed with magnetic beads and incubated for 1h on a four-dimensional rotating apparatus, after which all the magnetic beads were attracted with a magnet and the DNA concentration in the supernatant was determined and recorded as C2. The C2/C1 values can be used to determine the immobilization efficiency of ssDNA libraries. If C2/C1 is less than 0.5, insufficient library fixation is demonstrated.

(2) Screening: after the incubation is finished, adsorbing the magnetic beads by strong magnets, washing the magnetic beads by screening buffer solution, and removing unbound DNA sequences; and then incubating the magnetic beads and 100pmol of target toxin DA on a four-dimensional rotating instrument at room temperature, carrying out magnetic separation after incubation is finished to obtain ssDNA eluent, measuring the concentration of ssDNA in the eluent by using a fluorimeter, and calculating the retention rate.

(3) Amplification: the eluted ssDNA was used as a template, and a 50. mu.L PCR reaction was as follows:

amplification conditions: pre-denaturation at 95 ℃ for 5 min; at 95 ℃, denaturation is carried out for 30s, 60 ℃, annealing is carried out for 45s, extension is carried out for 72 ℃ for 30s, and 20 cycles are carried out; extension for 5min at 72 ℃.

(4) Single-stranded secondary library preparation: adding a sample buffer solution into the dsDNA of the PCR amplification product, fully and uniformly mixing, then performing renaturation treatment, namely performing thermal denaturation at 95 ℃ for 10min, performing ice bath quenching for 5min, and standing at room temperature for 5 min; adding the processed sample into a sample hole on polyacrylamide gel denatured by 12% urea; switching on an electrophoresis apparatus, and carrying out electrophoresis at a constant voltage of 300V; when the bromophenol blue migrates to the lower end 1/3 of the gel, the power supply is cut off; add 15ml ddH to clean dishes ₂ O and 5 mu L of DNA dye, fully and uniformly mixing, placing the gel in the gel, slightly shaking on a horizontal shaking table, and dyeing for 20 min; placing the polyacrylamide gel in a gel imaging system for imaging, and taking short-chain ssDNA as a target fragment for coagulationThe gel was purified by extraction and quantified as a secondary library for the next round.

(5) Repeating the above screening method, performing the next round of screening, and up to round 12, cloning and sequencing the enriched library, summarizing the obtained sequences in table 1, and analyzing the binding affinity constant Kd value of the sequences in table 1 and the target toxin DA by a biomembrane interference technology.

TABLE 1 aptamer sequences and their affinity constants K _d Value of

Example 3 truncation optimization of aptamer C1

In order to remove unnecessary nucleotides from the aptamer and obtain its core sequence to further study the mechanism of binding of the aptamer to DA, truncation optimization of the aptamer is required. The library used for screening the aptamer is in a closed-loop structure, self-complementation of a primer sequence is convenient for fully exposing a random sequence to be combined with a target, and because the primer does not participate in folding, the primer part of the aptamer C1 is directly removed to obtain C1-s, the affinity of the C1-s to DA is not reduced, and therefore the primer part is proved to be not a key sequence for combining the aptamer and the toxin. The secondary structure of C1-s was then analyzed by mfold web server software (FIG. 3), and the individual stem-loop structures were truncated to obtain the aptamer sequences shown in Table 2. The affinity results of the biomembrane interference technology show that the affinity of C1-d with the length of 19 nucleotides, which is obtained by truncation of the aptamer C1, with DA is remarkably improved, and the corresponding stem loop forms an essential structure for binding with the target DA.

TABLE 2 truncated sequence of aptamer C1 and its affinity constant K _d Value of

Example 4 determination of the interaction between aptamer and target by means of biomembrane interference technique

The biomembrane interference technology is a non-labeling technology based on the principle of light interference, namely a technology for detecting the surface reaction of the sensor by detecting the displacement change of an interference spectrum; when a visible light beam is emitted from the spectrometer, two reflection spectra are formed on two interfaces of the optical film layer at the tail end of the sensor, and an interference spectrum is formed. Any change in the thickness and density of the film layer due to molecular association or dissociation can be reflected by the shift value of the interference spectrum, and a real-time response monitoring map is made through the shift value.

The method is used for detecting the affinity and specificity of C1-d and DA, and comprises the following specific detection steps:

(1) preparation of aptamers: aptamer C1-d with biotin label is synthesized, and is prepared into 2 mu M solution by using screening buffer solution and is subjected to renaturation treatment (10 min at 95 ℃, 5min in ice bath and 5min at room temperature) for later use. Meanwhile, a random sequence with a biotin tag is prepared as a negative control.

(2) Preparation of target toxin: the affinity between DA and aptamer was determined by interaction with the aptamer at different concentrations (10. mu.M, 5. mu.M, 2.5. mu.M and 1.25. mu.M) and is shown in FIG. 4. In addition, in order to verify the specificity of the aptamer, DA analogue KA and non-specific toxins (GTX, STX, TTX, NOD-R, PLTX, DTX, OA) with the concentration of 5 μ M and a mixed solution of the toxins (the final concentration is 5 μ M) are prepared at the same time, and the interaction between the aptamer and the toxins is respectively detected, so that as shown in FIG. 5, C1-d can only be specifically bound with the DA, is a typical fast-binding slow-dissociation ligand of the DA and has great practical application value.

(3) A superstavidin (SSA) probe was used. Aptamer, DA solution and buffer solution with corresponding concentrations are respectively added into two 96 wells, and the concentration is 100-200 mu.L/well. The program settings were as follows: balancing the sensor (1 min); ② aptamer coupling (2 min); thirdly, rebalancing the sensor (1 min); fourthly, combining (3 min); dissociation (3 min). Importing the collected real-time combination and dissociation data into Octet data analysis softwareIn the method, a 1:1 binding mode is adopted for fitting analysis processing so as to obtain a fitting curve and an affinity constant K _d Values, etc.

The present invention is not limited to the embodiments described above, and those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are included in the scope defined by the claims of the present application.

Sequence listing

<110> China people liberation army navy military medical university

<120> aptamer specifically bound to domoic acid and application thereof

<130> specification of claims

<160> 17

<170> SIPOSequenceListing 1.0

<210> 1

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

attggcactc cacgcatagg ccaacatgat gttccgtcat tttgaggtgt gtacaccgtg 60

cctatgcgtg ctaccgtgaa 80

<210> 2

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

attggcactc cacgcatagg ggataacggg ttgatggtac ttctatctat cgcgttgtgc 60

cctatgcgtg ctaccgtgaa 80

<210> 3

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

attggcactc cacgcatagg gacatcgaga agaatcctga tacgacttgg ctttgctggc 60

cctatgcgtg ctaccgtgaa 80

<210> 4

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

attggcactc cacgcatagg gtaaagtacg tatgtcatgc acatgccgta ttctctttgc 60

cctatgcgtg ctaccgtgaa 80

<210> 5

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

attggcactc cacgcatagg ccaaggtgtc aatcttgaat agctgtgtaa cgtctatgtg 60

cctatgcgtg ctaccgtgaa 80

<210> 6

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

attggcactc cacgcatagg ggtggtcttc tgggtaatcc ccgactaccc ttatgccggc 60

cctatgcgtg ctaccgtgaa 80

<210> 7

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

attggcactc cacgcatagg ggaagctgct ctcatcaata aaaaatgaga cggaggttac 60

cctatgcgtg ctaccgtgaa 80

<210> 8

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

attggcactc cacgcatagg gaatggaccc ggtataattc cctcaagagt gccaatttca 60

cctatgcgtg ctaccgtgaa 80

<210> 9

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

attggcactc cacgcatagg gatctcataa ccagtctctt tgactgatgt tagtaaggtc 60

cctatgcgtg ctaccgtgaa 80

<210> 10

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ccaacatgat gttccgtcat tttgaggtgt gtacaccgtg 40

<210> 11

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

caacatgatg ttccgtcatt ttg 23

<210> 12

<211> 12

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ggtgtgtaca cc 12

<210> 13

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ccaacatgat gttccgtcat tttgagtg 28

<210> 14

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ccgaggtgtg tacaccgtg 19

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

attggcactc cacgcatagg 20

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ttcacggtag cacgcatagg 20

<210> 17

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

aaaaaaaaaa aaaaaaaaaa ttcacggtag cacgcatagg 40

Claims (10)

1. An aptamer specifically binding to domoic acid, wherein the sequence of the aptamer has the general formula: 5' -ATTGGCACTCCACGCATAGG-N ₄₀ -CCTATGCGTGCTACCGTGAA-3'; wherein N is any one of A, T, G, C four deoxyribonucleotide bases, and N is ₄₀ Representing a random sequence of length 40 nt.

2. The aptamer of claim 1 that specifically binds domoic acid, wherein:

wherein the sequence of the aptamer is shown in any one of SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 and SEQ ID NO. 8.

3. The aptamer specifically binding to domoic acid according to claim 1, wherein the sequence of the aptamer is as shown in any one of SEQ ID No.10, SEQ ID No.12 and SEQ ID No. 14.

4. Use of an aptamer specifically binding to domoic acid as claimed in any one of claims 1 to 3 in the preparation of a domoic acid separation and enrichment reagent, a purification reagent, an antagonist or a neutralizing agent.

5. Use of an aptamer specifically binding to domoic acid as claimed in any one of claims 1 to 3 in the preparation of a domoic acid detection reagent, kit or sensor.

6. The use of an aptamer specifically binding to domoic acid as claimed in any one of claims 1 to 3 for the preparation of a medicament for the treatment of domoic acid poisoning, wherein the aptamer comprises: the medicament for treating domoic acid poisoning is an aptamer specifically bound with domoic acid as the only active ingredient or a pharmaceutical composition containing the aptamer specifically bound with domoic acid.

7. Use of an aptamer specifically binding to domoic acid as claimed in any one of claims 1 to 3 in the preparation of a reagent for removing domoic acid from seafood or water.

8. The use of an aptamer specifically binding to domoic acid as claimed in any one of claims 1 to 3 in the rapid detection of domoic acid in an aquatic product or water.

9. A composition characterized by: the aptamer according to any one of claims 1 to 3 as an active ingredient, further comprising a pharmaceutically or detectably acceptable carrier.

10. A domoic acid detecting agent comprising the composition according to claim 9.

Images