KR102029803B1 - DNA Aptamer Specifically Binding to Prathyroid Hormone and Uses Thereof - Google Patents

Abstract

The present invention relates to a DNA aptamer specifically binding to parathyroid hormone and uses thereof. Specifically, the present invention relates to a DNA aptamer specifically binding to parathyroid hormone, the apatamer being an oligonucleotide having any one nucleotide sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 14, and SEQ ID NO: 16; and a composition, kit, chip and microarray for detecting parathyroid hormone using the same. The present invention also relates to a method for producing the DNA aptamer specifically binding to parathyroid hormone, a composition for diagnosing parathyroid hormone-related diseases comprising the DNA aptamer, and a method for providing information necessary for the diagnosis of parathyroid hormone-related diseases.

Description

DNA Aptamer Specific Binding to Prathyroid Hormone and Uses Thereof}

The present invention relates to DNA aptamers and their use which specifically bind to parathyroid hormone.

Parathyroid hormone (PTH) is a hormone secreted primarily by the parathyroid gland and is a peptide consisting of 84 amino acids. Parathyroid hormone increases calcium absorption in the gastrointestinal tract and kidneys, inhibits phosphorus reabsorption, increases blood calcium levels, and reduces phosphate levels. It is also known to function to increase the conversion of vitamin D. Calcium is also a major component of bone, but also plays an important role in constricting muscles and blood clotting, including the heart. Most of the calcium is stored in the bones, but parathyroid hormone is known to regulate the calcium concentration in the blood through the action of vitamin D and calcium from the bones to the blood. In general, calcium-sensing receptors present in the parathyroid gland react sensitively to minute changes in serum calcium. When the calcium level in the blood decreases, parathyroid hormone secretion increases and conversely, calcium in the blood When the concentration rises, the parathyroid hormone secretion is lowered through the action to lower the concentration of calcium plays a role in maintaining a constant blood calcium concentration in the body.

On the other hand, abnormalities in the secretion of parathyroid hormone, which plays a key role in regulating blood calcium levels, cause various diseases.In case of excessive secretion of parathyroid hormone due to hyperparathyroid function, excessive calcium from the bone to the blood is excessive. It causes a lot of abnormalities in the metabolism of calcium and phosphorus, leading to osteoporosis, and when the level of phosphate is higher than the normal level, it causes cardiovascular abnormalities or extraosseous calcification. On the other hand, when the parathyroid gland is removed and shrunk mainly due to external factors caused by surgery or internal factors such as genetic mutations cause hypoparathyroidism, a decrease in the parathyroid hormone secreted by the blood in bone Calcium is reduced to hypocalcemia, hyperphosphatemia, or tetany, osteosclerosis, or exostosis due to increased excitability of nerves or muscles.

Therefore, since parathyroid hormone plays a central role in the regulation of calcium metabolism, blood parathyroid hormone is measured from the blood of a patient to determine the exact cause in the diagnosis of various diseases caused by calcium metabolism control problems.

Parathyroid hormone in the blood is measured by using an antibody that binds directly to the parathyroid hormone to detect the parathyroid hormone from the sample, and is proceeding to confirm the luminescence reaction in the presence of the parathyroid hormone. Depending on the degree of luminescence that appears, the process of calculating the concentration of parathyroid hormone present in the sample.

Antibodies that specifically bind to parathyroid hormone may not only be used as specimens to analyze the cause of a disease, but may also serve as an indicator to evaluate the function of a therapeutic agent. When parathyroid gland problems occur due to internal and external factors, parathyroid hormone secretion may occur. When parathyroid hormone secretion decreases, artificially prescribed parathyroid hormone may be treated. To this end, a cell therapy agent that overexpresses parathyroid hormone may be utilized.However, a method for evaluating the parathyroid hormone acts as one of the functional evaluation indexes by checking the light emission using an antibody to confirm that the parathyroid hormone is properly produced. have.

However, parathyroid hormone is generally known to be present in very small amounts in the blood and has a very short half-life and is converted into various forms in the blood. Therefore, even when testing a patient suspected of abnormalities in calcium regulation, blood is collected, and it is known that the test is performed by using an antibody specific for parathyroid hormone. However, given the fact that the secretion of parathyroid hormone is very small and has a short half-life, and that it exists in various forms in the body, there is a problem in that a false positive reaction that binds nonspecifically occurs. In addition, in evaluating the function of a cell therapeutic agent that overexpresses parathyroid hormone, there is a problem in that it is difficult to accurately evaluate the result of nonspecific binding when luminescence is checked by using an antibody.

Therefore, development of a test method other than the use of antibodies is required as a method for accurate detection and diagnosis of parathyroid hormone.

On the other hand, aptamer is a short-length single-stranded nucleic acid having a stable tertiary structure has a characteristic of binding to the target material with high affinity and specificity. Aptamers are generally compared to antibodies, which are difficult to produce due to their large molecular structure and limitations of modification, whereas aptamers are composed of short nucleic acids, resulting in relatively small molecular structures. By using the mass production in a short time and low cost as well as has the advantage that a variety of deformation is easy according to the purpose. In addition, aptamers are composed of DNA or RNA, so they have higher stability to the surrounding environment such as pH and temperature than antibodies made of proteins. Therefore, aptamers are used in various industries including the detection of target substances and the development of disease diagnosis sensors. Applicability in the field is highly appreciated.

Republic of Korea Patent Publication No. 10-2016-0142446

Therefore, the present inventors synthesized DNA aptamers having specificity for parathyroid hormone during research to develop a technology that can detect parathyroid hormone more accurately, faster and more easily than the method for detecting parathyroid hormone using an antibody. The present invention was completed by confirming that these aptamers can replace antibody-based parathyroid hormone detection methods and can be used for examination and diagnosis of diseases caused by parathyroid hormone.

It is therefore an object of the present invention to provide a DNA aptamer that specifically binds to parathyroid hormone (PTH) protein.

Another object of the present invention to provide a composition for detecting parathyroid hormone (parathyroid hormone, PTH) comprising the DNA aptamer (aptamer) of the present invention as an active ingredient.

Another object of the present invention is to provide a parathyroid hormone (PTH) detection kit comprising the parathyroid hormone detection composition of the present invention as an active ingredient.

Another object of the present invention is a chip for detecting parathyroid hormone (parathyroid hormone, PTH), characterized in that the DNA aptamer of the present invention is immobilized on a substrate to specifically react to a sample containing parathyroid hormone protein. It is to provide a microarray.

Another object of the present invention is to provide a method for detecting parathyroid hormone proteins using the DNA aptamer of the present invention.

Another object of the present invention is to provide a composition for diagnosing parathyroid hormone-related diseases comprising the DNA aptamer of the present invention as an active ingredient.

Another object of the present invention is to provide a method for preparing a DNA aptamer that specifically binds to parathyroid hormone.

Another object of the present invention is to provide a method for providing information necessary for the diagnosis of parathyroid hormone-related diseases using the DNA aptamer of the present invention.

In order to achieve the above object, the present invention is a DNA aptamer that specifically binds to parathyroid hormone (PTH) protein, among the group consisting of SEQ ID NO: 9, SEQ ID NO: 14 and SEQ ID NO: 16 It provides a DNA aptamer (aptamer) characterized in that the oligonucleotide having any one selected nucleotide sequence.

In one embodiment of the present invention, the DNA aptamer may specifically bind to a human parathyroid hormone protein or His fusion parathyroid hormone protein.

In one embodiment of the present invention, the DNA aptamer may be to further include a label.

In one embodiment of the present invention, the labeling material is any one of a labeling material selected from the group consisting of a fluorescent material, an amine group, a biotin, a thiol group, and digoxigenin (digoxigenin) or the 5 'end of the DNA aptamer or It may be labeled at the 3 'end.

In addition, the present invention provides a composition for detecting parathyroid hormone (parathyroid hormone, PTH) comprising the DNA aptamer (aptamer) of the present invention as an active ingredient.

In addition, the present invention provides a kit for detecting parathyroid hormone (parathyroid hormone, PTH) comprising the composition of the present invention as an active ingredient.

In addition, the present invention is a chip for detecting parathyroid hormone (parathyroid hormone, PTH) characterized in that the DNA aptamer (aptamer) of the present invention is fixed on the substrate to specifically react to a sample containing parathyroid hormone protein Provide a microarray.

In addition, the present invention, (1) contacting the biological sample isolated from the living body with the DNA aptamer of the present invention; And (2) determining the presence or content of parathyroid hormone in the biological sample through a parathyroid hormone (PTH) protein specific binding reaction between the biological sample and the DNA aptamer. It provides a detection method of.

The present invention also provides a composition for diagnosing parathyroid hormone-related diseases comprising the DNA aptamer of the present invention as an active ingredient.

In one embodiment of the present invention, the disease is calcium hypotension, osteopenia, osteoporosis, hyperparathyroidism, parathyroidism, fracture, hyperparathyroidism-related calcium hyperemia, malignant calcium hyperemia, kidney failure, urolithiasis And hypertension may be selected from the group consisting of.

In addition, the present invention comprises the steps of (1) amplifying DNA aptamers using PCR and asymmetric PCR techniques, and selecting single-stranded DNA aptamers; (2) inducing binding of the single-stranded DNA aptamer and parathyroid hormone; (3) binding parathyroid hormone bound to Ni-NTA agarose resin with single stranded DNA aptamer; (4) removing DNA aptamers that failed to bind parathyroid hormone; And (5) recovering and selecting DNA aptamers that specifically bind to parathyroid hormone through the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technique. It provides a manufacturing method.

In one embodiment of the present invention, step (5) comprises a real-time PCR step for SELEX round selection having an optimal affinity; PCR and cloning steps for selection of parathyroid hormone binding DNA aptamer candidates in the optimal SELEX round; Measuring affinity for parathyroid hormone of the parathyroid hormone binding DNA aptamer candidate group; And DNA sequencing of selected parathyroid hormone-binding DNA aptamer candidate groups.

In addition, the present invention, (1) contacting the biological sample isolated from the living body with the DNA aptamer of the present invention; And (2) determining the presence or content of parathyroid hormone in the biological sample through a parathyroid hormone (PTH) protein specific binding reaction between the biological sample and the DNA aptamer. It provides a method of providing information necessary for the diagnosis of a disease.

In one embodiment of the present invention, the disease is calcium hypotension, osteopenia, osteoporosis, hyperparathyroidism, parathyroidism, fracture, hyperparathyroidism-related calcium hyperemia, malignant calcium hyperemia, kidney failure, urolithiasis And hypertension may be selected from the group consisting of.

The present invention relates to a DNA aptamer capable of specifically binding to parathyroid hormone and its use. Since the DNA aptamer of the present invention has a specific binding property to parathyroid hormone protein, it is used not only for detecting parathyroid hormone protein but also for measuring the level of parathyroid hormone protein in the blood to diagnose and diagnose diseases caused by parathyroid hormone. It can be used more effectively in examining prognosis. In addition, the DNA aptamer having a specific binding ability of the parathyroid hormone of the present invention can be used as a means for confirming the over-expression of the parathyroid hormone as a cell therapy, which is more accurate and effective detection and diagnosis than the existing antibody It is expected to provide a clear standard of judgment.

1 shows a result of amplifying random DNA aptamers using PCR and asymmetric PCR, followed by selective recovery of ssDNA using streptavidin beads, lane M: 100 bp DNA marker, lane 1: parathyroid gland. After amplifying the hormone specific binding DNA aptamer using PCR, the result of recovering only the parathyroid hormone specific binding DNA aptamer using a PCR purification kit. Lane 2: After amplifying the parathyroid hormone-specific binding DNA aptamer using PCR, the results show that only the parathyroid hormone-specific binding DNA aptamer was recovered using a PCR purification kit.
Figure 2 shows the amount quantitatively measured the concentration of parathyroid hormone-binding DNA aptamer recovered in each round in the SELEX process for the production of parathyroid hormone-specific binding DNA aptamer using nanodrops.
Figure 3 shows the results of a quantitative measurement of the affinity test of the aptamer candidate group secured in order to select DNA aptamers that bind optimally to parathyroid hormone using nanodrops.
Figure 4 shows the secondary structure of a total of three parathyroid hormone-binding DNA aptamers selected to have a specific binding force with parathyroid hormone.
Figure 5 schematically shows the process of binding the parathyroid hormone to the surface of the CM5 chip coated with dextran carboxyl group bound.
6 is a result of confirming that the parathyroid hormone can be detected directly by using aptamer blotting technique using parathyroid hormone binding aptamer and Dig binding aptamer, A: Figure 6A is a DNA aptamer binding to parathyroid hormone (PTH9) The result is the detection of parathyroid hormone using Dig binding DNA aptamer, lane M: western blotting marker (Elpis biotech, Korea), lane 1: recombinant parathyroid hormone protein, B: Figure 6B is a DNA app that binds to parathyroid hormone Parathyroid hormone was detected using tammer (PTH14) and Dig binding DNA aptamer. Lane M: Western blotting marker (Elpis biotech, Korea), Lane 1: Recombinant parathyroid hormone protein, C: Figure 6C shows parathyroid hormone Parathyroid hormone is detected using binding DNA aptamer (PTH16) and Dig binding DNA aptamer. Lane M: western blotting marker (Elpis biotech, Korea), lane 1: Recombinant parathyroid hormone protein.
7 is a result of confirming that the parathyroid hormone can be directly detected from the whole cell activating protein of parathyroid hormone overexpressing cells by using aptamer blotting technique using parathyroid hormone binding aptamer and Dig binding aptamer, FIG. Is the result of the detection of parathyroid hormone directly from the whole cell activating protein of parathyroid hormone overexpressing cells using DNA aptamer (PTH9) and Dig binding DNA aptamer binding to parathyroid hormone, Lane M: western blotting Marker (Elpis biotech, Korea), Lane 1: active protein of parathyroid hormone-expressing cells, lane 2: active protein of parathyroid hormone-expressing cells cultured on 7 days, lane 3: activity of cells that induce parathyroid hormone overexpression for 7 days Type protein. FIG. 7B is a result of confirming whether parathyroid hormone can be directly detected from whole cell-activated protein of parathyroid hormone overexpressing cells using DNA aptamer binding to parathyroid hormone (PTH14) and Dig binding DNA aptamer. western blotting marker (Elpis biotech, Korea), lane 1: active protein of parathyroid hormone-expressing cells, lane 2: active protein of parathyroid hormone-expressing cells cultured on 7 days, lane 3: cell inducing parathyroid hormone overexpression for 7 days It shows an active protein of. FIG. 7C shows that it is possible to detect parathyroid hormone directly from whole cell-activated protein of parathyroid hormone overexpressing cells using DNA aptamer binding to parathyroid hormone (PTH16) and Dig binding DNA aptamer. western blotting marker (Elpis biotech, Korea), lane 1: active protein of parathyroid hormone-expressing cells, lane 2: active protein of parathyroid hormone-expressing cells cultured on 7 days, lane 3: cell inducing parathyroid hormone overexpression for 7 days It shows an active protein of.

The present invention relates to DNA aptamers and their use which specifically bind to parathyroid hormone proteins.

The inventors have discovered an aptamer having a specific binding ability to parathyroid hormone as a method for solving the problems of the target material detection technique based on the existing antibody.

 DNA aptamers specifically binding to the parathyroid hormone protein of the present invention can be confirmed that the specific binding to the parathyroid hormone protein can easily detect the parathyroid hormone protein from biological samples, and to diagnose diseases related to the parathyroid hormone protein It was confirmed that it can be used to predict prognosis.

The DNA aptamer specifically binding to the parathyroid hormone protein of the present invention refers to a single strand of DNA capable of binding with high specificity and affinity to a target substance, and may be manufactured through SELEX (Systematic Evolution of Ligand of Exponential enrichment) technique. Can be.

As used herein, the term "DNA aptamer" refers to a DNA nucleic acid molecule capable of binding to a particular molecule with high affinity and specificity. As used herein, "DNA aptamer" is used interchangeably with "DNA oligonucleotide". Aptamers are short oligomers that form stable tertiary structures and have specific binding properties with target materials. In addition, aptamers are composed of nucleic acids, such as DNA or RNA, which are more stable than antibodies made of proteins, and have the advantage of being able to specifically bind to various target substances (proteins, peptides, metals, chemicals, etc.). In addition, since it can be manufactured using chemical synthesis techniques, it can be mass-produced in a short time and at low cost, and has an excellent advantage of continuously producing aptamers having the same ability after production and production once. In addition, since it is very stable at the surrounding pH and temperature, the possibility of application in various fields such as the detection of target substance and the development of disease diagnosis sensor is widely appreciated.

In one embodiment of the present invention, the DNA aptamer specifically binding to the parathyroid hormone protein has a nucleotide sequence selected from the group consisting of the nucleotide sequence of SEQ ID NO: 9, SEQ ID NO: 14 and SEQ ID NO: 16, It may be an oligonucleotide having a nucleotide sequence having at least 90% identity with such a nucleotide sequence. The DNA aptamer may be substituted with any one selected from the group consisting of a hydrogen atom, a fluorine atom, -OR, -COOR and an amino group of the ribose 2 'position of one or more nucleotides constituting the DNA aptamer.

In addition, the DNA aptamer may be configured to further include a label, wherein the label is any one selected from the group consisting of fluorescent material, amine group, biotin, thiol group and digoxigenin (digoxigenin) is It can be labeled at the 5 'end or 3' end of the DNA aptamer.

As used herein, the term “oligonucleotide” generally refers to a nucleotide polymer having less than about 200 in length, and may include DNA and RNA, preferably a DNA nucleic acid molecule. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or analogs thereof, or any substrate that can be introduced into the polymer by DNA or RNA polymerase or by a synthetic reaction. If modifications to the nucleotide structure are present, such modifications may be added before or after the synthesis of the oligonucleotide polymer. Nucleotide sequences can be interrupted by non-nucleotide components. Oligonucleotides can be further modified after synthesis, for example by binding to a label.

DNA aptamers of the present invention are typically obtainable by in vitro selection methods for binding of target molecules. Methods of selecting aptamers that specifically bind to target molecules are known in the art. For example, organic molecules, nucleotides, amino acids, polypeptides, marker molecules on the cell surface, ions, metals, salts, polysaccharides can be suitable target molecules that separate aptamers that can specifically bind to each ligand. . The selection of aptamers can use in vivo or in vitro selection techniques known by the SELEX method (Ellington et al., Nature 346, 818-22, 1990; and Tuerk et al., Science 249, 505-10, 1990 ). Specific methods for the selection and preparation of aptamers are described in US Pat. No. 5,582,981, WO 00/20040, US Pat. No. 5,270,163, Lorsch and Szostak, Biochemistry, 33: 973 (1994), Mannironi et al., Biochemistry 36: 9726 (1997), Blind, Proc. Natl. Acad. Sci. USA 96: 3606-3610 (1999), Huizenganand Szostak, Biochemistry, 34: 656-665 (1995), WO 99/54506, WO 99/27133, WO 97/42317 and US Pat. No. 5,756,291. It is hereby incorporated by reference.

The DNA aptamer specifically binding to the parathyroid hormone protein of the present invention is preferably an oligonucleotide having any one nucleotide sequence selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 9, 14 and 16.

Such DNA aptamers having parathyroid hormone protein specific binding ability as identified in the present invention form the tertiary structure shown in FIG. 4 herein.

In addition, the DNA aptamer specifically binding to the parathyroid hormone protein of the present invention and any one of the base sequence selected from the group consisting of the base sequence of SEQ ID NO: 9, 14 and 16, while maintaining the properties of binding to the parathyroid hormone protein It is also interpreted to include oligonucleotides having base sequences showing substantial identity.

The substantial identity above aligns the nucleotide sequence of the present invention with any other sequence to the maximum correspondence, and the algorithms commonly used in the art (Smith and Waterman, Adv. Appl. Math. 2: 482 (1981) Needleman and Wunsch, J. Mol. Bio. 48: 443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73: 237-44 (1988) Higgins and Sharp, CABIOS 5: 151-3 (1989); Corpet et al., Nuc.Acids Res. 16: 10881-90 (1988); Huang et al., Comp.Appl. BioSci. 8: 155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24: 307-31 (1994)), when analyzing aligned sequences, at least 90% identity, more preferably at least 95% By nucleotide sequence exhibiting the above identity, most preferably at least 98% identity.

In addition, the parathyroid hormone protein to which the DNA aptamer of the present invention can bind is preferably a human-derived parathyroid hormone protein or His fusion parathyroid hormone protein, the amino acid sequence of the human-derived parathyroid hormone protein is shown in SEQ ID NO: 25, The amino acid sequence of the recombinant parathyroid hormone protein fused with His is shown in SEQ ID NO: 26.

Furthermore, the present invention can provide a method for preparing DNA aptamers that specifically binds to parathyroid hormone, which method preferably comprises: (1) amplifying DNA aptamers using PCR and asymmetric PCR techniques; Selecting single-stranded DNA aptamers; (2) inducing binding of the single-stranded DNA aptamer and parathyroid hormone; (3) binding parathyroid hormone bound to Ni-NTA agarose resin with single stranded DNA aptamer; (4) removing DNA aptamers that failed to bind parathyroid hormone; And (5) recovering and selecting DNA aptamers specifically binding to parathyroid hormone through SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technique.

Wherein step (5) is specifically a real-time PCR step for SELEX round selection with optimal affinity; PCR and cloning steps for selection of parathyroid hormone binding DNA aptamer candidates in the optimal SELEX round; Measuring affinity for parathyroid hormone of the parathyroid hormone binding DNA aptamer candidate group; And DNA sequencing of selected parathyroid hormone-binding DNA aptamer candidate groups.

In one embodiment of the present invention, asymmetric PCR was performed to amplify only ssDNA (single strand DNA) out of dsDNA (double strand DNA) amplified by PCR, for DNA aptamer selection of the present invention. Asymmetric PCR yielded ssDNA by performing PCR using 10 μl forward primer and 2 μl reverse primer at a 10: 2 ratio, for example, at the same concentration (25 uM). In addition, the screening method of ssDNA aptamer, amplifies dsDNA by attaching biotin to the reverse primer during PCR, and treating the streptavidin at 3 'of the amplification product to form a biotin-streptavidin complex to selectively select the complex. Removal allowed only the opposite ssDNA aptamer to which biotin was not bound.

Thereafter, DNA aptamers having parathyroid hormone-binding ability were selected by SELEX using recombinant human-derived parathyroid hormone proteins for selected ssDNA aptamers. According to one embodiment of the present invention, the aptamer sequence is amplified and secured. Then fixing the parathyroid hormone to the resin; Binding the immobilized parathyroid hormone and DNA aptamers; A round of recovering only parathyroid-specific binding DNA aptamers reactive with parathyroid hormone was performed to select an optimal SELEX round having high affinity and specificity.

In addition, the screening process of the optimal SELEX round is the optimal round selection step through quantification analysis using nanodrop (NanoDrop spectrophotometer, USA) and real-time PCR; PCR and cloning steps to secure parathyroid hormone specific binding DNA aptamer candidates in the optimal SELEX round; And DNA sequencing of the acquired parathyroid hormone-specific binding DNA aptamer candidate group.

In the present invention, the “SELEX method” refers to a method of determining a DNA binding sequence of a molecule by selecting and amplifying a DNA having a high binding strength to a specific molecule in a collection of randomly synthesized DNA (Louis et al. 1992. Nature 355, 564-566).

The present invention can select the parathyroid hormone-specific binding DNA aptamer recovered in each round through the nanodrop for the continuous progress of SELEX and the optimal round selection after completing the SELEX process up to 10 times for the optimal SELEX round selection. Thereafter, PCR and cloning were performed to secure the sequence of the parathyroid hormone-specific binding DNA aptamer candidate group in the selected optimal SELEX round, and the DNA sequence of the selected parathyroid hormone-specific binding DNA aptamer candidate group was determined.

DNA aptamer of the present invention that specifically binds to parathyroid hormone (PTH) protein discovered by the above method is used to directly detect the parathyroid hormone protein or detect the parathyroid hormone protein in the biological sample of the patient It can be used to diagnose diseases related to parathyroid hormone.

Parathyroid hormone detection of the present invention is based on a method for detecting a complex of parathyroid hormone protein and DNA aptamer specifically binding to parathyroid hormone protein. In one embodiment of the present invention, the DNA aptamer specifically binding to the parathyroid hormone protein of the present invention is a fluorescent material (e.g., fluorescein (Foreorescein, Cy3, Cy5 or HRP), radioactive materials, or nucleotides labeled with chemicals such as biotin or modified with primary amines.

In addition, the DNA aptamer of the present invention may be manufactured in the form of biosensors, kits, and chips including an immobilized substrate and used to detect parathyroid hormone proteins.

As used herein, the term “sensors and chips” refers to sensors and chips in which a certain material is attached to a specific area of a substrate with high density. As used herein, the term "substrate" of biosensor and sensor chip means a support having suitable firmness or semi-rigidity, for example glass, membrane, slide, filter, chip, wafer, fiber, magnetic beads or Non-magnetic beads, gels, tubing, plates, polymers, microparticles, capillaries, and the like. DNA aptamers of the invention can be arranged and immobilized on the substrate. This immobilization can be accomplished by chemical bonding methods or by covalent methods such as UV. For example, DNA oligonucleotides can be bound to glass surfaces modified to include epoxy compounds or aldehyde groups, and can also be bound by UV at the polylysine coating surface. In addition, the DNA oligonucleotide may be bound to the substrate via a linker (eg, ethylene glycol oligomer and diamine).

DNA aptamers that specifically bind to the parathyroid hormone proteins of the present invention can be biotinylated, for example, and can be successfully immobilized on a streptavidin-coated substrate. . The DNA aptamer specifically binding to the parathyroid hormone protein of the present invention immobilized on the substrate may bind to and capture the parathyroid hormone protein, and the captured parathyroid hormone protein thus binds specifically to the parathyroid hormone protein. DNA aptamers can be used to visualize capture.

In addition, the composition for detecting parathyroid hormone protein in the present invention may be provided in the form of a kit. In the present invention, the kit includes a DNA oligonucleotide having a nucleotide sequence of SEQ ID NO: 9, 14 or 16 or a nucleotide sequence having at least 90% identity with the sequence as an active ingredient. Kits in the present invention may further comprise instructions or labels for using the kit to detect parathyroid hormone proteins in a sample.

According to another aspect of the present invention, the present invention may provide useful information for diagnosing or prognostic diseases of parathyroid hormone through specific detection of parathyroid hormone proteins.

As described in the prior art, parathyroid hormone is responsible for increasing calcium absorption in the gastrointestinal tract and kidneys, inhibiting resorption of phosphorus to increase blood calcium concentration, and reducing the concentration of phosphate, and also vitamins. It is known to function to increase the conversion of D.

Hyperparathyroidism, one of the disorders caused by abnormal control of these parathyroid hormones, is the most common cause of hypercalcemia, and due to improperly excessive secretion of parathyroid hormone due to impaired function of one or more parathyroid glands It is a disease that causes abnormalities in phosphorus and bone metabolism.

In addition, hypoparathyroidism is an endocrine deficiency disease caused by hypocalcemia, hyperphosphatemia, and reduction of serum parathyroid hormone.

In the present invention, the parathyroid hormone-related diseases include, but are not limited to, all diseases that can be caused by parathyroid hormone regulation abnormalities, calcium hypotension, osteopenia, osteoporosis, hyperthyroidism, parathyroidism, fracture, Hyperparathyroidism-related calcium hyperemia, malignant calcium hyperemia, renal failure, urolithiasis and hypertension. Therefore, the present invention comprises the steps of (1) contacting a biological sample isolated from the living body with the DNA aptamer of claim 1; And (2) determining the presence or content of parathyroid hormone in the biological sample through a parathyroid hormone (PTH) protein specific binding reaction between the biological sample and the DNA aptamer. It is possible to provide a detection method of.

Furthermore, the present invention can provide a composition for diagnosing parathyroid hormone-related diseases comprising the DNA aptamer of the present invention as an active ingredient and a method for providing information necessary for diagnosing parathyroid hormone-related diseases using the DNA aptamer of the present invention. have.

The information providing method for diagnosing parathyroid hormone-related diseases using the DNA aptamer of the present invention comprises the steps of: (1) contacting a biological sample isolated from a living body with the DNA aptamer of claim 1; And (2) determining the presence or content of parathyroid hormone in the biological sample through a parathyroid hormone (PTH) protein specific binding reaction between the biological sample and the DNA aptamer.

Here, when the level or content of parathyroid hormone (PTH) protein contained in the sample is higher than that of the normal control group (normal sample), it can be predicted or diagnosed that a disease due to hyperparathyroidism is developed, When the level or content of parathyroid hormone (PTH) protein contained in the sample is lower than that of the normal control group (normal sample), it may be predicted or diagnosed as a disease caused by hypothyroidism.

Furthermore, the DNA aptamer of the present invention can also be utilized as a means for confirming whether the cells expressing and secreting parathyroid hormone when the cell therapeutic agent is used as a therapeutic agent for a disease caused by hypothyroid hormone secretion.

The term “biological sample” may include blood, saliva, tear fluid, urine, synovial fluid, mucus, cells, tissues, and other tissues and body fluids, including cell culture supernatants, ruptured eukaryotic cells, and bacterial expression systems. However, the present invention is not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited to these examples.

< Example  1>

His Fusion Human-derived Recombinant Parathyroid Hormone Protein Preparation

Human-derived parathyroid hormone sequence information (NP_001303281.1) was obtained from NCBI for the production of aptamers for human-derived parathyroid hormone, and to obtain recombinant parathyroid hormone protein with His-tag attached to the terminal based on the obtained sequence information. It was. Finally, the recombinant parathyroid hormone (Cloud-clone corp, USA) with His-tag attached to the terminal was selected and used as a target material for the DNA aptamer of the present invention.

<Example 2>

DNA aptamers specific for parathyroid hormone

<2-1> DNA Random Library Amplification Using PCR Technique

76 random sequences containing 40 random sequences in dA: dG: dC: dT = 1.5: 1.15: 1.25: 1 ratios to produce single-stranded DNA aptamers that specifically bind to parathyroid hormone (PTH) bp template DNA (5'-ATACCAGCTTATTCAATT N40-AGATAGTAAGTGCAATCT-3 ') (SEQ ID NO: 20) and a pair of primers capable of amplifying it to 76 bp were customized to Bioneer (Korea) (forward primer: 5'-ATACCAGCTTATTCAATT). -3 '(SEQ ID NO: 21), biotinylated reverse primer: 5'-Biotin-AGATAGTAAGTGCAA TCT-3' (SEQ ID NO: 22).) Any DNA library was prepared using PCR (Bioneer, Korea). Amplified. The reaction composition for amplification of 76 bp DNA library by PCR was 1 μl template DNA, 5 μl 10X PCR buffer, 4 μl of each 2.5 mM dNTP mixture, 2 μl 25 uM forward primer, 25 uM biotinylated reverse primer 2 ㎕, 0.25 μl of Ex Taq polymerase (TaKaRa, Japan) (1 unit / μl) and 35.75 μl of distilled water. PCR reaction conditions were first denatured at 95 ° C. for 5 minutes, repeated 20 cycles for 30 seconds at 95 ° C., 30 seconds at 55 ° C., and 30 seconds at 72 ° C., and then further extended at 72 ° C. for 7 minutes. Was used.

After the PCR reaction, 4 μl was taken and the amplification product was confirmed by 2% agarose gel electrophoresis (see FIG. 1). The DNA library obtained through PCR was purified using a PCR purification kit (Qiagen, USA). 50 µl of distilled water was used for recovery.

<2-2> ssDNA Amplification Using Asymmetric PCR Technique

Asymmetric PCR was performed to amplify only ssDNA out of the amplified dsDNA using the PCR technique. The asymmetric PCR reaction composition consisted of 7 μl of template DNA obtained through <2-1>, 10 μl of 10X PCR buffer, 8 μl of 2.5 mM dNTP mixture, 10 μl of 25 uM forward primer, 25 uM biotinylated reverse primer 2 Μl, Ex Taq polymerase (Takara, Japan) 0.5 μl (1 unit / μl) and 62.5 μl of distilled water. The asymmetric PCR reaction conditions were first denatured at 95 ° C. for 5 minutes, repeated 15 cycles for 30 seconds at 95 ° C., 30 seconds at 55 ° C., and 30 seconds at 72 ° C., and then further extended at 72 ° C. for 7 minutes. Was performed. After the PCR reaction, 4 µl was taken to confirm that the band of the correct size appeared by using a 2% agarose gel, and the rest of the DNA was used for the usual PCI extraction and ethanol precipitation to recover the pure DNA aptamer pool. . The reaction solution was treated with the same volume of PCI (Phenol: Chloroform: Isoamylalcohol = 25: 24: 1) solution, stirred vigorously, and centrifuged at 13,000 rpm for 15 minutes at 4 ° C to recover only the supernatant. / 100 volumes of tRNA (sigma aldrich, USA), 3 volumes of 100% ethanol was added and reacted for more than 1 hour at -70 ℃. After the reaction, only DNA was recovered by centrifugation at 13,000 rpm for 20 minutes at 4 ° C. The recovered DNA was dried at 65 ° C. and then dissolved in 50 μl of distilled water. 4 μl of the recovered DNA was taken to confirm that a band of the correct size appeared through 2% agarose gel electrophoresis.

<2-3> ssDNA Fabrication and Recovery Using Heating-Cooling Technique

In order to remove dsDNA and ssDNA with biotin from the PCR product amplified by asymmetric PCR and to secure only pure forward ssDNA, 50 μl of distilled water was added to 50 μl of DNA obtained in <2-2>, followed by heating- The dsDNA was denatured to ssDNA using a heating-cooling technique. After dsDNA was reacted at 85 ° C. for 5 minutes to denature to ssDNA, immediately after completion of the reaction, the reaction solution was cooled to 4 ° C. to obtain ssDNA.

Subsequently, 50 μl of streptavidin (Pierce, USA) was added thereto and reacted at room temperature for 1 hour. After the reaction, the reaction solution was centrifuged for 10 minutes using a centrifugal separator at 4 ° C and 13,000 rpm, and only the supernatant was recovered to secure ssDNA. In order to secure pure ssDNA in the reaction solution, PCI extraction and ethanol precipitation were performed. That is, the reaction solution was treated with the same volume of PCI (Phenol: Chloroform: Isoamylalcohol = 25: 24: 1) solution, and stirred vigorously. Only the supernatant was recovered by centrifugation at 13,000 rpm for 15 minutes at 4 ° C. 1/100 volume of tRNA (sigma aldrich, USA) and 3 volumes of 100% ethanol were added to the supernatant and reacted at -70 ° C for at least 1 hour. After the reaction, centrifuged at 13,000 rpm for 20 minutes at 4 ° C to recover only ssDNA. The recovered ssDNA was dried at 65 ° C. and then dissolved in 50 μl of distilled water. 10 μl of the recovered ssDNA was taken and 10% acrylamide gel electrophoresis confirmed that the band of the correct size appeared.

<Example 3>

Screening of ssDNA Aptamers Specific for Parathyroid Hormone Using SELEX Technique

<3-1> Composition of each solution used in SELEX

The composition of the solution used in SELEX is as follows.

1X his binding buffer: 5 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl (pH 7.9)

1X his washing buffer: 60 mM imidazole, 500 mM NaCl, 20 mM Tris-HCl (pH 7.9)

1X Aptamer Screening Solution: 10 mM PBS (pH 7.4)

2X Aptamer Screening Solution: 20 mM PBS (pH 7.4)

<3-2> Preparation of aptamer structure of ssDNA for SELEX and induction of binding of parathyroid hormone and ssDNA aptamer

In order to select DNA aptamers that specifically bind to parathyroid hormone, 60 μl of distilled water was added to 40 μl of ssDNA obtained in the above example, 100 μl of 2X DNA aptamer selection solution was added, and then 5 minutes at 85 ° C. After boiling, denaturation was performed and cooled slowly at room temperature to form a stable three-dimensional structure of ssDNA aptamer.

10 μl of the parathyroid hormone prepared in Example 1 was added to the ssDNA aptamer stably completed three-dimensional structure to induce binding between the ssDNA aptamer and parathyroid hormone for 12 hours at 4 ℃.

DNA binding to parathyroid hormone Aptamers  Parathyroid hormone binding for screening Ni - NTA Agarose  Screening of ssDNA Aptamers Using Agarose Resin Activation and SELEX Method

In order to select DNA aptamers that specifically bind to parathyroid hormone, Ni-NTA agaroes resin (QIAGEN, German) that specifically binds to his-tag of recombinant protein was used. First, 500 µl of 1X his binding buffer was added to 200 µl of Ni-NTA agarose resin, followed by stirring. Then, after removing the supernatant by centrifugation (4 ℃, 13,000 rpm, 10 minutes), the parathyroid hormone coupled with the ssDNA aptamer prepared in the above <3-2> to the activated Ni-NTA agarose resin Ni The binding of -NTA agarose resin was induced at 4 ° C. for 1 hour. The supernatant was removed by centrifugation (4 ° C, 13,000 rpm, 10 minutes) to remove recombinant parathyroid hormone that did not bind to Ni-NTA agarose resin. Then, by washing three times with 1X his washing buffer to remove all of the ssDNA aptamer non-specifically bound to parathyroid hormone. SsDNA aptamers specifically bound to parathyroid hormone were denatured by adding 200 μl of 1X DNA aptamer elution solution at 85 ° C. for 5 minutes, and then recovered by centrifugation (4 ° C., 13,000 rpm, 10 minutes). The recovery process was performed twice, and the PCI extraction and ethanol precipitation were performed to recover the pure DNA aptamer pool from the parathyroid hormone-specific binding DNA aptamer elution solution, and then recovered in 50 μl of distilled water.

<3-4> Nonspecific ssDNA Aptamer Removal Through Negative SELEX

To remove ssDNA aptamers that do not bind to parathyroid hormone but adsorb specifically to Ni-NTA agarose resin, negative SELEX was performed using Ni-NTA resin with no parathyroid hormone fixed between 6 and 7 times of SELEX. . After reacting the ssDNA aptamer cooled at room temperature to the activated Ni-NTA resin column using 1X PBS buffer for 1 hour, the ssDNA aptamer solution without binding to the column was used for 7 times SELEX. Since then, SELEX has been performed up to 10 times, and the binding condition of SELEX decreases the amount of ssDNA aptamer and reaction time as the number of times increases, and as the number of times increases, the reaction condition roughens the reaction condition to specifically bind to parathyroid hormone, a target substance. Only tamers were obtained.

<Example 4>

Affinity test for optimal SELEX round selection specifically binding to parathyroid hormone

Nanodrop the concentration of ssDNA specifically bound to parathyroid hormone recovered in each round to quantitatively confirm the progress of SELEX and the thyroid hormone affinity for ssDNA aptamers eluted in each round after SELEX completion. It was measured using (Nano-drop).

As a result of measuring the concentration of ssDNA aptamer eluted in each round using a nano-drop, the concentration of 9 rounds was 802.3 ng / μl and the concentration of 10 rounds was 706.3 ng / μl. It was confirmed that the concentration was lower than the results. Therefore, these results confirmed that the optimal aptamer pool that binds most specifically to the parathyroid hormone is a 9-round pool (see FIG. 2).

Example 5

Selection and cloning of DNA aptamer candidates that specifically bind to parathyroid hormone

Forward primer: 5'-ATACCAGCTTATTCAATT-3 '(SEQ ID NO: 23), reverse primer: 5'- DsDNA was obtained by PCR using AGATAGTAAGTGCAATCT-3 ′ (SEQ ID NO: 24). The dsDNA thus obtained was cloned using Solgent's T-Blunt Cloning Kit. Cloning was carried out by mixing 1 μl of T-vector (10 ng / μl), 4 μl of PCR product (20 ng / μl), and 1 μl of 6 × T-blont buffer and reacting at 25 ° C. for 5 minutes. 6 μl of the T-blunt cloning reaction solution was mixed with 100 μl of DH5α, transformed by applying a heat shock at 42 ° C. for 30 seconds, and then reacted on ice for 2 minutes. Then 900 μl of SOC medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl 2, 10 mM MgSO 4, 20 mM glucose) was added thereto, followed by incubation at 37 ° C. for 40 minutes. do. After incubation, 200 μl of solution was taken to contain ampicillin (ampicillin, 50 μg / ml), kanamycin (kanamycin, 50 μg / ml), X-gal (50 μg / ml), and IPTG (5 μg / ml). Spreading in an LB culture plate (LB plate) and incubated at 37 ℃ for 15 hours, only white colonies were selected and asked to Solgent (Solgent, Korea) to determine the sequence of the aptamer. Through sequencing analysis, 19 non-overlapping parathyroid hormone-binding DNA aptamer sequences could be obtained, and Table 1 shows 19 sequences of parathyroid hormone-binding DNA aptamers.

< Example  6>

Nanodrops  DNA that specifically binds to parathyroid hormone Aptamers  Affinity testing and structure determination of candidates

In order to select DNA aptamers that bind optimally to parathyroid hormone, ssDNA aptamers were prepared using a clone having an aptamer sequence obtained in Example 5. The aptamers having the respective sequences were prepared in the same concentration, and the concentration of the recovered aptamer was measured by reproducing SELEX using nanodrops.

 As a result, DNA aptamers of SEQ ID NOs: 9, 14, and 16 were found to have higher concentrations than other DNA aptamers (see FIG. 3), and the structure for these selected aptamers was provided by Rensselear polytechnic institute. The structure was confirmed by imaging using the DNA mfold program (see FIG. 4).

<Example 7>

Detection of Parathyroid Hormone Binding DNA Aptamers by SPR

DNA binding to parathyroid hormone Aptamers  Fabrication Experiment of Parathyroid Hormone Protein Coated Sensor Chip CM5 for Each Affinity Test of Candidates

In order to quantify the affinity between the parathyroid hormone and the three parathyroid hormone protein-binding DNA aptamer candidate groups identified in Example 6, the present inventors have used surface plasma resonance using SIA detection system BIAcore X100 (BIACORE). Resonance (SPR) experiment was performed.

The affinity confirmation of the parathyroid hormone protein and three parathyroid hormone protein binding DNA aptamer candidate groups was carried out using the sensor chip CM5 (GE Healthcare) whose surface was coated with a carboxyl group. The sensor chip CM5 is activated by flowing 0.1M N-hydroxysuccinimide (NHS) and 0.4M N-ethyl-N '-(dimethyl aminopropyl) carbodiimide (EDC) mixture into the chip for 10 minutes at a rate of 5 μl / min to activate the carboxyl group. This better N-hydroxysuccinimide ester was converted to parathyroid hormone protein dissolved in 10 mM NaOAc (pH4.0) to coat the parathyroid hormone protein on the surface of the sensor chip CM5 activated with NHS-ester. Was treated for 7 minutes at a rate of 35 μl / min to fix parathyroid hormone protein on the chip surface. The sensor chip to which the parathyroid hormone protein was immobilized was inactivated with 1 M ethanolamine hydrochloride (pH 8.5) to prevent other reagents and ssDNA aptamers from sticking directly to the chip, and the rate variable was obtained by the BIA evaluation program (BIACORE). . A sensor chip CM5 schematic coated with parathyroid hormone protein is shown in FIG. 5.

<7-2> Evaluation of Affinity with Target Protein of Optimal Parathyroid Hormone Protein Binding DNA Aptamer

For quantitative evaluation of the parathyroid hormone binding aptamers PTH9 (SEQ ID NO: 9), PTH14 (SEQ ID NO: 14), and PTH16 (SEQ ID NO: 16), the ssDNA aptamer of each candidate group was HBS-EP buffer (GE Healthcare). Parathyroid hormone protein-binding DNA aptamer group prepared by dissolving at various concentrations (300nM, 200nM, 100nM, 50nM, 10nM, 0nM), and the sensor chip (channel 1) and parathyroid hormone protein are fixed. by injecting in sequence a (CH 2) a sensor chip, the present inventors have analyzed a PTH protein binding DNA aptamer candidates and parathyroid hormone dissociation constant (K _D, dissociation) between the protein, i.e. affinity. Dissociation constant ( KD _, dissociation) analysis of the parathyroid hormone protein binding DNA aptamer candidate group is shown in Table 2 below.

DNA Aptamer Dissociation Constant (K _D ) M PTH9 1.435 × 10 ^-9 PTH14 3.622 × 10 ^-11 PTH16 8.895 × 10 ^-10

<Example 8>

Optimal Parathyroid Hormone Protein Binding DNA Aptamer digoxigenin  Dig modification and HRP (Horseradish peroxidase) Attached Digoxigenin Binding DNA Aptamer

<8-1> digoxigenin modification of optimal parathyroid hormone protein-binding DNA aptamers

In order to confirm the parathyroid hormone protein detection ability of the three optimal parathyroid hormone protein-binding DNA aptamers selected in Example 7, it was synthesized by IDT (Integrated DNA Technologies, America) and 5 of the optimal parathyroid hormone protein-binding DNA aptamers. 'Digoxigenin (Dig) attached to the end. The synthesized optimal parathyroid hormone protein-binding DNA aptamers were suspended in 1 × PBS (pH 7.2) buffer and adjusted to the same concentration (190 ng / μl).

Dispense 50 µl of optimal parathyroid hormone-binding DNA aptamer prepared at the same concentration, boil it at 85 ° C for 5 minutes, denature it, and cool it slowly at room temperature to form a stable three-dimensional structure of ssDNA aptamer. I was.

<8-2> HRP Attachment Experiment of Dig-bound Dig-1 Aptamer

In order to proceed with HRP attachment of Dig-1 aptamer with Dig, Dig binding Dig-1 aptamer was obtained by requesting synthesis to IDT (Integrated DNA Technologies, America). Afterwards, HRP modification of Dig-1 aptamer conjugated with Dig was performed using a Horseradish peroxidase (HRP) conjugation kit (Micro) sold by Alpha diagnostics (USA). Dig-1 aptamer was used to create an environment for transforming aptamer to HRP using 1X conjugation buffer (0.1M NaHCO3, pH 9.5), and then reacted for 3 hours at 4 ° C. after adding a preactivated HRP solution. After the reaction was completed, the blocking buffer was added 200 ul per 1ml of the sample, and then reacted at room temperature for 1 hour or more to complete the HRP transformation process. After dialysis with PBS at 4 ℃, stabilizing buffer was added to 1 ml per 500 ul of sample to ensure sample stability.

Example 9

Parathyroid hormone specific binding DNA Aptamers  And Dig binding Aptamer  Detection Experiments on Recombinant Parathyroid Hormone and Parathyroid Hormone Overexpressing Cells

<9-1> Optimal Parathyroid Hormone Protein Binding DNA Attached with Dig Aptamers and HRP  Attached Dig Binding DNA Aptamer  Detection of parathyroid hormone

The parathyroid hormone protein prepared in Example 1 was loaded into SDS-PAGE (15%) in three portions, and then transferred to nitrocellulose membrane (GE Healthcare, Sweden) using Mini Trans Blot Cell of Bio rad (USA). Parathyroid hormone-bound membranes were washed three times for 10 minutes using 1X TBS-T buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% (v / v) Tween 20). Then, the membrane was blocked using a blocking buffer (5% bovine serum albumin, 1X TBS-T (pH 7.2)) at 4 ℃ for 1 hour to block the reaction with nonspecific proteins. Thereafter, the plate was washed three times for 10 minutes using TBS-T buffer. In Example <8-1>, 4 µl of 26 μl of an optimal parathyroid hormone-binding DNA aptamer having a stable tertiary structure was attached. After adding to blocking buffer (5% bovine serum albumin, 1X TBS-T (pH 7.2)), reaction was performed with parathyroid hormone-bound membrane for 3 hours at 4 ℃ for optimal parathyroid hormone-binding DNA app. Induced binding of the parathyroid hormone attached to the tammer and the membrane. Thereafter, washing was performed six times for 20 minutes using TBS-T buffer, and 4 mL of blocking buffer (5%) of the HRP-attached Dig binding dig-1 aptamer prepared in Example <8-2> was used. bovine serum albumin, 1X TBS-T (pH 7.2)), followed by reaction at 4 ° C. for 12 hours, where the dig- and RP-linked dig-1 aptamers at the ends of the optimal parathyroid hormone-binding DNA aptamers were attached. Of binding was induced. Finally, washing was performed 12 times for 20 minutes using TBS-T buffer, and Western blotting detection kit (Advansta, USA) was used to confirm the signal for each reaction. It was confirmed that the signal can be obtained at the target size of the parathyroid hormone (see FIG. 6).

<9-2> Optimal Parathyroid Hormone Protein Binding DNA Attached with Dig Aptamers and HRP  Attached Dig Binding DNA Aptamer  Detection of parathyroid hormone

Whole cell activated protein was obtained to perform parathyroid hormone identification experiments from parathyroid hormone overexpressing cells using DNA aptamers and HRP modified Dig binding aptamers. In this case, the whole cell active protein means a protein of the whole cells contained in the cell lysate in which the parathyroid hormone overexpressing cells are lysed using the lysis buffer. The whole cell-activated protein of the parathyroid hormone-overexpressed cells obtained above was confirmed by SDS-PAGE (15%), and the protein identified through SDS-PAGE was determined by using Nitro Mini Blot Cell of Bio rad (USA). Transfer to nitrocellulose membrane (GE Healthcare, Sweden). Parathyroid hormone-bound membranes were washed three times for 10 minutes using 1X TBS-T buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% (v / v) Tween 20). Then, the membrane was blocked using a blocking buffer (5% bovine serum albumin, 1X TBS-T (pH 7.2)) at 4 ℃ for 1 hour to block the reaction with nonspecific proteins. Thereafter, washing was performed three times for 10 minutes using TBS-T buffer. In Example <8-1>, 26 μl of an optimal parathyroid hormone-binding DNA aptamer with 4 digs forming a stable tertiary structure was added. After adding to ㎖ blocking buffer (5% bovine serum albumin, 1X TBS-T (pH 7.2)), reacted with parathyroid hormone-bound membrane for 3 hours at 4 ℃ for optimal parathyroid hormone binding. The binding of DNA aptamers and parathyroid hormones attached to the membrane was induced. Thereafter, washing was performed six times for 20 minutes using TBS-T buffer, and 4 mL of blocking buffer (5%) was applied to the HRP-attached Dig-bonded dig-1 aptamer prepared in Example <8-2>. bovine serum albumin, 1X TBS-T (pH 7.2)), followed by reaction at 4 ° C. for 12 hours, where the dig- and RP-linked dig-1 aptamers at the ends of the optimal parathyroid hormone-binding DNA aptamers were attached. Of binding was induced. Finally, washing was performed 12 times for 20 minutes using TBS-T buffer, and then visualized using each Western blotting detection kit (Advansta, USA).

As a result, as shown in Figure 7, it was found that the signal of the parathyroid hormone can be detected at 10 ~ 15 kDa by using the aptamer-based aptamer blotting technique.

Therefore, the present inventors can easily and accurately detect the parathyroid hormone protein when using the DNA aptamer having the parathyroid hormone-specific overlap ability prepared in the present invention, and accurately measure the quantification of the parathyroid hormone protein in the blood. It can be seen that it can be effectively used to diagnose and test diseases caused by parathyroid hormone.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

<110> Industry Academic Cooperation Foundation of Chungbuk National University and Ewha Womans University <120> DNA Aptamer Specific Binding to Prathyroid Hormone and Uses          Thereof <130> NPDC68492 <160> 26 <170> KoPatentIn 3.0 <210> 1 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PTH1 DNA aptamer sequence <400> 1 cactcgtcga agctttactt tccatagcac tcggtttatg 40 <210> 2 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PTH2 DNA aptamer sequence <400> 2 ccctgcctta tccattaata aacctatatt acatctacgg 40 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PTH3 DNA aptamer sequence <400> 3 cgaagtgagc atgttggctg tattttacgc gttttacccg 40 <210> 4 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PTH4 DNA aptamer sequence <400> 4 ccgttaaatc tacatgttca ttatcctgtt aagagcgtgg 40 <210> 5 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PTH5 DNA aptamer sequence <400> 5 gcccgtcact attaacttga tatttattaa ccgccctacg 40 <210> 6 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PTH6 DNA aptamer sequence <400> 6 ccaccacgtc tacttattac tagtccgttt cgctcttgta 40 <210> 7 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PTH7 DNA aptamer sequence <400> 7 cactctagtg tattattcat tacttcctca atatgtgtcg 40 <210> 8 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PTH8 DNA aptamer sequence <400> 8 ccctgttaat acacgccatt ataatccatc acttattttg 40 <210> 9 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PTH9 DNA aptamer sequence <400> 9 ccactataac tagcttctat tattaactag agtagtatgg 40 <210> 10 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PTH10 DNA aptamer sequence <400> 10 ccacacggct caattatcct ttatttactt gattttacgg 40 <210> 11 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PTH11 DNA aptamer sequence <400> 11 ccacgatcat atcaacgatt acttgccagc agatttatgg 40 <210> 12 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PTH12 DNA aptamer sequence <400> 12 ccacgtccca ttggtgccag ttaattcaat acgtttttgg 40 <210> 13 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PTH13 DNA aptamer sequence <400> 13 ccacttatac gtcttattat cacgcttatg tttattatgg 40 <210> 14 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PTH14 DNA aptamer sequence <400> 14 cacgaaagat caattacatg cttatcattt tattcattgg 40 <210> 15 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PTH15 DNA aptamer sequence <400> 15 gcacattcct gactctattt agttatatcc cttgttgaca 40 <210> 16 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PTH16 DNA aptamer sequence <400> 16 ccgtgatcac gttttaatct tccactcgct agctgcctcg 40 <210> 17 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PTH17 DNA aptamer sequence <400> 17 ccaatactac tcttacttgt tctaccgtgt tagcttcgtg 40 <210> 18 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PTH18 DNA aptamer sequence <400> 18 ccacagagct catattactt gattctcgac ttcctgcttg 40 <210> 19 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PTH19 DNA aptamer sequence <400> 19 acctgagtag aggataacat taccccgaat ttacatggta 40 <210> 20 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> template DNA sequence (N40) <400> 20 ataccagctt attcaattna gatagtaagt gcaatct 37 <210> 21 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> F primer <400> 21 ataccagctt attcaatt 18 <210> 22 <211> 18 <212> DNA <213> Artificial Sequence <220> R primer (5 'biotinylation) <400> 22 agatagtaag tgcaatct 18 <210> 23 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 9 round ssDNA aptamer F primer <400> 23 ataccagctt attcaatt 18 <210> 24 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 9 round ssDNA aptamer R primer <400> 24 agatagtaag tgcaatct 18 <210> 25 <211> 84 <212> PRT <213> Artificial Sequence <220> P223 amino acid sequence <400> 25 Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn   1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His              20 25 30 Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser          35 40 45 Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu      50 55 60 Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys  65 70 75 80 Ala Lys Ser Gln                 <210> 26 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> His-tagged PTH amino acid sequence <400> 26 Met Gly His His His His His His Ser Gly Ser Glu Phe Arg Ser Val   1 5 10 15 Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn Ser Met              20 25 30 Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His Asn Phe          35 40 45 Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser Gln Arg      50 55 60 Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu Lys Ser  65 70 75 80 Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys Ala Lys                  85 90 95 Ser gln        

Claims (15)

DNA aptamer consisting of the nucleotide sequence of SEQ ID NO: 14 that specifically binds to parathyroid hormone (PTH) protein. The method of claim 1,
The DNA aptamer is characterized in that specifically binding to the human parathyroid hormone protein or His fusion parathyroid hormone protein, DNA aptamer (aptamer). The method of claim 1,
The DNA aptamer is characterized in that it further comprises a label, DNA aptamer (aptamer). The method of claim 3, wherein
The labeling material may be any one labeling material selected from the group consisting of a fluorescent material, an amine group, a biotin, a thiol group, and digoxigenin, labeled at the 5 'end or 3' end of the DNA aptamer. DNA aptamers. Claim 1 composition for detecting parathyroid hormone (parathyroid hormone, PTH) comprising the DNA aptamer (aptamer) as an active ingredient. Parathyroid hormone (parathyroid hormone, PTH) detection kit comprising the composition of claim 5 as an active ingredient. The chip for detecting parathyroid hormone (parathyroid hormone, PTH), characterized in that the DNA aptamer of claim 1 is immobilized on a substrate to specifically react to a sample containing parathyroid hormone protein. (1) contacting the biological sample isolated from the living body with the DNA aptamer of claim 1; And
(2) measuring the presence or content of parathyroid hormone in the biological sample through a parathyroid hormone (PTH) protein specific binding reaction between the biological sample and the DNA aptamer;
Method for detecting parathyroid hormone proteins. Claim 1 diagnostic composition for parathyroid hormone-related diseases comprising the DNA aptamer (aptamer) as an active ingredient. The method of claim 9,
The disease is characterized in that selected from the group consisting of hypocalcemia, hyperparathyroidism, parathyroidism, parathyroidism-related calcium hyperemia, malignant calcium hyperemia, renal failure, urolithiasis and hypertension, parathyroid hormone A composition for diagnosing related diseases. delete delete (1) contacting the biological sample isolated from the living body with the DNA aptamer of claim 1; And
(2) measuring the presence or content of parathyroid hormone in the biological sample through a parathyroid hormone (PTH) protein specific binding reaction between the biological sample and the DNA aptamer;
Method of providing information necessary for the diagnosis of parathyroid hormone-related diseases. The method of claim 13,
The disease is characterized in that selected from the group consisting of hypocalcemia, hyperparathyroidism, parathyroidism, parathyroidism-related calcium hyperemia, malignant calcium hyperemia, renal failure, urolithiasis and hypertension, parathyroid hormone Method of providing information necessary for the diagnosis of related diseases. The microarray for detecting parathyroid hormone (PTH), characterized in that the DNA aptamer of claim 1 is immobilized on a substrate to specifically react to a sample containing parathyroid hormone protein.

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