WO2010018870A1 - 動脈硬化診断用ポリペプチドマーカー、及び該マーカー等を用いる動脈硬化の検出方法、並びに動脈硬化診断用キット - Google Patents
動脈硬化診断用ポリペプチドマーカー、及び該マーカー等を用いる動脈硬化の検出方法、並びに動脈硬化診断用キット Download PDFInfo
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5302—Apparatus specially adapted for immunological test procedures
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/323—Arteriosclerosis, Stenosis
Definitions
- the present invention relates to a polypeptide marker for diagnosing arteriosclerosis, a gene marker for diagnosing atherosclerosis, an antibody, a probe for detecting an atherosclerotic marker gene, a DNA microarray or DNA chip for detecting an atherosclerotic marker gene, a method for detecting atherosclerosis, and an artery
- the present invention relates to a curing diagnosis kit.
- Arteriosclerosis is a disease that occurs frequently in the aorta, coronary artery, cerebral artery or carotid artery and is the main cause of myocardial infarction and cerebral infarction. At present, the existence of atherosclerosis is said to be important for the basis of the onset of ischemic organ diseases such as ischemic heart disease and cerebrovascular disorder, which are the top causes of death.
- the pathomorphological features of arteriosclerotic lesions include fatty linears with accumulated cholesterol esters (foamed cells) in the subendothelium, and infiltration of advanced smooth muscle cells, macrophages, T cells, etc. It is in a fibrous plaque with cell necrosis and fat accumulation.
- Non-Patent Document 1 The site where fat accumulates shows structural fragility, the hemodynamic force triggers, the hard spot breaks down, and a thrombus is rapidly formed by the reaction between tissue factor and blood coagulation factor. It has been clarified that the occurrence of thrombotic occlusion due to the collapse of a hard spot in the coronary artery is closely related to the development of so-called acute coronary syndromes such as acute myocardial infarction, unstable angina pectoris, and sudden cardiac death (Non-Patent Document 1).
- Arteriosclerosis progresses gradually without subjective symptoms and suddenly attacks myocardial infarction, cerebral infarction, angina pectoris, etc., so early detection is necessary.
- diagnosis of arteriosclerotic lesions has been widely performed by ultrasonic examination, angiography, MRI (nuclear magnetic resonance imaging apparatus) imaging examination, electrocardiogram, electroencephalogram measurement, etc.
- MRI magnetic resonance imaging apparatus
- electrocardiogram electroencephalogram measurement
- Diagnosis of arteriosclerotic lesions by biochemical examination is related to accumulation of vascular wall lipid such as LDL (low density lipoprotein), lipoprotein (Lp- ⁇ ), remnant lipoprotein, oxidized LDL in serum or plasma
- LDL low density lipoprotein
- Lp- ⁇ lipoprotein
- remnant lipoprotein oxidized LDL in serum or plasma
- CRP C-reactive protein
- the following methods are known as methods for diagnosing arteriosclerotic lesions by measuring these atherosclerosis-induced lipoproteins.
- serum or plasma neutrophils such as lactoferrin, myeloperoxidase, or granulocyte elastase that form a complex with oxidized LDL present in serum or plasma, monocytes / macrophages, etc.
- Patent Document 1 For measuring the inflammatory cell-derived component of an animal by immunological methods (Patent Document 1), immunology using a fusion polypeptide comprising an extracellular region of oxidized LDL receptor and a constant region of an immunoglobulin heavy chain Using a conventional assay method (Patent Document 2), using an anti-human aldehyde-modified ⁇ 1 antitrypsin monoclonal antibody that specifically recognizes oxidized LDL and ⁇ 1 antitrypsin complex (Patent Documents 3 and 4), A method comprising measuring the degree of oxidative modification of LDL with an oxidizing agent comprising an azo compound such as V-70 ( Patent document 5) etc. are shown.
- Patent Document 11 Diagnosis by measuring VII coagulation factor-active protease (FSAP) as a risk factor for atherosclerosis (Patent Document 12), vascular endothelial cell / smooth muscle cell-derived neuropilin-like molecule (ESDN), and its gene
- FSAP VII coagulation factor-active protease
- ESDN vascular endothelial cell / smooth muscle cell-derived neuropilin-like molecule
- Patent Document 13 For detecting arteriosclerosis by detecting the expression of erythrocytes (Patent Document 13), using anti-human hepatic triglyceride lipase antibody (Patent Document 14), and diagnosing arteriosclerotic lesions using serotonin concentration in plasma samples as a marker (Patent Documents 15 and 16) and the like are shown.
- the MAD isoform sMAD3 polypeptide required for signaling of DPP a member of the TGF-family of cytokines / growth factors, chronic renal failure, Diagnosis of atherosclerosis (patent document 17), measurement of a complex of Lp- ⁇ and ⁇ 2-macroglobulin / interleukin 6 in blood by an immunological method using the antibody (Patent document 18), using a monoclonal antibody against paraoxonase (patent document 19), assaying arteriosclerosis by measuring saturated very long fatty acid (patent document 20), RC-9 protein, and A technique for diagnosing atherosclerosis using the antibody (Patent Document 21) is shown.
- Patent Document 22 as a marker for specifically detecting a lesion of arteriosclerosis, a polypeptide marker for diagnosing arteriosclerosis, a gene marker for diagnosing arteriosclerosis, and a polypeptide marker for diagnosing arteriosclerosis are specifically mentioned.
- An antibody to be bound, a probe for detecting a genetic marker for diagnosis of arteriosclerosis, and a method for detecting an arteriosclerotic lesion are shown.
- JP 2002-48790 A JP 2002-17353 A Japanese Patent Laid-Open No. 2000-184885 Japanese Patent Laid-Open No. 10-142226 JP-A-9-203736 JP 2002-181820 A JP 2002-142781 A JP 2002-55106 A Japanese Patent Laid-Open No. 11-346782 JP-A-8-160042 JP 2003-310281 A JP 2003-304873 A JP 2003-189872 A JP 2002-308900 A JP 2002-277461 A JP 2002-131313 A JP 2002-238589 A JP 2001-249128 A Japanese Patent Laid-Open No. 2000-333694 JP 2000-206113 A Special table 2000-503764 gazette JP 2005-168498 A
- arteriosclerotic lesions can be detected by a simple operation, and early detection of arteriosclerosis can be expected, but it is not sufficient. It is desired to detect arteriosclerotic lesions with higher accuracy using more arteriosclerosis diagnosis polypeptide markers, arteriosclerosis diagnosis gene markers, and the like. Therefore, the present invention provides a polypeptide marker for diagnosing arteriosclerosis, a genetic marker for diagnosing arteriosclerosis, an antibody, a probe for detecting an arteriosclerosis marker gene, and for detecting an arteriosclerosis marker gene. It is an object of the present invention to provide a DNA microarray or DNA chip, a method for detecting arteriosclerosis, and a kit for diagnosing arteriosclerosis.
- the present inventor conducted a search for proteins expressed in the serum of patients suffering from arteriosclerotic lesions in order to further find a marker for diagnosing arteriosclerosis that can specifically detect arteriosclerotic lesions.
- a novel polypeptide marker for diagnosing arteriosclerosis that can specifically detect arteriosclerotic lesions has been found, and the present invention has been completed.
- sera of patients suffering from arteriosclerotic lesions who were admitted to hospitals and cooperating facilities were screened as follows with the consent of the person or family.
- the obtained cDNA clone was incorporated into the plasmid pBluescriptII and the nucleotide sequence was determined.
- the plasmid was recombined with the plasmid pGEX, introduced into Escherichia coli, and the protein was expressed in a large amount by IPTG (isopropyl ⁇ -D-thiogalactoside) to prepare a protein extract.
- IPTG isopropyl ⁇ -D-thiogalactoside
- the protein extract was solid-phased in a 96-well plate, and the antibody serum level was measured by the ELISA method in the arteriosclerosis patient group and the healthy subject group, and the significant difference was examined from the measured value. A significant difference was observed between the patient group and the healthy subject group for the 13 clones as markers.
- the reaction between the protein extract and a number of patient sera was examined by Western blotting.
- the present invention comprises the following polypeptide markers for diagnosing arteriosclerosis, genetic markers for diagnosing arteriosclerosis, antibodies, probes for detecting atherosclerotic marker genes, DNA microarrays or DNA chips for detecting atherosclerotic marker genes, and arteriosclerosis A detection method and an arteriosclerosis diagnostic kit are included.
- a gene marker for diagnosing arteriosclerosis comprising a gene represented by the amino acid sequence of the polypeptide marker for diagnosing arteriosclerosis according to [1] or a base sequence encoding a partial amino acid sequence thereof.
- Arteriosclerosis marker gene detection DNA microarray wherein the arteriosclerosis marker gene detection probe according to [5] and / or the arteriosclerosis marker gene detection probe according to [6] is immobilized on a substrate, or DNA chip.
- [8] A method for detecting arteriosclerosis, wherein the antibody according to [4] is used and the expression of an arteriosclerosis marker polypeptide that specifically binds to the antibody is detected in a test sample.
- [10] Detection of arteriosclerosis using the probe for detecting an arteriosclerosis marker gene according to [5] or [6], and detecting the expression of a gene that hybridizes with the probe for detecting the atherosclerosis marker gene in a test cell Method.
- a method for detecting arteriosclerosis comprising constructing a primer, performing PCR using the primer, and detecting the expression of an atherosclerotic marker gene.
- a kit for diagnosing arteriosclerosis comprising at least one selected from the group consisting of:
- Polypeptide marker for diagnosing arteriosclerosis gene marker for diagnosing arteriosclerosis of the present invention, antibody, probe for detecting atherosclerotic marker gene, DNA microarray or DNA chip for detecting atherosclerotic marker gene, method for detecting arteriosclerosis, and arteriosclerosis
- the diagnostic kit for detecting an arteriosclerotic lesion the detection can be performed with higher accuracy.
- the polypeptide marker for diagnosing arteriosclerosis and the gene marker for diagnosing arteriosclerosis of the present invention are specifically expressed in arteriosclerotic lesions, they can be used for detection of arteriosclerosis.
- the polypeptide serving as the polypeptide marker for arteriosclerosis diagnosis of the present invention is SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 in the sequence listing. Or a partial amino acid sequence thereof.
- the partial amino acid sequence is a partial sequence of the amino acid sequences shown in the respective sequence numbers in the sequence listing, and is 4 or more, preferably 5 or more, more preferably 6 or more, still more preferably It is a sequence consisting of 7 or more amino acids.
- amino acid sequence information of the polypeptide of the present invention is as follows: Accession numbers: XM_001129783 (SEQ ID NO: 1), NM_006088 (SEQ ID NO: 3), NM_014878 in NCBI gene database as of July 31, 2008 (2008).
- the gene marker for diagnosing arteriosclerosis of the present invention comprises a gene represented by a base sequence encoding a polypeptide having the amino acid sequence and a partial amino acid sequence thereof.
- the gene of the gene marker for diagnosing arteriosclerosis may be a gene having a base sequence capable of expressing the polypeptide.
- Examples of the gene serving as a genetic marker for arteriosclerosis diagnosis according to the present invention are the genes of the following clones, and SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 in the sequence listing. , 22, 24, 26, 28, 30, 32, 34 or 36, or a gene having a partial base sequence thereof.
- Each of these gene information can be approached by each accession number described below in NCBI gene database.
- LIMS1 is an adapter protein containing five LIM domains or double zinc fingers. LIMS1 may also act on cell adhesion or spreading mediated by integrins.
- the partial base sequence is a partial sequence of the base sequences shown in the respective sequence numbers in the sequence listing, and is 12 or more, preferably 15 or more, more preferably 18 or more, and still more preferably 20 It is a sequence consisting of more than one base (hereinafter the same). Table 1 summarizes the genes that serve as the aforementioned genetic markers for diagnosing arteriosclerosis.
- the antibody of the present invention is an antibody that specifically binds to the polypeptide having the polypeptide that serves as a polypeptide marker for arteriosclerosis diagnosis as an antigen.
- the antibody may be a monoclonal antibody or a polyclonal antibody. These antibodies can be produced by a conventional method using the polypeptide as an antigen.
- the antibody of the present invention may be labeled with a labeling substance.
- a labeling substance an enzyme, a radioisotope, a fluorescent dye, biotin, digoxygenin, or the like can be used.
- the enzyme is not limited as long as it satisfies conditions such as a large turner number (rotation number), stability in a state where it is bound to an antibody, and the ability to specifically color a substrate.
- EIA Peroxidase normal EIA Peroxidase, ⁇ -galactosidase, alkaline phosphatase, glucose oxidase, acetylcholinesterase, glucose-6-phosphate dehydrogenase, malate dehydrogenase and the like used in (enzyme immunoassay) can be used.
- an enzyme inhibitor, a coenzyme, etc. can also be used.
- These enzymes and antibodies can be bound by a known method using a crosslinking agent such as a maleimide compound.
- a known substance can be used according to the type of enzyme used.
- radioisotope As a radioisotope, what is used by normal RIA (radioimmunoassay), such as 125 I and 3 H, can be used.
- fluorescent dye those used in usual fluorescent antibody methods such as fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) can be used.
- these labeled antibodies may be bound with metals such as manganese and iron.
- metals such as manganese and iron.
- the arteriosclerosis marker gene detection probe of the present invention is a probe having all or part of DNA that hybridizes under stringent conditions with the gene serving as the above-described gene marker for arteriosclerosis diagnosis.
- stringent conditions in the present invention are, for example, 50% formamide, 5 ⁇ SSC, 5 ⁇ Denhardt's solution, 0.1 M sodium dihydrogen phosphate (pH 6.5), 0.5% SDS, Hybridization at 42 ° C.
- condition 2 a cleaning process
- Condition 2 the latter (Condition 2) is more preferable.
- stringency of hybridization there are various factors that influence the stringency of hybridization. Those skilled in the art can combine various elements to obtain a string equivalent to the above-described hybridization stringency. It is possible to realize a genie.
- the probe for detecting an atherosclerotic marker gene of the present invention includes SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, Examples include probes having all or part of antisense DNA for the gene represented by the nucleotide sequence represented by 32, 34 or 36. That is, it is a probe comprising a DNA having a base sequence complementary to the base sequence shown in the sequence number or a partial base sequence thereof.
- These arteriosclerosis marker gene detection probes may be appropriately labeled with a fluorescent label or the like.
- the DNA microarray or DNA chip for detecting the atherosclerotic marker gene of the present invention is used for detecting the expression of a gene that serves as the genetic marker for diagnosing atherosclerosis, and for diagnosing an arteriosclerotic lesion by the detection.
- the above-mentioned probe for detecting atherosclerosis marker gene is immobilized. Only one of the above-described probes for detecting an arteriosclerosis marker gene may be fixed, or two or more probes may be used.
- the DNA microarray or DNA chip can be manufactured by a conventional method.
- the DNA microarray can be obtained, for example, by spotting a solution containing each of the arteriosclerosis marker gene detection probes prepared in advance on a substrate and drying it.
- the DNA chip can be obtained, for example, by synthesizing DNA having a desired base sequence on a substrate by photolithography.
- the arteriosclerosis detection method of the present invention uses the above-mentioned polypeptide marker for diagnosing arteriosclerosis, and an antibody (arteriosclerosis marker antibody) that specifically binds to the polypeptide marker for diagnosing arteriosclerosis in the blood of a subject.
- This is a method for detecting expression (hereinafter referred to as “detection method (1)”).
- the arteriosclerosis marker antibody is induced by the expression of a polypeptide serving as a polypeptide marker for arteriosclerosis diagnosis.
- Arteriosclerosis can be detected by detecting a sclerosing marker antibody.
- serum obtained from a subject is brought into contact with a solid-phased polypeptide marker for arteriosclerosis diagnosis, an antibody in the serum is bound, and the unbound protein is removed.
- a labeled antibody secondary antibody
- a method of detecting the signal of the labeled antibody, and the like can be mentioned.
- the labeling substance for the labeled antibody the enzymes, radioisotopes, fluorescent dyes, biotin, digoxygenin, and the like mentioned for labeling the antibody of the present invention can be used.
- the amount of antibody can be calculated by comparing with Alternatively, a substrate that emits light by enzymatic action can be added, and measurement can be performed with high sensitivity using a weak luminescence measuring device (luminometer).
- a radioisotope is used as the labeling substance
- the amount of antibody can be calculated by measuring the radiation dose emitted from the radioisotope with a scintillation counter or the like.
- the amount of antibody can be calculated by measuring the amount of fluorescence with a measuring device equipped with a fluorescence spectrometer.
- Western blotting can be used.
- polypeptide conjugates for diagnosing arteriosclerosis, antibodies in the subject's blood, and conjugates of labeled antibodies can be separated by known separation means (chromatography, salting out, alcohol precipitation, enzymatic method, in-phase method, etc.) After the separation, the signal of the labeled antibody may be detected.
- an arteriosclerosis marker antibody is present in the blood of a subject obtained from the subject, and at least one kind of the arteriosclerosis marker antibody is present.
- the subject can be determined as a patient suffering from arteriosclerotic lesion or as a high-risk person suffering from arteriosclerotic lesion.
- the method for detecting arteriosclerosis uses the above-described probe for detecting an arteriosclerosis marker gene, and in a test cell, expresses a gene (arteriosclerosis marker gene) that hybridizes with the probe for detecting the arteriosclerosis marker gene.
- This is a detection method (hereinafter referred to as “detection method (2)”). Since arteriosclerosis marker gene is expressed in test cells obtained from patients suffering from arteriosclerotic lesions, arteriosclerosis is detected by detecting the expression of the arteriosclerosis marker gene with a probe for detecting arteriosclerosis marker gene Can do.
- the detection method (2) comprises preparing a probe for detecting an arteriosclerosis marker gene having a chain length suitable for hybridization having the base sequence as described above, and appropriately assigning a label such as a fluorescent label, Examples include a method in which this is brought into contact with a sample obtained from a test cell, a hybridization reaction is performed, and the expression of a gene serving as a gene marker for arteriosclerosis diagnosis is detected.
- the detection method (2) a known detection method can be used except that the probe for detecting an arteriosclerosis marker gene of the present invention is used.
- a Northern blot method can be used.
- any of the aforementioned arteriosclerosis marker gene detection probes may be used, or a plurality of types of arteriosclerosis marker gene detection probes may be used. It may be.
- a DNA microarray or a DNA chip in which the atherosclerosis marker gene detection probe is immobilized on a substrate may be used.
- PCR Polymerase Chain Reaction
- RT-PCR reverse transcription PCR
- real-time RT-PCR method a primer comprising a sense primer and an antisense primer for amplifying the arteriosclerosis marker gene is used.
- the primer is constructed by the base shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 or 36 in the sequence listing. This can be appropriately performed based on the arrangement.
- the detection sensitivity can be improved by performing the quantitative or semi-quantitative PCR to amplify the atherosclerotic marker gene and then detecting the expression of the atherosclerotic marker gene using the sample. it can.
- the amount of detection is higher than that of a healthy person by detecting an arteriosclerosis marker gene in a subject and comparing it with the result of a healthy person detected by the same method.
- Many subjects can be determined as patients suffering from arteriosclerotic lesions or high-risk individuals with arteriosclerotic lesions.
- the method for detecting arteriosclerosis according to the present invention uses an antibody whose antigen is a polypeptide serving as a polypeptide marker for arteriosclerosis diagnosis according to the present invention, and a polypeptide that specifically binds to the antibody in a test sample ( This is a method for detecting the expression of an arteriosclerosis marker polypeptide (hereinafter referred to as “detection method (3)”).
- detection method (3) a method for detecting the expression of an arteriosclerosis marker polypeptide.
- arteriosclerosis marker polypeptide In a test sample obtained from a patient suffering from arteriosclerotic lesion, arteriosclerosis marker polypeptide is expressed. Therefore, arteriosclerosis is detected by detecting the arteriosclerosis marker polypeptide using the antibody of the present invention. Can do.
- the detection method (3) can be carried out using a known immunoassay method except that the antibody of the present invention is used.
- the immunological measurement method include a Western blot method, an ELISA (Enzyme-Linked Immunosorbent Assay) method, a radioimmunoassay method, and a fluorescent antibody method.
- the antibody used in the detection method (3) may be a monoclonal antibody or a polyclonal antibody.
- the direct fluorescent antibody method may be used or the indirect fluorescent antibody method may be used.
- an antibody used for detection is fluorescently labeled, and the antibody is contacted and bound to a sample cell as a test sample to label a cell expressing an arteriosclerosis marker polypeptide.
- the indirect fluorescent antibody method is a method in which an unlabeled antibody of the present invention is contacted and bound to a sample cell, and then a secondary antibody (anti-immunoglobulin antibody) labeled with the antibody is further bound. This is a method for labeling cells expressing an atherosclerotic marker polypeptide.
- the arteriosclerosis marker polypeptide is directly expressed using TOF-MASS.
- a detection method or a method using a chip in which a protein that interacts with an arteriosclerosis marker polypeptide is immobilized on a substrate may be used.
- an arteriosclerosis marker polypeptide is present in a test sample obtained from a subject, and one or more types of the arteriosclerosis marker polypeptide are used.
- An existing subject can be determined as a patient suffering from an arteriosclerotic lesion or a high-risk person atherosclerotic lesion.
- Arteriosclerosis marker gene detection probe used for detection of arteriosclerosis lesion of the present invention arteriosclerosis marker gene detection DNA microarray or DNA chip to which the arteriosclerosis marker gene detection probe is fixed, and arteriosclerosis marker poly of the present invention
- the antibody for peptide detection can be commercialized as an arteriosclerosis diagnosis kit including one or more of them and capable of detecting and diagnosing the above-mentioned arteriosclerosis.
- the polypeptide marker for diagnosing arteriosclerosis of the present invention can also be commercialized as an arteriosclerosis diagnostic kit that can detect and diagnose arteriosclerosis.
- the polypeptide marker for diagnosing arteriosclerosis By using the polypeptide marker for diagnosing arteriosclerosis, the genetic marker for diagnosing arteriosclerosis, the antibody, and the probe for detecting the arteriosclerotic marker gene of the present invention described above in combination with those of Patent Document 22, detection of arteriosclerosis is further enhanced. Can be done with precision.
- Example preparation For patients with carotid artery stenosis who visited hospitals and research cooperation facilities, the gender and age ( ⁇ 5 years) were matched from outpatients, and normal subjects were selected as criteria (control). At the time of outpatient visit or at the time of hospitalization for neurosurgery, 1) Explain the purpose of the study and the voluntary nature of research cooperation to the family, and obtain informed consent. 2) Family history, medical history, drinking, smoking, etc. Interviews were conducted regarding lifestyle and working mode. 3) Blood was collected and serum and blood cell components were separated and stored frozen.
- the plasmid was purified using Plasmid Miniprep kit (Sigma). 9) The base sequence of the obtained clone was determined by a sequencer, homology search with a public database was performed, the gene was identified, and it was used as an antigen protein candidate.
- ELISA method (4) ELISA Method as Secondary Screening
- pBluescript containing an antigen protein candidate insert was recombined into a protein expression and purification vector pGEX-4T (Amersham Bioscience).
- Select restriction enzymes suitable for recombination from pBluescript to pGEX-4T from the obtained base sequence information, and treat with restriction enzymes such as BamH I, Sal I, Not I, EcoR I, Xho I, and Sma I. It was.
- restriction enzymes such as BamH I, Sal I, Not I, EcoR I, Xho I, and Sma I. It was.
- the target band was excised using the purification kit GeneElute Minus EtBr Spin Columns (SIGMA), and the restriction enzyme-treated insert and pGEX-4T were recovered.
- SIGMA GeneElute Minus EtBr Spin Columns
- the nucleotide sequence of the detected clone gene was determined by sequencing. As a result of collating the nucleotide sequence obtained thereby with the nucleotide sequence on the publicly available gene database, the gene of the obtained clone matched the nucleotide sequence of the gene encoding the polypeptide for diagnosis of arteriosclerosis. .
- Example 1 [Measurement of antibody titer by ELISA method] Using the experimental procedure of the above production example, the antibody titer measurement by ELISA method was performed on 13 candidate clones of the detected 18 types of genetic markers for atherosclerosis diagnosis to confirm the increase of serum antibody in arteriosclerosis patients did. The measurement results are shown in Table 2.
- Example 2 [Confirmation of atherosclerosis marker gene expression by Western blotting]
- the expression of the arteriosclerosis marker gene in arteriosclerosis patients was confirmed by Western blotting.
- the detection result positive signal by Western blotting: arrow in the figure
- FIG. 1 (a) is clone name: S16D2 (SEQ ID NO: 22 in the sequence listing)
- FIG. 1 (b) is clone name: S27D3 (SEQ ID NO: 24 in the sequence listing)
- FIG. 1 (c) is clone name: S36E1 (sequence listing).
- FIG. 1 shows the result of clone name: S39D6 (SEQ ID NO: 28 of the sequence listing), and FIG. 1 (e) shows the result of clone name: TS27B2 (SEQ ID NO: 30 of the sequence listing). It is.
- Polypeptide marker for diagnosing arteriosclerosis gene marker for diagnosing arteriosclerosis of the present invention, antibody, probe for detecting atherosclerotic marker gene, DNA microarray or DNA chip for detecting atherosclerotic marker gene, method for detecting arteriosclerosis, and arteriosclerosis
- arteriosclerosis can be detected easily and with high accuracy, and thus can be suitably used for early diagnosis of arteriosclerosis.
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Abstract
Description
本願は、2008年8月15日に、日本に出願された特願2008-209210号に基づき優先権を主張し、その内容をここに援用する。
このように、動脈硬化の病変の診断に関する生化学的検査については多くの方法が示されているが、それらの検出マーカーはリスクマーカーがほとんどである。より本質的である動脈硬化の病変の特異的診断のためには、該病変を特異的に検出できるマーカーの更なる開発が望まれていた。
そこで本発明は、動脈硬化病変の検出の精度をさらに向上させることのできる動脈硬化診断用ポリペプチドマーカー、動脈硬化診断用遺伝子マーカー、抗体、動脈硬化マーカー遺伝子検出用プローブ、動脈硬化マーカー遺伝子検出用DNAマイクロアレイ又はDNAチップ、及び動脈硬化の検出方法、並びに動脈硬化診断用キットを提供することを目的とする。
具体的な探索方法としては、病院、及び協力施設に入院した動脈硬化性病変罹患患者の血清を本人、又は家族の了解を得て下記のようにスクリーニングを行った。得られたcDNAクローンはプラスミドpBluescriptIIに組み込み塩基配列を決定した。その後プラスミドpGEXに組換えて大腸菌に導入し、IPTG(isopropyl β-D-thiogalactoside)により蛋白質を大量発現させて蛋白質抽出液を調製した。次いで、その蛋白質抽出液を96穴プレートに固相化し、動脈硬化患者群と、健常者群でELISA法により抗体血清レベルの測定を行い、その測定値より有意差を調べたところ、本発明のマーカーである13クローンについて、患者群と健常者群の間で有意差が見られた。さらに、蛋白質抽出液と多数の患者血清との反応をウェスタンブロット法により調べた。その結果、本発明のマーカーである5クローンが新たに動脈硬化病変罹患患者血清と陽性反応を示し動脈硬化性病変の存在、もしくは不安定性プラークの特異的マーカーとして有用であることを見い出した。そして、これらのクローンの抗原性や遺伝子のプローブを利用し、動脈硬化の検出方法を開発し、更には該検出方法に用いる診断キットの作製が可能となった。
[2][1]に記載の動脈硬化診断用ポリペプチドマーカーのアミノ酸配列又はその部分アミノ酸配列をコードする塩基配列で示される遺伝子からなる動脈硬化診断用遺伝子マーカー。
[3]配列表の配列番号2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34もしくは36に示される塩基配列、又はその部分塩基配列で示される遺伝子からなる動脈硬化診断用遺伝子マーカー。
[6][3]に記載の遺伝子に対するアンチセンスDNAの全部又は一部を有する、[5]に記載の動脈硬化マーカー遺伝子検出用プローブ。
[9][1]に記載の動脈硬化診断用ポリペプチドマーカーを用い、被験体血中における、該動脈硬化診断用ポリペプチドマーカーに特異的に結合する抗体の発現を検出する動脈硬化の検出方法。
[10][5]又は[6]に記載の動脈硬化マーカー遺伝子検出用プローブを用い、被検細胞における、該動脈硬化マーカー遺伝子検出用プローブとハイブリダイズする遺伝子の発現を検出する動脈硬化の検出方法。
[12][5]又は[6]に記載の動脈硬化マーカー遺伝子検出用プローブ、[7]に記載の動脈硬化マーカー遺伝子検出用DNAマイクロアレイ又はDNAチップ、及び[4]に記載の抗体からなる群から選択される少なくとも1つ以上を具備する動脈硬化診断用キット。
ここで、部分アミノ酸配列とは、前記配列表の各配列番号に示されるアミノ酸配列のうちの一部分の配列であり、4個以上、好ましくは5個以上、より好ましくは6個以上、更に好ましくは7個以上のアミノ酸からなる配列である。
また、部分塩基配列とは、前記配列表の各配列番号に示される塩基配列のうちの一部分の配列であり、12個以上、好ましくは15個以上、より好ましくは18個以上、更に好ましくは20個以上の塩基からなる配列である(以下、同じ。)。
前述の動脈硬化診断用遺伝子マーカーとなる遺伝子を表1にまとめる。
基質としては、使用する酵素の種類に応じて公知の物質を用いることができる。例えば、酵素としてペルオキシダーゼを使用する場合には基質として3,3’,5,5’-テトラメチルベンジシンを用いることができ、酵素としてアルカリフォスファターゼを用いる場合には基質としてパラニトロフェノールを用いることができる。
蛍光色素としては、フルオレセインイソチオシアネート(FITC)やテトラメチルローダミンイソチオシアネート(TRITC)等の通常の蛍光抗体法に用いられるものを使用することができる。
これらの動脈硬化マーカー遺伝子検出用プローブは、適宜蛍光標識等の標識を付与しておいてもよい。
本発明の動脈硬化の検出方法は、前述の動脈硬化診断用ポリペプチドマーカーを用い、被験体血中における、該動脈硬化診断用ポリペプチドマーカーに特異的に結合する抗体(動脈硬化マーカー抗体)の発現を検出する方法(以下、「検出方法(1)」という。)である。動脈硬化病変罹患患者から得られる被験体血中では、動脈硬化診断用ポリペプチドマーカーとなるポリペプチドの発現により前記動脈硬化マーカー抗体が誘導されているため、動脈硬化診断用ポリペプチドマーカーにより該動脈硬化マーカー抗体を検出することにより動脈硬化を検出することができる。
標識物質として酵素を用いる場合は、酵素作用によって分解して発色する基質を加え、該基質の分解量を光学的に測定することにより酵素活性を求め、これを結合抗体量に換算し、標準値と比較することにより抗体量を算出することができる。あるいは酵素作用によって発光する基質を加え、微弱発光測定装置(ルミノメーター)で高感度に測定できる。
標識物質として放射性同位体を用いる場合は、該放射性同位体から発せられる放射線量をシンチレーションカウンター等により測定することにより抗体量を算出することができる。標的物質として蛍光色素を用いる場合は、蛍光分光計を具備する測定装置等により蛍光量を測定することにより抗体量を算出することができる。
また、検出方法(2)では、該動脈硬化マーカー遺伝子検出用プローブが基板に固定化されたDNAマイクロアレイ又はDNAチップを用いてもよい。
このように、前記定量的又は半定量的PCRを行って動脈硬化マーカー遺伝子を増幅した後、その試料を用いて該動脈硬化マーカー遺伝子の発現の検出を行なうことにより、検出感度を向上させることができる。
検出方法(3)に用いる抗体は、モノクローナル抗体であってもポリクローナル抗体であってもよい。
また、本発明の動脈硬化診断用ポリペプチドマーカーについても、前述の動脈硬化の検出及び診断が行なえるような動脈硬化診断用キットとして製品化しておくことができる。
<製造例>
(試料の調製)
(1)病院及び研究協力施設を受診した頚動脈狭窄症患者を対象とし、外来受診者の中から性別・年齢(±5歳)を一致させ、正常被験者を基準(コントロール)として選択した。外来受診時又は脳神経外科入院時に、1)研究目的や研究協力の任意性等を家族に説明し、インフォームド・コンセントを得た上で、2)家族歴・既往歴・飲酒や喫煙等のライフスタイル、作業態様に関する問診を行い、3)採血を行って血清と血球成分を分離凍結保存した。さらに、4)医師の診療録を基に、臨床的重症度、治療経過、血液検査所見等を記載した。解析対象の患者血液は血清分離後マイナス80度にて研究開始まで凍結保存した。抗体及び診療録は暗号匿名化した後、使用した。
(2)血清によるスクリーニング対象は、ヒト毛細血管内皮由来のcDNAライブラリーを組み込んだ市販λZAP IIファージベクター(STRATAGENE)を用いた。
(3)発現クローニング法によるスクリーニングは、St John, T(Science, 231, 845-850, 1986)らの方法に準じて実施した。
1)上記(2)のファージベクターを大腸菌(XL1-Blue)に感染させ、NZYagar培地(φ15cm dish)上で培養した。
2)プラークが出現したのを確認後、IPTG処理したニトロセルロース膜を培地上に載せ、該ニトロセルロース膜にファージ由来蛋白質を発現、転写させた。
3)1%BSA/PBSで2000倍希釈した患者血清とニトロセルロース膜とを一夜インキュベートし、発現蛋白質と血清中抗体(血清IgG)を反応させた。
4)洗浄後、二次抗体としてアルカリフォスファターゼ標識ヤギ抗ヒトIgG抗体をニトロセルロース膜と反応させた。
5)発色試薬としてNBT(nitroblue terazolium)、BCIP(5-bromo-4-chloro-3-indolyl-phosphate)を用いて血清IgG認識クローンを同定した。
6)陽性クローンは二次、三次スクリーニング(φ10cm dish)を行い、偽陽性クローンを排除した。
7)選択されたファージをExAssist helper phage system(Stratagene, La Jolla, CA)によりpBlueScriptに変換した。
8)Plasmid Miniprep kit(Sigma)を用いプラスミドを精製した。
9)得られたクローンはシークエンサーにより塩基配列を決定し、公開されているデーターベースと相同性検索を行い、遺伝子を同定し、抗原蛋白質候補とした。
(4)二次スクリーニングとしてのELISA法
1)抗原蛋白質候補のインサートを含むpBluescriptの、蛋白質発現、精製用ベクターpGEX-4T(Amersham Bioscience)への組換えを行った。得られた塩基配列情報よりpBluescriptからpGEX-4Tへの組換えに適した制限酵素を選択し、BamH I, Sal I, Not I, EcoR I, Xho I, Sma Iなどの制限酵素により処理を行った。
2)アガロースゲル電気泳動後、目的のバンドを精製キットGeneElute Minus EtBr Spin Columns(SIGMA)を用い切り出し、制限酵素処理されたインサート及びpGEX-4Tを回収した。
3)Ligation-Convenience Kit(ニッポンジーン)を用い、前記インサートとpGEX-4Tをライゲーションし、インサートを含むプラスミドを作製した。
4)クローンにより適当な制限酵素が存在しない場合には、PCR法によりインサートを作製した。あらかじめ制限酵素認識部位を持つプライマーを作製し、動脈瘤組織より抽出したRNAを鋳型に逆転写PCRを行ってcDNAを作製し、さらにPCRを行い全長のインサートを得た。以下は同様に制限酵素、ライゲーションキットによる処理を行った。
5)真核細胞蛋白質発現用に改良された大腸菌コンピテントセルBL21(ニッポンジーン)に、得られたpGEX-4Tの組換え体を形質転換した。
6)アンピシリン入りLB培地で培養し、IPTGでインサートDNA由来蛋白質の発現を誘導した。
7)大腸菌を遠心分離回収し、超音波破砕後、可溶画分と不溶画分に分離した。
8)目的蛋白質が不溶画分に入る場合は、尿素を用い可溶化を行った。
9)抗原蛋白質はglutathione-Sepharose(Amersham Pharmacia)を用いて精製した。
10)96穴ELISAプレートに10μg/mlの濃度で抗原蛋白質を注入し、一晩4℃で保存し、固相化させた。
11)PBS洗浄、10%ウシ胎児血清PBS溶液でブロッキング後、患者血清、及び対照血清(健常者の血清)を2000倍希釈し、固相化した蛋白質と反応させた。
12)PBS洗浄後、HRP標識ヤギ抗ヒトIgG抗体を加えた。
13)基質を加えて発色させ、プレートリーダーにてOD490nmの吸収を測定した。
(5)ウェスタンブロット法による二次スクリーニング
1)上記で得られた抗原蛋白質候補のインサートを含むpBluescript又はpGEX-4Tにより形質転換した大腸菌(SOLR、JM109、BL21)をLBアンピシリン(50μg/ml)2mlで1夜培養した。
2)LBアンピシリン20mlに移し換えた後、1時間培養後、IPTGを終濃度1mMとなるように加えたものと、コントロール(IPTGを加えないもの)とをさらに3時間培養した。
3)培養液を遠心後、大腸菌を回収し、SDSサンプルバッファーにて溶解、ウェスタン法のサンプルとした。
4)10%ポリアクリルアミドゲル上で前記サンプルを電気泳動後、転写装置でニトロセルロース膜に蛋白質を転写した。
5)1%スキムミルクでブロッキング後、2000倍希釈した患者血清を一次抗体として加え、1夜インキュベートした。
6)PBST(20 mM Tris-HCl, (pH 7.6), 50 mM NaCl, 0.1% Tween-20)で洗浄後、3万倍希釈HRP標識ヤギ抗ヒトIgG抗体を加え、20分間反応させた。
7)PBSTで洗浄後、発光試薬Immobilon Western(MILLIPORE)を用いて発光させた。
8)フィルムに露光させ現像した。
9)IPTG処理に依存して検出されるバンドを抗原蛋白質と推定した。
検出したクローンの遺伝子(動脈硬化診断用遺伝子マーカー)の塩基配列決定をシークエンシングにより行った。それにより得られた塩基配列を、公開されている遺伝子データーベース上の塩基配列と照合した結果、得られたクローンの遺伝子は、前記動脈硬化診断用ポリペプチドをコードする遺伝子の塩基配列と一致した。
[ELISA法による抗体価の測定]
上記製造例の実験手順を用いて、検出した18種類のうち13種類のクローンの動脈硬化診断用遺伝子マーカー候補について、ELISA法による抗体価の測定を行い、動脈硬化患者における血清抗体の上昇を確認した。その測定結果を表2に示す。
[ウェスタンブロット法による動脈硬化マーカー遺伝子発現の確認]
上記発現クローニング法により同定されたマーカー候補遺伝子のうち実施例1で用いたものを除く5種のクローンについて、ウェスタンブロット法により、動脈硬化患者における動脈硬化マーカー遺伝子の発現を確認した。その検出結果(ウェスタンブロット法による陽性シグナル:図中の矢印)を図1に示す。図1(a)がクローン名:S16D2(配列表の配列番号22)、図1(b)がクローン名:S27D3(配列表の配列番号24)、図1(c)がクローン名:S36E1(配列表の配列番号26)、図1(d)がクローン名:S39D6(配列表の配列番号28)、図1(e)がクローン名:TS27B2(配列表の配列番号30)の結果を示したものである。
また、図1に示すように、本実施例のスクリーニングで得られた5種類については、ウェスタンブロット法により特異的反応陽性シグナルが確認された。
Claims (13)
- 配列表の配列番号1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33もしくは35に示されるアミノ酸配列、又はその部分アミノ酸配列で示されるポリペプチドからなる動脈硬化診断用ポリペプチドマーカー。
- 請求項1に記載のアミノ酸配列又はその部分アミノ酸配列をコードする塩基配列で示される遺伝子からなる動脈硬化診断用遺伝子マーカー。
- 配列表の配列番号2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34もしくは36に示される塩基配列、又はその部分塩基配列で示される遺伝子からなる動脈硬化診断用遺伝子マーカー。
- 請求項1に記載のポリペプチドを抗原とする、該ポリペプチドに特異的に結合する抗体。
- 請求項2又は3に記載の遺伝子とストリンジェントな条件下でハイブリダイズするDNAの全部又は一部を有する動脈硬化マーカー遺伝子検出用プローブ。
- 請求項3に記載の遺伝子に対するアンチセンスDNAの全部又は一部を有する、請求項5に記載の動脈硬化マーカー遺伝子検出用プローブ。
- 基板に、請求項5に記載の動脈硬化マーカー遺伝子検出用プローブ、及び/又は請求項6に記載の動脈硬化マーカー遺伝子検出用プローブが固定化された動脈硬化マーカー遺伝子検出用DNAマイクロアレイ又はDNAチップ。
- 請求項4に記載の抗体を用い、被検試料における、該抗体に特異的に結合する動脈硬化マーカーポリペプチドの発現を検出する動脈硬化の検出方法。
- 請求項1に記載の動脈硬化診断用ポリペプチドマーカーを用い、被験体血中における、該動脈硬化診断用ポリペプチドマーカーに特異的に結合する動脈硬化マーカー抗体の発現を検出する動脈硬化の検出方法。
- 請求項5又は6に記載の動脈硬化マーカー遺伝子検出用プローブを用い、被検細胞における、該動脈硬化マーカー遺伝子検出用プローブとハイブリダイズする動脈硬化マーカー遺伝子の発現を検出する動脈硬化の検出方法。
- 配列表の配列番号2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34もしくは36に示される塩基配列に基づいてプライマーを構築し、該プライマーを用いてPCRを行い、動脈硬化マーカー遺伝子の発現を検出する動脈硬化の検出方法。
- 請求項5又は6に記載の動脈硬化マーカー遺伝子検出用プローブ、請求項7に記載の動脈硬化マーカー遺伝子検出用DNAマイクロアレイ又はDNAチップ、及び請求項4に記載の抗体からなる群から選択される少なくとも1つ以上を具備する動脈硬化診断用キット。
- 配列表の配列番号1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33もしくは35に示されるアミノ酸配列、又はその部分アミノ酸配列で示されるポリペプチドからなる動脈硬化診断用ポリペプチドマーカーを具備する動脈硬化診断用キット。
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JP2010524759A JP5742002B2 (ja) | 2008-08-15 | 2009-08-14 | 動脈硬化診断用ポリペプチドマーカー、及び該マーカー等を用いる動脈硬化の検出を補助する方法、並びに動脈硬化診断用キット |
US13/058,456 US20110287955A1 (en) | 2008-08-15 | 2009-08-14 | Polypeptide marker for diagnosis of arteriosclerosis, method for detection of arteriosclerosis by using the maker or the like, and kit for diagnosis of arteriosclerosis |
CN200980128162.0A CN102099470B (zh) | 2008-08-15 | 2009-08-14 | 用于诊断动脉硬化的多肽标记、用该标记等检测动脉硬化的方法以及用于诊断动脉硬化的试剂盒 |
KR1020117001433A KR101506314B1 (ko) | 2008-08-15 | 2009-08-14 | 동맥경화 진단용 폴리펩티드 마커, 및 상기 마커 등을 이용한 동맥 경화의 검출방법, 및 동맥경화 진단용 키트 |
EP09806761.4A EP2319924B1 (en) | 2008-08-15 | 2009-08-14 | Polypeptide marker for diagnosis of arteriosclerosis, method for detection of arteriosclerosis by using the maker or the like, and kit for diagnosis of arteriosclerosis |
US13/783,892 US9366681B2 (en) | 2008-08-15 | 2013-03-04 | Polypeptide marker for diagnosis of arteriosclerosis, method for detection of arteriosclerosis by using the maker or the like, and kit for diagnosis of arteriosclerosis |
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KR101735891B1 (ko) * | 2013-09-04 | 2017-05-16 | 이화여자대학교 산학협력단 | 신규한 심혈관계 질환의 진단 및 치료용 펩티드 |
KR102525734B1 (ko) * | 2014-11-07 | 2023-04-27 | 후지쿠라 가세이 가부시키가이샤 | 데옥시하이푸신·신타제 유전자를 지표로서 사용하는 동맥 경화 및 암의 검출 방법 |
KR101895767B1 (ko) * | 2016-06-16 | 2018-09-07 | 충남대학교 산학협력단 | 동맥경화증 진단용 바이오 마커 조성물 |
CN106822869B (zh) * | 2017-03-16 | 2020-10-30 | 北京热休生物技术有限公司 | DEF8蛋白的多肽与热休克蛋白gp96的复合物在制备治疗与预防癌症药物中的应用 |
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CN117384271A (zh) * | 2023-10-17 | 2024-01-12 | 青岛赛诺基因科技有限公司 | 与含赖氨酸甲基化修饰肽具有高亲和力的变体Chromo结构域 |
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KR20110027793A (ko) | 2011-03-16 |
EP2319924A1 (en) | 2011-05-11 |
EP2319924A4 (en) | 2012-01-25 |
JPWO2010018870A1 (ja) | 2012-01-26 |
KR101506314B1 (ko) | 2015-04-06 |
EP2319924B1 (en) | 2016-03-09 |
EP3021121A1 (en) | 2016-05-18 |
CN102099470A (zh) | 2011-06-15 |
EP3021121B1 (en) | 2018-06-06 |
US9366681B2 (en) | 2016-06-14 |
US20110287955A1 (en) | 2011-11-24 |
JP5742002B2 (ja) | 2015-07-01 |
US20130183691A1 (en) | 2013-07-18 |
CN102099470B (zh) | 2014-11-12 |
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