KR20200027243A - Biomarker for diagnosing radiation exposure and method using thereof - Google Patents

Abstract

The present invention relates to a biomarker for diagnosing radiation exposure and a method using the same. According to a composition and a kit for diagnosing radiation exposure, and a method for diagnosing radiation exposure using the same in one aspect, radiation exposure time or exposure dose can be easily diagnosed and a radiation treatment effect can be predicted. The composition comprises a formulation for measuring an expression level of a G-patch domain and KOW motifs protein or a fragment thereof.

Description

Biomarker for diagnosing radiation exposure and method using thereof

It relates to a biomarker for the diagnosis of radiation exposure and a method using the same.

Radiation exposure increases the risk of developing heart disease or cancer. Cytologic and genetic measurements have been used for decades to measure the extent of human damage caused by radiation exposure. However, these methods can only confirm whether the human gene or cell is damaged by radiation, but do not provide information on the molecular mechanism of the human response to radiation. Therefore, much effort is still being made to find markers that can conveniently and accurately diagnose radiation exposure.

Although research on radiation exposure markers has been mainly focused on cancer for the past several decades, it has recently been expanded to research on exposure markers that can be found in normal tissue or samples exposed to radiation. Although some protein markers have been studied as radiation exposure markers so far, it has been difficult to quantitatively predict the degree of radiation exposure.

In general, a method of calculating the radiation exposure level of a dosimeter using a thermoluminum dosimeter (TLD) used by radiation workers during work and a method of mathematically calculating the radiation dose by reconstructing the exposure situation are used. Due to the indirect or theoretical measurement of the culture of the medium, there is a limitation that the reliability of the measurement of the dose to the individual human body is poor.In addition, the measurement of micronuclei formation, the measurement of the mutation of glycophorin A in red blood cells, the degree of DNA cleavage, And methods such as ESR (Electron Spin Resonance) using tooth enamel have been tried, but due to individual differences, the error range of the indicators is very large and radiation dose measurement is almost impossible. For the measurement, the patient was exposed by the chromosome aberration method. Research on an optimal gold standard method for collecting lymphocytes and culturing lymphocytes and determining the frequency of occurrence of dicentrics and rings over the chromosomes formed in the lymphocytes through optical microscope and microstructure observation equipment However, it takes a lot of time and money, and it is rarely used as a limitation that only a small number of experts can diagnose.

Therefore, there is a need for research on biomarkers capable of accurately and rapidly diagnosing radiation exposure by quantitatively responding to radiation exposure dose in a liquid sample that is easy to collect.

Since the expression is quantitatively changed upon exposure to radiation, the present invention relates to a biomarker composition capable of predicting radiation exposure dose and diagnosing a radiation treatment effect, and is a GPKOW (G-patch domain and KOW motifs, G-patch domain and KOW motif) ) Provides a composition, kit for diagnosing radiation exposure, and a method for diagnosing radiation exposure using the protein, including a preparation for measuring the expression level of a protein or fragment thereof.

The present invention has been studied extensively to find protein markers for radiation exposure, and as a result of exposure to radiation, the specific protein, that is, the expression of GPKOW and its mRNA is specifically increased in blood cells, and the use of GPKOW as a radiation exposure marker Invented.

One aspect provides a composition for diagnosing radiation exposure comprising an agent that measures the expression level of a G-patch domain and KOW motifs (GPKOW) protein or fragment thereof.

The term "diagnosis" in this specification is meant to identify the presence or characteristics of the pathophysiology. The diagnosis in the present invention is to confirm whether and / or the degree of radiation exposure.

When exposed to radiation, somatic cells may be damaged, resulting in acute side effects such as damage to hematopoietic tissue, damage to the digestive system, damage to the central nervous system, or skin, or chronic side effects such as cataracts, tumors, and decreased fertility. In addition, it may cause damage to germ cells caused by chromosomal abnormalities. Therefore, there is a need for a marker capable of quantitatively diagnosing radiation exposure.

In addition, radiation therapy plays a very important role in the treatment of anti-cancer, but radiation treatment irresistibly causes damage to normal tissues around the tumor by radiation exposure. Damage to normal tissue around these tumors is a barrier to improving radiation therapy. In addition, when radiation above the threshold dose is irradiated to normal organs due to radiation treatment, the organ's intrinsic function may be partially lost or permanently impaired, leading to chronic side effects. Therefore, markers capable of monitoring damage to normal and cancerous tissues responsive to radiation exposure are needed, and such markers allow patient-specific treatment.

As used herein, the term "marker (marker)" is a substance that can be diagnosed by distinguishing between a normal group of individuals and a radiation-exposed individual, a polypeptide, protein, or nucleic acid showing an increase or decrease in a group exposed to radiation compared to a normal group , Organic biomolecules such as genes, lipids, glycolipids, glycoproteins or sugars. The marker for diagnosing radiation exposure in the present invention is a GPKOW gene and a protein encoded by the GPKOW gene that exhibits a specifically low level of expression in a sample of the radiation exposure group as compared to a sample of the normal control group.

The GPKOW (G-patch domain and KOW motifs) protein is a protein body constituting a spliceosome responsible for pre-mRNA splicing of genes that are continuously transcribed to maintain cell homeostasis. The GPKOW protein is expressed in major blood cells, but the association with radiation exposure or the effect of radiation exposure is unknown. GPKOW protein is mainly expressed in the mitochondria (mitochondria), cytosol (cytosol), cytoskeleton (cytoskeleton), there is an advantage that does not require the separation or decomposition of organs in the cell to monitor the expression or function of GPKOW.

The GPKOW protein may also be expressed as T54, Spp2, GPATC5, GPATCH5, and Genebank Accession No. NM_015698.5, BC003148.1, BC000397.2, AB209806.1, XM_017029415.2 NCBI Reference Sequence: may be a protein encoded from a nucleic acid comprising the nucleotide sequence of NG_021310.2. The GPKOW protein may be a GPKOW isoform protein or a precursor thereof. The term "isoform" refers to different forms of the same protein. The GPKOW protein is, for example, GenBank Accession No. XP_016884904.1, NP_056513.2, Q92917.2, EAW50694.1, AAH03148.1, AAH00397.1 or UniprotKB Accession No. It may be a protein comprising the amino acid sequence of Q92917.

The GPKOW protein may be a protein comprising the amino acid sequence of SEQ ID NO: 1 or 2.

The fragment of the GPKOW protein may be an immunogenic fragment. It may be a fragment having one or more epitopes that can be recognized by an antibody against the GPKOW protein.

Measurement of the expression level of the GPKOW protein can be performed by measuring the expression level of the GPKOW protein or the polynucleotide encoding the GPKOW protein, for example, mRNA expression level.

In the present specification, the term "measurement of expression level" is a process of confirming the expression level of a protein expressed in a radiation exposure diagnostic gene in a biological sample in order to diagnose radiation exposure, and according to one embodiment of the present invention, specific for the protein Proteins can be identified using antibodies or antigen-binding fragments that bind antigenically. As an analysis method for this, western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, radioimmunodiffusion, Ouchterlony ) Immuno-diffusion method, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS (Fluorescence Activated Cell Sorter), fluorescence activated cell sorter (FACS), protein chip (protein) chip) and the like, but is not limited thereto.

The term "antibody" as used herein refers to a specific immunoglobulin directed against an antigenic site as a term known in the art. The antibody in the present invention refers to an antibody that specifically binds to the radiation exposure marker GPKOW of the present invention, and the GPKOW gene is cloned into an expression vector to obtain a GPKOW protein encoded by the GPKOW gene, and is obtained from the obtained GPKOW protein. Antibodies can be prepared according to conventional methods in the art. The form of the antibody includes a polyclonal antibody or a monoclonal antibody, and all immunoglobulin antibodies are included. The antibody refers to a complete form with two full-length light chains and two full-length heavy chains. In addition, the antibody also includes special antibodies such as humanized antibodies.

According to one embodiment, the agent for measuring the mRNA expression level encoding the GPKOW protein is a primer, probe, or antisense nucleotide that specifically binds to the mRNA of the gene, and the nucleic acid sequence of the GPKOW gene is known. Based on the sequence, a primer or probe that specifically binds to mRANA of these genes can be designed.

The term "measurement of mRNA expression level" in the present specification is a process of determining the mRNA expression level of a radiation exposure marker gene in a biological sample to diagnose radiation exposure, according to an embodiment of the present invention, using a primer or probe for mRNA Can be measured. As an analysis method for this, RT-PCR (Reverse Transctiption-Polymerase Chain Reaction), competitive RT-PCR (competitive RT-PCR), real-time RTPCR (Real-time RT-PCR), RNase protection assay (RPA: RNase protection assay), Northern blotting, or DNA chip, but is not limited thereto.

The term “primer” herein is a short nucleic acid that can form a complementary template and base pair with a nucleic acid sequence having a short free 3- 'hydroxyl group and acts as a starting point for template strand copying. Refers to the sequence. The primer 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. According to an embodiment of the present invention, PCR amplification may be performed using the sense and antisense primers of the mRNA of the GPKOW gene to diagnose radiation exposure through measurement of a desired product production amount. PCR conditions, sense and antisense primer length can be appropriately selected according to techniques known in the art.

The term "probe" as used herein refers to a nucleic acid fragment such as RNA or DNA corresponding to short to several bases to hundreds of bases that can specifically bind other than mRNA, and is labeled to indicate the presence or absence of specific mRNA. You can check the amount.

The probe may be produced in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, or an RNA probe. According to one embodiment of the present invention, hybridization is performed using a probe complementary to the mRNA of the GPKOW polynucleotide, and the expression and expression level of the mRNA can be measured through the degree of hybridization to measure whether and how much radiation is exposed. The appropriate probe selection and hybridization conditions may be appropriately selected according to techniques known in the art.

The primer or probe of the present invention can be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known methods. Such nucleic acid sequences can also be modified through a variety of methods known in the art. Examples of such modifications are methylation, encapsulation, substitution with one or more homologs of natural nucleotides, and modifications between nucleotides, such as uncharged linkages (eg methyl phosphonate, phosphotriester, phosphoroamidate, carbamate Or the like) or a modified linker (eg, phosphorothioate, phosphorodithioate, etc.).

Since the mRNA expression level of the GPKOW protein and its gene increases specifically during blood exposure, the composition for detecting markers for diagnosing radiation exposure according to the present invention is particularly easy to obtain blood cells among individuals suspected of radiation exposure. It can be used to measure the level of protein expression in samples obtained from blood.

The composition may further include a sample necessary for diagnosis of radiation exposure.

Another aspect provides a kit for diagnosing radiation exposure comprising an agent that measures the expression level of a GPKOW protein or fragment thereof.

Measurements of GPKOW protein, fragments of GPKOW protein, expression level, and expression level are as described above.

The kit may further include a sample necessary for diagnosis of radiation exposure. The kit may include an antibody, a substrate for immunological detection of the antibody, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or fluorescent substance, or a chromogenic substrate.

In other embodiments, the kit may be an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit.

Another aspect includes the steps of (a) measuring the expression level of a GPKOW protein or fragment thereof in a biological sample isolated from an individual; And (b) comparing the measured expression level with the protein expression level of a normal control.

Measurements of GPKOW protein, fragments of GPKOW protein, expression level, and expression level are as described above.

The method includes measuring the expression level of a GPKOW protein or fragment thereof in a biological sample isolated from an individual.

The step (a) may include the step of incubating the antibody specifically binding to the biological sample and the GPKOW protein or a fragment thereof. The incubation can be performed in vitro.

The step (a) may include the step of performing mass spectrometry of the biological sample by measuring the peptide derived from the GPKOW protein.

The step (a) may include the step of performing hybridization using a probe complementary to the mRNA of the GPKOW polynucleotide, and measuring the mRNA expression level of the GPKOW polynucleotide through the degree of hybridization.

The subject may be an individual suspected of radiation exposure, or may be a mammal, including a human.

The term "biological sample" refers to a sample obtained from an organism. The biological sample may be blood, plasma, platelets, serum, ascites fluid, bone marrow fluid, lymph fluid, saliva, tear fluid, mucosal fluid, amniotic fluid, or a combination thereof. When the biological sample is blood or plasma, since blood or plasma that is easily collected is used as a sample, the organ tissues of the individual are not extracted, so it can be easily analyzed without burdening the individual. In addition, since blood circulates through the human body at a high speed, it can react sensitively by exposure to radiation, and thus, the accuracy and sensitivity of diagnosis is high, and thus it can be usefully used for diagnosis of radiation exposure.

The biological sample may include an intact protein. The intact protein may be a protein isolated from a biological sample without modification of a separate protein. The intact protein can be a protein isolated from a biological sample without proteolysis, for example, by proteolytic enzymes.

The measurement method is, for example, Western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA:

Radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay

(Complement Fixation Assay), FACS, protein chips, and the like.

The method includes comparing the measured expression level with the GPKOW protein expression level of a normal control.

The normal control may be an individual who has not been exposed to radiation, or a biological sample isolated from the same individual before being exposed to radiation.

Since the expression level of the measured protein increases in proportion to the time and / or dose exposed to radiation, the expression level of the GPKOW protein is compared to the expression level of the GPKOW protein in the normal control group, thereby increasing the expression level of the GPKOW protein. Depending on the extent of this increase, and / or the extent of radiation exposure, for example, the radiation exposure dose is predicted, the effect on radiation therapy is predicted, and the damage to normal organs other than the object to be treated by irradiation during radiation therapy The degree can be diagnosed and predicted.

According to a composition, kit for diagnosing radiation exposure according to an aspect, and a method for diagnosing radiation exposure using the same, it is possible to easily diagnose a radiation exposure time or a radiation dose, and predict a radiation treatment effect.

1 is a result of quantifying a protein body in a non-labeled quantitative method for protein expression pattern changes in blood cells upon irradiation, and classifying proteins according to the quantitative value.
2 is a result of 2Gy irradiation of x-rays to blood cell lines GM10832 and GM10860 and confirmation of GPKOW mRNA expression after 0, 4, and 9 hours by RT-PCR.
Figure 3 is a blood cell line GM010833, GM10834, and GM10860 x-ray irradiation at 0, 0.25, 0.5, 1, 2, 4 Gy dose, and after 9 hours GPKOW mRNA expression is the result confirmed by RT-PCR.
Figure 4 is a graph showing the change in the expression value of GPKOW protein according to the irradiation time.

It will be described in more detail through the following examples. However, these examples are intended to illustrate one or more embodiments by way of example, and the scope of the present invention is not limited to these examples.

Example 1. Screening of radiation exposure biomarkers

After irradiating blood cells, biomarker candidates were selected for each sample using mass spectrometry-based proteome profiling and label-free quantification (LFQ). Specifically, 2Gy radiation was irradiated to the blood cell line, and protein changes in protein expression patterns after 0, 1, 5, and 24 hours were quantified through non-labeled quantitation, and groups of proteins were grouped according to pattern changes between samples. It was classified as a cluster. The classification results are shown in FIG. 1. Among the 6 clusters, cluster 3 was selected in which the expression level was consistently increased over time.

Among the 123 proteins belonging to cluster 3, GPKOW, a protein whose expression level is continuously increased even after 24 hours after radiation exposure, was selected as a biomarker.

Example 2. Evaluation of expression change of GPKOW mRNA according to radiation exposure

In order to evaluate whether the GPKOW selected in Example 1 functions as a biomarker of radiation exposure, a change in expression of GPKOW mRNA was confirmed through RT-PCR after irradiation with blood cell lines.

Specifically, the lymphoblastoid cell line (lymphoblastoid cell line) was distributed from GM10832, GM10833, GM10834, and GM10860 cell lines from Coriell Cell Repositories (NJ, USA). The pre-distributed cell line was suspended in RPMI 1640 medium containing 2 mM L-glutamine and 15% untreated fetal bovine serum (FBS) and cultured in a suspended state in a T75 flask.

The lymphoblastic cell lines were irradiated with X-RAD 320 Biological X-Ray Irradiator (Precision X-Ray, Inc., CT, USA) at 2 Gy dose, and then sampled after 0, 4, and 9 hours. In addition, the lymphoblastic cell lines were irradiated with x-rays at doses of 0, 0.25, 0.5, 1, 2, 4, and 8 Gy and sampled 9 hours later.

 For the sampled cell line, PBS (Phosphate Buffer Solution, PBS containing 0.01% Triton X-100, complete protease inhibitor cocktail solution) and 1% PMSF (phenylmethylsulfonyl fluoride) Phosphate buffer) was used to obtain a protein homogenate for proteomic analysis through a 20% probe type sonication process 3 times for 5 seconds in cell lysate.

Total RNA was extracted from the protein homogenate through a known method used in the art. The extracted total RNA was purified to a purity of 1.95 or higher at 260/280 nm and 1.2 or higher at 260/230 nm. To synthesize cDNA from 1 μg of purified mRNA, 20 μl of cDNA was synthesized from 1 μg of purified mRNA using the method described in High Capacity RNA-to-cDNA Kit (Applied Biosystems, USA). Using the kit, 10 μl of 2X RT buffer, 1 μl of 20X Enzyme mix, and 1 μg total RNA were mixed up to 9 μl to construct a reaction composition and adjusted with nuclease-free H ₂ O so that the final volume was 20 μl. After the enzyme reaction at 37 ° C for 60 minutes, the reaction was stopped at 95 ° C for 5 minutes, and cDNA synthesis synthesized at 4 ° C was terminated. Completed cDNA until PCR was performed for the expression of each gene was stored and used in a freezer at -15 to -25 ° C.

After cDNA synthesis, a primer pair of GPKOW (NM_015698.5) (forward: 5'-accagatgaggagcaagaga-3 ', reverse: 5'-ctgctttctcactcttggct-3'; 377bp) was used, 94 ° C 4 minutes 1 cycle, 94 ° C 30 seconds PCR was performed at 57 ° C for 50 seconds, 72 ° C for 1 minute and 27 cycles, and 72 ° C for 10 minutes and 1 cycle. The PCR product obtained through PCR was electrophoresed on a 2% agarose gel to confirm mRNA expression.

2 is a result of confirming the expression of GPKOW mRNA after 0, 4, and 9 hours by x-ray irradiation on blood cell lines GM10832 and GM10860 by RT-PCR.

As shown in FIG. 2, it was confirmed that the expression level of GPKOW mRNA increased quantitatively over time after x-ray irradiation in two types of blood cells, and continued to increase even after 9 hours.

3 is a blood cell line GM010833, GM10834, and GM10860 x-ray irradiation at 0, 0.25, 0.5, 1, 2, 4 Gy dose, and after 9 hours GPKOW mRNA expression is the result confirmed by RT-PCR.

As shown in FIG. 3, it was confirmed that the expression level of GPKOW mRNA increased quantitatively in proportion to the dose irradiated with x-rays in three types of blood cells.

Therefore, it can be seen that the expression level of GPKOW mRNA increases quantitatively in proportion to the radiation dose and irradiation time.

Example 3. Evaluation of GPKOW protein expression change according to radiation exposure

In order to evaluate the change according to the radiation exposure of the GPKOW protein, quantification was performed through a non-labeled quantitative method that statistically analyzes the spectral intensity of peptides using a high-resolution mass spectrometer. Specifically, 1 × 10 ⁶ cells were lysed using 100 μl of 8M urea to which a protease / portatase inhibitor cocktail was added, and then disulfide bonds were reduced at 37 ° C. with 50 mM dithiothreitol. Subsequently, alkylation was performed in a light-free condition with 55 mM iodoacetamide, followed by quenching with 55 mM dithiothreitol. 50 mM ammonium bicarbonate was added up to 800 ml, treated at a ratio of protein: protease = 25: 1, and digested with O / N at 37 ° C. After decomposition, the reaction was stopped using 10% formic acid, and desalting was performed using a C18 reverse phase desalting cartridge. The desalted peptide was dried using a vacuum dryer (Speed Vac) and then dissolved in 0.1% formic acid to perform LC-MS analysis. Q-Exactive Plus BioPharm version (Thermo, USA) was used for MS equipment, UltiMate ™ 3000 RSLCnano System (Thermo, USA) was used for LC, and peptide samples were injected in 4 μl increments, and ions linked to NanoLC at a flow rate of 300 μl / min Analysis was carried out with a trap mass spectrometer. The LC is separated for a total of 150 minutes including the concentration gradient of Solution A (5% acetonitrile, 0.1% formic acid) and solution B (95% acetonitrile, 0.1% formic acid) for a total of 150 minutes.The analysis column used is AQUA C18 (5 μm , 125A), the peptide mixture was separated using a fused silica capillary column with an inner diameter of 75 μm, an outer diameter of 360 μm, and a length of 50 cm. The separated peptides were ion-sprayed using an electrospray ionization voltage of 2000 V, and the ion emitter was injected into a mass spectrometer through a 20 μm inner diameter trapped ion spray emitter to obtain spectral data. . Orbitrap MS analysis is a high energy collision-induced dissociation (HCD, 25) after one survey scan (resolution 30,000) with an ion trap MS for a range of 400 to 2000 m / z. % energy level) was used to perform 8 MS / MS (resolution 60,000) analysis using Orbit Lab MS. Duplicate peptide ion detection was minimized through a 5-minute dynamic exclusion option. The ion trap's Auto gain control target setting was 3E04 for Full MS and 2E5 for FT MS / MS. Qualitative analysis and unlabeled quantitative analysis were performed on the obtained RAW file using Proquestome Discoverer (Thermo, USA, version 2.2), a database analysis software based on Sequest algorithm. Cysteine carbamidomethylation was treated as a fixed modification to cysteine, and oxidation was performed as a variable modification to methionine. Trypsin missed cleavage uses a value of “1”, the sequence database uses the latest swissprot sequence DB, and quantitative analysis using peptide ion intensity is performed for unlabeled quantification. Using the identified proteins as the query for the Uniprot accession number, Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 (http://david.abcc.ncifcrf.gov), gene ontology (GO) Was analyzed for three areas of cellular component, molecular function and biological process. Heat map and clustering analysis and Volcano plot using Perseus (http://www.perseus-framework.org) Analysis was performed.

As a result of the experiment, Table 1 below shows the quantitative values of protein expression of GPKOW over time after radiation exposure. The quantitative value of GPKOW expression is an average of the values obtained by analyzing three times.

Time after 2Gy 0 hours 1 hours 5 hours 24 hours GPKOW protein
Quantitative expression value 9.2 27.4 41.1 322.3

As shown in Table 1, it was confirmed that the expression level of GPKOW protein increased in proportion to time after radiation exposure. 4 is a graph showing the change in the expression value of GPKOW protein according to the irradiation time.

As shown in FIG. 4, as a result of plotting the graph with the x-axis as the time and the y-axis as the quantitative expression value of the GPKOW protein, it was confirmed that the graph shows linearity and that the GPKOW protein increases in proportion to the time after irradiation.

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Claims (11)

GPKOW (G-patch domain and KOW motifs) composition for diagnosing radiation exposure comprising an agent for measuring the expression level of a protein or fragment thereof. The method according to claim 1, wherein the GPKOW protein is a composition for diagnosing radiation exposure comprising the amino acid sequence of SEQ ID NO: 1 or 2. The method according to claim 1, wherein the agent is a composition for diagnosing radiation exposure, which is an antibody or antigen-binding fragment thereof that specifically binds GPKOW protein or a fragment thereof. The method according to claim 3, wherein the antibody is a polyclonal antibody or monoclonal antibody composition for diagnosing radiation exposure. The method according to claim 1, wherein the agent is a composition for diagnosing radiation exposure, which is a primer, probe, or antisense nucleotide that specifically binds to a polynucleotide encoding a GPKOW protein. A kit for diagnosing radiation exposure comprising the composition according to claim 1. (a) measuring the expression level of a GPKOW protein or fragment thereof in a biological sample isolated from an individual or the expression level of a polynucleotide encoding a GPKOW protein; And
(B) a method for providing information necessary for the diagnosis of radiation exposure, comprising comparing the measured expression level with the protein expression level of a normal control. The method of claim 7, wherein the biological sample is blood, plasma, platelets, serum, ascites fluid, bone marrow fluid, lymph fluid, saliva, tear fluid, mucous fluid, amniotic fluid, or a combination thereof. The method of claim 7, wherein the step (a) comprises incubating an antibody specifically binding to the biological sample and the GPKOW protein or a fragment thereof. The method of claim 7, wherein step (a) comprises measuring the peptide derived from the GPKOW protein, thereby performing mass spectrometry of the biological sample. The method according to claim 7, wherein step (a) comprises a hybridization (hybridization) using a probe complementary to the mRNA of the GPKOW polynucleotide, and measuring the mRNA expression level of the GPKOW polynucleotide through the degree of hybridization How would that be.

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