KR101086026B1 - DNA aptamers binding to visfatin with specificity and production method thereof - Google Patents

Abstract

The present invention relates to a DNA aptamer that specifically binds to bispartin, and more particularly, to a DNA aptamer that specifically binds to visfatin selected from a random DNA library and a method of preparing the same. . According to the present invention, a DNA aptamer showing a high affinity for bispartin, a biomarker of type 2 diabetes, from a random DNA pool was selected using the FluMag-SELEX process, a modified method of the SELEX process. The sequence, properties, and binding capacity of the selected DNA aptamers were analyzed. Therefore, the type 2 diabetes can be diagnosed more effectively by using the DNA aptamer of the present invention.

Bispartin, DNA Aptamer, Type 2 Diabetes, Biomarker, Affinity, SELEX

Description

DNA aptamer specifically binding to bispartin and its preparation method {DNA aptamers binding to visfatin with specificity and production method

The present invention relates to a DNA aptamer that specifically binds to bispartin, and more particularly, to a DNA aptamer that specifically binds to visfatin selected from a random DNA library and a method of preparing the same. .

Adipokine is a protein secreted from adipose tissue and plays an important role in metabolism. Visfatin is a newly discovered visceral fat-derived protein that has been reported to be closely related to type 2 diabetes caused by obesity. Specifically, it was reported that the amount of bispartin expression in visceral fat was increased in obesity, and the blood concentration of bispartin was more closely related to the amount of visceral fat than subcutaneous fat. In addition, it was confirmed that the concentration of bispartin in the blood increased as obesity was expressed, and clinical results showed that the general level of blood bispatin (31.9 ± 31.7 ng / ml) in type 2 diabetic patients was 15.8 ± 16.7 ng / ml) (Miao-Pei Chen, et al., The Journal of Clinical Endocrinology & Metabolism 91 (1): 295-299). High levels of bispartin were expressed and blood levels were also higher than those of rats fed normal diet (Atsunori Fukuhara, et al., Science (2005), 307, 4426-4430). It is shown that the increase is closely related to type 2 diabetes caused by obesity, which also means that bispartin can be used as a biomarker for type 2 diabetes. Therefore, type 2 diabetes can be diagnosed quickly and accurately by measuring blood levels of bispartin derived from adipocytes.

Aptamers are structures of single-stranded DNA or RNA that have high specificity and affinity for specific targets. Aptamers are superior in cost to detection materials used in the field of sensors because of their high affinity for targets, good thermal stability, and in vitro synthesis. In addition, there are no restrictions on the target material, so aptamers can be synthesized for various targets such as proteins, biomolecules such as amino acids, small organic chemicals such as environmental hormones or antibiotics, and bacteria. Due to the properties of aptamers that specifically bind to target materials, a lot of research has recently been conducted to use aptamers in new drug development, drug delivery systems, and biosensors.

Among the various sensing materials used in the existing biosensor field, the superiority in sensitivity is the detection of the target material using the antibody. The first problem is that the injection of the target substance (antigen) to be detected into the body of the animal to secure the antibody made through the immune system of the living body and then undergo a purification process is time-consuming and expensive. In addition, the target material that can be used as an antigen is limited compared to the aptamer which has little restrictions on the target material, which makes it difficult to produce antibodies against low molecular weight chemicals such as toxic substances. In general, since antibodies are very large proteins having a size of 100K Da or more, there are limitations in signal detection when applied to biosensors such as electrochemicals, and there is a problem that thermal stability is significantly lower than that of DNA or other chemicals. . Therefore, the existing technology using antibodies in the diagnosis of blood biomarkers is not cost and time efficient, the range of application is limited, there are limited problems in the application as a biosensor. As a new sensing material to improve these problems, a lot of research has been made using aptamers, nucleic acid constructs that show high affinity specifically for various target materials.

Accordingly, the present inventors have developed a DNA aptamer which is a nucleic acid construct showing a particularly high affinity for bispartin, a biomarker of type 2 diabetes, as a result of intensive research to overcome the problems of the prior arts. In addition, it was determined that type 2 diabetes could be effectively diagnosed using a composition comprising a DNA aptamer specifically bound to bispartin, thereby completing the present invention.

Accordingly, a main object of the present invention is to provide a DNA aptamer specifically binding to bispartin, a biomarker of type 2 diabetes, and a method of manufacturing the same.

Another object of the present invention is to provide a composition for diagnosing type 2 diabetes using DNA aptamer specifically binding to bispartin.

According to one aspect of the present invention, the present invention provides a DNA aptamer capable of specifically binding to visfatin. In the present invention, the DNA aptamer specifically binding to the target material was selected using the SELEX process for the DNA aptamer specifically binding to the bispartin.

The term 'SELEX process' used in the present invention is a method of identifying a DNA binding sequence of a molecule by selecting and amplifying a DNA or RNA having a high binding capacity to a specific molecule in a collection of randomly synthesized DNA or RNA (Louis C Bock, et al., 1992. Nature 355, 564-566.

'FluMag-SELEX process' used in the present invention is a fluorescent band in the PCR product separation process (double-stranded DNA to single-stranded DNA) using a polyacrylamide gel followed by using a fluorescently labeled primer in the DNA amplification step It is a modified method to take advantage of the drought, and it is a SELEX method using magnets to distinguish DNA that binds to or does not bind to a target. In the case of other SELEX, radioactive label, capillary electrophoresis, membrane filter, etc. were used, which is expensive and difficult to handle. In order to separate PCR products, chromatography and affinity columns are used, but they are expensive and have low efficiency (R. Stoltenburg, et al., (2005) Anal Bioanal Chem 383: 83-91).

In the DNA aptamer of the present invention, the DNA aptamer may be any DNA sequence aptamer specifically binding to bispartin, but preferably in SEQ ID NO: 1 to SEQ ID NO: 36 selected by the FluMag-SELEX method It can have any one base sequence.

According to another aspect of the present invention, the present invention provides a method for preparing a DNA aptamer specifically binding to bispartin (visfatin) comprising the following steps:

a) reacting bispartin and magnetic beads in a borate buffer solution to induce covalent bonds;

b) inducing binding at room temperature by mixing a bispartin-magnetic bead obtained by the covalent bond with a DNA pool having a primer base for PCR at both ends and having 30-50 random bases in the center;

c) separating the DNA bound to the bispartin-magnetic beads using a magnet;

d) separating DNA from the separated bispartin-magnetic beads; And

e) amplifying the DNA specifically binding to bispartin by performing PCR using a primer pair complementary to the PCR primer region.

In the method of the present invention, the covalent bond of step a) is characterized in that the covalent bond of the amino group of the bispartin (amino group) and the magnetic bead tosyl group (tosyl group).

In the method of the present invention, after step a), the tosyl group of the magnetic beads that are not bound to the bispartin in the covalently bound bispartin-magnetic beads is 3-A at 35-40 ° C. with a BSA (Bovine Serum Albumin) solution. Preferably, the method further comprises inactivating for 5 hours. After step a), if the tosyl group remains active, non-specific binding due to various interactions between molecules, such as van der Waals bonds, hydrophobic bonds, etc., may occur. Block the actual machine.

In the method of the present invention, the DNA separation in step d) is characterized by separating the DNA from the bispartin-magnetic beads by placing for 3-10 minutes at 70-90 ℃.

In the method of the present invention, after performing PCR using a primer to which fluorescein is attached to one of the primer pairs in step e), single-stranded DNA denatured by the fluorescein is subjected to electrophoresis. It is preferred to further comprise the step of separating.

The polyacrylamide gel used in the electrophoresis includes urea and formamide, so that two bands are formed after the electrophoresis. In the electrophoresis, two strands of DNA are denatured to attach fluorescein. DNA strands are placed above and non-attached DNA strands are placed below. Therefore, the DNA band to which the fluorescein is attached is cut and subjected to gel extraction, and then separated by ethanol precipitation to obtain denatured single-stranded DNA.

According to another aspect of the present invention, the present invention provides a composition for diagnosing type 2 diabetes, comprising a DNA aptamer specifically bindable to visfatin. The DNA aptamer may preferably have a nucleotide sequence of any one of SEQ ID NOs: 1 to 36.

In the composition of the present invention, the type 2 diabetes is characterized by diagnosing by measuring the level of bispartin in the blood binding to the DNA aptamer. Accurately measuring blood levels of bispartin, an important biomarker of type 2 diabetes, is very important in diagnosing type 2 diabetes. Therefore, using the composition comprising the DNA aptamer of the present invention, which is nonspecific among various biological materials in blood samples and can specifically bind to a small amount of bispartin, it can be used to diagnose type 2 diabetes more quickly and accurately. Can be. For example, the DNA aptamer of the present invention is spotted on a chip with a fluorescent material, a chromophore or an antibody, and then reacted with a blood sample to rapidly measure the level of trace bispartin in blood. Can be. In another method, when the DNA aptamer is immobilized on magnetic beads to bind bispartin, the bound DNA aptamer-bispartin complex can be separated using a magnet. By isolating only bispartin can be selectively detected. In addition to the method described in the present invention, a method for detecting bispartin in blood using a sensor connected with a DNA aptamer and a connector of the present invention can be used.

Hereinafter, a method of preparing and characterizing a DNA aptamer specifically binding to bispartin of the present invention will be described in more detail step by step.

1) Fixing Visfatin on Magnetic Beads

The amino group of bispartin and the tosyl group on the surface of the magnetic bead form a covalent bond. For this, the bispartin and the magnetic bead are reacted in a buffer solution. After the reaction, the magnetic beads bound to bispartin can be separated using a magnet. The beads are washed with the same buffer solution to remove unbound or weakly bound bispartin.

2) Development of Bispatin-binding Specific DNA Aptamers

Generally, SELEX (Systematic Evolution of Ligand by Exponential Enrichment) is widely used as a method for developing target-specific aptamers. In the present invention, a bispartin binding specific DNA aptamer was developed using a FluMag-SELEX process modified from a general SELEX. To this end, we first synthesized a 76 mer DNA pool with 40 random bases in the middle of the primer binding site for PCR amplification at both ends, and the DNA pool was added to the buffer solution with magnetic beads fixed with bispartin. After mixing, reacting, and using a magnet to remove unbound DNA. After high temperature treatment, the DNA bound to bispartin is separated from the magnetic beads, and the eluted DNA is obtained by ethanol precipitation.

PCR was performed to amplify the amount of bispartin binding specific DNA, and the PCR reaction was purified, followed by electrophoresis on a polyacrylamide gel containing a high concentration of urea. Isolate into strand DNA. After gel extraction of the original single-stranded DNA band, the separated DNA is obtained by ethanol precipitation. At this time, the obtained DNA pool is mixed with the first bispatin-fixed magnetic bead solution and reacted with bispartin. By repeating this process, more than 90% of the DNA pool is bound to bispatin, and then the DNA pool is cloned using the TOPO cloning kit, and DNA is extracted from the colonies.

3) DNA aptamer analysis that specifically binds to bispartin

Surface magnetic resonance devices (SPR) were used to compare the affinity of aptamers specifically binding to bispartin and the magnitude of binding force depending on the concentration of bispartin. For this purpose, the surface of the bare gold chip is modified and each DNA aptamer is immobilized on the chip. Then, bispartin is reacted by concentration on the chip, the binding force is confirmed, and the other edipocaine is analyzed for specificity. The binding to (bispatin, adiponectin) and human serum albumin (HSA) is also analyzed.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example 1 Fixation of Bispartin in Magnetic Beads

Bispatin (adipogen Inc.) and magnetic beads (M-280 Tosylactivated magnetic beads, Dynal Biotech ASA, Norway) were added to a buffer solution (0.1 M borate buffer, pH 9.5) and reacted at room temperature for 48 hours. Magnetic beads bound to bispartin can be separated using a magnet, and the unbound bispartin was removed by washing the beads with the same buffer solution. Bispartin-coupled magnetic beads (hereinafter referred to as 'bispartin-magnetic beads') are not reacted with bispartin by washing at 37 ° C for 4 hours with 0.1% BSA (Bovine Serum Albumin, Sigma Co.) solution after washing. Not tosyl groups were inactivated. After washing several times, the magnetic beads were mixed with the buffer solution (0.1 M PBS, pH 7.4) and stored at 4 ° C.

Example 2 DNA Pool Synthesis with Arbitrary Sequences

As a 76mer DNA pool, a primer region for PCR at both ends and a DNA pool having 40 random bases in the center were synthesized as follows. The DNA pool used in the present invention was commissioned chemically from Genotech Inc. (Korea).

ATACCAGCTTATTCAATT-N40-AGATAGTAAGTGCAATCT

Example 3 Screening of DNA Aptamers Bound to Bispartin

The random DNA pool synthesized in Example 2 was mixed with bispartin-fixed magnetic beads (bispartin-magnetic beads) (20 mM Tris-Cl buffer, pH 7.6 contained 100 mM NaCl, 2 mM MgCl _2). , 5 mM KCl, 1 mM CaCl ₂ and 0.02% Tween 20), mixed, and reacted at room temperature for 30 minutes. The tube containing the mixed solution was fixed to a magnet to remove DNA not bound to bispatin. DNA that did not bind to bispartin was removed and washed five times with buffer solution to remove weak binding DNA. In order to elute the DNA bound to bispartin, the tube was placed at 80 ° C. for 5 minutes to separate the DNA from the magnetic beads, and the eluted DNA was obtained by ethanol precipitation. The amount of DNA bound to bispartin thus obtained was measured. In FIG. 2, the amount of DNA that eluted ssDNA binds to bispartin obtained in each selection round is shown.

Example 4 Preparation of DNA Aptamer Pool Binding to Bispartin

PCR was performed using known primer regions to increase the amount of bispartin binding specific DNA. PCR products are double-stranded DNA, so in order to separate them into single-stranded, fluorescein (FP) was fixed to one primer as follows.

forward FP-fluorescein-ATACCAGCTTATTCAATT

reverse RP-AGATTGCACTTACTATCT

The PCR reaction was purified using a purification kit and then polyacrylamide gel electrophoresis was performed to make double-stranded DNA into single strands. The 10% polyacrylamide gel contains 6 M of urea and 20% formamide, resulting in two bands after electrophoresis, in which two strands of DNA are denatured during electrophoresis. The attached DNA strands are on top and the unattached DNA strands are on bottom.

DNA bands with fluorescein were cut out and subjected to gel extraction to obtain DNA separated by ethanol precipitation. At this time, the secured DNA pool was reacted with bispartin by mixing with the first bead-fixed magnetic bead solution. A schematic diagram of this process is shown in FIG. 1, and a series of processes (FluMag-SELEX process) is one selection, and a total of seven selection processes result in a DNA pool in which 90% or more finally binds to bispartin. Got. After the 3rd and 7th screening, HSA (human serum albumin, Sigma Co.), BSA (Bovine Serum Albumin, Sigma Co.) and other edupokines, adiponectin (adipogen Inc.) and bispartin (Retinol, respectively) Counter selection with Binding Protein 4, Adipogen Inc.) blocked DNA that binds nonspecifically and secured a DNA pool that specifically binds to bispatin with high affinity. In FIG. 2 the percentage of ssDNA eluted from bispartin-fixed magnetic beads in each selection is shown.

Finally, the DNA pool was cloned using a TOPO cloning kit (Invitrogen), and DNA was extracted from the colonies, and then subjected to basic analysis. As a result, 36 different nucleic acid constructs specifically binding to bispartin (DNA apps Tamers).

Example 5. Characterization of DNA Aptamers Specific to Bispatin

Table 1 shows the results of analyzing the base sequences of 36 different DNAs that specifically bind to bispatin with high affinity. 3 to 38 show the results of predicting the secondary structure of the 36 bispartin aptamers using the m-fold program.

TABLE 1-1. 36 different DNA aptamer sequences that specifically bind to bispatin with high affinity

TABLE 1-2.

Experiments were conducted to show that 36 aptamers that bind strongly to bispartin bind specifically to bispartin but not to other blood proteins. Since bispartin is one of the edipocaines, the best control to show that bispartin aptamer specifically binds only to bispartin was another adipokine, adiponectin, bispartin, and HSA. .

For this purpose, SPR (Surface Plasmon Resonance) device was used. First, a carboxyl group (COOH group) is formed on the surface of a bare gold chip with 50 mM 3,3'-dithiodipropionic acid, and then EDC / NHS (N-ethyl-N '-(dimethylaminopropyl) carbodiimide; EDC / Sigma Co And N-hydroxysuccinimid; NHS / Fluka) to form Self Assenmbly Monolayer. Streptavidin was immobilized thereon and each aptamer with biotin was coated with 1 μM. 1 μM of bispartin, adiponectin, bispartin, HSA was added to the buffer solution (20 mM Tris-Cl buffer, pH 7.6 contained 100 mM NaCl, 2 mM MgCl ₂ , 5 mM KCl, 1 mM CaCl ₂ and 0.02%). After reaction for 30 minutes in Tween 20), the unbound protein was washed out with the same buffer solution. Finally, SPR results can be analyzed to confirm that bispartin aptamer binds to bispatin better than other proteins. As a result of this, the No. The binding force test results of 19 aptamers are shown in FIG. 39.

Example 6 Analysis of Specificity and Avidity of DNA Aptamers Specific to Bispatin

Among 36 different aptamers that specifically bind to bispartin, binding assays with bispartin were performed on No. 19 aptamers having the highest specificity and avidity. In the same manner as in Example 5, the aptamer was immobilized at 100 nM on the gold chip, and then bispartin at different concentrations from 25 nM to 400 nM was added to the buffer solution (20 mM Tris-Cl buffer, pH 7.6 contained 100 mM). NaCl, 2 mM MgCl ₂ , 5 mM KCl, 1 mM CaCl ₂ and 0.02% Tween 20) for 30 minutes. To obtain the dissociation constants, each reaction degree was plotted using Sigmaplot 10.0 for nonlinear regression and single site saturation ligand binding. Maximum binding position, Kd is dissociation constant, X is unbound bispartin).

As a result, it was confirmed that the Kd value of No. 19 aptamer was very strongly bound to bispatin as 123.5 ± 28.9 nM, and analytical data thereof is shown in FIGS. 40A to 40C.

As described above, according to the present invention, the use of a DNA aptamer that specifically binds to bispartin is expected to measure the concentration of bispartin in blood more sensitively and accurately than the antibody-based assays used in the prior art. In addition, the DNA aptamer has a lower production cost than the antibody and is easy to be immobilized on the surface, which is advantageous for producing a biosensor chip. Therefore, the development of a biosensor capable of sensitively and accurately measuring the level of bispartin in the blood using DNA aptamers that can specifically bind to bispartin may help increase the accuracy of diagnosis of type 2 diabetes. It is expected.

1 is a schematic diagram of a FluMag-SELEX process for the development of a nucleic acid construct aptamer specifically binding to bispartin according to the present invention.

Figure 2 is an increase in the ratio of ssDNA binding to bispartin in the ssDNA pool of each selection in the SELEX process according to the present invention.

3 to 38 are schematic diagrams of DNA aptamer secondary structures of 36 nucleic acid construct aptamers specifically binding to bispartin according to the present invention using a web server-based m-fold program. to be.

FIG. 39 shows the results of specificity analysis for other edipocaine and HSA of No. 19, a DNA aptamer specifically binding to bispartin according to the present invention. FIG.

40 shows the result of binding assay with bispartin of No. 19 aptamer having the largest binding specificity according to the present invention.

<110> Korea University Industrial & Academic Collaboration Foundation <120> DNA aptamers binding to visfatin with specificity and production          method <160> 36 <170> KopatentIn 1.71 <210> 1 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 1 ataccagctt attcaattgg ccagggtcag ggcagtagga tatgctgggg acgggcgtga 60 gatagtaagt gcaatct 77 <210> 2 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 2 ataccagctt attcaattac gtagtcaacg ttgatactgc cactcgtgga tctgcggtag 60 atagtaagtg caatct 76 <210> 3 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 3 ataccagctt attcaattac gggcgggtcg acgtacgatt gcgggctggt gtgtgtccag 60 atagtaagtg caatct 76 <210> 4 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 4 ataccagctt attcaattgt aggtggcaac aggcggcccg ctaatagtat tgtatgtgca 60 gatagtaagt gcaatct 77 <210> 5 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 5 ataccagctt attcaattac 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<220> <223> DNA aptamer <400> 11 ataccagctt attcaattgg gcacggcgag tgagcaatgg ttagggctgg gcgtgtggag 60 atagtaagtg caatct 76 <210> 12 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 12 ataccagctt attcaattga cagcgatacg gtggcggata taaggtgggg tgggcgtgag 60 atagtaagtg caatct 76 <210> 13 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 13 ataccagctt attcaattgg gcaggacagg tgtcggcttg ataggctggg gtgtgtgtag 60 atagtaagtg caatct 76 <210> 14 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 14 ataccagctt attcaattag tcgtgtgtgc tgatcggtgg gcgcggatac ccccttggta 60 gatagtaagt gcaatct 77 <210> 15 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 15 ataccagctt attcaattgg gcgggacaag tgtcggcttg ataggctggg gtgtgtggag 60 atagtaagtg caatct 76 <210> 16 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 16 ataccagctt attcaattgc ggggacgaac ggactgcaca ttgcatatgg tggcgtcaag 60 atagtaagtg caatct 76 <210> 17 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 17 ataccagctt attcaattgg cgtcatatac agtaccgacg acgttgtcac cgacttatag 60 atagtaagtg caatct 76 <210> 18 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 18 ataccagctt attcaattgg ggcgggacag gtgtcggctt gataggctgg ggtgtgtgga 60 gatagtaagt gcaatct 77 <210> 19 <211> 77 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 19 ataccagctt attcaattgg ggcagcggca caatggcgga aggtgggtat gggtcgtgga 60 gatagtaagt gcaatct 77 <210> 20 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 20 ataccagctt attcaattgg ttgtgttatg ccaccctact gcatcactat atcagcctag 60 atagtaagtg caatct 76 <210> 21 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 21 ataccagctt attcaattgt ggtgcaacgg tcacatatca cgacgggtcc cgcagcctag 60 atagtaagtg caatct 76 <210> 22 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 22 ataccagctt 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Artificial Sequence <220> <223> DNA aptamer <400> 28 ataccagctt attcaattac agtagtaagg ggtccgtcgt ggggtagttg ggtcgtggag 60 atagtaagtg caatct 76 <210> 29 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 29 ataccagctt attcaattgg gtacgtatga ccatcacagc tctatcctac acccctctag 60 atagtaagtg caatct 76 <210> 30 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 30 ataccagctt attcaattac ggacggcaca cagtgtacag tcacctgcat cggcgtgtag 60 atagtaagtg caatct 76 <210> 31 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 31 ataccagctt attcaattat atgcggccca tcacctacca gctgtgttgt ttgcccctag 60 atagtaagtg caatct 76 <210> 32 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 32 ataccagctt attcaattca ctcgagcgaa gtgttgttat gtccctttac cgcgatgtag 60 atagtaagtg caatct 76 <210> 33 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 33 ataccagctt attcaattac agtagtgagg ggtccgtcgt ggggtagttg ggtcgtggag 60 atagtaagtg caatct 76 <210> 34 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 34 ataccagctt attcaattcg agtacacgag ccatggatca cacccgtctg tttggtcgag 60 atagtaagtg caatct 76 <210> 35 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 35 ataccagctt attcaattcc acgcgcctat ccaccacaga tcatcattcc gtgcatgtag 60 atagtaagtg caatct 76 <210> 36 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> DNA aptamer <400> 36 ataccagctt attcaattgt acgatgacca ggccttccac gctccattca tccgttgtag 60 atagtaagtg caatct 76  

Claims (10)

Bispartin (visfatin) detection composition comprising a DNA aptamer having any one of SEQ ID NO: 1 to 32, and 34 to 36. delete Method for preparing a DNA aptamer specifically bindable to visfatin (visfatin) having the base sequence of any one of SEQ ID NOS: 1 to 32, and 34 to 36, including the following steps: a) reacting bispartin and magnetic beads in a borate buffer solution to induce covalent bonds; b) inducing binding at room temperature by mixing a bispartin-magnetic bead obtained by the covalent bond with a DNA pool having a primer base for PCR at both ends and having 30-50 random bases in the center; c) separating the DNA bound to the bispartin-magnetic beads using a magnet; d) separating DNA from the separated bispartin-magnetic beads; And e) amplifying the DNA specifically binding to bispartin by performing PCR using a primer pair complementary to the PCR primer region. The method of claim 3, wherein the covalent bond of step a) is a covalent bond between an amino group of bispartin and a tosyl group of magnetic beads. According to claim 3, After the step a), the tosyl group of the magnetic beads in the covalently bonded bispartin-magnetic beads not bound to the bispatin 3- at 35-40 ° C with Bovine Serum Albumin (BSA) solution Inactivating for five hours. 4. The method of claim 3, wherein the separation of DNA in step d) is performed at 70-90 ° C. for 3-10 minutes to separate DNA from bispartin-magnetic beads. The method of claim 3, wherein the PCR is performed using a primer to which fluorescein is attached to one of the primer pairs in step e), and then the single-stranded DNA denatured by the fluorescein is subjected to electrophoresis. Further comprising the step of separating. A composition for diagnosing type 2 diabetes, comprising a DNA aptamer having any one of SEQ ID NOs: 1 to 32, and 34 to 36. delete The method of claim 8, wherein the type 2 diabetes is diagnosed by measuring the level of bispartin in the blood binding to the DNA aptamer having any one of SEQ ID NO: 1 to 32, and 34 to 36 Composition.

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