Aptamer specifically binding to α-conotoxin MI and its application

Abstract

The present invention relates to the field of biomedical engineering technology, and provides an aptamer that specifically binds to α-conotoxin MI (CTX-MI) and its applications. The general formula of the aptamer sequence is: 5'-ATTGGCACTCCACGCATAGG-N 40 -CCTATGCGTGCTACCGTGAA-3'; wherein, N is any one of the four deoxyribonucleotides A, T, G, and C, 40 represents the number of random nucleotides, preferably a sequence shown in any one of SEQ ID NO.1 to SEQ ID NO.6, and most preferably a sequence shown in any one of SEQ ID NO.7 to SEQ ID NO.9. It can be prepared into a CTX-MI separation and enrichment reagent, a purification reagent, an antagonist or a neutralizing agent, and can also be prepared into a CTX-MI detection reagent, a detection kit or a detection sensor, and applied to the detection of CTX-MI in water samples. It can also lay a foundation for the preparation of drugs for preventing or treating CTX-MI poisoning and the removal of CTX-MI in water or aquatic products.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical engineering, and in particular to an aptamer specifically binding to alpha-conotoxin MI (CTX-MI) and an application thereof. Background Art

[0002] Conotoxins (CTXs) are a general term for polypeptide toxins secreted by the gastropod mollusk cone snail. They are secreted by the venom glands on the inner wall of the cone snail's venom tube and venom sac, and are stored in the venom sac in the form of a cocktail of various toxic peptides. They can be quickly injected into the body of the opponent during predation or defense, causing instant paralysis, convulsions, seizures and even death. In addition, CTXs also have multiple activities such as analgesia, anti-tumor, antiviral, antibacterial, and insecticide, and are one of the main sources of marine drug research and development. CTXs are of various types and are the smallest nucleic acid-encoded animal neurotoxin peptides discovered so far. They can act on a variety of voltage-gated ion channels and ligand-gated ion channels.

[0003] α-Conotoxin MI (CTX-MI) is the most widely distributed and most toxic type of conotoxin. It can specifically act on muscle nicotinic acetylcholine receptors (AChR) to block nerve signal transmission. Its LD 50 The concentration of CTX-MI in a cone snail can reach 15-20 μg / kg, and the CTX-MI in a cone snail is enough to kill an adult. Since its mechanism of action is not yet fully understood, and there is a lack of corresponding therapeutic drugs and anti-venom serum in clinical practice, this brings great difficulties to the safety protection of underwater workers and the treatment after poisoning. In addition, CTX-MI is highly toxic, has a small molecular weight and is easy to synthesize, which poses a risk of causing bioterrorism, so it needs to be quickly identified and sensitively detected.

[0004] However, its detection methods are relatively scarce at present, mainly relying on chromatographic and mass spectrometric techniques such as high performance liquid chromatography (HPLC), copper chelated nanomagnetic beads combined with biomass spectrometry (MALDI-TOF-MS). This type of method has high sensitivity and can accurately analyze the composition, structure and content of the target compound; however, the detection instrument is expensive, the sample processing process is cumbersome and time-consuming, the sample purity requirement is high, and the operation by professionals is required, which makes it difficult to be widely popularized. Immunoassays such as enzyme-linked immunosorbent assay (ELISA) are currently recognized as sensitive, accurate and fast detection methods, but because CTX-MI is a small molecule polypeptide, it is antigenic but not immunogenic, and the corresponding antibodies cannot be directly obtained from the immune animal. Therefore, there is currently a lack of CTX-MI immunoassay methods based on monoclonal antibodies. On the other hand, because no compounds that specifically recognize and bind to CTX-MI have been found, there is a lack of targeted drugs or leads for the treatment or prevention of poisoning. It can be seen that the lack of specific molecular recognition elements is one of the huge obstacles to the rapid detection and detoxification treatment of CTX-MI.

[0005] Nucleic acid aptamers are tertiary structures formed by curling and folding single-stranded DNA or RNA. The classic screening method is the systematic evolution of ligands by exponential enrichment (SELEX) technology. Nucleic acid aptamers can bind to a variety of target substances such as cells, proteins, nucleic acids, lipids, and small biological molecules with high affinity and strong specificity through intermolecular interactions such as hydrogen bonds and van der Waals forces. Because they have the excellent characteristics of antibodies, and have the advantages of high stability, easy synthesis and modification, convenient storage and transportation, low immunogenicity, and no need to use immune animals in the preparation process, they are called "chemical antibodies". As a new type of molecular recognition element, they have attracted much attention in the fields of diagnosis, detection, treatment, scientific research, etc.

[0006] However, there are currently no reports on molecular recognition probes that specifically bind to CTX-MI, high-affinity aptamers that specifically bind to CTX-MI, and related performance evaluations and applications. Summary of the invention

[0007] The present invention is conducted to solve the above technical problems, and aims to provide an aptamer that specifically binds to CTX-MI and its application.

[0008] The first object of the present invention is to provide a plurality of single-stranded DNA aptamers that can specifically bind to CTX-MI with high affinity, and to test the affinity between the plurality of aptamers and CTX-MI to obtain an affinity constant (K D) values ​​are smaller and the response values ​​are higher for six aptamers: MBMI-01, MBMI-02, MBMI-03, MBMI-39, MBMI-92, and MBMI-94.

[0009] The second object of the present invention is to optimize the above aptamers in a truncated manner to provide CTX-MI aptamers MBMI-1C, MBMI-2C, and MBMI-92C with equivalent or even better binding ability but shorter length. Among them, the affinity constant (K D ) was the lowest, at 0.87μM.

[0010] The third purpose of the present invention is to provide the application of aptamers, such as the application of aptamers in the preparation of CTX-MI separation and enrichment reagents, purification reagents, neutralizers or antagonists; the application of aptamers in the preparation of CTX-MI detection reagents, kits or sensors; the application of aptamers in the preparation of drugs for preventing or treating CTX-MI poisoning; and the application of aptamers in the rapid detection of CTX-MI in water bodies or aquatic products. At the same time, it lays a foundation for the preparation of drugs for preventing or treating CTX-MI poisoning, the removal of CTX-MI in water bodies or aquatic products, and the application in exploring the biological functions and mechanisms of action of CTX-MI.

[0011] The main technical scheme of the present invention is: the magnetic bead SELEX technology is combined with the high-throughput sequencing technology (HT-MB-SELEX method) to screen and obtain six high-affinity aptamers that specifically bind to CTX-MI. According to the prediction results of its secondary structure by the online tool the mfoldweb server, the six aptamers are truncated to obtain three truncated aptamers with equivalent binding ability. By combining with the biosensor platform, a CTX-MI aptamer sensor can be prepared and used for the rapid detection of CTX-MI. In addition, the CTX-MI aptamer can also lay the foundation for the preparation of drugs for preventing or treating CTX-MI poisoning, the removal of CTX-MI in water bodies or aquatic products, and the trace enrichment or purification of CTX-MI.

[0012] The first aspect of the present invention provides an aptamer that specifically binds to CTX-MI, the general sequence formula of which is: 5'-ATTGGCACTCCACGCATAGG- 40 -CCTATGCGTGCTACCGTGAA-3'; wherein N is any one of the four deoxyribonucleotides A, T, G, and C, and 40 represents the number of random nucleotides.

[0013] The following representative sequences were obtained by screening with magnetic beads-SELEX technology:

[0014] MBMI-01: as shown in SEQ ID NO: 1;

[0015] MBMI-02: as shown in SEQ ID NO: 2;

[0016] MBMI-03: as shown in SEQ ID NO: 3;

[0017] MBMI-39: as shown in SEQ ID NO:4;

[0018] MBMI-92: as shown in SEQ ID NO: 5;

[0019] MBMI-94: as shown in SEQ ID NO:6.

[0020] The second aspect of the present invention is to optimize the above aptamers in a truncated manner to provide three CTX-MI aptamers with equivalent binding ability but shorter length, which are named aptamer MBMI-01C, aptamer MBMI-02C and aptamer MBMI-92C, and their sequences are shown in SEQ ID NO. 7 to SEQ ID NO. 9. The three aptamers can bind to CTX-MI, and the affinity is equivalent to that before truncation.

[0021] Preferably, the aptamer can also be chemically modified, such as biotin, fluorescent molecules, isotopes, electrochemistry, enzymes or thiol chemical modifications, insertion, truncation, mutation, cyclization and other modifications can be performed on its 5' end or 3' end.

[0022] The third aspect of the present invention provides the application of aptamers: such as the application in the preparation of CTX-MI separation and enrichment reagents, purification reagents, neutralizers or antagonists; the application in the preparation of CTX-MI detection reagents, kits or sensors for rapid detection of CTX-MI in water bodies or aquatic products; the application in the preparation of drugs for preventing or treating CTX-MI poisoning.

[0023] Preferably, the drug for treating CTX-MI poisoning has an aptamer that specifically binds to CTX-MI as the sole active ingredient or is a pharmaceutical composition comprising an aptamer that specifically binds to CTX-MI.

[0024] In a fourth aspect, the present invention provides a pharmaceutical composition of an aptamer that specifically binds to CTX-MI, which contains the aptamer that specifically binds to CTX-MI as an active ingredient and further comprises a medically acceptable drug carrier.

[0025] The pharmaceutical composition can be used to neutralize or antagonize CTX-MI, and can also be used to alleviate or cure symptoms such as organ numbness, vomiting, dizziness, respiratory failure, convulsions, convulsions, amnesia, etc. caused by CTX-MI poisoning.

[0026] Preferably, the pharmaceutical composition is a preparation for removing CTX-MI from water or aquatic products. The preparation can completely remove CTX-MI from water or aquatic products, or reduce the content of CTX-MI in water or aquatic products to below the standards prescribed and recommended by the World Health Organization and the Food and Agriculture Organization of the United Nations.

[0027] The pharmaceutical composition of the present invention and pharmaceutically acceptable excipients together constitute a pharmaceutical preparation composition, thereby exerting therapeutic effects more stably. These preparations can ensure the conformational integrity of the aptamer core sequence disclosed in the present invention.

[0028] The fifth aspect of the present invention specifically provides the use of aptamers named MBMI-01C, MBMI-02C and MBMI-92C in the rapid detection of CTX-MI in tap water samples, so as to prevent people from being poisoned mainly by drinking contaminated water and accidentally eating contaminated aquatic products.

[0029] Beneficial protection and effects of the present invention:

[0030] In view of the good stability and water solubility of CTX-MI and the free amino group at its N-terminus, the present invention screens and obtains single-stranded DNA aptamers with high affinity and strong specific binding to CTX-MI based on the HT-MB-SELEX method. As molecular recognition elements that specifically bind to CTX-MI, these aptamers have the advantages of high affinity, strong specificity, good stability, low immunogenicity, and easy preparation and modification. They can be used for the separation and enrichment of trace CTX-MI in samples, qualitative and quantitative analysis of CTX-MI, research and development of drugs for relieving or treating CTX-MI poisoning, removal of CTX-MI in water bodies and aquatic products, and exploration of the biological functions and mechanisms of action of CTX-MI.

[0031] Through experimental verification, the aptamer of the present invention can quickly and specifically bind to CTX-MI, wherein the affinity between the aptamer MBMI-01C and CTX-MI is extremely high and the length is short. Therefore, the aptamer screened by the present invention can be prepared into an aptamer sensor or detection reagent, and applied to the detection of CTX-MI in drinking water samples. In addition, these aptamers can also lay the foundation for the preparation of drugs for preventing or treating CTX-MI poisoning and the removal of CTX-MI in water bodies or aquatic products.

[0032] In addition, according to the characteristics of CTX-MI molecules, the present invention designs magnetic beads-SELEX, and obtains single-stranded DNA aptamers that can specifically bind to CTX-MI with high affinity through forward screening and reverse screening, which has the characteristics of simple operation and high repeatability, greatly simplifies the construction technology route, low production cost and short purification cycle. As a new type of molecular recognition probe, aptamers have the advantages of low cost, stable properties and convenient modification, and are suitable for large-scale application in industrial production of biopharmaceuticals. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a schematic diagram of aptamer screening based on magnetic beads;

[0034] Figure 2 It is a summary analysis of the recovery rate of aptamer binding to the target molecule CTX-MI in each round of screening;

[0035] Figure 3 It is the analysis of high-throughput sequencing results;

[0036] Figure 4 It is the secondary structure diagram predicted by Mfold software for the initial sequence;

[0037] Figure 5 It is the secondary structure diagram predicted by Mfold software after the sequence of the truncated primer;

[0038] Figure 6 The affinity (A) and specificity (B) identification diagrams of the aptamer MBMI-01C. DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0040] The experimental methods in the following examples, for which specific conditions are not specified, are generally carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for exemplary purposes only.

[0041] Example 1. Toxin Immobilization

[0042] The free amino group at the N-terminus of CTX-MI is coupled with the carboxyl modification on the surface of the magnetic beads. Taking the fixation of 100 μL of magnetic beads as an example, the specific process is as follows:

[0043] (1) Prepare a 25 mM MES solution and adjust the pH to 5.0; use the MES solution to prepare 50 mg / mL EDC and NHS solutions respectively and store them on ice; prepare 50 mM Tris and adjust the pH to 8.0.

[0044] (2) Take 100 μL of magnetic beads, fully adsorb them on a magnetic rack to remove the supernatant, then wash them three times with an equal volume of MES solution and remove the supernatant.

[0045] (3) Add 50 μL each of EDC and NHS solution, mix thoroughly, and incubate at room temperature with slow rotation for 30 min to activate the magnetic beads.

[0046] (4) The supernatant was removed by fully adsorbing the sample on a magnetic rack, and the sample was washed three times with an equal volume of MES solution, and the supernatant was removed.

[0047] (5) Dilute the toxin to 20 μM using MES solution, add 100 μL to the magnetic beads, mix thoroughly, and incubate at room temperature with slow rotation for 1 h to fix the toxin on the surface of the magnetic beads.

[0048] (6) The supernatant was removed by full adsorption on the magnetic rack, and the cells were washed three times with an equal volume of MES solution, and the supernatant was removed.

[0049] (7) Add 100 μL of Tris solution, mix thoroughly, and incubate at room temperature with slow rotation for 15 min. Repeat this step three times to block the unbound free carboxyl groups on the surface of the magnetic beads.

[0050] (8) The supernatant was fully adsorbed by the magnetic stand, and the beads were washed three times with equal volumes of MES solution and screening buffer, respectively, and resuspended with equal volumes of screening buffer, and stored at 4° C. In this example, CTX-MI and α-conotoxin GI (CTX-GI) were immobilized for the forward screening and reverse screening in Example 3, respectively. In addition, blank magnetic beads without any target immobilized were prepared according to the above steps for the reverse screening in Example 3.

[0051] Example 2. Construction of random ssDNA library and primers thereof

[0052] 1. Construction of a random ssDNA library with a length of 80 nt

[0053] 5'-ATTGGCACTCCACGCATAGG-N 40 -CCTATGCGTGCTACCGTGAA-3′;

[0054] Wherein, N is any one of the four deoxyribonucleotides A, T, G, and C, and 40 represents the number of random nucleotides.

[0055] 2. Primer construction

[0056] Primer construction was performed according to Table 1:

[0057] Table 1 Summary of primer design

[0058]

[0059] Example 3. Screening of CTX-MI nucleic acid aptamers (HT-MB-SELEX method)

[0060] In order to obtain CTX-MI high affinity and high specificity binding aptamers, we screened CTX-MI aptamers based on magnetic bead SELEX technology. The process is as follows: Figure 1 As shown. In order to improve the screening efficiency, the pressure of positive screening can be increased by reducing the amount of magnetic beads, shortening the incubation time, and extending the rinsing time as the number of rounds increases. In order to improve the specificity of the screening, negative magnetic beads were introduced for reverse screening starting from the 7th round, where empty magnetic beads were used for rounds 7-9 and magnetic beads fixed with CTX-GI were used for rounds 10-12. In each round of screening, the recovery rate of the aptamer binding to the target toxin CTX-MI and the recovery rate of the reverse screening are shown in Figure 2 As shown, after the 12th round of screening, the recovery rate of ssDNA entered a plateau, so the screening was stopped. The specific steps are as follows:

[0061] (1) Take 1.3 nmol of the initial ssDNA library, add 200 μL of screening buffer (0.9 mM CaCl2, 2.7 mM KCl, 1.5 mM KH2PO4, 0.6 mM MgCl2·6H2O, 0.1 M NaCl and 20 mM Na2HPO4, pH = 7.4), perform denaturation treatment (i.e., 95°C water bath for 10 min, ice bath quench for 5 min, and room temperature for 5 min), add 200 μL of CTX-MI magnetic beads, and incubate at room temperature with slow rotation for 2 h. After incubation, rinse with screening buffer 6 times, including the fifth slow rotation rinse for 15 min to remove non-specifically bound ssDNA. Collect the magnetic beads bound to ssDNA, add 200 μL of screening buffer, and bathe at 95°C for 20 min to recover the DNA sequence that specifically binds to CTX-MI.

[0062] (2) The eluted ssDNA was used as a template, and the 50 μL PCR reaction system was as follows:

[0063]

[0064] Amplification conditions: 94°C for 10 min; denaturation at 94°C for 30 s; annealing at 60°C for 30 s; denaturation at 72°C for 30 s; 20 cycles; 72°C for 10 min.

[0065] (3) Preparation of single-stranded secondary library: Add DNA urea denaturation loading buffer to the PCR amplified library, mix well and perform denaturation treatment. Spot the treated sample into the loading well of 12% urea denaturing polyacrylamide gel and electrophoresed at a constant voltage of 200V for 30 minutes. After staining, cut off the shorter single-stranded part at the bottom and use a gel recovery kit to recover the ssDNA.

[0066] (4) Repeat the next round of screening according to the above screening method, the input amount of the secondary library is uniformly set to 100 pmol, and the screening pressure is increased as appropriate. The screening is stopped after the 12th round. The recovery rate of each round is as follows Figure 2 As shown. The secondary libraries of rounds 2, 4, 6, 9, and 12 were cloned and purified using different upstream primers (upstream primers 2-6) and downstream primer 2, respectively, and mixed in equal proportions for high-throughput sequencing. Data analysis is shown in Figure 3 As shown, as the number of screening rounds increases, the richness of the sequences decreases ( Figure 3 A), the enrichment of ssDNA with high affinity increases ( Figure 3 BC), proving that the method of the present invention can effectively enrich the high-affinity aptamer of CTX-MI in the random library. Sequences with an enrichment degree greater than 1‰ in the 12th round of sequencing results were extracted, totaling 98, and homology clustering and phylogenetic tree analysis were performed on them, dividing them into four families I-IV, and selecting the one with the highest enrichment degree from several sequences with close genetic relationships, and a total of 20 candidate aptamers were obtained.

[0067] Example 4. Determination of molecular interactions using biomembrane interferometry

[0068] Biofilm interferometry is a label-free molecular interaction analysis technology. Its principle is that the instrument emits white light to the sensor surface and collects the reflected light. The reflection spectra of different frequencies are affected by the thickness of the optical film layer of the biosensor. The reflection spectra of some frequencies are affected by the thickness of the optical film layer of the biosensor. The reflected light of some frequencies forms constructive interference, while others form destructive interference. These interference lights are detected by the spectrometer to form an interference spectrum, which is displayed as the phase shift intensity (nm) of the interference spectrum. Therefore, once there is a change in the number of molecules bound to the sensor surface, the spectrometer will detect the displacement of the interference spectrum in real time, and this displacement directly reflects the thickness of the biofilm on the sensor surface.

[0069] The above method is used to optimize the aptamer, and the specific detection steps are as follows:

[0070] (1) The aptamer labeled with 5' biotin was dissolved in screening buffer and diluted to 2 μM. All aptamers were subjected to denaturation treatment before use to promote their refolding to form the optimal spatial structure.

[0071] (2) 20 aptamer suspensions selected in Example 3, target toxin molecules diluted with screening buffer, and 200 μL of buffer were added to the corresponding reaction wells, and the streptavidin chip (SSA) was immersed in each reaction well in turn according to the set program (equilibration for 2 min, aptamer fixation for 3 min, re-equilibration for 2 min, binding for 2 min, and dissociation for 3 min).

[0072] (3) Each chip with an aptamer fixed was subjected to binding and dissociation with different concentrations of the target toxin molecule CTX-MI (1.25, 2.5, 5, 10, 20 μM) or non-specific target toxin molecules domoic acid (DA), okadaic acid (OA), saxitoxin (STX), tetrodotoxin (TTX) and CTX-GI (10 μM). The results showed that each aptamer could only specifically bind to CTX-MI and could not specifically bind to other substances.

[0073] (4) The 20 candidate aptamers were interacted with 10 μM CTX-MI to obtain the response value (Response) and affinity constant (K D ), get K D The value is not higher than 10 -6 The aptamers of M are MBMI-01, MBMI-02, MBMI-03, MBMI-39, MBMI-92, and MBMI-94, and their sequence information and affinity constants (K D ) values ​​are shown in Table 2, and the secondary structure information is shown in Figure 4 A~ Figure 4 As shown in F, MBMI-01 has the highest affinity, with a binding K D The value is 0.85μM and the response value is 0.2778nm.

[0074] (5) Select one of the primers with higher affinity and response value in each family, namely MBMI-01, MBMI-02 and MBMI-92, and cut off the primers at both ends (20 nt each). The secondary structures after truncation are as follows: Figure 5 A~ Figure 5 C, and the affinity and response values ​​are shown in Table 2. The results show that the response value increased significantly after truncation, while K DThe value decreased significantly, indicating that the removal of the primers did not affect the interaction between the two and could significantly increase the affinity, among which MBMI-01C had the highest affinity. Subsequently, five concentration gradients of CTX-MI were used to fit the interaction between MBMI-01C and CTX-MI to obtain a more accurate affinity constant. The binding and dissociation curves are shown in Figure 6 As shown in A, the combined K D The value was 0.52μM. At the same time, MBMI-01C did not bind to other nonspecific target toxins or the response value and K D The values ​​were significantly different in magnitude compared with those of CTX-MI ( Figure 6 B). It can be seen that the aptamer MBMI-01C can bind to CTX-MI with high affinity and strong specificity. The buffer used in the biomembrane interference technique is a screening buffer. Each experiment is equipped with a control chip that interacts with the buffer to eliminate the influence of the buffer composition on the response value.

[0075] Table 2. Sequence information and affinity test values ​​of each aptamer

[0076]

[0077]

[0078] The examples of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the creative spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

[0079]

[0080]

[0081] Sequence Listing <110> Chinese People's Liberation Army Naval Medical University <120> Aptamer specifically binding to α-conotoxin MI and its application <130> Claim description <160> 17 <170> SIPOSequenceListing 1.0 <210> 1 <211> 80 <212> DNA <213> Artificial Sequence <400> 1 attggcactc cacgcatagg tttggggatg ggcaacggta aaaagggtca aaaggctttt 60 cctatgcgtg ctaccgtgaa 80 <210> 2 <211> 80 <212> DNA <213> Artificial Sequence <400> 2 attggcactc cacgcatagg atttgggttt cgggataagg ggcaaaagga caggttttgt 60 cctatgcgtg ctaccgtgaa 80 <210> 3 <211> 80 <212> DNA <213> Artificial Sequence <400> 3 attggcactc cacgcatagg ggcgcttgtg gtgtaagggt tgggtatatg tcgtaatagg 60 cctatgcgtg ctaccgtgaa 80 <210> 4 <211> 80 <212> DNA <213> Artificial Sequence <400> 4 attggcactc cacgcatagg gtaggggatt taaggagggg ccttaggagg gggggggtaa 60 cctatgcgtg ctaccgtgaa 80 <210> 5 <211> 80 <212> DNA <213> Artificial Sequence <400> 5 attggcactc cacgcatagg ctcgcggagg gaggggtttt ggggggcggg atggtattgg 60 cctatgcgtg ctaccgtgaa 80 <210> 6 <211> 80 <212> DNA <213> Artificial Sequence <400> 6 attggcactc cacgcatagg agcggggttg gtttgcggga gggggggtgg aggtaagctc 60 cctatgcgtg ctaccgtgaa 80 <210> 7 <211> 40 <212> DNA <213> Artificial Sequence <400> 7 tttggggatg ggcaacggta aaaagggtca aaaggctttt 40 <210> 8 <211> 40 <212> DNA <213> Artificial Sequence <400> 8 atttgggttt cgggataagg ggcaaaagga caggttttgt 40 <210> 9 <211> 40 <212> DNA <213> Artificial Sequence <400> 9 ctcgcggagg gaggggtttt ggggggcggg atggtattgg 40 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <400> 10 attggcactc cacgcatagg 20 <210> 11 <211> 26 <212> DNA <213> Artificial Sequence <400> 11 aaagcaattg gcactccacg catagg 26 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <400> 12 aacgccattg gcactccacg catagg 26 <210> 13 <211> 26 <212> DNA <213> Artificial Sequence <400> 13 aaggcgattg gcactccacg catagg 26 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <400> 14 acaggaattg gcactccacg catagg 26 <210> 15 <211> 26 <212> DNA <213> Artificial Sequence <400> 15 accggcattg gcactccacg catagg 26 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <400> 16 ttcacggtag cacgcatagg 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <400> 17 ttcacggtag cacgcatagg 20

Claims

1. An aptamer that specifically binds to CTX-MI, characterized in that: The sequence of the aptamer is shown in any one of SEQ ID NO.1 to SEQ ID NO.

9.

2. The aptamer that specifically binds to CTX-MI according to claim 1, characterized in that: in, The 5' end or 3' end of the aptamer is modified with biotin, fluorescent molecules, isotopes, electrochemistry, enzymes or thiol chemistry.

3. Use of the aptamer that specifically binds to CTX-MI according to claim 1 in the preparation of a CTX-MI separation and enrichment reagent, antagonist or neutralizer.

4. Use of the aptamer that specifically binds to CTX-MI according to claim 1 in the preparation of a CTX-MI detection reagent, a detection kit or a detection sensor.

5. Use of the aptamer that specifically binds to CTX-MI according to claim 1 in the preparation of an agent for removing CTX-MI from aquatic products or water bodies.

6. Use of the aptamer that specifically binds to CTX-MI according to claim 1 in rapid detection of CTX-MI in water.

7. A composition, characterized in that: The aptamer according to claim 1 is used as an active ingredient, and also includes a pharmaceutically or diagnostically acceptable carrier.

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