KR20140125553A - Composition for diagnosing obesity - Google Patents

Abstract

The present invention relates to a composition for diagnosing obesity including a drug measuring a level of protein encoded by mRNA of a gene AW544981 or the gene, a kit for obesity diagnosis including the composition, and a screening method of an obesity drug. The gene AW544981 according to the present invention is highly expressed in an obesity-induced group significantly increased compared to a normal group (control group) and the expression thereof reduces during obesity therapy, thereby being usefully used for diagnosing obesity.

Description

Composition for diagnosing obesity [0002]

The present invention relates to an obesity diagnostic composition comprising an agent for measuring mRNA of the AW544981 gene or a protein level encoded by the gene, an obesity diagnostic kit comprising the composition, and a method for screening an obesity treating agent.

In recent years, the obesity population has been rapidly spreading due to the urbanized living environment and excessive nutrition. Obesity is not only a mere external problem but also a cause of insulin resistance, type 2 diabetes, hypertension, hyperlipidemia, arteriosclerosis, fatty liver, cirrhosis It is known to be associated with an increase in the incidence of metabolic syndrome, which is a social problem. According to the 2010 World Health Organization (WHO), the overweight adult population is estimated to be about 1.6 billion people worldwide, and the obesity population is 400 million people, and 2.6 million people die each year from obesity and overweight. In 2015, 2.3 billion people worldwide will be overweight and 700 million will become obese. In Korea, the prevalence of obesity is increasing by an average of 400,000 a year. According to the analysis of the 2010 National Health and Nutrition Survey data of the Ministry of Health and Welfare and the Disease Control Center, the obesity rate (BMI≥25 kg / m ² ) increased from 26.0% in 1998 to 30.8% in 2010 (36.3% for male and 24.8% for female) in 2010, and it was higher than that of normal weight in hypertension, diabetes, abnormal lipid (Hypertension 2.5 times, diabetes 2 times, hypercholesterolemia 2.3 times, hypertriglyceridemia 2.4 times). As a result of the increase in the number of patients with metabolic syndrome such as hypertension caused by obesity and diabetes mellitus, the cost of health insurance treatment has steadily increased. As of 2010, major chronic disease treatment costs account for 31% (National Health Insurance Corporation, 2010) Not only Korea but the entire world is declaring war on obesity.

Obesity means a state of excessive accumulation of fat in the body, and it occurs not only as a cause of one cause but as a combination of genetic and environmental factors. Bouchard et al. (1990) investigated whether there is a correlation between individual differences in body weight gain and genotype. In a study of identical twins, we found that individual differences in body weight and body fat change patterns for overeating were due to genetic factors. Levine et al. (2005) suggested that non-excercise activity thermogenesis is a major factor in individual differences in weight control programs, and approximately 40-70% of human obesity-related phenotypes are genetic (Comuzzie, 1998). Therefore, it is very important to find gene markers that can diagnose obesity quickly and accurately.

Accordingly, the present inventors have continued to develop a gene marker capable of diagnosing obesity, and as a result, the present invention has been completed by confirming the AW544981 gene that is specifically expressed in an obesity model with increased body weight.

It is an object of the present invention to provide an obesity diagnostic composition comprising an agent for measuring mRNA of the AW544981 gene or a protein level encoded by the gene.

It is still another object of the present invention to provide an obesity diagnostic kit comprising the composition.

It is still another object of the present invention to provide a method of providing information for predicting the risk of obesity, which comprises measuring the expression level of mRNA of AW544981 gene or a protein encoded by the gene from a biological sample.

Yet another object of the present invention is to provide a method for producing a recombinant vector comprising: (a) culturing cells into which the AW544981 gene has been introduced; (b) treating the cells of step (a) with an agent for increasing or inhibiting the expression of the AW544981 gene; And (c) comparing the expression level of the AW544981 gene with the cells treated in the step (b) in a control.

In order to solve the above problems, the present invention provides an obesity diagnostic composition comprising an agent for measuring mRNA of AW544981 (NM_145986.1) gene or protein level encoded by the gene.

The present invention also provides an obesity diagnostic kit comprising the composition.

In addition, the present invention provides a method for providing information for predicting obesity occurrence risk comprising measuring the expression level of mRNA of AW544981 gene or a protein encoded by the gene from a biological sample.

The present invention also relates to a method for producing a recombinant vector comprising the steps of: (a) culturing cells into which AW544981 gene has been introduced; (b) treating the cells of step (a) with an agent for increasing or inhibiting the expression of the AW544981 gene; And (c) comparing the expression level of the AW544981 gene with the cells treated in the step (b) in a control.

The AW544981 gene according to the present invention has a remarkably increased expression in the obesity induction group compared to the normal group (control group), and the expression is reduced in the treatment of obesity, and thus it can be usefully used for the diagnosis of obesity.

Figure 1 shows changes in body weight of each mouse during the experimental period (statistical significance between obesity induction group and normal group; ^* p <0.05, ^** p <0.01, ^*** p <0.001. statistical ^{significance, & p <0.05, && p} <0.01, &&& p <0.001.).
FIG. 2 is a diagram showing liver tissues in the normal group, the obesity induction group, and the group ingested with luteolin.
Fig. 3 is a diagram showing adipose tissues in the normal group, the obesity induction group, and the group ingested with luteolin.

Hereinafter, the present invention will be described in more detail.

The present invention provides an obesity diagnostic composition comprising an agent for measuring mRNA of the AW544981 (NM_145986.1) gene or a protein level encoded by the gene.

In the present invention, "diagnosis" means confirming a pathological condition. For the purpose of the present invention, the diagnosis is to ascertain whether or not obesity develops.

The agent for measuring mRNA level in the present invention includes a sense and antisense primer complementary to the mRNA of the AW544981 gene, or a probe.

As used herein, the term "primer" refers to a primer that is hybridized under appropriate conditions in a suitable buffer solution (for example, four different nucleoside triphosphates and a polymer such as DNA, RNA polymerase or reverse transcriptase) Refers to single stranded oligonucleotides that can serve as a starting point for DNA synthesis. The appropriate length of the primer may vary depending on the intended use, but is usually 15 to 30 nucleotides. Short primer molecules generally require a lower temperature to form a stable hybrid with the template. The primer sequence need not be completely complementary to the template, but should be sufficiently complementary to hybridize with the template. The primers of the present invention can be chemically synthesized using methods known in the art such as, for example, the phosphoramidite solid support method. Further, it can be modified by methylation, capping or the like by a known method.

The term &quot; probe &quot; in the present invention means a nucleic acid fragment such as RNA or DNA corresponding to a few nucleotides or hundreds of nucleotides that can specifically bind to mRNA and is labeled to confirm the presence or absence of a specific mRNA . The probe may be prepared in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, or an RNA probe.

In the present invention, the agent for measuring the level of the protein encoded by the AW544981 gene comprises an antibody specific to the protein encoded by the gene.

&Quot; Antibody &quot; in the present invention means a specific protein molecule indicated for an antigenic site, and includes both polyclonal antibodies, monoclonal antibodies and recombinant antibodies.

The polyclonal antibody can be produced by a method well known in the art for obtaining an antibody-containing serum by injecting an antigen of the gene into an animal and collecting it from an animal. Such polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, cows, dogs, and the like. Monoclonal antibodies may also be prepared using hybridoma methods (see Kohler and Milstein (1976) European Jounal of Immunology 6: 511-519) or phage antibody libraries (Clackson et al, Nature, The antibodies of the present invention can also be prepared using two full length antibodies of the full length (SEQ ID NO: Functional fragment of an antibody molecule refers to a fragment having at least an antigen binding function, and Fab, F (ab ') 2, ), F (ab ') 2, and Fv.

The AW544981 gene according to the present invention has a remarkably increased expression in the obesity induction group compared to the normal group (control group), and the expression is reduced in the treatment of obesity, and thus it can be usefully used for the diagnosis of obesity.

The present invention also provides an obesity diagnostic kit comprising the composition.

The diagnostic kit of the present invention comprises one or more other component compositions, solutions or devices suitable for the assay method

For example, the kit of the present invention can be used to perform PCR using genomic DNA derived from a sample to be analyzed, a primer set specific for the marker gene of the present invention, a suitable amount of DNA polymerase (for example, Taq polymerase) , a dNTP mixture, a PCR buffer solution, and water. The PCR buffer may contain KCl, Tris-HCl and MgCl _2. In addition, components necessary for conducting electrophoresis to confirm whether the PCR product is amplified can be further included in the kit of the present invention.

In addition, the kit of the present invention may be a kit containing essential elements necessary for performing RT-PCR. RT-PCR kits can be used in combination with test tubes or other appropriate containers, reaction buffers (varying in pH and magnesium concentration), deoxynucleotides (dNTPs), Taq polymerase and reverse transcriptase , DNase, RNase inhibitor, DEPC-water, sterile water, and the like. It may also contain a primer pair specific to the gene used as a quantitative control.

In addition, the kit of the present invention may be a kit containing essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and the substrate may include a cDNA corresponding to a quantitative structural gene or a fragment thereof.

In addition, the kit of the present invention may be in the form of a microarray having a substrate on which the marker gene of the present invention is immobilized.

In addition, the kit of the present invention may be a kit comprising essential elements necessary for performing ELISA. An ELISA kit comprises an antibody specific for a marker protein and comprises an agent that measures said protein level. The ELISA kit may comprise a reagent capable of detecting an antibody forming an "antigen-antibody complex", such as a labeled secondary antibody, chromopores, an enzyme (eg, conjugated to an antibody) have. In addition, antibodies specific for the quantitative control protein may be included.

The term "antigen-antibody complex" in the present invention means a combination of a protein encoded by the AW544981 gene and an antibody specific thereto, and the amount of the antigen-antibody complex formed is quantitatively determined through the size of a signal of a detection label Can be measured. Such detection labels may be selected from the group consisting of enzymes, chromophores, ligands, emitters, microparticles, redox molecules, and radioisotopes, but are not necessarily limited thereto.

In addition, the present invention provides a method for providing information for predicting obesity occurrence risk comprising measuring the expression level of mRNA of AW544981 gene or a protein encoded by the gene from a biological sample.

Biological samples in the present invention include, but are not limited to, tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid and urine.

In the present invention, the level of mRNA expression is determined by PCR, RT-PCR, Competitive RT-PCR, Realtime RT-PCR, RNase protection But are not limited to, methods such as RPA (RNase protection assay), Northern blotting and DNA chip.

The level of protein expression in the present invention is determined by Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (ELISA), and immunohistochemistry using an antigen-antibody binding reaction using an antibody that specifically binds to the protein encoded by AW544981 gene. (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immuno staining, immunoprecipitation assay, complement fixation assay, But are not limited to, methods such as fluorescence activated cell sorter (FACS) and protein chip.

Through the above methods, the mRNA of the AW544981 gene or the mRNA or protein of the AW544981 gene as compared to the normal group (control) can be determined by comparing the mRNA level of the AW544981 gene or the protein level encoded by the gene with the mRNA or protein expression level in the normal (control) Significant increases in protein expression levels can be used to diagnose obesity and obesity risk.

The present invention also relates to a method for producing a recombinant vector comprising the steps of: (a) culturing cells into which AW544981 gene has been introduced; (b) treating the cells of step (a) with an agent for increasing or inhibiting the expression of the AW544981 gene; And (c) comparing the expression level of the AW544981 gene with the cells treated in the step (b) in a control.

According to one embodiment of the present invention, the expression of AW544981 gene was significantly increased in the obese induction group compared to the normal group (control group), and the expression of AW544981 gene was significantly decreased in the group treated with obesity treatment. Therefore, it is possible to screen the therapeutic agent for obesity by confirming the expression control of AW544981 gene.

Hereinafter, the present invention will be described more specifically based on examples. It will be apparent to those skilled in the art that the embodiments are only for describing the present invention in more detail and that the scope of the invention is not limited by these embodiments in accordance with the gist of the present invention.

Example 1. Obesity animal model design

Four-week-old male C57BL / 6J mice were used for the experiment. After a week of adaptation period, the mice were fed a normal diet (randomized complete block design) (n = 10) (N = 13), which was supplemented with 20% fat, 1% cholesterol, and luteolin, an obesity treatment, in HFD For a total of 16 weeks. The environment of the animal breeding room was kept constant at constant temperature (25 ± 2 ° C), humidity (50 ± 5%) and cancer / light cycle from 6 to 18, food and drinking water were ad libitum, The diet was refrigerated at 4 ° C. Body weights were measured at intervals of one week. After the end of the experiment, the animals were fasted for 12 hours, followed by light primary anesthesia through ether inhalation. Secondary anesthesia was performed by intramuscular injection of ketamine, and fasting blood was collected from the inferior vena cava. Liver and adipose tissues of experimental animals were separated and rinsed several times with phosphate buffered saline (PBS) solution. The surface of the rats was weighed and the weight was measured. The sample was quenched into liquid nitrogen and stored at -70 ° C until analysis . Some of the liver and adipose tissue were also fixed in 10% formaldehyde solution for microscopic analysis.

Example 2. Analysis of weight change

The mean weight change of the mice during the experimental period is shown in Fig.

As shown in FIG. 1, during the entire experimental period, the body weight of the group induced by obesity was significantly higher than that of the normal group, confirming that the obesity was successfully induced. In addition, the experimental group consumed together with ruteolin showed weight reduction effect from the 12th week after the diet, compared to the obese induction group.

Example 3. Morphological analysis of liver and adipose tissue

For morphological observation of the tissues, liver tissue and adipose tissue were fixed in 10% formaldehyde solution for 24 hours, then exchanged twice with the same solution, dehydrated with 2-fold ethanol and embedded in paraffin. Later, poly-L-lysine-treated tissue sections with a thickness of 5 μm were prepared, stained with H & E (hematoxylin-eosin) according to a known method, and observed with an optical microscope at 200 magnifications. The results are shown in Fig. 2 and Fig.

As shown in FIG. 2, the obesity-induced group showed a large amount of lipid droplets accumulating around the portal vein of the liver tissue, In the experimental group consumed with luteolin, fat area size and number were decreased compared with obese induction group.

In addition, as shown in FIG. 3, the adipose tissue size of obesity induction group was larger than that of normal group, and the adipose tissue volume of luteolin group was higher than that of obesity induction group Small.

Example 4. Analysis of gene expression

4-1. Isolation of total RNA

1 mL of TRIzol (Invitrogen, Grand Island, NY) was added per 0.1 g of liver tissue, homogenized using tissue lyzer (QIAGEN, germany) and centrifuged at 3,250 x g for 10 min at 4 ° C. The supernatant was taken, and 200 μL of chloroform was added, followed by centrifugation at 3,250 × g at 4 ° C. for 20 minutes. The supernatant was again taken, and the same amount of isopropanol was added. The mixture was centrifuged at 3,000 × g for 10 minutes at 4 ° C. The obtained RNA pellet was obtained. The RNA pellet was washed twice with 75% ethanol, dried and dissolved in DEPC (diethyl pyrocarbonate) -treated water. The resulting total RNA was diluted to a concentration of 0.5 to 10 μg / μL and stored at -70 ° C. until analysis. Total RNA was isolated from adipose tissue in the same manner.

4-2. Microarray experiment

4-2-1. RNA QC and total cRNA synthesis

RNA synthesis and purification were performed using the Illumina Total Prep RNA Amplification Kit (Ambion, Austin, Tex.). More specifically, first strand cDNA synthesis was obtained by reverse transcription using 500 ng of the T7 oligo (dT) primer to the RNA obtained in Example 3-1, and the second strand cDNA synthesis was performed using DNA polymer Strand cDNA into double-stranded cDNA by the reaction of RNase H with Rasase. After in vitro transcription cRNA synthesis step, biotin-NTP labeled cRNA was obtained and quantified with ND-1000 spectrophotometer (NanoDrop, Wilmington, DE).

4-2-2. Whole-gene expression microarray experiment Whole-Genome Gene Expression Microarray Experiment

Microarray experiments were performed using Whole-genome gene expression with IntelliHyb Seal System (Illumina, Inc., San Diego, USA) according to manufacturer's recommendations. 750 ng of the labeled cRNA sample obtained in Example 3-2-1 was hybridized to the MouseWG-6 V2 Expression Beadchip and reacted in a 58 ° C thermostat for 16-18 hours. Detection of array signals was performed using Amersham fluorolink streptavidin-Cy3 (GE Healthcare Bio-Sciences, Little Chalfont, UK) according to the bead array manual.

4-2-3. Derive scan and extract image data

Bead chip scanning was performed by the manufacturer using the Illumina BeadArray Reader Confocal Scanner, and the bead chip image derived from the scanner was analyzed with raw data using the Gene Expression Module using Illumina BeadStudio Software (Illumina BeadStudio v3.1.3 (Gene Expression Module v3.3.8)).

4-2-4. Standardization of Microarrays

The Illumina Mouse WG-6 V2 Expression Bead Chip has been standardized for analysis. The standardization method is based on the assumption that the distribution of genes is almost the same in all time-series because the number of genes is sufficiently large. For convenience of analysis, a non-parametric method using the distribution of genes as an index of standardization Quantile Normalization was used.

4-3. Analysis of microarray results and correlation with body weight

In order to analyze obesity-associated genes, we used a Biocondoctor R program to correlate the gene expression values and body weights obtained from the results of microarray of liver tissue and adipose tissue in the normal, obese, and luteolin- Respectively. In both tissues, genes with correlation to body weight were selected at p value <0.001. The results are shown in Table 1 below.

TargetID Symbol
(liver) Liver Adipose tissue Symbol
(adipose tissue) C.C. HFD / ND
FC LU / HFD
FC C.C. HFD / ND
FC LU / HFD
FC ILMN_2763245 Cxcl1 0.761 4.49 -1.25 0.966 2.83 -1.80 Prc1 ILMN_1221503 Ccnd1 0.855 2.80 -1.19 0.955 2.70 -1.73 Lgmn ILMN_2776850 Gas7 0.805 2.44 1.12 0.938 2.62 -1.42 AW544981 ILMN_1252076 Lyz2 0.806 2.28 -1.14 0.933 2.32 -1.83 Lyz2 ILMN_2686975 Fam129b 0.914 2.23 -1.20 0.949 2.14 -1.56 Mcm5 ILMN_2718217 Tubb6 0.837 2.16 -1.13 0.912 2.01 -1.65 Clec4a3 ILMN_2733356 Endod1 0.847 2.09 -1.19 0.876 2.00 -1.08 Cyp2c70 ILMN_2757125 Prc1 0.767 2.06 -1.47 0.869 1.94 -1.85 Gas7 ILMN_1227579 Acot11 0.882 1.94 -1.11 0.935 1.92 -1.24 Fabp3 ILMN_3059326 Sparc 0.867 1.90 -1.42 0.903 1.87 -1.43 P2ry14 ILMN_1219200 P2ry14 0.902 1.85 -1.46 0.917 1.81 -1.41 Fam129b ILMN_2631423 H2-Ab1 0.800 1.84 -1.12 0.853 1.76 -1.50 F2r ILMN_1231138 E2f1 0.940 1.80 -1.26 0.956 1.75 -1.33 Ehd4 ILMN_2759499 AW544981 0.831 1.78 -1.02 0.885 1.74 -1.49 Tubb6 ILMN_1245514 Cyp2c70 0.788 1.75 -1.03 0.899 1.70 -1.29 Cxcl1 ILMN_2896768 Cbr3 0.883 1.75 -1.19 0.912 1.69 -1.21 Col4a2 ILMN_2887630 Fabp3 0.778 1.74 -1.38 0.844 1.68 -1.63 Arhgdib ILMN_2822579 Col4a2 0.863 1.70 -1.19 0.872 1.66 -1.03 E2f1 ILMN_2597868 Lgmn 0.797 1.64 -1.22 0.928 1.63 -1.22 Endod1 ILMN_2742849 Mcm5 0.832 1.58 -1.19 0.933 1.62 -1.13 Scamp5 ILMN_2740852 F2r 0.909 1.58 -1.18 0.919 1.60 -1.34 Pip4k2a ILMN_1222036 Paqr7 0.848 1.58 -1.24 0.819 1.60 -1.09 Ephx1 ILMN_1224768 Ehd4 0.851 1.52 -1.05 0.928 1.58 -1.11 Ccnd1 ILMN_2827072 Slc13a4 0.864 1.51 -1.44 0.905 1.57 -1.24 Acot11 ILMN_2717439 Pip4k2a 0.789 1.51 -1.05 0.875 1.52 -1.29 Hcls1 ILMN_2417863 Tnip1 0.805 1.50 -1.03 0.846 1.51 -1.05 Paqr7 ILMN_2612206 Cdc20 0.765 1.50 -1.28 0.780 1.50 -1.60 H2-Ab1 ILMN_2789948 Arhgdib 0.883 1.50 -1.31 0.834 1.50 -1.31 Sparc ILMN_2652482 Dfna5h 0.802 1.46 -1.00 0.967 1.48 -1.24 Cdc20 ILMN_2685516 Hcls1 0.776 1.45 -1.02 0.763 1.48 -1.06 Dfna5h ILMN_1250907 Mcm2 0.904 1.44 -1.21 0.821 1.47 -1.04 Slc13a4 ILMN_1242246 Scamp5 0.819 1.42 -1.02 0.897 1.47 -1.26 Mcm2 ILMN_2664224 Ephx1 0.936 1.42 -1.08 0.931 1.44 -1.06 2310005E10Rik ILMN_2733753 2310005E10Rik 0.786 1.42 -0.99 0.931 1.44 -1.18 Tnip1 ILMN_2606921 Klb -0.798 -1.42 1.09 -0.827 -1.47 1.21 3110043O21Rik ILMN_1213026 9630058J23Rik -0.773 -1.44 1.03 -0.834 -1.51 1.04 Dapk1 ILMN_2438727 3110043O21Rik -0.778 -1.45 1.03 -0.812 -1.54 1.12 9630058J23Rik ILMN_2619697 B3gnt9 -0.824 -1.47 1.11 -0.810 -1.56 1.18 Irs2 ILMN_2777986 Pck1 -0.875 -1.49 1.09 -0.957 -1.58 1.15 B3gnt9 ILMN_3155915 Dapk1 -0.832 -1.51 1.16 -0.893 -1.69 1.28 Klb ILMN_3144164 Irs2 -0.881 -2.46 1.75 -0.942 -2.39 1.65 Pck1

(CC), correlation coefficient (HFD / ND FC), high-fat diet / normal diet fold change, LU / HFD FC,

As shown in Table 1, the AW544981 gene, whose function has not been fully known yet, shows a strong positive correlation with body weight, and it is confirmed that the AW544981 gene is greatly increased in the obesity induction group. In addition, it was confirmed that the expression of AW544981 gene was decreased in liver and adipose tissues in rats fed with rhea olein, which has the effect of obesity treatment. Especially, the expression level of the AW544981 gene was significantly decreased in adipose tissues that best reflects obesity Respectively.

Through the above experimental results, it was confirmed that obesity can be diagnosed by measuring the level of mRNA of AW544981 gene or the protein encoded by the gene, and the substance that regulates the expression of the gene can be screened.

Claims (9)

A composition for the diagnosis of obesity comprising an agent for measuring mRNA of the AW544981 (NM_145986.1) gene or a protein level encoded by the gene.
The composition for diagnosing obesity according to claim 1, wherein the agent for measuring the mRNA level is a sense and antisense primer complementary to the mRNA of the gene, or a probe.
The composition for diagnosing obesity according to claim 1, wherein the agent for measuring the protein level is an antibody specific to a protein encoded by the gene.
An obesity diagnostic kit comprising the composition of any one of claims 1 to 3.
Measuring the expression level of the mRNA of the AW544981 gene or the protein encoded by the gene from the biological sample; and providing the information for predicting obesity risk.
6. The method according to claim 5, wherein the biological sample is at least one selected from the group consisting of tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid and urine.
6. The method according to claim 5, wherein the mRNA expression level is selected from the group consisting of a polymerase chain reaction (PCR), a reverse transcription polymerase chain reaction (RT-PCR), a competitive reverse transcription polymerase chain reaction (PCR) ), An RNase protection assay (RPA; RNase protection assay, Northern blotting, and a DNA chip). Way.
6. The method of claim 5, wherein the protein expression level is determined by Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (ELISA), or immunohistochemistry through an antigen-antibody binding reaction using an antibody that specifically binds to the protein encoded by AW544981 gene Immunoassay, radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immuno staining, immunoprecipitation assays, complement fixation Wherein the protein is measured by at least one method selected from the group consisting of a fluorescence activated cell sorter (FACS) and a protein chip.
(a) culturing cells into which the AW544981 gene has been introduced;
(b) treating the cells of step (a) with an agent for increasing or inhibiting the expression of the AW544981 gene; And
(c) comparing the expression level of the AW544981 gene with the cells treated in the step (b) in the control.

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