KR101240207B1 - Proteinic markers for early diagnosing Alzheimer's disease - Google Patents

Abstract

The present invention is a protein marker for early diagnosis of Alzheimer's disease, Alzheimer's disease early diagnosis composition comprising a substance for detecting the change of the proteinaceous marker, Alzheimer's disease early diagnosis kit comprising the composition for the early diagnosis, Alzheimer's disease for early diagnosis The present invention relates to a method for detecting proteinaceous markers and to a screening method for treating Alzheimer's disease using the proteinaceous markers.

Description

Protein markers for early diagnosing Alzheimer's disease

The present invention relates to protein markers for early diagnosis of Alzheimer's disease, and more specifically, protein markers for early diagnosis of Alzheimer's disease, which are less present in the blood of individuals in early stages of Alzheimer's disease than in normal individuals' blood, and Alzheimer's disease comprising the same. It relates to a composition for early diagnosis.

Alzheimer's disease is the most common form of neurodegenerative disease and is a protein that stabilizes amyloid plaque or senile plaque and intracellular microtubule on which the abnormal protein beta amyloid condenses. It is characterized by the generation of neurofibrillary tangles (NFTs), which are aggregates, and symptoms such as decreased memory and lack of recognition. It is estimated that there are more than 30 million Alzheimer's patients worldwide, with an estimated 100 million patients by 2050. As aging accelerates, the number of patients gradually increases and is a serious social problem. However, diagnosis and treatment are difficult because simple, convenient and efficient diagnostic markers have not been developed yet.

Currently, the diagnosis of Alzheimer's disease depends on clinical diagnosis such as medical history and neuropsychological examination. However, the accuracy of clinical diagnosis of Alzheimer's disease is 50-82%. As the number of patients with Alzheimer's disease increases and societal costs are incurred, there is a need for reliable diagnostic methods to prevent, treat, and predict the progress of the disease, and the need for early diagnosis markers is increasing.

Many studies have been made to diagnose Alzheimer's disease, and methods for predicting the onset of Alzheimer's disease have been developed through the concentration of tau protein and amyloid-beta. The following is a diagnosis method of Alzheimer's disease developed to date.

1) Q & A

1-1) The mini-mental state examination (MMSE)

1-2) Neurocognitive function test (SNSB)

2) Brain imaging test

2-1) Volumetric magnetic resonance imaging (MRI)-measures the decrease in volume of the brain at the stage of mild cognitive impairment (MCI) and Alzheimer's disease, which are classified as early stages of Alzheimer's disease.

2-2) Positron emission tomography (PET)-Identify loss of function of temporal lobe

2-3) PiB PET (Pittsburgh Compound B positron emission tomography)-Detects amyloid plaques in the brain using 11C Pittsburgh Compound B (11C-PIB), a contrast agent that adheres to beta amyloid plaques

2-4) Computed tomography (CT)-brain atrophy

2-5) Single photon emission computed tomography (SPECT)-Check cerebral blood flow

However, the above methods are expensive or lack the sensitivity of the accuracy and specificity to distinguish the specific disease from other diseases, and most of the methods cannot be diagnosed after the disease progresses. It has the limitation of being possible. Currently, the only way to confirm the diagnosis is to check for plaque and nerve fiber bundles by post-mortem examination of brain tissue after autopsy. As such confirmation and treatment are difficult, the importance of accurate early diagnosis is highlighted.

The development of biomarkers for neurodegenerative diseases is essential for early diagnosis, monitoring progress, and effective treatment of the disease. Although neurodegenerative diseases appear mostly in older patients, studies show that there is a "preclinical" stage in which the patient's behavior is normal but pathological changes occur in the brain prior to the onset of the disease. If the diagnosis can be made, more effective treatment will be possible, so early detection and differentiation of Alzheimer's disease can be made by measuring the level of protein that is specifically increased or decreased in the blood or cerebrospinal fluid of Alzheimer's disease patients before the onset of symptoms. Developing new clinical markers with high clinical specificity and sensitivity, as well as monoclonal antibodies that can detect them, can lead to the development of kits that can predict early diagnosis and progression of Alzheimer's disease.

Cerebrospinal fluid involves pain in the sampling process, is not easy to collect, there are concerns about medical side effects, and it is not particularly suitable for the elderly patients with Alzheimer's disease. Blood samples were easier to collect than cerebrospinal fluid, and 500 ml of cerebrospinal fluid is absorbed into the blood every day, and plasma suitable for the development of effective neurodegenerative biomarkers was used as a sample. Because humans and mice have similar structure and function of genetic factors and use human tau protein to make transgenic mice, studies of mouse plasma can provide awareness of human diseases.

Accordingly, the present inventors have made a thorough research to overcome the problems of the prior art, and as a result of inserting the human tau gene, overexpressing the tau, using a transgenic mouse that caused Alzheimer's disease, By using the two-dimensional electrophoresis technique to identify the biomarker proteins that decrease and increase in the plasma of Alzheimer's disease individuals than in the plasma of normal control individuals, and completed the present invention.

Therefore, the main object of the present invention is to prescribe Alzheimer's disease for application to the diagnosis and treatment of Alzheimer's disease through protein studies that can identify the difference in expression between the blood of normal individuals and the blood of early stages of Alzheimer's disease. It is to provide a diagnostic proteinaceous marker.

Another object of the present invention is to provide a composition for early diagnosis of Alzheimer's disease comprising a substance for detecting a change in the proteinaceous marker.

Another object of the present invention is to provide an Alzheimer's disease early diagnosis kit comprising the composition for early diagnosis of Alzheimer's disease.

Another object of the present invention is to provide a method for detecting the proteinaceous marker for early diagnosis of Alzheimer's disease.

Another object of the present invention is to provide a screening method for treating Alzheimer's disease using the proteinaceous marker.

According to one aspect of the invention, the invention provides a protein marker for early diagnosis of Alzheimer's disease, characterized in that one or more combinations selected from the group consisting of the following polypeptides:

ATP synthase subunit beta having an amino acid sequence of SEQ ID NO: 1 and having a molecular weight of 55-57 kD and a pi of 4.7-5.7;

Adenosine kinase (Isoform Long) having the amino acid sequence of SEQ ID NO: 2 and having a molecular weight of 39-41 kD and a pi of 5.3-6.3; And

Regucalcin a molecular weight of 32-34 kD and a pi of 4.7-5.7 with the amino acid sequence of SEQ ID NO: 3.

In the present invention, the protein marker for early diagnosis of Alzheimer's disease is detected from a blood sample of an individual requiring Alzheimer's disease to diagnose Alzheimer's disease, and blood is easier to collect than cerebrospinal fluid and a portion of the cerebrospinal fluid is blood. As a result, the plasma separated from blood was considered to be suitable as a sample for the development of an effective neurodegenerative diagnosis marker. In addition, in the present invention, since humans and mice have a similar structure and function of genetic factors and human tau protein is used to make transgenic mice, studies on mouse plasma may provide awareness of human diseases. .

In the present invention, most of the neurodegenerative diseases are present in elderly patients, but since pathological changes occur in the brain before the symptoms of the disease, more effective treatment will be possible if Alzheimer's disease can be diagnosed at this stage. Alzheimer's disease can be diagnosed early by measuring protein levels that specifically increase or decrease in the blood of Alzheimer's disease patients before they appear. Therefore, "early diagnosis" of the present invention means diagnosing Alzheimer's disease at a stage before the symptoms of the disease appear.

The present inventors analyze the proteome of proteins obtained from the blood of a normal subject and the blood of an Alzheimer's disease subject to detect proteins characteristically reduced in Alzheimer's disease subjects as compared to a normal subject, and thus can be used for the diagnosis or treatment of Alzheimer's disease. Castle markers were excavated.

As used herein, the term 'proteome' refers to the totality of all proteins that can be made from the genome, so that the pattern of the proteome always changes depending on the specific physiological or pathological condition in a cell or tissue. Being a dynamic concept. Proteomics is a generic term for methods and techniques for studying these proteomes. Cellular cells are studied by studying the properties of proteins, focusing on gene expression, post-translational modification, and binding to other proteins. It refers to the field of research that you want to understand holistically by linking my transformation process and network formation with the course of disease. As such, the proteome represents a physiological or pathological condition in a cell or tissue, and thus is most suitable as a method of finding a diagnostic marker that can be directly used for diagnosis of a disease. In addition, in the case of Alzheimer's disease, if the expression of a specific gene is found to promote Alzheimer's disease by participating in the degree of disease progression, the presence of the protein may be identified and identified as a target protein for the development of an inhibitor of Alzheimer's disease. The development of diagnostics and therapeutics using them has been progressing due to the superior sensitivity of Genomics and the easy amplification of genes, but changes in DNA or mRNA levels do not directly lead to changes in proteins that are actually active in cells. Theoretical problems remain in that it is not possible, and in reality, proteomics is the only research method for body fluids without genetic material. Currently, non-invasive approaches are used for diagnosis of body fluids such as plasma, serum, urine, cerebrospinal fluid, amniotic fluid, and secretion, and many researchers use proteomic methods to identify disease-specific proteins as diagnostic markers. I introduce it.

As used herein, the term “proteinaceous marker for early diagnosis of Alzheimer's disease” refers to a substance which is a protein as a main component, among the substances which are a standard for distinguishing between normal blood and Alzheimer's disease blood. In the present invention, the proteinaceous marker is characterized in that the amount of abundance in the blood of an individual with Alzheimer's disease is large or small compared to the amount in the tissue of a normal individual.

Since the present invention is to analyze the proteome of the tissues of individuals with Alzheimer's disease with the proteome of the tissues of normal individuals, the protein markers thus identified can be said to be specific for Alzheimer's disease, and thus can be usefully used to diagnose Alzheimer's disease. Can be. Furthermore, given that the proteinaceous markers thus identified are prominent in Alzheimer's disease, the physiological function of the proteinaceous markers may be directly related to the onset of Alzheimer's disease, and thus the proteinaceous markers. May be usefully used as a target protein for studying the mechanism of the development of Alzheimer's disease or for the development of Alzheimer's disease therapeutics.

In particular, since the protein marker for early diagnosis of Alzheimer's disease is found at the level of proteome in the blood of an actual Alzheimer's disease individual, it is used more clinically than the specific markers of Alzheimer's disease found at the level of conventional DNA or mRNA. It is a valuable marker and is very useful as a target of Alzheimer's disease in that it is limited to Alzheimer's disease.

In the present invention, the proteinaceous marker may be part of the full-length protein having the sequence number as long as it has the molecular weight, in which case it has trypsin digested peptide sequences shown in red in the MS / MS results of FIGS. 7A to 7C. The trypsin digested peptide refers to a peptide of protein digested with trypsin for MALDI-TOF or MS / MS analysis as a method of proteome analysis. The molecular weight also includes a generally accepted experimental error range as a value identified on two-dimensional electrophoresis.

According to another aspect of the present invention, the present invention provides a composition for the early diagnosis of Alzheimer's disease comprising a substance which detects the presence or absence of the proteinaceous marker and its amount, pattern or both.

In the Alzheimer's disease early diagnosis composition of the present invention, the presence or absence of the proteinaceous marker and specific detection of the pattern or the specificity of the Alzheimer's disease early diagnosis of the present invention, and the amount and pattern to confirm For example, a process of confirming the presence and amount or pattern thereof using an antibody specifically binding to the proteinaceous marker may be utilized. As used herein, the term “biological sample” refers to a cell or tissue capable of detecting the presence and amount or pattern of a proteinaceous marker for early diagnosis of Alzheimer's disease, the expression of the gene related to the proteinaceous marker, and the amount or expression pattern thereof. And the like. In addition, in the present specification, "antibody" refers to a protein that can specifically bind to epitopes of antigens, and is a concept including both polyclonal antibodies, monoclonal antibodies, and recombinant antibodies.

In the present invention, the polyclonal antibody may be prepared by injecting an antigenic proteinaceous marker or a fragment having the epitope into an external host according to conventional methods known to those skilled in the art. External hosts include mammals such as mice, rats, sheep, rabbits. Immunogens are injected by intramuscular, intraperitoneal or subcutaneous injection and are usually administered with an adjuvant to increase antigenicity. Blood is periodically collected from external hosts to collect serum showing improved titers and specificity for antigens or to separate and purify antibodies from them.

In addition, the monoclonal antibodies can be prepared by the technology of generating immortalized cell lines by fusion known to those skilled in the art (Koeher and Milstein (1975) Nature, 256: 495). The manufacturing method is briefly described as follows. First, 20 μg of pure protein is immunized to Balb / C mice or the peptide is synthesized and combined with bovine serum albumin to immunize mice. Thereafter, antigen-producing lymphocytes isolated from mice are fused with myeloma in humans or mice to produce immortalized hybridomas, and the ELISA method is used to select and propagate only hybridoma cells that produce the desired monoclonal antibody. The monoclonal antibody is isolated and purified from the culture.

Methods for measuring the presence, amount or pattern of the protein using the antibody include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay, radioimmunodiffusion, But are not limited to, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assays, complement fixation assays, FACS, and protein chips. no.

Through these analytical methods, it is possible to compare the amount of antigen-antibody complex formation in a biological sample of a normal individual with the amount of antigen-antibody complex formation in a biological sample of an Alzheimer's disease individual, and thus for the early diagnosis of Alzheimer's disease of the present invention. The presence or absence and the amount or pattern of proteinaceous markers can be determined, and ultimately, early diagnosis can be made for patients suspected of Alzheimer's disease.

Here, the term “antigen-antibody complex” means a combination of a protein marker for early diagnosis of Alzheimer's disease and an antibody specific thereto. The amount or pattern of formation of the antigen-antibody complex is usually a detection label associated with a secondary antibody. Measurements can be made by detecting the magnitude and pattern of the signal. Such detection labels include, but are not limited to, enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules, radioisotopes, and the like. When enzymes are used as detection labels, available enzymes include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholinese Therapase, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase, luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphphenolpyruvate deca Carboxylase, β-latamase, and the like, but are not limited thereto. When fluorescent materials are used as detection labels, available fluorescent materials include fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, etc. However, this is not limiting. When a ligand is used as a detection label, available ligands include, but are not limited to, biotin derivatives. When a luminescent material is used as the detection label, available luminescent materials include, but are not limited to, acridinium ester, luciferin, luciferase, and the like. When the microparticles are used as the detection label, the microparticles available include, but are not limited to, colloidal gold and colored latex. When redox molecules are used as detection labels, the available redox molecules include ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, K ₄ W (CN ) ₈ , [Os (bpy) ₃ ] ²⁺ , [RU (bpy) ₃ ] ²⁺ , [MO (CN) ₈ ] ^4-, etc., but are not limited to these. When radioisotopes are used as detection labels, the available radioisotopes include ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ^131. I or ¹⁸⁶ Re and the like, but is not limited thereto.

In the composition for early diagnosis of Alzheimer's disease of the present invention, the "substance that specifically detects the presence and amount or pattern of proteinaceous markers for early diagnosis of Alzheimer's disease" detects the presence and amount or pattern of the proteinaceous markers. It may be any substance that can be used specifically for the proteinaceous marker in the assay method to be, but is not necessarily limited to the antibody. Since the composition for early diagnosis of Alzheimer's disease according to the present invention has technical characteristics in detecting the difference between the presence or absence of the proteinaceous marker and the pattern or pattern in a biological sample of a normal individual and a biological sample of a suspected Alzheimer's disease, Thus, any substance capable of the presence or absence of a proteinaceous marker and its amount or pattern can be used as "a substance which specifically detects the presence or absence and the quantity or pattern of a proteinaceous marker for early diagnosis of Alzheimer's disease." This desired technical effect can be achieved, and those skilled in the art will be able to select and use a suitable material without particular difficulty according to specific embodiments with reference to the average knowledge and well-known techniques in the art.

According to another aspect of the present invention, the present invention provides a composition for early diagnosis of Alzheimer's disease comprising a substance for detecting the expression of the gene encoding the proteinaceous marker, and the amount of expression, expression pattern or both.

In the Alzheimer's disease early diagnosis composition of the present invention, the presence and amount or pattern of the proteinaceous marker, in addition to using the method for detecting the protein itself, expression of the gene encoding the proteinaceous marker in the tissue It is also possible to use a method of inferring the presence or absence and the amount or pattern of the proteinaceous marker by detecting the presence and the amount or expression pattern thereof.

Here, whether the gene is expressed and the amount of expression or expression pattern detection can be generally performed by a process of confirming the presence and amount or pattern of mRNA generated by the transcription of the gene. Assays to determine the presence and amount or pattern of these mRNAs include RT-PCR, competitive RT-PCR, Real-time RT-PCR, and RNase protection assays. RNase protection assays, northern blots, DNA chips, and the like, but is not limited thereto.

Through these assay methods, the presence and amount or pattern of mRNA in a biological sample of a normal individual can be compared with the presence or amount or pattern of mRNA in a biological sample of an individual suspected of Alzheimer's disease, To determine whether the gene encoding the proteinaceous marker for early diagnosis of Alzheimer's disease of the present invention and the difference in the expression amount or expression pattern, and ultimately diagnose the onset of Alzheimer's disease in the tissues of patients suspected of Alzheimer's disease It becomes possible.

In the composition for early diagnosis of Alzheimer's disease of the present invention, the kit for measuring the presence and amount or pattern of the mRNA by RT-PCR includes a primer specific for the mRNA for the proteinaceous marker of the present invention. As used herein, a "primer" refers to a nucleic acid sequence having a free 3 'hydroxyl group capable of complementarily binding to a template and allowing reverse transcriptase or DNA polymerase to initiate replication of the template. Means. A primer is a nucleotide having a sequence complementary to the nucleic acid sequence of a particular gene, and may be used in a length of about 7 bp to 50 bp, preferably about 10 bp to 30 bp. Other RT-PCR kits may include test tubes or other suitable containers, reaction buffers, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC-water (DEPCwater), depending on the specific embodiment. , Sterile water and the like. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures. The primer may also include additional other base sequences that do not change the basic properties of the primer that serve as the starting point for DNA synthesis. Primers can be chemically synthesized using well known methods, and such nucleic acid sequences can also be modified using many means known in the art.

In the composition for early diagnosis of Alzheimer's disease of the present invention, the "substance that specifically detects expression of the gene encoding the proteinaceous marker for early diagnosis of Alzheimer's disease and its expression amount or expression pattern" is a gene encoding the proteinaceous marker. May be any substance that can be specifically used for the proteinaceous marker in the expression method and the expression amount or expression pattern analysis method, and is not necessarily limited to the primer for RT-PCR. The composition for early diagnosis of Alzheimer's disease according to the present invention detects whether the gene encoding the proteinaceous marker for early diagnosis of Alzheimer's disease and the difference in its expression amount or expression pattern in a biological sample of a normal individual and a biological sample of a suspected Alzheimer's disease Because of the technical characteristics of the present invention, any substance capable of detecting such a difference may be described as “a specific method for detecting the expression of the gene encoding the proteinaceous marker for early diagnosis of Alzheimer's disease and its expression amount or expression pattern. Material ”and the invention can achieve the desired technical effect, and those skilled in the art will be able to select and use a suitable material according to specific embodiments with reference to the average knowledge in the art.

According to another aspect of the present invention, the present invention provides a kit for early diagnosis of Alzheimer's disease comprising the composition for early diagnosis of Alzheimer's disease.

Alzheimer's disease early diagnosis kit of the present invention is a substance that specifically detects the presence and amount or pattern of Alzheimer's disease early diagnosis proteinaceous marker included in the Alzheimer's disease early diagnosis composition, or Alzheimer's disease early proteinaceous marker As long as it is suitable for the presence or absence of a protein and a method for analyzing the amount or pattern, or for the expression of a gene and the expression amount or expression pattern, in addition to a substance for detecting the expression of the gene encoding the expression and its expression amount or expression pattern. It may further comprise a kind or more of other components, solutions or devices. For example, if the diagnostic kit is a diagnostic kit for detecting the presence of the protein and its amount or pattern, the diagnostic kit may be, for example, a diagnostic kit including essential components necessary for performing an ELISA. Such ELISA kits include components capable of detecting bound antibodies, such as labeled secondary antibodies, chromopores, enzymes (eg, enzymes conjugated with antibodies) and substrates thereof, and quantitative control proteins. It may include an antibody specific to, and the like. On the other hand, when the diagnostic kit is a diagnostic kit for detecting the expression of the gene and its expression amount or expression pattern, the diagnostic kit may be a diagnostic kit containing the essential components necessary to perform RT-PCR, These RT-PCR kits can be used in addition to individual primers specific for the marker gene, depending on the specific embodiment, such as, for example, test tubes or other suitable containers, reaction buffers, deoxynucleotides (dNTPs), Taq-polymerases and reverse transcriptases. Enzymes, DNAse, RNAse inhibitor DEPC-water (DEPCwater), sterile water, primer pairs specific for the gene used as a quantitative control, and the like. In addition, according to a specific embodiment, the Alzheimer's disease early diagnosis kit may include a DNA chip or a protein chip.

According to another aspect of the present invention, the present invention provides a method for detecting a protein marker for early diagnosis of Alzheimer's disease from a patient in order to provide information necessary for early diagnosis of Alzheimer's disease.

The method for detecting a protein marker for early diagnosis of Alzheimer's disease comprises treating the Alzheimer's disease early diagnosis composition of the present invention to a biological sample collected from a patient suspected of Alzheimer's disease, and from the treatment result, for early diagnosis of Alzheimer's disease. It can be carried out by detecting the difference between the presence or absence and the amount or pattern of proteinaceous markers. Further, the Alzheimer's disease early diagnosis composition of the present invention is treated to a biological sample collected from a patient suspected of Alzheimer's disease, and from the result of the treatment, the expression of the gene encoding the proteinaceous marker for early diagnosis of Alzheimer's disease, and its It can be carried out by detecting the difference in the expression amount or expression pattern.

According to another aspect of the invention, the present invention comprises the steps of treating a test compound to cells and animals expressing the proteinaceous marker, and identifying whether the test compound promotes the physiological activity of the proteinaceous marker. It provides a screening method for treating Alzheimer's disease comprising.

In the present invention, whether the test compound promotes the physiological activity of the proteinaceous marker may be determined by confirming whether to increase the expression of the proteinaceous marker.

The protein marker for early diagnosis of Alzheimer's disease may be a protein that is directly involved in the development of Alzheimer's disease because the change in blood of Alzheimer's disease individuals is prominent. Therefore, the mechanism of the development of Alzheimer's disease or the treatment of Alzheimer's disease It can be usefully used as a target protein for development. That is, since the proteinaceous marker for early diagnosis of Alzheimer's disease solves an important premise of the development of a therapeutic agent for Alzheimer's disease, a method for screening a therapeutic agent for Alzheimer's disease using this proteinaceous marker also belongs to the scope of the present invention.

As a method for screening such a therapeutic agent, a method for fixing the Alzheimer's disease early diagnosis proteinaceous marker of the present invention in an affinity column and contacting it with a sample for purification [Pandya et al, Virus Res 87: 135-143, 2002], Methods using the two-hybrid method [Fields, S and Song, O., Nature 340: 245-246, 1989], Western Blot ["Molecular Cloning-A Laboratory Manual" Cold Spring Habor Laboratory, NY, Maniatis, T. at al. (1982) section 18.30-18.74], a high throughput screening method [Aviezer et al, J Biomol Screen 6: 171-7, 2001], and the like, and a number of known methods can be used. The appropriate method can be selected accordingly. Samples containing test compounds for use in screening include, but are not limited to, tissue extracts, expression products of gene libraries, synthetic compounds, synthetic peptides, natural compounds, and the like.

As described above, according to the present invention Alzheimer's disease early diagnosis kit comprising a proteinaceous marker for early diagnosis Alzheimer's disease, a composition for detecting Alzheimer's disease including a substance for detecting a change in the protein marker, the Alzheimer's disease early diagnosis composition The present invention provides a method for detecting a protein marker for early diagnosis of Alzheimer's disease, and a method for screening an Alzheimer's disease therapeutic agent using the protein marker.

FIG. 1 is a PCR analysis image confirming the presence or absence of a transgene using genomic DNA extracted from tails of 4 week-old normal mice (NTg; Non-Transgenic) and transgenic mice (Tg; Transgenic).
FIG. 2A is a 2-D electrophoresis image showing reduced and increased protein in plasma of 2 months normal mice.
FIG. 2B is a 2-D electrophoresis image showing reduced and increased protein in plasma of Alzheimer's disease induced transgenic mice than in plasma of 2 months normal mice.
FIG. 2C is a 2-D electrophoresis image showing reduced and increased protein in plasma of Alzheimer's disease induced transgenic mice than in plasma of 4 months normal mice.
FIG. 3 shows four months of transgenic mice in plasma of two-month normal mice (2N; 2-month non-transgenic) and two-month transgenic mice (2T; 2-month transgenic) among the proteins shown in FIGS. This is an enlarged image detailing proteins with significantly reduced expression in 4T (4-month transgenic) plasma.
Figure 4a is an image of the results of Western blot ATP synthase subunit beta, a protein that is significantly reduced in the plasma of 4 months transgenic mice (4N; 4-month non-transgenic).
4B is a result showing the relative strength of FIG. 4A.
Figure 5a is an experimental result of Western blot of adenosine kinase, a protein that is significantly reduced in the plasma of 4 months transgenic mice,
5B is a result showing the relative strength of FIG. 5A.
Figure 6a is an experimental result of Western blot of Regucalcin, a protein that is significantly reduced in the plasma of 4 months transgenic mice,
Figure 6b is a result showing the relative strength of Figure 6a,
7A to 7D show results of identifying proteins expressed differently in plasma of normal mice and Alzheimer's disease-induced mice using LC / MS / MS.

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

Example 1. Sample preparation

Alzheimer's disease-induced tau-transduced mice and littermate controls produced by the Korea Food and Drug Administration were used. Tau transgenic mice microinject the pNSE / htau23 gene, which connects the human wild type tau cDNA (htau23) (GenBank accession No. J03778) to the NSE promoter, into the male pronucleus in the fertilized egg of BDF1 mice. The pups born after implantation in the ICR mice fertility were produced by crossing wild type BDF1 mice. Transgenic mice designed to overexpress human Tau genes cause Alzheimer's disease, which was confirmed by expression of the transgene and observation of behavioral symptoms. The mice received were maintained under a 12-h light-dark cycle, a temperature of 23 ± 1 ° C., and a humidity standard of 50 ± 10%.

The genomic DNA isolated from the tail of the 4 week old mouse was analyzed by PCR to confirm the expression of the transgene. After 25 cycles (amplification), the expression of 1,112 bp htau23 was confirmed using 1% agarose gel.

1 is a PCR analysis image confirming the presence or absence of the transgene using genomic DNA extracted from the tail of 4 weeks old mice (NTg; Non-Transgenic) and transgenic mice (Tg; Transgenic).

Mice were euthanized using carbon dioxide gas at 2 and 4 months, respectively, and blood was collected by directly inserting a syringe coated with EDTA as an anticoagulant to the heart. The number of two-month normal control individuals was eight, the number of two-month Alzheimer's disease individuals was 13, the number of four-month normal control individuals was six, and the number of four-month Alzheimer's disease individuals was seven. The obtained blood was centrifuged at 12000 rpm for 10 minutes at 4 ° C and then plasma was separated. The separated plasma was quantitated by Bradford method in a sample buffer solution and stored in -70 ℃ freezer. The composition of the sample buffer is as follows: 7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris-Cl, pH 8.5, 65 mM DTT, 1 mM EDTA, protease inhibitor cocktail (PIC).

Example 2 Two-Dimensional Electrophoresis

2-1. Isoelectroforcusing (IEF)

One-dimensional electrophoresis was performed with a protein amount of 250 μg of the sample treated with the sample buffer. 24 cm strip for one-dimensional electrophoresis by mixing rehydration buffer (8 M urea, 2% CHAPS, 1% IPG buffer (pH 4-7), 13 mM DTT, bromophenol blue) to a total volume of 450 μl Drystrip (GE Healthcare, Uppsala, Sweden) in the pH 4-7 range was mounted in the strip holder after being placed in a strip holder. After 5 hours of rehydration at 80 ° C. for 5 hours at 20 ° C., one-dimensional electrophoresis was performed under the following conditions: 200 V 30 minutes, 500 V 1 minute, 500-8000 V 1 hour, 8000 V 146,000 Vhr. One-dimensional electrophoresis was performed with the IPGphor IEF system (GE Healthcare, Uppsala, Sweden).

2-2. Two-dimensional Electrophoresis (SDS-PAGE)

For two-dimensional electrophoresis, place a strip of one-dimensional electrophoresis into a cap tube and place in a first equilibration solution (50 mM pH 8.8 Tris-HCl buffer, 6 M urea, 20% glycerol, 2% SDS, 1% DTT). After reaction for 10 minutes in 10 ml and discard, add 10 ml of the second equilibration solution (50 mM pH 8.8 Tris-HCl buffer, 6 M urea, 20% glycerol, 2% SDS, 2.5% idoacetamide (IAA)) Reacted. The reaction strip was mounted on a 12.5% homogenous SDS-PAGE gel (26 × 20 cm) and sealed with 0.5% agarose. Two-dimensional electrophoresis was performed by adding electrophoresis buffer (24 mM Tris, 192 mM glycine, 0.1% SDS) to an Ettan DALT II system (GE Healthcare, Uppsala, Sweden). The electrophoretic conditions are as follows: 55 V 1 hour, 160 V 1 hour, 330 V 4 hours.

2-3. Staining

After two-dimensional electrophoresis, silver staining was performed to visualize the gel. The silver staining process is as follows: Fixation-Reaction with a solution of 50% methanol, 12% acetic acid for at least 2 hours, Washing-3 times for 20 minutes with 50% ethanol, Sensitizing-0.2 Reaction with 1% sodium thiosulfate solution for 1 minute, Washing-3 times for 20 seconds, Improving-0.1% Silver nitrate, 0.075% formaldehyde (37%) solution Reaction, Washing-3 times in 20 seconds, Visualization-Reaction-Immobilization (Fixation)-50 minutes with pre-chilled 6% Sodium carbonate, 0.05% Formaldehyde (37%) solution Stop the reaction by treatment with 12% methanol and 12% acetic acid solution.

2-4. Image Analysis

The analyzed gels were first scanned with ImageScanner (GE Healthcare, Uppsala, Sweden). In order to analyze differences in protein expression, the analysis was performed with Image Master 2D Elite Software (GE Healthcare, Uppsala, Sweden), a program dedicated to two-dimensional electrophoresis analysis.

FIG. 2A is a 2-D electrophoresis image showing reduced and increased protein in plasma of two-month normal mice, and FIG. 2B shows reduced and increased protein in plasma of Alzheimer's disease-induced transgenic mice than in plasma of two-month normal mice. 2-D electrophoresis image, FIG. 2C is a 2-D electrophoresis image showing reduced and increased protein in plasma of Alzheimer's disease-induced transgenic mice than in plasma of 4 months normal mice. Spot differences in expression in the 2-D electrophoresis image results were analyzed below.

Example 3. Mass Spectrometry (LC / MS / MS Analysis)

In order to identify the spots found with the image analysis program, the spots were cut out from the gel after two-dimensional electrophoresis. First, 100 μl of a destaining solution mixed with 30 mM Potassium Ferricyanide and 100 mM Sodium Thiosulfate solution in a 1: 1 ratio was applied to the gel pieces for 5 minutes to remove silver. It was then washed three times with 400 μl of water.

After reacting with 50 mM ammonium bicarbonate for 20 minutes, the mixture was washed with water and 100 μl of acetonitrile was added to dehydrate the gel until it turned white. The gel was placed in a speed vacuum centrifuge to completely remove the solution from the gel. The dried gel pieces were reacted overnight at 37 ° C. after adding 20 μl of 50 mM ammonium bicarbonate containing 0.2 μg of trypsin (Promega Corp., WI, USA). Peptides were extracted by reacting 50% acetonitrile with 40 μl of a trifluotoacetic acid (TFA) solution at 37 ° C. for 1 hour on a trypsin-treated gel piece. This peptide mixture was used to identify proteins by LC / MS / MS by desalting and concentrating prior to mass spectrometry. LC / MS / MS was conducted at the Korea Center for Basic Science, Seoul Center.

In gel digestion finished samples were dissolved in 200 μl of A buffer and analyzed using a Thermo Finnigan LCQ DeCa Xp Max mass spectrometer. A column of C18 cartridge was used, and helium gas was analyzed under capillary temperature of 200 ° C, voltage spray volt-2.3KV, and capillary ion spray sorce-23V. A) Dw (0.5% acetic acid, 0.02% formic acid), B) 80% CH ₃ CN (0.5% acetic acid, 0.02% formic acid) Solvent was used and loaded at 200 μl / min for 80 minutes. The analysis program used Xcalibur (sequest search).

To identify proteins, all MS / MS spectra were examined for protein sequences in the NCBInr database using the MASCOT search program (www.matrixscience.com).

Example 4. Western blotting

4-1. SDS-PAGE Electrophoresis

Twenty μg of plasma proteins (ATP synthase subunit beta and adenosine kinase) or 35 μg (regucalcin) were subjected to 12% SDS-PAGE electrophoresis. Plasma samples were separated by molecular weight (loading at 100 V on a separating gel) under reducing conditions in mini gel (6 × 8 cm). The composition of the running buffer used was 0.025 M Tris-Cl, 0.192 M Glycine, 1% SDS (pH 8.3).

4-2. Blotting

After SDS-PAGE, gel proteins were transferred to semi-dry transfer kits (GE Healthcare, Uppsala, Sweden) using ATP synthase subunit beta and adenosine kinase for PVDE (polyvinylidene fluoride) membrane and regucalcin for NitroCellulose membrane. This was done at 50 mA for 1 hour 30 minutes.

4-3. Antibody reaction

ATP synthase subunit beta, adenosine kinase and regucalcin were performed. The membrane was blocked with 5% w / v nonfat dry milk at 4 ° C. for 1 hour. Primary antibodies include chicken polyclonal to ATP synthase Subunit Beta (1: 5000 dilution, Abcam, Cambridge, UK), rabbit polyclonal to Adenosine kinase (1: 200 dilution; Abcam, Cambridge, UK), goat polyclonal to Regucalcin (1 : 70 dilution, Santa Cruz Biotechnology, CA, USA) was used overnight at 4 ℃. After washing with TBS / T (0.1%, Tris-buffered saline-tween 20), it was reacted with a secondary antibody at room temperature for 1 hour. Secondary antibodies include ATP synthase Subunit Beta, goat anti-chicken IgY-HRP conjugated antibody (1: 10000 dilution; Santa Cruz Biotechnology, CA, USA), and adenosine kinase stabilized goat anti-rabbit HRP-conjugated antibody (1: 500 dilution). Concentration, PIERCE, IL, USA) and regucalcin were used as donkey anti-goat IgG-HRP conjugated antibody (1: 500 dilution; Santa Cruz Biotechnology, CA, USA). After washing with TBS / T after the secondary antibody reaction, it was visualized with Immobilon ^TM Western (Chemiluminescent HRP Substrate) (Millipore Corporation, Billerica, Mass., USA) and Luminescent Image Analyzer (LAS) -3000 imaging system (Fujifilm Life) Science, CT, USA) and analyzed by Multi Gauge image analysis software (Fuji Film Life Science, CT, USA).

As a result of performing the two-dimensional electrophoresis experiment as described above, proteins with reduced and increased expression in the plasma of Alzheimer's disease individuals were identified as follows.

Significantly Reduced Expression in Plasma of Alzheimer's Disease Transgenic Mice

18 proteins with reduced or increased expression in plasma of 2-month Alzheimer's disease-transduced mice and plasma of 4-month Alzheimer's disease-transduced mice (# 300, # 440, # 511, # 614, # 628, # 629, # 631, # 685, # 709, # 852, # 855, # 860, # 891, # 908, # 914, # 942, # 1051, # 1070) 2c). Reduced expression of the protein was reduced by 50% or more than that of the normal control group, and the increased protein was increased by 100% or more.

LC / MS / MS identified proteins with decreased and increased expression during Alzheimer's disease. The protein was found to be closely related to Alzheimer's disease and similar levels were found in plasma of normal control and 2-month Alzheimer's disease-transduced mice but markedly decreased in plasma levels of 4-month Alzheimer's-transduced mice. Three proteins (# 440, # 511, # 629) that were not identified on the gel were selected and these proteins are shown in Table 1 below, and the expression level of these three proteins on the two-dimensional electrophoresis gel was shown in FIG. See Figure 3. Here, the reason why the plasma protein of the 4-month mouse was selected as a marker is that the tau protein is at an earlier stage than the 6-month period when the behavioral abnormality appears in the transgenic mouse (the behavioral symptom, that is, no symptoms of Alzheimer's disease). This is because it is a time when it is sufficiently overexpressed (ie, the disease is progressing due to sufficient accumulation of substances causing Alzheimer's disease).

[Table 1]

2. Determination of the amino acid sequence of a protein

As described above, proteins of similar levels in plasma of the normal control group and the 2-month Alzheimer's disease-transduced mouse but markedly decreased in the plasma of 4-month Alzheimer's disease-transduced mouse were identified using LC / MS / MS. To obtain the results of FIGS. 7A to 7C. Red in the figure shows the sequence of matched trypsin digesting peptides. The results are summarized in Table 2 below.

[Table 2]

The amino acid sequences of these proteins were searched in the NCBInr database (http://www.ncbi.nlm.nih.gov/) to search for ATP synthase subunit beta (SEQ ID NO: 1), adenosine kinase (SEQ ID NO: 2), regucalcin (SEQ ID NO: 3) was confirmed to have.

3. Western blotting and ELISA test results

The results of Western blotting of ATP synthase subunit beta, adenosine kinase, and regucalcin, the proteins of which expression was changed, are shown in FIGS. 4A, 5A, and 6A, and their relative intensities are shown in FIGS. 4B, 5B, and 6B. .

As shown in the results, the amount of ATP synthase subunit beta, adenosine kinase, and regucalcin decreased significantly as Alzheimer's disease progressed early. This result was the same as the result of the second electrophoresis. These results suggest that ATP synthase subunit beta, adenosine kinase, and regucalcin may be very useful for early diagnosis of Alzheimer's disease, and have also shown potential as a composition for treating and preventing Alzheimer's disease.

<110> Korea University Industrial and Academic Collaboration Foundation <120> Proteinic markers for early diagnosing Alzheimer's disease <160> 3 <170> Kopatentin 1.71 <210> 1 <211> 529 <212> PRT <213> Tau transgenic mouse <400> 1 Met Leu Ser Leu Val Gly Arg Val Ala Ser Ala Ser Ala Ser Gly Ala   1 5 10 15 Leu Arg Gly Leu Ser Pro Ser Ala Ala Leu Pro Gln Ala Gln Leu Leu              20 25 30 Leu Arg Ala Ala Pro Ala Gly Val His Pro Ala Arg Asp Tyr Ala Ala          35 40 45 Gln Ala Ser Ala Ala Pro Lys Ala Gly Thr Ala Thr Gly Arg Ile Val      50 55 60 Ala Val Ile Gly Ala Val Val Asp Val Gln Phe Asp Glu Gly Leu Pro  65 70 75 80 Pro Ile Leu Asn Ala Leu Glu Val Gln Gly Arg Asp Ser Arg Leu Val                  85 90 95 Leu Glu Val Ala Gln His Leu Gly Glu Ser Thr Val Arg Thr Ile Ala             100 105 110 Met Asp Gly Thr Glu Gly Leu Val Arg Gly Gln Lys Val Leu Asp Ser         115 120 125 Gly Ala Pro Ile Lys Ile Pro Val Gly Pro Glu Thr Leu Gly Arg Ile     130 135 140 Met Asn Val Ile Gly Glu Pro Ile Asp Glu Arg Gly Pro Ile Lys Thr 145 150 155 160 Lys Gln Phe Ala Pro Ile His Ala Glu Ala Pro Glu Phe Ile Glu Met                 165 170 175 Ser Val Glu Gln Glu Ile Leu Val Thr Gly Ile Lys Val Val Asp Leu             180 185 190 Leu Ala Pro Tyr Ala Lys Gly Gly Lys Ile Gly Leu Phe Gly Gly Ala         195 200 205 Gly Val Gly Lys Thr Val Leu Ile Met Glu Leu Ile Asn Asn Val Ala     210 215 220 Lys Ala His Gly Gly Tyr Ser Val Phe Ala Gly Val Gly Glu Arg Thr 225 230 235 240 Arg Glu Gly Asn Asp Leu Tyr His Glu Met Ile Glu Ser Gly Val Ile                 245 250 255 Asn Leu Lys Asp Ala Thr Ser Lys Val Ala Leu Val Tyr Gly Gln Met             260 265 270 Asn Glu Pro Pro Gly Ala Arg Ala Arg Val Ala Leu Thr Gly Leu Thr         275 280 285 Val Ala Glu Tyr Phe Arg Asp Gln Glu Gly Gln Asp Val Leu Leu Phe     290 295 300 Ile Asp Asn Ile Phe Arg Phe Thr Gln Ala Gly Ser Glu Val Ser Ala 305 310 315 320 Leu Leu Gly Arg Ile Pro Ser Ala Val Gly Tyr Gln Pro Thr Leu Ala                 325 330 335 Thr Asp Met Gly Thr Met Gln Glu Arg Ile Thr Thr Thr Lys Lys Gly             340 345 350 Ser Ile Thr Ser Val Gln Ala Ile Tyr Val Pro Ala Asp Asp Leu Thr         355 360 365 Asp Pro Ala Pro Ala Thr Thr Phe Ala His Leu Asp Ala Thr Thr Val     370 375 380 Leu Ser Arg Ala Ile Ala Glu Leu Gly Ile Tyr Pro Ala Val Asp Pro 385 390 395 400 Leu Asp Ser Thr Ser Arg Ile Met Asp Pro Asn Ile Val Gly Asn Glu                 405 410 415 His Tyr Asp Val Ala Arg Gly Val Gln Lys Ile Leu Gln Asp Tyr Lys             420 425 430 Ser Leu Gln Asp Ile Ile Ala Ile Leu Gly Met Asp Glu Leu Ser Glu         435 440 445 Glu Asp Lys Leu Thr Val Ser Arg Ala Arg Lys Ile Gln Arg Phe Leu     450 455 460 Ser Gln Pro Phe Gln Val Ala Glu Val Phe Thr Gly His Met Gly Lys 465 470 475 480 Leu Val Pro Leu Lys Glu Thr Ile Lys Gly Phe Gln Gln Ile Leu Ala                 485 490 495 Gly Glu Tyr Asp His Leu Pro Glu Gln Ala Phe Tyr Met Val Gly Pro             500 505 510 Ile Glu Glu Ala Val Ala Lys Ala Asp Lys Leu Ala Glu Glu His Gly         515 520 525 Ser     <210> 2 <211> 360 <212> PRT <213> Tau transgenic mouse <400> 2 Met Ala Ala Ala Asp Glu Pro Lys Pro Lys Lys Leu Lys Val Glu Ala   1 5 10 15 Pro Gln Ala Leu Ser Glu Asn Val Leu Phe Gly Met Gly Asn Pro Leu              20 25 30 Leu Asp Ile Ser Ala Val Val Asp Lys Asp Phe Leu Asp Lys Tyr Ser          35 40 45 Leu Lys Pro Asn Asp Gln Ile Leu Ala Glu Asp Lys His Lys Glu Leu      50 55 60 Phe Asp Glu Leu Val Lys Lys Phe Lys Val Glu Tyr His Ala Gly Gly  65 70 75 80 Ser Thr Gln Asn Ser Met Lys Val Ala Gln Trp Leu Ile Gln Glu Pro                  85 90 95 His Lys Ala Ala Thr Phe Phe Gly Cys Ile Gly Ile Asp Lys Phe Gly             100 105 110 Glu Ile Leu Lys Arg Lys Ala Ala Asp Ala His Val Asp Ala His Tyr         115 120 125 Tyr Glu Gln Asn Glu Gln Pro Thr Gly Thr Cys Ala Ala Cys Ile Thr     130 135 140 Gly Gly Asn Arg Ser Leu Val Ala Asn Leu Ala Ala Ala Asn Cys Tyr 145 150 155 160 Lys Lys Glu Lys His Leu Asp Leu Glu Arg Asn Trp Val Leu Val Glu                 165 170 175 Lys Ala Arg Val Tyr Tyr Ile Ala Gly Phe Phe Leu Thr Val Ser Pro             180 185 190 Glu Ser Val Leu Lys Val Ala Arg Tyr Ala Ala Glu Asn Asn Arg Val         195 200 205 Phe Thr Leu Asn Leu Ser Ala Pro Phe Ile Ser Gln Phe Phe Lys Glu     210 215 220 Ala Leu Met Asp Val Met Pro Tyr Val Asp Ile Leu Phe Gly Asn Glu 225 230 235 240 Thr Glu Ala Ala Thr Phe Ala Arg Glu Gln Gly Phe Glu Thr Lys Asp                 245 250 255 Ile Lys Glu Ile Ala Lys Lys Ala Gln Ala Leu Pro Lys Val Asn Ser             260 265 270 Lys Arg Gln Arg Thr Val Ile Phe Thr Gln Gly Arg Asp Asp Thr Ile         275 280 285 Val Ala Ala Glu Asn Asp Val Thr Ala Phe Pro Val Leu Asp Gln Asn     290 295 300 Gln Glu Glu Ile Ile Asp Thr Asn Gly Ala Gly Asp Ala Phe Val Gly 305 310 315 320 Gly Phe Leu Ser Gln Leu Val Ser Asp Lys Pro Leu Thr Glu Cys Ile                 325 330 335 Arg Ala Gly His Tyr Ala Ala Ser Val Ile Arg Arg Thr Gly Cys             340 345 350 Thr Phe Pro Glu Lys Pro Asp Phe         355 360 <210> 3 <211> 299 <212> PRT <213> Tau transgenic mouse <400> 3 Met Ser Ser Ile Lys Val Glu Cys Val Leu Arg Glu Asn Tyr Arg Cys   1 5 10 15 Gly Glu Ser Pro Val Trp Glu Glu Ala Ser Gln Ser Leu Leu Phe Val              20 25 30 Asp Ile Pro Ser Lys Ile Ile Cys Arg Trp Asp Thr Val Ser Asn Gln          35 40 45 Val Gln Arg Val Ala Val Asp Ala Pro Val Ser Ser Val Ala Leu Arg      50 55 60 Gln Leu Gly Gly Tyr Val Ala Thr Ile Gly Thr Lys Phe Cys Ala Leu  65 70 75 80 Asn Trp Glu Asn Gln Ser Val Phe Val Leu Ala Met Val Asp Glu Asp                  85 90 95 Lys Lys Asn Asn Arg Phe Asn Asp Gly Lys Val Asp Pro Ala Gly Arg             100 105 110 Tyr Phe Ala Gly Thr Met Ala Glu Glu Thr Ala Pro Ala Val Leu Glu         115 120 125 Arg His Gln Gly Ser Leu Tyr Ser Leu Phe Pro Asp His Ser Val Lys     130 135 140 Lys Tyr Phe Asp Gln Val Asp Ile Ser Asn Gly Leu Asp Trp Ser Leu 145 150 155 160 Asp His Lys Ile Phe Tyr Tyr Ile Asp Ser Leu Ser Tyr Thr Val Asp                 165 170 175 Ala Phe Asp Tyr Asp Leu Gln Thr Gly Gln Ile Ser Asn Arg Arg Ile             180 185 190 Val Tyr Lys Met Glu Lys Asp Glu Gln Ile Pro Asp Gly Met Cys Ile         195 200 205 Asp Ala Glu Gly Lys Leu Trp Val Ala Cys Tyr Asn Gly Gly Arg Val     210 215 220 Ile Arg Leu Asp Pro Glu Thr Gly Lys Arg Leu Gln Thr Val Lys Leu 225 230 235 240 Pro Val Asp Lys Thr Thr Ser Cys Cys Phe Gly Gly Lys Asp Tyr Ser                 245 250 255 Glu Met Tyr Val Thr Cys Ala Arg Asp Gly Leu Asn Ala Glu Gly Leu             260 265 270 Leu Arg Gln Pro Asp Ala Gly Asn Ile Phe Lys Ile Thr Gly Leu Gly         275 280 285 Val Lys Gly Ile Ala Pro Tyr Ser Tyr Ala Gly     290 295

Claims (8)

An adenosine kinase (Isoform Long) having an amino acid sequence of SEQ ID NO: 2 having a molecular weight of 39-41 kD and a pi of 5.3-6.3, wherein the proteinaceous marker composition for early diagnosis of Alzheimer's disease.
The composition for early diagnosis of Alzheimer's disease, comprising a substance for detecting the presence or absence of the proteinaceous marker of claim 1, and an amount, a pattern, or both.
The method of claim 2, wherein the substance that detects the presence of the proteinaceous marker and its amount, pattern, or both is an antibody that specifically recognizes the proteinaceous marker for early diagnosis of Alzheimer's disease. Composition.
A composition for early diagnosis of Alzheimer's disease comprising a substance for detecting whether the gene encoding the proteinaceous marker of claim 1 is expressed, and its expression amount, expression pattern, or both.
The method of claim 4, wherein the expression of the gene encoding the proteinaceous marker, and the amount of expression, expression pattern, or a substance for detecting both are used for detecting mRNA of the gene encoding the proteinaceous marker for early diagnosis of Alzheimer's disease. Alzheimer's disease early diagnosis composition, characterized in that the primer for RT-PCR.
Alzheimer's disease early diagnosis kit comprising the composition for early diagnosis of Alzheimer's disease according to any one of claims 2 to 5.
A method for detecting the proteinaceous marker of claim 1 in order to provide information necessary for early diagnosis of Alzheimer's disease.
A method of screening an Alzheimer's disease therapeutic agent comprising the step of treating a test compound to a cell or animal expressing the proteinaceous marker of claim 1 and confirming whether the test compound promotes the physiological activity of the proteinaceous marker.

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