KR102574799B1 - Bio-marker for drug addiction diagnosis and kit for drug addiction diagnosis - Google Patents

Abstract

The present invention relates to a biomarker composition capable of diagnosing drug addiction or withdrawal symptoms and a kit capable of diagnosing drug addiction or withdrawal symptoms. According to the present invention, the RAVER2 protein is specific in animal models showing drug addiction or withdrawal symptoms As it was confirmed that it had the effect of regulating the biomarker miR-137, which can diagnose drug addiction or withdrawal symptoms, drug addiction or withdrawal symptoms can be diagnosed using the RAVER2 protein In addition, it can be usefully used in related pharmaceutical and medical businesses.

Description

Biomarker composition for diagnosing drug addiction or withdrawal symptoms and kit for diagnosing drug addiction or withdrawal symptoms {Bio-marker for drug addiction diagnosis and kit for drug addiction diagnosis}

The present invention relates to a biomarker composition capable of diagnosing drug addiction or withdrawal symptoms and a kit capable of diagnosing drug addiction or withdrawal symptoms, and more particularly, to a biomarker composition for diagnosing drug addiction or withdrawal symptoms based on RAVER2 protein it's about

In general, "narcotics" means compounds having effects similar to opium and its derivatives, and prior to this, opium poppy, including habit-forming or addictive drugs such as morphine, cocaine, noscapine, papaverine, thebaine, heroin, etc. It is defined as an opioid or opioid drug that is a natural combination derived from plants. Demonstrate that in order for a compound to be classified as a narcotic, it must be capable of causing "significant changes in mood and behavior", must be capable of causing a state of "coma analgesia", and can present serious risks such as dependence, tolerance and addiction. shall.

Abuse of these narcotics leads to dependence on the drug, and tolerance in which the amount must be increased each time it is used to be effective. The phenomenon is called drug addiction.

Drug addicts are in a state where they cannot live a normal life without using addictive drugs, and in addition to showing a tendency to become immersed in a certain part, paranoid schizophrenia, heart failure, increased blood pressure, vomiting, difficulty breathing, loss of sensory ability, Symptoms include loss of consciousness, nightmares, and confusion.

So far, appearance and behavioral analysis, urine, blood tests, or eye tests have been performed as diagnostic methods to determine these drug addiction or withdrawal symptoms, but these are low in accuracy, , it can be applied only when pathological symptoms are present.

Therefore, several studies have been conducted to identify the cause of these pathological symptoms, but research and development for solving these causes and diagnosing drug withdrawal symptoms has not been conducted.

In order to solve this problem, literature has been published that attempts to determine drug addiction using microRNA, but this also requires a fairly limited and complicated pretreatment experiment process, along with the problem that a lot of processing time and labor is required. There is a limitation that most of the symptoms are related to the condition of continuous exposure.

Patent Document 1. Korean Patent Publication No. 10-2017-0125493

The present invention has been made to solve the above problems, and an object of the present invention is a composition for diagnosing drug addiction or withdrawal symptoms, and a kit for diagnosing drug addiction or withdrawal symptoms, which can quickly and accurately diagnose and analyze drug addiction or withdrawal symptoms. And it is intended to provide a method of providing information for diagnosing drug addiction or withdrawal symptoms.

The present invention has been made to solve the above problems, and an object of the present invention is to provide a method for screening a candidate substance capable of preventing or treating drug addiction or withdrawal symptoms.

In order to achieve the above object, the present invention provides a biomarker composition for diagnosing drug addiction or withdrawal symptoms containing RAVER2 protein as an active ingredient.

The RAVER2 protein may be any one selected from the amino acid sequence represented by SEQ ID NO: 1.

The drug may be any one selected from the group consisting of methamphetamine, amphetamine, cocaine, ecstasy and methylphenitate, preferably methamphetamine.

Expression of the RAVER2 protein may be decreased in biological samples isolated from animals suffering from drug addiction or drug withdrawal symptoms.

In order to achieve the above object, the present invention provides a composition for diagnosing drug addiction or withdrawal symptoms comprising an agent for measuring the expression of a RAVER2 protein or a gene encoding the RAVER2 protein.

The agent for measuring the expression of the RAVER2 protein comprises any one selected from the group consisting of oligopeptides, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, ligands and aptamers that specifically bind to the RAVER2 protein it could be

An agent for measuring the expression of the gene encoding the RAVER2 protein may be an antisense oligonucleotide, a primer pair or a probe.

In order to achieve the above object, the present invention provides a kit for diagnosing drug addiction or withdrawal symptoms comprising the composition.

The kit may be a RT-PCR kit, a DNA chip kit, or a protein chip kit.

The present invention provides a method for providing information necessary for diagnosing drug addiction or withdrawal symptoms in order to achieve the above object.

a) measuring the expression level of the RAVER2 protein or the gene encoding it in a sample isolated from the subject; and

b) comparing the expression level of the RAVER2 protein or gene encoding the RAVER2 protein selected in a) with the expression level of the corresponding protein or gene in a control sample.

The sample may be brain tissue.

In order to achieve the above object, the present invention provides a method for screening drug addiction or withdrawal treatment candidates comprising the following steps.

a) processing a candidate substance in a cell or animal model expressing the RAVER2 protein;

b) measuring the expression level of RAVER2 protein from cells or animal models treated with the candidate substance in step a); and

c) selecting a candidate material that increases the expression level of the RAVER2 protein measured in step 2) compared to a control group not treated with the candidate material.

According to the present invention, the RAVER2 protein showed a specific expression pattern in animal models showing drug addiction or withdrawal symptoms, and it was confirmed that it had the effect of regulating the biomarker miR-137 that can diagnose drug addiction or withdrawal symptoms. Bar, drug addiction or withdrawal symptoms can be diagnosed using the RAVER2 protein, and it can be usefully used in related pharmaceutical and medical businesses.

1A is data obtained by measuring the expression levels of miR-135a, miR-135b, miR-137, miR-139-5p, and miR-132 in the striatum for each group according to Experimental Example 1. B. of the present invention.
Figure 1b is for analyzing the miR-137 gene associated with withdrawal symptoms for each group according to Experimental Example 1. C of the present invention, 1-2 weeks, 3 methamphetamine through a gradual ramp-up protocol - The time elapsed after injection was stopped in the exposure group injected for 4 weeks and the group in which withdrawal symptoms were induced by methamphetamine (1-2 weeks, 3-4 weeks, 5-6 weeks, 7-8 weeks) This is a result of comparing the expression level of miR-137 from the withdrawal test group (withdrawal) in which blood was collected according to ).
In the drawing, it was calculated as a continuous time from the injection process of 4 weeks, and marked as 5-6W, 7-8W, 9-10W, 11-12W.
FIG. 1C is a graph showing the miR-137 expression level in the blood of FIG. 1B relative to the miR-137 expression level in the striatum of FIG. 1A.
Figure 1d is for analyzing the miR-132 gene expression pattern for each group according to Experimental Example 1. C of the present invention, methamphetamine was administered for 1-2 weeks, 3-4 weeks through a ramp-up protocol. After injection was stopped in the exposure group injected weekly and the group in which withdrawal symptoms were induced by methamphetamine, the elapsed time (1-2 weeks, 3-4 weeks, 5-6 weeks, 7-8 weeks) This is the result of comparing the expression level of miR-132 from the withdrawal test group (withdrawal) in which blood was collected according to the results.
In the drawing, it was calculated as a continuous time from the injection process of 4 weeks, and marked as 5-6W, 7-8W, 9-10W, 11-12W.
Figure 1e is for analyzing the miR-135a gene expression pattern from the blood of each group according to Experimental Example 1. C of the present invention.
Figure 2a is a schematic diagram showing the experimental design of the mouse animal model in which withdrawal symptoms are induced according to Experimental Example 2 of the present invention.
Figure 2b is a result of comparing the expression level of miR-137 from striatum after inducing methamphetamine withdrawal symptoms for each group according to Experimental Example 2. B of the present invention for 1, 3, and 6 weeks.
Figure 2c is for analyzing the miR-137 gene associated with withdrawal symptoms for each group according to Experimental Example 2. C of the present invention, after injecting methamphetamine for 2 weeks through a gradual ramp-up protocol , This is the result of comparing the expression level of miR-137 from the blood of animal models when 1, 3, and 6 weeks elapsed after stopping the infusion (withdrawal).
FIG. 2d is a graph showing the miR-137 expression level in the blood of FIG. 2c relative to the miR-137 expression level in the striatum of FIG. 2b.
Figure 3a is to identify proteins related to withdrawal symptoms for each group according to Experimental Example 3. B of the present invention, after inducing withdrawal symptoms, hnRNPA1, hnRNPA3, hnPNPDL, hnRNPU, PTBP-1, RAVER2 from the striatum , It is a result of comparing the mRNA expression levels of SYNCRIP and YBX1.
Figure 3b is to identify proteins related to withdrawal symptoms for each group according to Experimental Example 4. B of the present invention, after inducing withdrawal symptoms (1 week, 3 weeks, 6 weeks), RAVER2 protein expression from the striatum The result of confirming the amount through Western blot (left) and the graph of qPCR result (right).
Figure 4a shows the nucleotide sequence of RAVER2 mRNA and the nucleotide sequence of miR-137.
4b is a result of measuring luciferase activity in HEK293TN cells according to Experimental Example 5 of the present invention. For each cell, the expression level of RAVER2 3'UTR was measured in the group (miR-137) and control group through the luciferase vector.
Figure 4c is a diagram showing the schematic structure of the lentiviral vector and the experimental design for inhibiting intracellular RAVER2 protein expression using the lentiviral vector.
Figure 4d is a fluorescence microscopy image of SH-SY5Y cells according to Experimental Example 5 of the present invention. , For each cell group (shRAVER2 group) and control group via a lentiviral vector.
Figure 4e is a graph showing the amount of RAVER2 mRNA expression measured in a group (shRAVER2 group) and a control group through a lentiviral vector according to Experimental Example 5 of the present invention.
Figure 4f is a graph (right) of the result of measuring the expression level of RAVER2 protein by Western blot in the group (shRAVER2 group) and control group through the lentiviral vector according to Experimental Example 5 of the present invention (left) and qPCR (right).
4g and h are graphs showing the amount of miR-137 expression measured in a group (shRAVER2 group) and a control group through a lentiviral vector according to Experimental Example 5 of the present invention.
Figure 5a is a graph analyzing the miR-137 expression level in the blood of patients undergoing a methamphetamine withdrawal period by qPCR, and Figure 5c is a graph analyzing the miR-137 expression level in the blood of patients undergoing a methamphetamine withdrawal period by qPCR. am.
5b, 5d and 5e are results of ROC curve analysis using miR-137 in the blood of patients undergoing a methamphetamine withdrawal period.

In the following, various aspects and various embodiments of the present invention will be described in more detail.

One aspect of the present invention relates to a biomarker composition for diagnosing drug addiction or withdrawal symptoms comprising RAVER2 protein as an active ingredient.

In the present invention, "RAVER2 protein" is composed of 678 amino acids in humans and 673 amino acids in mice, and is a protein with a molecular weight of 74 kDa and can be represented by SEQ ID NO: 1.

In addition, the mRNA sequence of the RAVER2 protein has been registered with NCBI (National Center for Biotechnology Information) (human NM_018211.3) (SEQ ID NO: 2), (mouse NM_183024.1) (SEQ ID NO: 3).

In the present invention, "diagnosis" refers to confirming the presence or characteristics of a pathological state, and the diagnosis includes not only drug addiction but also confirming drug withdrawal symptoms that appear after stopping drugs. For purposes of this invention, diagnosis means identifying a state or characteristic of addiction or withdrawal from a narcotic.

In addition, "biomarker" can be used as a diagnostic marker or diagnostic marker, and is a substance that can be diagnosed by distinguishing whether drug addiction or withdrawal symptoms occur in biological samples. Compared to normal samples, drug withdrawal symptoms are induced in animal models and organic biomolecules such as polypeptides or nucleic acids (mRNA) that show a decrease in RAVER2 protein expression.

The term "drug addiction" comprehensively refers to poisoning caused by administering and consuming drugs, which are highly dependent drugs, and is a type of drug dependence. It can be defined as a state of lack of self-control ability to take drugs, and a state of long-term exposure to drugs.

In addition, "drug withdrawal" means repeated administration of a drug, which is a drug with high dependence, and craving to take a drug is induced during a withdrawal period in which the drug is not taken in a state of high dependence on the drug , it refers comprehensively to symptoms such as various mental disorders that occur as a result.

The drug may be any one selected from the group consisting of methamphetamine, amphetamine, cocaine, ecstasy and methylphenitate, preferably methamphetamine.

In one embodiment of the present invention, it was confirmed that RAVER2 was specifically reduced in an animal model in which drug addiction or drug withdrawal symptoms were induced. It can be utilized as a marker composition.

Another aspect of the present invention relates to a composition for diagnosing narcotic addiction or withdrawal symptoms, which measures the expression of the RAVER2 protein or the gene encoding it, and can be used to diagnose drug addiction or withdrawal symptoms from a subject.

In a specific embodiment of the present invention, the present inventors produced an animal model in which drug withdrawal symptoms were induced by discontinuing the drug after long-term administration. The produced animal model was found to decrease RAVER2 in brain tissue.

Accordingly, the present inventors compared the expression patterns of miR-137 related to drug addiction and withdrawal symptoms according to the expression patterns of RAVER2 in the animal model, and as a result, it was confirmed that the expression of miR-137 can be controlled by RAVER2. This indicates that when the expression of RAVER2 is lowered, drug addiction or withdrawal symptoms can be diagnosed in animal models. That is, since the RAVER2 protein shows the same tendency as miR-137, which diagnoses drug addiction or withdrawal symptoms, it can be seen that drug addiction or withdrawal symptoms can be diagnosed and confirmed through an abnormal decrease in RAVER2 protein.

In the present invention, "an agent for measuring the expression of a gene encoding RAVER2 protein or RAVER2" is a protein of RAVER2, a marker whose expression is increased in a biological sample of an animal or animal model in which drug addiction or drug withdrawal symptoms are induced as described above, or It refers to a molecule that can be used for detection of a marker by specifically binding to the mRNA encoding it and confirming its expression level.

An agent for measuring the expression of the RAVER2 protein is an oligopeptide, monoclonal antibody, polyclonal antibody, chimeric antibody, ligand, PNA (peptide nucleic acid) or aptamer that specifically binds to the RAVER2 protein (aptamer).

In the present invention, "antibody" is a term known in the art and refers to a specific protein molecule directed against an antigenic site. For the purpose of the present invention, an antibody refers to an antibody that specifically binds to the RAVER2 protein, which is a marker of the present invention, and such an antibody is cloned into an expression vector according to a conventional method and encoded by the marker gene. Obtain a protein to be, and can be prepared from the obtained protein by a conventional method. This includes partial peptides that can be made from the protein. The form of the antibody of the present invention is not particularly limited, and polyclonal antibodies, monoclonal antibodies, or any antibody having antigen-binding properties are also included in the antibody of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies.

Antibodies used for detecting markers for diagnosing drug addiction or drug withdrawal symptoms of the present invention include functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and includes Fab, F(ab'), F(ab') 2 and Fv.

An agent for measuring the expression of the gene encoding the RAVER2 protein may be an antisense oligonucleotide, a primer pair or a probe.

The "primer" is a nucleic acid sequence having a short free 3' hydroxyl group, capable of forming a base pair with a complementary template, and serving as a starting point for template strand copying Refers to a short nucleic acid sequence. Primers can initiate DNA synthesis in the presence of reagents for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature. In the present invention, PCR amplification is performed using sense and antisense primers of the RAVER2 gene, and cancer can be diagnosed through whether a desired product is produced. PCR conditions and lengths of sense and antisense primers can be modified based on those known in the art.

The "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to as short as several bases to as long as hundreds of bases capable of specific binding to mRNA, and is labeled so that the presence or absence of a specific mRNA can be confirmed. The probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like. In the present invention, hybridization is performed using a probe complementary to the RAVER2 gene, and cancer can be diagnosed through hybridization. Selection of suitable probes and hybridization conditions can be modified based on those known in the art.

The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences can also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, "capping", substitution of one or more homologues of a natural nucleotide, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) or to charged linkages (eg phosphorothioates, phosphorodithioates, etc.).

In addition, the present invention provides a kit for diagnosing drug addiction or withdrawal symptoms comprising the composition.

The kit may be a RT-PCR kit, a DNA chip kit, or a protein chip kit.

The kit may include not only a preparation for measuring the expression of a RAVER2 protein or a gene encoding the RAVER2 protein, but also tools and reagents commonly used in the art for immunological analysis.

Examples of such tools or reagents include, but are not limited to, suitable carriers, labeling substances capable of generating a detectable signal, chromophores, solubilizers, detergents, buffers, stabilizers, and the like. When the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator. The carrier includes a soluble carrier and an insoluble carrier, and an example of the soluble carrier is a physiologically acceptable buffer known in the art, such as PBS, and an example of the insoluble carrier is polystyrene, polyethylene, polypropylene, polyester, poly polymers such as acrylonitrile, fluororesin, crosslinked dextran, polysaccharide, latex-coated magnetic microparticles, other paper, glass, metal, agarose, and combinations thereof.

Since the kit of the present invention includes a biomarker composition and a diagnostic composition, and the description thereof has been specifically described above, the overlapping contents may refer to the above contents.

According to the present invention, when the expression level of the RAVER2 protein and the gene encoding it is reduced in a sample isolated from a subject, it can be determined that he or she is experiencing drug addiction or drug withdrawal symptoms.

That is, the present invention, a) measuring the expression level of the RAVER2 protein or the gene encoding it in a sample isolated from the subject; and

b) Gene detection method for providing information necessary for diagnosing drug addiction or withdrawal symptoms by comparing the expression level of the RAVER2 protein selected in a) or the gene encoding it with the expression level of the corresponding protein or gene in the control sample can provide.

Here, the measurement of the expression level of the RAVER2 protein or the gene encoding it is to confirm the presence and expression level of an expressed protein capable of diagnosing drug addiction or drug withdrawal symptoms in a sample isolated from a subject. Alternatively, it may be confirmed using a biomolecule that specifically binds to the gene encoding it.

The measurement of the expression level of the RAVER2 protein or the gene encoding it is not particularly limited as long as it is a method commonly used in the art.

Specifically, protein expression levels were determined by immunoprecipitation, radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunohistochemistry, RT-PCR, Western blotting and flow cytometry (FACS). It can be measured by any one method selected from the group consisting of

The gene encoding the RAVER2 protein can be identified through the expression level of mRNA, and the mRNA expression level is reverse transcriptase polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (real-time PCR), It can be measured by any one method selected from the group consisting of next-generation sequencing (NGS).

The sample is not particularly limited thereto, but may be blood, plasma, serum, urine, saliva or tissue, and the tissue may be brain tissue, preferably brain tissue.

In addition, another aspect of the present invention is a) processing a candidate substance in a cell or animal model expressing the RAVER2 protein;

b) measuring the expression level of RAVER2 protein from cells or animal models treated with the candidate substance in step a); and

c) Candidates for drug addiction or withdrawal symptoms can be screened through the step of selecting a candidate substance that increases the expression level of the RAVER2 protein measured in step 2) compared to the control group not treated with the candidate substance.

The test substance may be any one selected from the group consisting of natural compounds, synthetic compounds, RNA, DNA, polypeptides, enzymes, proteins, ligands, antibodies, antigens, bacterial or fungal metabolites, and bioactive molecules.

Hereinafter, the present invention will be described in more detail through examples and the like, but the scope and contents of the present invention are reduced or limited by the examples below and cannot be interpreted. In addition, based on the disclosure of the present invention including the following examples, it is clear that a person skilled in the art can easily practice the present invention for which no experimental results are specifically presented, and the patents to which such variations and modifications are attached. It goes without saying that it falls within the scope of the claims.

In addition, the experimental results presented below are only representative experimental results of the above examples and comparative examples, and each effect of various embodiments of the present invention that is not explicitly presented below will be described in detail in the corresponding section.

Experimental example 1. Methamphetamine withdrawal monkey animal model

go. Preparation of experimental animals and methamphetamine withdrawal animal models

In this experiment, 2-3 year old crab-eating monkeys ( Macaca Fascicularis ) were prepared for use as experimental animals, and the experimental process was limited to 120 g of standard mammalian diet per 12 hours and allowed to consume water as desired, and the Animal Protection and Use Committee of the Korea Institute of Toxicology It was treated according to the strict authorization of

The experimental animals were allowed to have an adaptation period of 1 week or more, and animals with abnormalities observed by observing general behavioral abnormalities were not used in this experiment. All experimental animals were allowed to consume drinking water and feed freely during the experimental period under conditions of 12-hour lighting and 12-hour shading in a breeding farm capable of constant temperature and humidity.

In the present invention, five animals were randomly assigned to two groups (two control groups and three experimental groups) as follows.

Control group: After repeated intravenous injection of 0.5 ml/kg of sterile saline, not methamphetamine, at the same time 6 days a week for 4 weeks, the injection was stopped for 8 weeks (control).

1-2 week experimental group: To induce addiction or exposure to methamphetamine, a gradual ramp-up protocol was used. Specifically, in the first week, 6 days per week, 0.1 to 0.3 mg/kg once a day. Injection, 0.2-0.4mg/kg twice a day, 6 days per week in the second week (1-2W Exposure).

3-4 week experimental group: To induce addiction or exposure to methamphetamine, a ramp-up protocol was used. Specifically, in the first week, 0.1 to 0.3 mg/kg was administered once a day, 6 days per week. Injections, 0.2-0.4 mg/kg 2 times a day, 6 days per week in the second week, 0.3-0.5 mg/kg 3 times a day, 6 days per week in the third week, and 6 days per week in the fourth week, A group injected with 0.6 mg/kg three times a day (3-4W Exposure).

Withdrawal experimental group: After injecting methamphetamine for 4 weeks using a gradual ramp-up protocol like the 3-4 week experimental group to induce addiction or exposure, withdrawal symptoms due to methamphetamine were discontinued for 8 weeks This derived group (Meth or Withdrawal).

me. The caudate of each group and from the putamen qPCR Through miR -135a, miR -135b, miR -137, miR -139-5p, miR Comparison of expression patterns of -132

Expression of miR-135a, miR-135b, miR-137, miR-139-5p, and miR-132 after RNA was isolated from the striatum of the same area from the two groups prepared in A. Quantities were confirmed via qPCR.

Figure 1a is for analyzing genes related to withdrawal symptoms for each group according to Experimental Example 1. B of the present invention, after inducing withdrawal symptoms, miR-135a, miR-135b, miR-137, miR from the striatum This is the result of comparing the expression levels of -139-5p and miR-132. According to this, it was confirmed that miR-137 among miRNAs was increased through qPCR in the striatum of a monkey animal model (experimental group) in which withdrawal symptoms were induced by methamphetamine.

all. blood from each group qPCR Through miR -135a, miR -135b, miR -137, miR -139-5p, miR Comparison of expression patterns of -132

From the two groups prepared in A. above, blood was taken and RNA was isolated, and the expression levels of miR-135a, miR-135b, miR-137, miR-139-5p, and miR-132 were confirmed by qPCR. .

Figure 1b is for analyzing the miR-137 gene associated with withdrawal symptoms for each group according to Experimental Example 1. C of the present invention, 1-2 weeks, 3 methamphetamine through a gradual ramp-up protocol - The time elapsed after injection was stopped in the exposure group injected for 4 weeks and the group in which withdrawal symptoms were induced by methamphetamine (1-2 weeks, 3-4 weeks, 5-6 weeks, 7-8 weeks) This is a result of comparing the expression level of miR-137 from the withdrawal test group (withdrawal) in which blood was collected according to ).

In the drawing, it was calculated as a continuous time from the injection process of 4 weeks, and marked as 5-6W, 7-8W, 9-10W, 11-12W.

FIG. 1c is a graph showing the miR-137 expression level in the striatum of FIG. 1a and the miR-137 expression level in the blood.

Figure 1d is for analyzing the miR-132 gene expression pattern for each group according to Experimental Example 1. C of the present invention, methamphetamine was administered for 1-2 weeks, 3-4 weeks through a ramp-up protocol. After injection was stopped in the exposure group injected weekly and the group in which withdrawal symptoms were induced by methamphetamine, the elapsed time (1-2 weeks, 3-4 weeks, 5-6 weeks, 7-8 weeks) This is the result of comparing the expression level of miR-132 from the withdrawal test group (withdrawal) in which blood was collected according to the results.

In the drawing, it was calculated as a continuous time from the injection process of 4 weeks, and marked as 5-6W, 7-8W, 9-10W, 11-12W.

Figure 1e is for analyzing the miR-135a gene expression pattern from the blood of each group according to Experimental Example 1. C of the present invention.

As shown in FIGS. 1B to 1E , it was confirmed that miR-137 was reduced in the blood of animal models after withdrawal symptoms were induced. In addition, as a result of comparing the expression patterns of miR-137 in the striatum and blood in the experimental group, it was confirmed that an inverse correlation was shown.

Furthermore, in the striatum of the experimental group, miR-132 and miR-135a, whose expression patterns were changed due to withdrawal symptoms, were not detected in blood samples.

Experimental example 2. Methamphetamine withdrawal mouse animal model

go. Preparation of experimental animals and methamphetamine withdrawal animal models

In this experiment, mice over 7 weeks of age were prepared for use as experimental animals, and the experimental process allowed standard rodent diet and water to be consumed as desired, and animal protection and use by the Korea Institute of Science and Technology It was processed according to the strict approval of the committee.

The experimental animals were allowed to have an adaptation period for 1 week, and general behavior abnormalities were observed, and animals with abnormalities were not used in this experiment. All experimental animals were allowed to consume drinking water and feed freely during the experimental period under conditions of 12-hour lighting and 12-hour shading in a breeding farm capable of constant temperature and humidity.

In the present invention, 16 or more animals were randomly assigned to two groups (8 or more control groups and 8 or more experimental groups) as follows (see FIG. 2a).

Control group: 5.0 ml/kg of sterile saline, not methamphetamine, was injected intravenously 6 days a week, at the same time every day for 2 weeks, and the injection was stopped for 1, 3, and 6 weeks (control),

Withdrawal experimental group: To induce addiction or exposure to methamphetamine, a ramp-up protocol was used. 1.5-2.5 mg/kg twice daily on days 4-6 of week 1, 2.5-3.5 mg/kg 3 times daily on days 1-3 of week 2, and 4.0 mg on days 4-6 of week 2 /kg was injected 3 times a day, and then the injection was stopped for 1, 3, and 6 weeks (1, 3, 6 withdrawal time) to induce withdrawal symptoms by methamphetamine (meth)

me. of each group from the dorsal striatum qPCR Through miR Comparison of expression patterns of -137

From the two groups prepared in A. above, dorsal striatum parts of the same area were taken and RNA was isolated, and the expression level of miR-137 was confirmed through qPCR.

Figure 2b is a result of comparing the expression level of miR-137 from striatum after inducing methamphetamine withdrawal symptoms for each group according to Experimental Example 2. B of the present invention for 1, 3, and 6 weeks. According to this, it was confirmed that among the miRNAs, miR-137 was continuously increased in the striatum of the mouse animal model (experimental group) in which withdrawal symptoms were induced by methamphetamine.

all. blood from each group qPCR Through miR Comparison of expression patterns of -137

From the two groups prepared in A. above, blood was taken and RNA was isolated, and the expression level of miR-137 was confirmed through qPCR.

Figure 2c is for analyzing the miR-137 gene associated with withdrawal symptoms for each group according to Experimental Example 1. C of the present invention, after injecting methamphetamine daily for 2 weeks, stopping the injection and 1, 3, 6 This is the result of comparing the expression level of miR-137 from the blood of animal models when the week has elapsed (withdrawal).

FIG. 2d is a graph showing the miR-137 expression level in the blood of FIG. 2c relative to the miR-137 expression level in the striatum of FIG. 2b.

As shown in FIG. 2c and d, it was confirmed that miR-137 was continuously reduced in the blood of the mouse animal model in which withdrawal symptoms were induced. In addition, as a result of comparing the miR-137 expression level in the striatum and blood of the mouse animal model in which withdrawal symptoms were induced, it was confirmed that an inverse correlation was shown.

In summary, through Experimental Examples 1 and 2, it was confirmed that the miR-137 gene is a useful biomarker that is related to withdrawal symptoms caused by drug withdrawal as well as addiction symptoms to which drugs (particularly methamphetamine) are continuously exposed.

Experimental example 3. Identification of proteins in relation to drug addiction or withdrawal symptoms-1

go. Preparation of laboratory animals and methamphetamine withdrawal monkey animal model

In this experiment, 2-3 year old crab-eating monkeys (Macaca Fascicularis) were prepared for use as experimental animals, and the experimental process was limited to 120 g of standard mammalian diet per 12 hours, allowed to consume water as desired, and safety evaluation It was processed according to the strict approval of the Animal Care and Use Committee of the Korea Institute of Toxicology.

The experimental animals were allowed to have an adaptation period of 1 week or more, and animals with abnormalities observed by observing general behavioral abnormalities were not used in this experiment. All experimental animals were allowed to consume drinking water and feed freely during the experimental period under conditions of 12-hour lighting and 12-hour shading in a breeding farm capable of constant temperature and humidity.

In the present invention, according to Experimental Example 1, 5 animals were randomly assigned to two groups (2 control groups and 3 experimental groups).

Control group: 0.5 ml/kg of sterile saline, not methamphetamine, was injected intravenously 6 days a week at the same time every day for 4 weeks, and the injection was stopped for 8 weeks (control),

Experimental group: A ramp-up protocol was used to induce addiction or exposure to methamphetamine. Week 6, 0.2-0.4mg/kg twice daily injection, 0.3-0.5mg/kg 6 days/week, 3 times daily injection during week 3, and 0.6mg/kg 6 days/week during week 4 A group in which withdrawal symptoms were induced by methamphetamine (Meth or Withdrawal) by injecting KG three times a day for 4 weeks and stopping the injection for 8 weeks (Meth or Withdrawal).

me. Discovery of new target genes related to withdrawal symptoms

From the two groups prepared in A. above, parts of the striatum of the same area were taken and RNA was isolated from the caudate and putamen, and then hnRNPA1, hnRNPA3, hnPNPDL, hnRNPU, PTBP-1, The mRNA expression levels of RAVER2, SYNCRIP and YBX1 were confirmed by qPCR.

Figure 3a is to identify proteins related to withdrawal symptoms for each group according to Experimental Example 3. B of the present invention, after inducing withdrawal symptoms, hnRNPA1, hnRNPA3, hnPNPDL, hnRNPU, PTBP-1, RAVER2 from the striatum , It is a result of comparing the mRNA expression levels of SYNCRIP and YBX1. According to this, it was confirmed that the mRNA of RAVER2 (a type of RNA-binding protein) was reduced through qPCR in the striatum of the monkey animal model (experimental group) in which withdrawal symptoms were induced by methamphetamine.

Experimental example 4. Identification of proteins in relation to drug addiction or withdrawal symptoms-2

go. Preparation of experimental animals and methamphetamine withdrawal mouse animal models

In this experiment, mice over 7 weeks of age were prepared for use as experimental animals, and the experimental process allowed standard rodent diet and water to be consumed as desired, and animal protection and use by the Korea Institute of Science and Technology It was processed according to the strict approval of the committee.

The experimental animals were allowed to have an adaptation period for 1 week, and general behavior abnormalities were observed, and animals with abnormalities were not used in this experiment. All experimental animals were allowed to consume drinking water and feed freely during the experimental period under conditions of 12-hour lighting and 12-hour shading in a breeding farm capable of constant temperature and humidity.

In the present invention, 16 or more animals were randomly assigned to two groups (8 or more control groups and 8 or more experimental groups) as in Experimental Example 2 above (see FIG. 2a).

Control group: 5.0 ml/kg of sterile saline, not methamphetamine, was injected intravenously 6 days a week for 2 weeks at the same time, and the injection was stopped for 1, 3, and 6 weeks (control),

Withdrawal experimental group: To induce addiction or exposure to methamphetamine, a ramp-up protocol was used. 1.5-2.5 mg/kg twice daily on days 4-6 of week 1, 2.5-3.5 mg/kg 3 times daily on days 1-3 of week 2, and 4.0 mg on days 4-6 of week 2 /kg was injected three times a day, and then injection was stopped for 1, 3, and 6 weeks (1, 3, 5 withdrawal time) to induce withdrawal symptoms by methamphetamine (meth)

me. of each group from the dorsal striatum qPCR Through miR Comparison of expression patterns of -137

From the two groups prepared in A. above, the same area of the striatum was taken and RNA was isolated, and then the amount of RAVER2 protein expression was confirmed through qPCR.

Figure 3b is to identify proteins related to withdrawal symptoms for each group according to Experimental Example 4. B of the present invention, after inducing withdrawal symptoms (1 week, 3 weeks, 6 weeks), RAVER2 protein expression from the striatum The result of confirming the amount through Western blot (left) and the graph of qPCR result (right). According to this, in the striatum of the mouse animal model (experimental group) in which withdrawal symptoms were induced by methamphetamine, the protein of RAVER2 was continuously reduced during the withdrawal period (1 week (W), 3 weeks (W), 6 weeks (W)) confirmed.

Experimental Example 5. Inhibition of intracellular RAVER2 protein expression using a lentiviral vector

go. cell culture, transfection and viral infection

HEK293TN, an immortalized human embryonic kidney cell line, was cultured and maintained in DMEM medium (Hyclone) containing 10% FBS and 1% penicillin/streptomycin. Then, in order to artificially reduce the expression of RAVER2 in each cell line, a lentiviral construct containing the shRAVER2 sequence (5'-GGATGAAGGTAGTTACGTTGG-3') was used. Lentivirus production was performed according to the instructions of the SBI manufacturer. Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.) was used to transfect the cells with pPACK plasmid, control or shRAVER2 lentiviral vectors, respectively. The cells were infected with control or shRAVER2-containing lentivirus, cultured for 2 days and then harvested.

Figure 4a shows the nucleotide sequence of RAVER2 mRNA and the nucleotide sequence of miR-137. According to this, it can be seen that RAVER2 and miR-137 are sequences that interact with each other.

me. luciferase assay ( luciferase assay)

Lentiviral vector for miR-137 expression Human pre-microRNA Expression Construct Lenti-miR-137 (System Biosciences, Mountain View, CA) and control vector, and luciferase vector miRNA 3ㅄUTR target expression clone for luciferase assay for Human RAVER2 (Genecopoeia, Rockville, MD) was used. Using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), HEK293TN cells were first transfected with a lentiviral vector for miR-137 expression or a control vector, followed by a luciferase vector containing the RAVER2 3'UTR sequence. were transfected with 2 mg of plasmid DNA. Cells were lysed 48 hours after transfection, and luciferase activity was measured using the Secrete-Pair Dual Luminescence Assay Kit (Genecopoeia, Rockville, MD, USA).

4b is a result of measuring luciferase activity in HEK293TN cells according to Experimental Example 5 of the present invention. For each cell, the expression level of RAVER2 3'UTR was measured in the group (miR-137) and control group through the luciferase vector.

As shown in FIG. 4b, as a result of measuring the luciferase activity, it was confirmed that miR-137 reduces RAVER2.

all. the lentivirus through shRAVER2 in cells into which RAVER2 Expression analysis

Figure 4c is a diagram showing the schematic structure of the lentiviral vector and the experimental design for inhibiting intracellular RAVER2 protein expression using the lentiviral vector.

In addition, Figure 4d is a fluorescence microscopy image in the SH-SY5Y neuronal cell line according to Experimental Example 5 of the present invention, for the group (shRAVER2 group) and the control group through the lentiviral vector for each cell, Figure 4e is the present invention According to Experimental Example 5 of the lentiviral vector through the group (shRAVER2 group) and the graph showing the amount of RAVER2 mRNA expression measured in the control group, Figure 4f is a group through the lentiviral vector (shRAVER2 group) according to Experimental Example 5 of the present invention ) and the result of measuring the expression of RAVER2 protein in the control group by Western blot (left) and a graph (right) measured by qPCR.

As shown in FIGS. 4D to 4F , it was confirmed that RAVER2 expression was successfully suppressed in the cells in which the expression of RAVER2 was artificially suppressed through the gene transfer technique using the lentivirus prepared in Experimental Example 6.

4g and h are graphs showing the amount of miR-137 expression measured in a group (shRAVER2 group) and a control group through a lentiviral vector according to Experimental Example 5 of the present invention, according to which the amount of miR-137 secreted from neurons As it was confirmed that this decrease, it was confirmed that the RAVER2 protein according to the present invention can effectively regulate miR-137.

In addition, it was confirmed that the amount of miR-137 was reduced when the RAVER2 protein was inhibited. Therefore, the RAVER2 protein according to the present invention is a parent substance that controls miR-137, and miR-137, as described above, is a drug addiction or drug withdrawal symptom Therefore, when looking at the overall results, it can be seen that drug addiction or drug withdrawal symptoms can be diagnosed through the RAVER2 protein.

In addition, since the effect of treating or preventing drug addiction or drug withdrawal symptoms can be evaluated through the activation level of the RAVER2 protein, a substance capable of treating drug addiction or drug withdrawal symptoms can be screened using this.

Experimental example 6. Withdrawal symptoms in the patient Gene expression pattern analysis

go. sample acquisition

Blood was collected from patients undergoing a methamphetamine withdrawal period. The patient was obtained from the Addiction Brain Center of Gangnam Eulji Hospital. Informed consent was provided in accordance with the Declaration of Helsinki, and written informed consent was obtained from all patients, and all experiments were conducted with approval from the Bugok National Hospital and the Korea Institute of Science and Technology.

me. miR -137 amount of expression analyze

Figure 5a is a graph analyzing the miR-137 expression level in the blood of patients undergoing a methamphetamine withdrawal period by qPCR, and Figure 5c is a graph of patients undergoing a methamphetamine withdrawal period for 3 months or less (WD≤0.25) and more than 3 months. (WD > 0.25) This is a graph of miR-137 expression level in blood analyzed by qPCR.

As shown in FIG. 5, miR-137 was significantly reduced in the blood of patients undergoing a methamphetamine withdrawal period, and there was no difference in the amount of miR-137 expression in the blood between patients divided into less than 3 months and more than 3 months of withdrawal, compared to the control group. It was confirmed by qPCR that it was equally reduced compared to

all. ROC analysis

5b, 5d and 5e are results of ROC curve analysis using miR-137 in the blood of patients undergoing a methamphetamine withdrawal period.

According to this, ROC curve analysis was performed using the miR-137 gene from the obtained sample. As a result, it was confirmed that blood miR-137 could detect methamphetamine withdrawal through the ROC curve.

In addition, it was confirmed that the expression level of miR-137 in the blood could detect methamphetamine withdrawal regardless of the withdrawal period.

<110> KOREA INSTITUTE OF SCIENCE AND TECHNOLOGY <120> Bio-marker for drug addiction diagnosis and kit for drug addiction diagnosis <130> HPC7733 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 514 <212> PRT <213> artificial sequence <220> <223> RAVER2 protein <400> 1 Val Thr Gly His Ser Lys Gly Tyr Gly Phe Val Glu Tyr Met Lys Lys 1 5 10 15 Asp Phe Ala Ala Lys Ala Arg Leu Glu Leu Leu Gly Arg Gln Leu Gly 20 25 30 Ala Ser Ala Leu Phe Ala Gln Trp Met Asp Val Asn Leu Leu Ala Ser 35 40 45 Glu Leu Ile His Ser Lys Cys Leu Cys Ile Asp Lys Leu Pro Ser Asp 50 55 60 Tyr Arg Asp Ser Glu Glu Leu Leu Gln Ile Phe Ser Ser Val His Lys 65 70 75 80 Pro Val Phe Cys Gln Leu Ala Gln Asp Glu Gly Ser Tyr Val Gly Gly 85 90 95 Phe Ala Val Val Glu Tyr Ser Thr Ala Glu Gln Ala Glu Glu Val Gln 100 105 110 Gln Ala Ala Asp Gly Met Thr Ile Lys Gly Ser Lys Val Gln Val Ser 115 120 125 Phe Cys Ala Pro Gly Ala Pro Gly Arg Ser Thr Leu Ala Ala Leu Ile 130 135 140 Ala Ala Gln Arg Val Met His Ser Asn Gln Lys Gly Leu Leu Pro Glu 145 150 155 160 Pro Asn Pro Val Gln Ile Met Lys Ser Leu Asn Asn Pro Ala Met Leu 165 170 175 Gln Val Leu Leu Gln Pro Gln Leu Cys Gly Arg Ala Val Lys Pro Ala 180 185 190 Val Leu Gly Thr Pro His Ser Leu Pro His Leu Met Asn Pro Ser Ile 195 200 205 Ser Pro Ala Phe Leu His Leu Asn Lys Ala His Gln Ser Ser Val Met 210 215 220 Gly Asn Thr Ser Asn Leu Phe Leu Gln Asn Leu Ser His Ile Pro Leu 225 230 235 240 Ala Gln Gln Gln Leu Met Lys Phe Glu Asn Ile His Thr Asn Asn Lys 245 250 255 Pro Gly Leu Leu Gly Glu Pro Pro Ala Val Val Leu Gln Thr Ala Leu 260 265 270 Gly Ile Gly Ser Val Leu Pro Leu Lys Lys Glu Leu Gly His His His 275 280 285 Gly Glu Ala His Lys Thr Ser Ser Leu Ile Pro Thr Gln Thr Thr Ile 290 295 300 Thr Ala Gly Met Gly Met Leu Pro Phe Phe Pro Asn Gln His Ile Ala 305 310 315 320 Gly Gln Ala Gly Pro Gly His Ser Asn Thr Gln Glu Lys Gln Pro Ala 325 330 335 Thr Val Gly Met Ala Glu Gly Asn Phe Ser Gly Ser Gln Pro Tyr Leu 340 345 350 Gln Ser Phe Pro Asn Leu Ala Ala Gly Ser Leu Leu Val Gly His His 355 360 365 Lys Gln Gln Gln Ser Gln Pro Lys Gly Thr Glu Ile Ser Ser Gly Ala 370 375 380 Ala Ser Lys Asn Gln Thr Ser Leu Leu Gly Glu Pro Pro Lys Glu Ile 385 390 395 400 Arg Leu Ser Lys Asn Pro Tyr Leu Asn Leu Ala Ser Val Leu Pro Ser 405 410 415 Val Cys Leu Ser Ser Pro Ala Ser Lys Thr Thr Leu His Lys Thr Gly 420 425 430 Ile Ala Ser Ser Ile Leu Asp Ala Ile Ser Gln Gly Ser Glu Ser Gln 435 440 445 His Ala Leu Glu Lys Cys Ile Ala Tyr Ser Pro Pro Phe Gly Asp Tyr 450 455 460 Ala Gln Val Ser Ser Leu Arg Asn Glu Lys Arg Gly Ser Ser Tyr Leu 465 470 475 480 Ile Ser Ala Pro Glu Gly Gly Ser Val Glu Cys Val Asp Gln His Ser 485 490 495 Gln Gly Thr Gly Ala Tyr Tyr Met Glu Thr Tyr Leu Lys Lys Lys Arg 500 505 510 Val Tyr <210> 2 <211> 4376 <212> RNA <213> artificial sequence <220> <223> Homo sapiens ribonucleoprotein, PTB binding 2 (RAVER2), mRNA <400> 2 cccgggcctc ctcccgcttt ctctctccgc ttcccctgga gcctccgagg agtccgcagc 60 cgctgggcgc ccgggaagat ggcggcggcg gcgggagacg gcggcggcga ggggggcgcg 120 ggcctgggca gcgcggcggg gctggggccg gggccggggc tgcgcgggca gggcccctca 180 gccgaagcgc acgagggcgc cccggacccg atgcccgccg cgctgcaccc tgaggaggtc 240 gccgcgcgac tgcagcggat gcagcgggag ctgagcaacc gcaggaaaat cctggtgaaa 300 aacctgcccc aggacagcaa ctgccaggaa gttcatgatt tgttaaaaga ctatgactta 360 aaatattgtt atgtggacag aaataaacga acagcttttg ttaccttat gaatggggaa 420 caagcccaga acgcaattca gatgtttcat caatattctt ttagaggaaa agacttaata 480 gtccagcttc agccaacaga tgctttgttg tgtattacca acgtgcccat ttcttttaca 540 tcagaagagt ttgaagaact tgttcgtgct tatggaaata ttgagagatg ttttctggtc 600 tatagtgaag ttactggcca ttccaaaggc tatggatttg tggaatacat gaaaaaggac 660 tttgctgcaa aggctagact ggagctattg ggtagacagt tgggagcatc agcactcttt 720 gcacaatgga tggatgttaa tctattggct tcagagctca ttcattctaa gtgcctttgt 780 attgataaac tccccagtga ctacagggat tcagaagagc tgttgcaaat tttttccagt 840 gtccataaac ctgtgttttg ccagcttgca caggatgaag gtagttacgt tggtggcttt 900 gcagtggttg aatatagcac tgcggagcag gctgaagagg tccagcaggc agcagacggt 960 atgaccatca agggcagcaa agtccaggtt tccttctgtg ctcctggagc gccagggcga 1020 agtacattag cagcattgat agcggctcaa cgtgtgatgc acagtaatca aaagggctta 1080 cttccagagc caaatccagt acaaattatg aaaagtttaa acaaccctgc catgttgcaa 1140 gttcttctac agccccagtt atgtggacga gctgttaaac cagccgttct tggaacacct 1200 cacagcttgc cacatctgat gaatccatcc atctctcctg catttttaca tttgaataaa 1260 gcacatcaga atctttctca tataccactg gcacaacaac aattaatgaa gtttgagaat 1320 attcatacta ataataaacc cggcttactt ggagagcccc cagctgtggt acttcagact 1380 gcactaggga tagggtcagt gcttccattg aaaaaggagt tgggacatca tcatggagaa 1440 gcacataaaa catctagcct gattccaact caaacaacga taacagctgg aatgggcatg 1500 ttaccattct ttccaaatca gcacattgct ggacaggctg ggccagggca cagtaatact 1560 caggagaaac agccagccac ggtgggaatg gcagaaggaa atttctcagg gtcgcagcct 1620 tatcttcaga gctttccaaa ccttgctgct ggaagcttgc tggtggggaca ccataagcag 1680 cagcaaagcc agccaaaagg cacagagata agttcagggg ctgcctctaa gaatcaaact 1740 tcactcttgg gagaaccacc aaaagaaatt cggctcagta aaaatccata cttgaatttg 1800 gcaagtgtgt tgcccagtgt gtgcttatca tcccctgcaa gtaaaaccac tcttcataag 1860 actggaattg caagcagcat tctggatgca atctctcagg gaagtgaatc acaacacgca 1920 ttggaaaagt gcattgctta ttctccacct tttggtgatt atgcacaggt gtcatcttta 1980 agaaatgaaa agcgaggctc atcatatctc atctctgccc cagaaggtgg ttcagtggaa 2040 tgtgttgacc agcattctca gggcacagga gcatattaca tggaaactta cttaaaaaag 2100 aagcgagtat actgagttaa gctctctcct aataaccccc taaggttctc tgcagtatct 2160 ctgctgttca tcgctgcctt aacttcattc aaactagcca tatcctttaa atacgggttt 2220 atagtttccc agaaggaact gtgttgtgca gcaggcagaa atgccaaata aaaaatagca 2280 ataagaagag acatcaattg aggacatttt ccactagagt cagatgcatc ttaatcagtg 2340 tgctaactta caatccattg gagaaactaa caatgttcca atattccttg atttaggact 2400 gggaataatt ctgacaccat atataaaaga agtttgaagc aataaatggg aaacagtgtt 2460 gtttcctgag ccttagaaga atgagtttct tataagccaa tgtgcatttc aatttacttt 2520 ccctagaagt aattgcattt atagattttt aggacttttc ctagaactac aagggtgcaa 2580 gtaccaaatg tgctgatggt aatattataa agatgctcta catgcacact caattacgcc 2640 cacaccagcc atgcaaaatt aaacccggta agagagtgac agtgatatcc aaggtatcca 2700 ctactaccga tgctttaaga tgttgtttac catttatacc ttctgtgaaa aatagaaggt 2760 attttaagaa aagtaacttt ggtgatctaa taaaagggtt tggcaaacta caccctgggt 2820 ccacatttgg tctaccaccc gcttttgtgc tgcctgcaag ctaagactaa tgtttacatt 2880 tttaaatggt tggaggaaaa aagaaaactt atgacatgaa atttatatga aattcagatt 2940 tcagtgtcca taaataaact tttatggga cgttgccatg ctcattcatt tacgtgttgt 3000 ctgtggcact cttacgctac aagggcagaa ctgattagct tcaacagaga ctgtatggcc 3060 tacaaagacc atatggccta aaatttcaag tatctggcca tttatatata gtttgctgat 3120 ccctgatcta atagcactaa ttctaatagc aactattcct ttcaattcac ctaaagacag 3180 acctttagcc tttagacaga cttgtaagtg gaaaggaaca tgggtttctg tgctcatttc 3240 atagcgtaga ctagtaatga tctaatgact agaatggggc ctattttgat cactaaatgc 3300 cctattattt ccacaatata gtatagtaaa gcagtcaata ctgcactttc tgatggagtc 3360 ttaaggagtc acagtattta taaccatttt gagctaataa gctatacctc actctctgct 3420 ttctctctca ctctttttga tagtgtgtta gggtgaaaaa tcagttattt aacccaattt 3480 acttgtgaaa gaagttttat ctttatggac tatgatgact tttgacatca ccatttcttg 3540 aagaccttgg tattaaatgc atgagaaacc ttttgccatc tagaaatttc taagtttgtt 3600 tatgttataa gactgtaatt aattgatagg gaatttggga tctcaatccg gggctttttg 3660 aaatcagatc attaaatatc tcgtcacttt ggggacttgt tatttttaac tatgatgtgg 3720 atatcctgtt ttaattatga agtctagaat tggtacatat gttgtatgct ttgtaaagaa 3780 tttcacttgg tctaatttat tgcatatact agcttctgtt ggaaaatgac tgacagctag 3840 aaagtttaag gtaatttcta agtatactac ctataagtaa ctgataagaa ttaggcatca 3900 ttcagtgata tagcagatga ctaatgggtt tttcaggccc tgcacttctc tttcccattt 3960 tcctgttgca ttcagcctct tcaatgctta gtcacacctc cactatagta caaacaaaca 4020 aacaaacaga agaaaggaaa gatggtgaat tttgcaacag agctgcaaaa agtcagtcag 4080 tcaaagcagg aagaggttga aggaactaaa acaaacaaac aaaggtgtct ggactacagc 4140 acagtggaca tgccatccat actggcctgt gccccgttat caatcactgg gcacaacagt 4200 gaagaagtgc aaatcaagtg gtgtttgttt gtttgttagt agtaaggacc gtaacccacc 4260 ccttcctccc cccaaaacta tactgttatt tttctatgta agcaaattgt ttgaaatgct 4320 ttgaaaatga aatcttaata ttaaaaggca tatgcatgta aaaaaaaaaa aaaaaa 4376 <210> 3 <211> 4006 <212> RNA <213> artificial sequence <220> <223> Mus musculus ribonucleoprotein, PTB-binding 2 (Raver2), mRNA <400> 3 cgccccccgcg cggcctctcc gctttctctc ccgcttcgct ccggcctctc cgagtctgca 60 gcggggcgcc gggaagatgg cggcgcgggg aggcggcgcg gggggcgcgg gctcgggatc 120 cggaccctct gctgggacgg cgggggaggc cgcggagccc gcgctgcgcc cgggggaggt 180 ggctgcgctg cacccccagg aggtggctgc acggctgcag cggatgcgac gggagctgag 240 caaccggcgg aaaatcctag tgaaaaacct gccccaggac agcagctccc aggaggttca 300 tgagttatta caggactatg agctaaagta ttgctatgtg gacaggaata aacgaacagc 360 ttttgttacc ttattgaatg gggagcaggc gcagagcgcg attcagaggt ttcatcagtt 420 ttctttccga ggcagagagt tgacagtgca gctgcagccc acagacgctc tcttgtgcat 480 caccaacctg cccatctcct tcaccttaga agagtttgaa gaactcgtcc gtgcctatgg 540 caacatcgag agatgttttt tggtctacag tgaagttact ggccactcca aaggctacgg 600 gtttgtggag tacatgaaga aggactttgc tgccaaggcc agactggagc tactgggcag 660 gcagatggga gcctcggcgc tcttcgcaca gtggatggat gttaatctct tggcctcgga 720 gctcatccat tctaagtgcc tttgtataga taaactcccc agtgactaca gcgattcgga 780 agagctcttg caactttttt ccggtatcca taaacctgtg ttttgccagc ttgcacagga 840 tgaaggaagt cacggtggtg gctttgcggt ggttgaatat agcactgcgg agcatgccga 900 agaggttcag caggtggcag atggcatcac catcaagggg agccaagttc aactgtcctt 960 ctgcgcccca ggagcaccag ggcgaagcac tctagcagtc ctcatagcag ctcagcgcgc 1020 gatgcacagt aatcaaaagg gattactgcc agaacccaat ccggtacaga ttatgaaaag 1080 tttaaacaac cctgccatgt tacaagttct tctgcagccc cagctatgcg gacgagccat 1140 gaagccagtt ctcggagtcg cgcccagctt atctcacctg ctgagtccgt cactcccctc 1200 cgccatctta cacttcagta aagcacagca gagctcagct gtgggtaata catcgtcctt 1260 aatccttcag aacctgtctc ccttgccctt gatacagcaa cagttgatga agtttgacaa 1320 tgcccacact aacaataagc ctggcttgct gggagagcct cctgccatgg tactgcagcc 1380 tgctctggcg atagggccac cgcttccact gaaaacggac ctgggacacc atggcgaagc 1440 acacaaaaca tctaacctga ttccacctca gacgacattg gcagccgggc tgggcatgct 1500 gccgttcttc tcaaaccagc tccctgctgg acaagccggc ccggggcgtg ggaccactca 1560 ggagaaacag tccgcttcgg tgagcattag cgaagctagc ttctcagggt cgcagcatta 1620 cctgcagacc ttcccaggcc ttcctgctgg aggcccgctg acgggcaacc agaagacacc 1680 gcagagccag ccaaaaggca cagaggttgc ctctaagaat cagacttcac tcttgggaga 1740 accaccaaaa gagatccggc tcagtaaaaa tccatatttg aatttggcaa gtgtgctacc 1800 cagtgtgtgc ctatccactg caggtaaagg catgcctccg aagactggaa ttgcgagcaa 1860 cattctggat gcgatctctc agggaagtga gtcccagcat gcactggaga agtgcatcgc 1920 ctattctcca tccatcgagg attatgccca ggcatcctcc ttgaggaatg agaagcgagg 1980 ctcctcctat ctcatctctg ccccagaagg aggcccggta gaactcgctg gccagcaccc 2040 tcaggacacc ggaggtgagct acacagaaac ctacttgaaa aagaagcgtg tgtactaagc 2100 tccgggtctc ctgcccgcct gctgcggcgc ggcgcctgct gctcatcgct gccttaactt 2160 cattcacact agccatatcc tttctatacg ggtgtctgac ttcccagaag gaactgtcat 2220 gcagcaggca gatcagccaa atacccacct agcaataaga aacaccactg gtcgagggca 2280 tttccactag cattagatgt gtcttaatcc acgtgataag gactccgctc acaggagagt 2340 cagcattcca atagtccttg tttcgggcct gggaacaatt ttgacatcat atataaagtc 2400 tgaagcaata actgggaaac tgttgtttcc ttagccttag aatgaatttc gtataagcca 2460 atgtgcattt accttactct cccctaaaaa tagtctcatc tgtagtcttt cctcaaactg 2520 caagggtggc agtaccaccc agtggtgaca acagatgctc atcatgcagc ctccattagc 2580 tctacaaagc cacataagac caacccagtg gcagagtgaa agtgatgtga cttcagatgc 2640 ttcacttccg gagtatcatt taccatatac accttttgcg gaaaacaaag tatttcagga 2700 aaagtaactt ttgtacataa tacaagggtt agcagaatac acactagggc cacatgtagt 2760 ccaccaccta gctttgtgct gcctgtgaat caagtgagga catttctaaa cgtctggggg 2820 taggggagga cttacaacat gaaactgcag tttcaatgtc cccaagatga tgttgggaac 2880 acagtgaggc ttgctcattt acatattatg tagttacttt tgtgtcacaa tgggaactga 2940 gtagcctcga gaaccacaga gcctgcaatg gcaggggcgt ttactctctt gcccttagtc 3000 tgtctcccta ttctttttat cttatgggag atgtgcatag caaggagcca gggtttccct 3060 gccgtctcat agcatacagc agtgaaggtc tcgtccctag acagggccga gccccagcag 3120 agcagtgcac acagcccttc ctgagggagg ccgtggacag cgtggggcag ttctgagcta 3180 acaggctcta cctcactctg gttatttact cactctttgg tagttgttaa gctgaaagga 3240 aatggccgct taatccaatt cacttctgaa agaacacttg tctttatttg tctgactttt 3300 gacttcaccc tttgccgaag atcttggtat taagtgagca acctttttcc atctagaaat 3360 ttctatgtgt tcattgttaa gagactacaa ctgatgaagt gtaggatctc aatgatccta 3420 gatcagatca ttaaaaaacc ttatttggag gacttatcat ttttgactga taatgctctc 3480 cagcattaat tataaaatct aaaactggta catatgttac atgttttata atgaatttta 3540 cctggcctaa tttattgcat ataccagctt tcattgaaaa aatgacagct aaaaaatgaa 3600 aggaagaccg tcctagacac gcttcctata ggtgatgacc agcacgaggt cccagcagat 3660 gactaatggt gtgctctgac cctgcacctc ctgcctctgg ctgcagctct aagtcacaga 3720 atagtggagg ctagagactg ctatctggga gctgcagtgt gaggaggaag cagtggaggg 3780 aacttagaga cacttcacat tccatgtggg tcctccacta tgccagcctg tgcccgttat 3840 tacaagacag aaatgcaaat caagtgatgt tagtacaagg tcattacaac caacccccca 3900 aagtctacgc tgttagtttt ctgtgtcagc taatggtttg aaatgctttt caagatgcaa 3960 gcttaatatt aaaaggcata tgcatgtgaa aaaaaaaaaa aaaaaa 4006

Claims (12)

A biomarker composition for diagnosing drug addiction or withdrawal symptoms containing RAVER2 protein as an active ingredient. According to claim 1,
The RAVER2 protein is a biomarker composition for diagnosing drug addiction or withdrawal symptoms, characterized in that the amino acid sequence represented by SEQ ID NO: 1. According to claim 1,
The drug is a biomarker composition for diagnosing drug addiction or withdrawal symptoms, characterized in that any one selected from the group consisting of methamphetamine, amphetamine, cocaine, ecstasy and methylphenitate. According to claim 1,
The RAVER2 protein is a biomarker composition for diagnosing drug addiction or withdrawal symptoms, characterized in that the expression is reduced in biological samples isolated from animals suffering from drug addiction or drug withdrawal symptoms. A composition for diagnosing drug addiction or withdrawal symptoms comprising an agent for measuring the expression of a RAVER2 protein or a gene encoding the RAVER2 protein. According to claim 5,
The agent for measuring the expression of the RAVER2 protein comprises any one selected from the group consisting of oligopeptides, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, ligands and aptamers that specifically bind to the RAVER2 protein A composition for diagnosing drug addiction or withdrawal symptoms. According to claim 5,
The composition for diagnosing drug addiction or withdrawal symptoms, characterized in that the agent for measuring the expression of the gene encoding the RAVER2 protein is an antisense oligonucleotide, a primer pair or a probe. A kit for diagnosing drug addiction or withdrawal symptoms comprising the composition according to claim 1 or 5. According to claim 8,
The kit is a kit for diagnosing drug addiction or withdrawal symptoms, characterized in that the RT-PCR kit, DNA chip kit or protein chip kit. a) measuring the expression level of the RAVER2 protein or the gene encoding it in a sample isolated from the subject; and
b) a method for providing information necessary for diagnosing drug addiction or withdrawal symptoms, comprising the step of comparing the expression level of the RAVER2 protein selected in a) or the gene encoding it with the expression level of the corresponding protein or gene in a control sample . According to claim 10,
The method for providing information necessary for diagnosing drug addiction or withdrawal symptoms, characterized in that the sample is brain tissue. delete