WO2017039359A1 - Composition for diagnosing infectious diseases or infectious complications by using tryptophanyl-trna synthetase and method for detecting diagnostic marker - Google Patents
Composition for diagnosing infectious diseases or infectious complications by using tryptophanyl-trna synthetase and method for detecting diagnostic marker Download PDFInfo
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- WO2017039359A1 WO2017039359A1 PCT/KR2016/009802 KR2016009802W WO2017039359A1 WO 2017039359 A1 WO2017039359 A1 WO 2017039359A1 KR 2016009802 W KR2016009802 W KR 2016009802W WO 2017039359 A1 WO2017039359 A1 WO 2017039359A1
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- tryptophanyl
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/112—Disease subtyping, staging or classification
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/9015—Ligases (6)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/26—Infectious diseases, e.g. generalised sepsis
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to a composition for diagnosing an infectious disease using tryptophanyl-tRNA synthetase (WRS) and a method for detecting a diagnostic marker, and more particularly, to tryptophanyl thialene synthase protein or mRNA.
- the present invention relates to a composition for diagnosing an infectious disease, a diagnostic kit, and a method for detecting WRS for providing information necessary for diagnosing an infectious disease, and a method for determining a death risk for an infectious disease using the WRS. .
- Infectious disease is a disease that occurs when foreign organisms such as bacteria, bacteria, and viruses start to live in the blood, body fluids, and tissues, and can cause life loss if they are not correctly identified and treated properly. to be.
- the threat of infectious diseases with fatal consequences is increasing.
- Current diagnostic tools for infections include polymerase chain react ion (PCR) to identify the infected organs and direct microbial culture from blood and urine, as well as empirical judgment by the clinician based on the patient's symptoms. ).
- systemic inflammation reactions differ depending on the cause, and the rapid diagnosis of systemic c inf lammatory response caused by a non-infectious cause and inflammation reaction caused by a pathogen infection is necessary for proper treatment. very important. This diagnosis is essential for the survival of acute infectious inflammatory patients, especially since it is important for patients with infectious inflammatory diseases caused by an infectious disease to begin appropriate antibiotic treatment as soon as possible, such as death.
- CRP C-reactive protein
- the present inventors have found that the level of tryptophany® tRNA synthetase (TrpRS or WRS, hereinafter WRS) in the body during infection infection caused by bacteria, virus, or fungi infection is rapid from the beginning of infection.
- WRS tryptophany® tRNA synthetase
- the level of WRS is significantly higher than that of normal people, especially in the case of infectious inflammatory diseases, and in the case of non-infectious inflammatory diseases, the level of WRS, which is not related to WRS levels, can rapidly and rapidly infect infectious diseases and their complications.
- the present invention has been completed by discovering that it can be used as a marker for accurate diagnosis.
- an object of the present invention is to provide an expression level of tryptophany 1-tRNA synthetase protein or tryptophanyl thialen synthase mRNA. It is to provide a composition for diagnosis of an infectious disease comprising an agent for measuring the. It is another object of the present invention to provide a kit for diagnosing an infectious disease comprising an agent for measuring the expression level of tryptophanyl thialN synthase protein or tryptophanyl thialA synthase mRNA.
- a further object of the present invention comprises the steps of providing a sample of W (a) the test body in order to provide information necessary for the diagnosis of infectious diseases; (b) measuring the expression level of tryptophany tRNA synthetase in the sample; And (c) comparing the level of the tryptophanyl thiANA synthase to a normal person and determining that the subject has an increased expression level of tryptophanyl thiANA synthase as having an infectious disease. To provide a method for detecting tryptophanyl tRNA synthetase.
- Still another object of the present invention is to provide a composition for determining the risk of death due to an infectious disease, including an agent for measuring the expression level of tryptophanyl thialN synthase protein or tryptophanyl thialN synthase mRNA.
- Another object of the present invention is to provide a sample of the subject (a); (b) measuring the expression level of tryptophanyl-tRNA synthetase in the sample; And (c) to provide a method for determining the risk of death due to infectious diseases comprising the step of determining that the higher the level of the tryptophanyl ThialN synthase is higher risk of death.
- Another object of the present invention is the use of an agent for measuring the expression level of tryptophany tRNA synthetase protein or tryptophanyl TN synthase mRNA for the preparation of a diagnostic agent for infectious diseases. To provide it.
- Another object of the present invention is to measure the level of expression of tryptophanyl-tRNA synthetase protein or tryptophanyl thial-NA synthase mRNA in a subject's sample. It provides a way to diagnose the problem.
- Another object of the present invention is to measure the expression level of tryptophanyl-tRNA synthetase protein or tryptophanyl-thial-NA synthase mRNA for preparing an agent for determining the risk of death due to an infectious disease. It is to provide a use of the formulation.
- the present invention is an infection comprising an agent for measuring the expression level of tryptophanyl-tRNA synthetase protein or tryptophanyl thial-A synthesis synthetic mRNA It provides a diagnostic composition for the disease.
- the present invention also provides a composition for diagnosing an infectious disease consisting of an agent for measuring the expression level of tryptophanyl-tRNA synthetase protein or tryptophanyl thial-A synthase mRNA.
- the present invention provides a composition for diagnosing an infectious disease consisting essentially of an agent for measuring the expression level of tryptophany tRNA synthetase protein or tryptophanyl thiARNA synthase mRNA.
- the present invention provides a kit for diagnosing an infectious disease comprising an agent for measuring the expression level of tryptophanyl thiaL synthase protein or tryptophanyl thiaL synthase mRNA. .
- the present invention provides a kit for diagnosing an infectious disease consisting of an agent for measuring the expression level of tryptophanyl thialN synthase protein or tryptophanyl thialA synthase mRNA.
- the present invention provides a kit for diagnosing an infectious disease, consisting essentially of an agent for measuring the expression level of tryptophanyl thiaL synthase protein or tryptophanyl thiaL synthase mRNA.
- the present invention comprises the steps of (a) providing a sample of the subject to provide information necessary for the diagnosis of an infectious disease; (b) measuring the expression level of tryptophanyl tRNA synthetase ( ⁇ 01) 11 ⁇ 1 1 13 ⁇ 41 ⁇ 2 synthetase in the sample; And (c) comparing the level of the tryptophanyl thiANA synthase with a normal person and determining that a subject having an increased expression level of tryptophanyl thiANA synthase is infected with an infectious disease.
- the present invention in the diagnosis of infectious diseases.
- A providing a sample of the subject to provide the necessary information;
- the trip Tryptophanyl tRNA synthetase comprising comparing the level of topanyl thial-A synthase with a normal person and determining that the subject with an increased expression level of tryptophanyl T-A synthase compared to a normal person is infected with an infectious disease Provides a way to hide it.
- the present invention essentially provides a sample of a subject to provide information necessary for the diagnosis of an infectious disease; (b) measuring the expression level of tryptophany tRNA synthetase in the sample; And (c) comparing the level of the tryptophanyl thiANA synthase with a normal person and determining that the subject has an increased expression level of tryptophanyl thiANA synthase as having an infectious disease. It provides a method for detecting the tryptophanyl tRNA synthetase.
- the present invention provides a risk of death from an infectious disease comprising an agent for measuring the expression level of tryptophanyl thialene or the expression level of this synthase protein or tryptophanyl thialene synthase niRNA. It provides a composition for determination. In addition, the present invention provides a composition for determining the risk of death by an infectious disease consisting of an agent for measuring the expression level of tryptophanyl thialN synthase protein or tryptophanyl thialN synthase mRNA.
- the present invention provides a composition for determining the risk of death due to an infectious disease consisting essentially of an agent for measuring the expression level of tryptophanyl thialN synthase protein or tryptophanyl thialN synthase mRNA.
- the present invention comprises the steps of (a) providing a sample of the subject; (b) measuring the expression level of tryptophany tRNA synthetase in the sample; And (c) provides a method for determining the risk of death due to infectious diseases comprising the step of determining that the higher the level of the tryptophanyl Thialase synthase is higher risk of death.
- the present invention comprises the steps of (a) providing a sample of the subject; (b) measuring the expression level of tryptophanyl—tRNA synthetase in the sample; And (c) determining that the higher the level of the tryptophanyl TNA synthase is, the higher the risk of death is.
- the present invention is essentially (a) ⁇ system for providing a sample of the subject; (b) above Measuring the expression level of tryptophany tRNA synthetase in the sample; And (c) determining that the higher the level of the tryptophanyl thiA-N synthase is, the higher the risk of death is.
- the present invention provides an expression of tryptophanyl-tRNA synthetase protein or tryptophanyl thial-A synthase mRNA for the preparation of a diagnostic agent for infectious diseases. Provide the use of the agent to determine the level.
- the present invention provides a method for measuring the expression level of tryptophanyl-tRNA synthetase protein or tryptophanyl thial-A synthase mRNA in a sample of a subject. It provides a method for diagnosing an infectious disease, characterized in that.
- the present invention is a tryptophanyl-tRNA synthetase protein or tryptophanyl thialene for preparing an agent for determining the risk of death by infectious diseases
- the use of an agent to measure the expression level of A synthase mRNA is provided.
- the present invention will be described in detail.
- the present invention provides a composition for diagnosing an infectious disease comprising an agent for measuring the expression level of tryptophanyl-tRNA synthetase protein or tryptophanyl thial-NA synthase tnRNA.
- WRS increased rapidly from the beginning of infection due to bacterial, viral or fungi infection, and significantly increased than normal controls when complications such as pneumonia or sepsis appeared as infection complications. It was confirmed for the first time.
- WRS expression levels are highly correlated with the severity and prognosis of sepsis, and because WRS increases only in infectious inflammation, it is possible to quickly and accurately distinguish between infectious and non-infectious inflammatory diseases.
- Uh it was confirmed that the value as a diagnostic marker in new infectious diseases and infectious complications is very high.
- the inventors have confirmed in connection with the diagnosis of WRS and infectious diseases in detail as follows.
- WRS was found to increase significantly in infections caused by bacteria, viruses, or fungi.
- PBMCs peripheral blood mononuclear cells
- PR8 WRS secreted out of these cells from these PBMCs early in the infection and at least 1 hour after infection. The level increases rapidly.
- the amount of WRS in the sera of patients with viral pneumonia was increased compared to the control group.
- the amount of WRS was significantly increased in the serum of sepsis or sepsis shock patients due to bacterial or fungi infection, compared to the serum of a healthy control group.
- GRS and KRS another type of aminoacyl tRNA synthetase (ARS) that are secreted out of cells, do not differ between sepsis patients and normal controls, and the increase in WRS due to infection is not a general phenomenon. It was found to be WRS specific.
- Gram-negative bacteria, Gram-positive bacteria, and Gomstone infections did not appear to be statistically significant in increasing trends in WRS in patients with sepsis due to infection. It was confirmed that both can be usefully used for diagnosing sepsis. In addition, there was no significant difference in blood WRS levels between patients with single infection and patients with multiple infections. Therefore, it could be useful for diagnosing sepsis due to single infection or multiple infections.
- the amount of WRS was higher than that of normal control group. There was no statistically significant difference. Therefore, the expression level of WRS was not increased in all inflammatory reactions, but only specifically in inflammatory reactions caused by bacterial, viral or fungi infection. In addition, the level of WRS was more increased in patients with septic shock than in patients with sepsis, and the expression level of WRS was related to the severity of sepsis. In other words, the higher the expression level of WRS, the more severe sepsis symptoms. You can judge that.
- SIRS systemi c inf l ammat ion react ive symptom
- the sensitivity (sensi t ivi ty) and accuracy (speci f i ci ty) were excellent in diagnosing the inflammation caused by the WRS through the ROC curve boom of the WRS.
- the correlation between the severity of sepsis expressed by SOFA score and WRS was higher than that of CRP. In other words, the higher the level of expression of WRS and the higher the SOFA score, the higher the probability of organ failure caused by sepsis.
- WRS levels in sepsis patients who died 28 days after sepsis diagnosis were significantly higher than those in surviving sepsis, further demonstrating that WRS correlates well with sepsis severity and prognosis.
- the higher the expression level of WRS the higher the severity of sepsis and the poorer the prognosis.
- the clinical trials were performed on serum of 120 normal persons, 18 SIRS patients (due to infectious diseases), 166 sepsis patients and 160 septic shock patients.
- the results of WRS are related to the results of procalcitonin, but WRS is specifically detected and detected only for complications of infectious diseases. , It could be confirmed that the screening of death patients is also possible.
- WRS increases specifically in sepsis and rapidly increases in the early stages of infection, thereby quickly distinguishing between infectious and non-infectious inflammatory diseases, thereby preventing early Daewoong delays caused by not knowing the cause of inflammation.
- the patient can be given the most appropriate treatment.
- the present invention provides a composition for diagnosing an infectious disease, including an agent for measuring the expression level of WRS, that is, WRS protein or WRS mRNA level.
- 'WRS' refers to tryptophanyl thialene synthase ⁇ ! ⁇ ⁇ ⁇ ⁇ synthetase, also known as tryptophan thialene ligase (t ryptophan-tRNA l igase), TrpRS, WARS, etc. have.
- WRS is an enzyme that mediates the aminoacyl at ion reaction of amino acid tryptophan and tRNA.
- WRS is encoded by the WARS gene in humans, and the amino acid and mRNA nucleotide sequences of the protein accession number NP_004175.2 (protein), Genbank access i on number NM_004184.3 (mRNA sequence).
- WRS has two subtypes (i soform): cytoplasmic c form (WARS or tryptophanyl-tRNA synthetase, cytoplasmic c) and mi tochondrial form (WARS2 or tryptophanyl-tRNA synthetase, mi tochondrial).
- WRS in is preferably cytopl asmi c form.
- the expression means that the protein or nucleic acid is produced in the cell.
- 'Protein' is used interchangeably with 'polypeptide' or 'peptide' and refers to a polymer of amino acid residues, for example as commonly found in natural proteins.
- 'Polynucleotide' or ' 1 nucleic acid' refers to deoxyribonucleotides (DNA) or ribonucleotides (RNA) in the form of single- or double-strands. Unless otherwise limited, known analogues of natural nucleotides are also included which are localized to nucleic acids in a manner similar to naturally occurring nucleotides.
- 'mRNA' is RNA that transfers genetic information (gene-specific nucleotide sequences) to ribosomes that specify amino acid sequences from specific genes during protein synthesis.
- the diagnosis in the present invention measures the expression level of the WRS gene, that is, the level of the WRS protein or WRS mRNA, to determine the presence or pathology of an infectious disease. To confirm.
- the agent for measuring the expression level of the WRS protein may be an antibody that specifically binds to the WRS protein.
- the WRS protein may be derived from a mammal including a human, and may preferably include a human WRS amino acid sequence represented by SEQ ID NO: 1.
- the antibody in the present invention is an antibody that specifically binds only to WRS protein without reacting with other proteins including other kinds of aminoacyl thiA-N synthase other than WRS.
- WRS antibodies can be produced by cloning the WRS gene into an expression vector to obtain a protein encoded by the gene, and from the protein obtained according to conventional methods in the art. Fragments of WRS proteins comprising WRS antigenic sites can also be used to prepare WRS protein specific antibodies.
- the form of the antibody of the present invention is not particularly limited and includes a polyclonal ant ibody or a monoclonal ant ibody.
- the antibody of the present invention includes all kinds of immunoglobulin antibodies that specifically bind to WRS.
- immunoglobulin antibodies that specifically bind to WRS.
- the antibodies of the present invention also include recombinant antibodies and special antibodies such as humanized antibodies and chimeric antibodies as long as they can specifically bind to WRS proteins.
- WRS protein in the present invention preferably comprises a human WRS amino acid sequence represented by SEQ ID NO: 1, the antibody specifically binding to the WRS protein in the present invention, preferably the amino acid represented by SEQ ID NO: 1 It may be an antibody that specifically binds to a protein having a sequence.
- the diagnostic composition of the present invention comprising the WRS-specific antibody as an agent for measuring the expression level of WRS may further include an agent necessary for a method for detecting a known protein, and using the present composition, a known protein may be used. Any method of detection can be used to determine the level of WRS protein in the subject.
- the agent for measuring the expression level of WRS mRNA may be a probe or primer set that specifically binds to WRS mRNA.
- the WRS mRNA may be derived from a mammal including a human, and preferably may include a human WRS mRNA nucleotide sequence represented by SEQ ID NO: 2.
- the diagnostic composition of the present invention comprising the WRS mRNA specific probe or primer set as an agent for measuring the expression level of WRS may further include an agent required for a method for detecting a known RNA.
- the present composition can be used to measure the level of WRS mRNA in a subject using any known method of detecting RNA.
- the 'primer' is a single stranded oligonucleotide that acts as a starting point for DNA synthesis.
- the primer specifically binds to a polynucleotide that is a template at suitable buffer and temperature conditions, and the DNA polymerase is linked to the primer by adding nucleoside triphosphate having a base complementary to the template DNA. DNA is synthesized by this.
- Primers typically consist of 15 to 30 nucleotide sequences, and vary in base composition and length. The temperature at which they bind to the mold strand (melting temperature, Tm) varies.
- the sequence of the primer need not have a sequence that is completely complementary to some nucleotide sequences of the template, and it is sufficient to have sufficient complementarity within a range that can be hybridized with the template to perform primer-specific functions. Therefore, the primers for measuring the expression level of the WRS mRNA in the present invention does not need to have a sequence completely complementary to the WRS gene sequence, and the amount of WRS mRNA by amplifying a specific section of the WRS mRNA or WRS cDNA through DNA synthesis. It is sufficient to have a length and complementarity for the purpose to be measured.
- the primer for amplification reaction consists of a set (pair) of complementary binding to the template (or sense) and the opposite (antisense, ant i sense) at each end of a specific section of the WRS mRNA to be amplified do. Primers can be easily designed by those skilled in the art with reference to WRS mRNA or cDNA sequences.
- the primer of the present invention may be preferably a set or a pair specifically binding to the WRS mRNA nucleotide sequence represented by SEQ ID NO: 2, and most preferably represented by the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4 It may be a primer set.
- Probe is a fragment of a polynucleotide, such as RNA or DNA, from short to several hundred bases in length that can specifically bind to mRNA or cDNA (complementary DNA) of a specific gene. Meaning, it is labeled (l abel ing) can be confirmed whether the presence of the target mRNA or cDNA to bind, expression amount and the like.
- probes complementary to WRS mRNA can be used to diagnose infectious inflammatory diseases by measuring the expression level of WRS mRNA by performing a hybr idi zat ion with a sample of a subject. The choice and probe conditions of the probe can be appropriately selected according to techniques known in the art.
- primers or probes of the present invention can be synthesized chemically using phosphoramidite solid support synthesis or other well known methods.
- primers or probes can be variously modified according to methods known in the art within a range that does not prevent the hybridization with WRS mRNA. Examples of such modifications include methylation, capping, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, for example, uncharged linkages (e.g., methyl phosphonates, phosphoriesters, phosphoramidates, Carbamate, etc.) or charged linkages (eg, phosphorothioate, phosphorodithioate), and binding of a labeling agent (fluorescence or enzyme).
- uncharged linkages e.g., methyl phosphonates, phosphoriesters, phosphoramidates, Carbamate, etc.
- charged linkages eg, phosphorothioate, phosphorodithioate
- the infection means that one or two or more kinds of exogenous bacteria (including bacteria, Gram-negative bacteria or Gram-positive bacteria), viruses, and bearish (bacteria) enter the body and settle, proliferate, and become parasitic.
- Infectious diseases can be any disease that results from infection of a pathogen and the resulting reaction in vivo. Reactions resulting from infectious diseases may include inflammation, pain, fever, fatigue, swelling, and lowering blood pressure.
- the infectious disease of the present invention is salmonellosis, food poisoning, typhoid fever, paratyphoid, pneumonia, pulmonary tuberculosis, tuberculosis, sepsis, septic shock, urinary tract infection, cystitis, pyelonephritis, It may be urethritis, prostatitis, upper respiratory tract infection, otitis media, more preferably salmonelosis, food poisoning, pneumonia, sepsis, septic shock.
- the sepsis is a systemic inflammatory syndrome, which is a complication of an infectious disease, and when the cause cannot be diagnosed quickly and accurately early, severe sepsis or septic shock, lung, kidney, liver , Multit organ dysfunct ion syndrome (MODS), disseminated endovascular syndrome (DIC), acute respiratory swelling syndrome (ARDS), or acute renal failure (AKI) It is a fatal disease that can progress and die.
- MODS Multit organ dysfunct ion syndrome
- DIC disseminated endovascular syndrome
- ARDS acute respiratory swelling syndrome
- AKI acute renal failure
- Sepsis as used herein includes sepsis, severe sepsis, septic shock and sepsis associated with the final stage of sepsis, mult iple organ dysfunct ion syndrome (MODS), disseminated intravascular arch Syndrome (DIC), Acute Respiratory Tension Syndrome (ARDS) or Acute Renal Failure (AKI), including, but not limited to, all stages of sepsis.
- MODS mult iple organ dysfunct ion syndrome
- DIC disseminated intravascular arch Syndrome
- ARDS Acute Respiratory Tension Syndrome
- Acute Renal Failure Acute Renal Failure
- the present invention also provides a kit for diagnosing an infectious disease comprising an agent for measuring the expression level of a tryptophanyl-tRNA synthetase (WRS) protein or WRS mRNA.
- WRS tryptophanyl-tRNA synthetase
- the diagnostic kit of the present invention includes one or more other component compositions suitable for analytical methods as well as primers or probes that selectively recognize WRS proteins as markers or primers and probes that recognize WRS proteins as markers. Or solutions or devices may be included.
- the diagnostic kit may be a diagnostic kit comprising essential elements necessary for performing reverse transcription polymerase reaction.
- the reverse transcription polymerase kit contains individual primer pairs specific for the marker gene. All.
- the primer is a nucleotide having a sequence specific to the nucleic acid sequence of each marker gene, which is about 7bp to 50bp in length, more preferably about 10bp to 30bp in length. It may also include primers specific for the nucleic acid sequence of the control gene.
- reverse transcriptase polymer reaction kits include test leuub or other suitable containers, reaction buffers (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNAse, RNAse Inhibitor DEPC-water, sterile water, and the like.
- reaction buffers pH and magnesium concentrations vary
- dNTPs deoxynucleotides
- enzymes such as Taq-polymerase and reverse transcriptase
- DNAse DNAse
- RNAse Inhibitor DEPC-water sterile water, and the like.
- the DNA chip kit may include a substrate on which a cDNA or oligonucleotide corresponding to a gene or a fragment thereof is attached, and a reagent, an agent, an enzyme, or the like for preparing a fluorescent probe.
- the substrate may also include cDNA or ligonucleotide corresponding to the control gene or fragment thereof.
- ELISA kits contain specific antibodies to the marker protein. Antibodies are antibodies that have high specificity and affinity for each marker protein and have little cross-reactivity to other proteins. They are monoclonal antibodies, polyclonal antibodies, or recombinant antibodies. .
- the ELISA kit can also include antibodies specific for the control protein.
- Other ELISA kits may bind to reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (as conjugated to antibodies) and substrates or antibodies thereof. Other substances, and the like.
- the kit of the present invention may include a washing solution or an eluent capable of removing the enzyme, the substrate to be reacted with, the unbound protein, and the like, and retaining only the bound protein marker.
- Samples used for analysis include biological samples capable of identifying infectious inflammatory disease specific proteins that can be distinguished from normal states of blood, serum, urine, tears and saliva.
- a biological liquid sample for example blood, serum, plasma.
- Samples may be prepared to increase the detection sensitivity of protein markers, for example serum samples obtained from patients may be anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, continuous It may be pretreated using a method such as extractive extract or gel electrophoresis.
- the present invention to provide information necessary for the diagnosis of infectious diseases
- WRS can function as a marker of a novel infectious disease and provide a method of measuring the expression level of WRS to provide information necessary for the diagnosis of an infectious disease.
- the method of the present invention will be described below step by step.
- Step (a) of the method of the present invention is a step of providing a sample of a subject.
- the sample may be used without limitation as long as it is collected from a subject to diagnose an infectious disease, for example, cells or tissues obtained by biopsy, blood, whole blood, serum, plasma, saliva, cerebrospinal fluid, various rainwater, urine. Or can be stool.
- an infectious disease for example, cells or tissues obtained by biopsy, blood, whole blood, serum, plasma, saliva, cerebrospinal fluid, various rainwater, urine.
- blood, plasma, or serum are preferably, or serum.
- Step (b) of the method of the present invention is a step of measuring the expression level of WRS in the sample provided in step (a).
- the expression level of the WRS may be the expression level of the WRS protein or WRS mRNA.
- WRS protein specific antibodies are as described in the diagnostic composition of the present invention.
- Methods for measuring the expression level of the WRS protein can be used without limitation, methods known in the art, such as Western blotting, dot blotting, enzyme immunoassay (enzyme) 1 inked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipi tat i on, complement fixation assay, Flow cytometry (FACS) or protein chip methods, but are not limited to these.
- an ELISA method can be used.
- WRS mRNA levels can be expressed by amplifying WRS mRNA or cDNA from a sample of a subject using a primer set or probe that specifically binds to WRS mRNA, or by localizing with a probe. The presence and expression of WRS mRNA in the subject sample can be measured using a hybridization ion. Primers and probes of the WRS are as described in the diagnostic composition of the present invention.
- Determination of WRS mRNA expression level can be used without limitation the conventional expression level confirmation method in the art, and examples of the analysis method is reverse transcript ion polymerase chain react ion (RT-PCR), competitive RT ⁇ PCR ( compet it ive RT-PCR, real-time RT-PCR, RNase protect ion assay (RPA), northern blotting, DNA microarray chip ), RNA sequencing, hybridization using nanostrings, in situ hybridization of tissue sections, and the like, but are not limited thereto.
- RT-PCR reverse transcript ion polymerase chain react ion
- competitive RT ⁇ PCR compet it ive ive RT-PCR, real-time RT-PCR, RNase protect ion assay (RPA), northern blotting, DNA microarray chip
- RNA sequencing hybridization using nanostrings, in situ hybridization of tissue sections, and the like, but are not limited thereto.
- Step (c) of the method of the present invention is to compare the level of the WRS of the subject sample measured in step (b) with a normal person, and to determine that the subject has an increased infectious disease level compared to the normal person as having an infectious disease. to be.
- the expression level of the WRS of the subject measured by the method of step (b) described above is compared with the WRS level of the normal person measured by the same method.
- Subjects with increased levels of expression of WRS compared to healthy normal subjects are determined to have an infectious disease.
- the infectious disease is sepsis
- the higher the expression level of WRS it can be determined that sepsis is more severe.
- the extent of WRS expression level is analyzed by analyzing the correlation between WRS expression level and sepsis severity according to techniques known in the art for the selected method of measuring WRS expression level. In some cases, an appropriate diagnosis may be provided to indicate the severity of sepsis.
- the expression of WRS in the serum of the deceased patient significantly increased compared to the surviving patient through a clinical trial of the investigator, which is a procalcitonin (marker used conventionally procalci tonin) was indistinguishable.
- the present invention provides a composition for determining the risk of death by an infectious disease comprising an agent for measuring the expression level of tryptophanyl-tRNA synthetase protein or tryptophanyl thial-NA synthase mRNA to provide.
- the risk of death refers to the risk of death due to an infectious disease, and the death due to infection shows some or all symptoms of inflammatory reaction, high fever pain, difficulty breathing, hypothermia, lowering blood pressure, shock, partial or multiple symptoms. This means death of organs due to organ failure.
- (C) provides a method for identifying the risk of death due to an infectious disease comprising the step of determining that the higher the level of the tryptophanyl TNA synthase is higher risk of death.
- the sample and the like in the determination method of the present invention are as described above.
- the present invention provides a use of an agent for measuring the expression level of tryptophany® tRNA synthetase protein or tryptophanyl thialene synthase mRNA for the preparation of a diagnostic agent for infectious disease.
- the present invention provides a method for diagnosing an infectious disease characterized by measuring the expression level of tryptophanyl-tRNA synthetase protein or tryptophanyl thial-NA synthase mRNA in a sample of a subject. to provide.
- the present invention is the use of an agent for measuring the expression level of tryptophanyl-tRNA synthetase protein or tryptophanyl thial-A synthase mRNA for the preparation of the agent for determining the risk of death by infectious diseases
- the subject of the present invention may be an animal, preferably a mammal, particularly an animal including a human, more preferably a human or a patient (pat i ent) in need of diagnosis.
- the method of diagnosing the perioral disease is to compare the WRS level of the measured subject sample with a normal person and express the protein or mRNA of the WRS compared to the normal person. Increasing levels of a subject are diagnosed as having an infectious disease.
- the comparison with the normal person is as described above.
- the term 'comprising' of the present invention is used in the same way as 'containing' or 'featured' and does not exclude additional component elements or method steps that are not mentioned in the composition or method. .
- the term 'consisting of' excludes additional elements, steps or components, etc., unless otherwise noted.
- the term 'essentially consisting of' means, in the scope of the composition or method, that the substance or step is included in addition to the substance or step described and does not substantially affect its basic properties. .
- WRS is increased only in an infectious disease caused by an infection, and thus is distinguished from a non-infectious disease and rapidly increases in the early stage of infection.
- Levels of WRS also correlate closely with the severity and prognosis of diseases or complications caused by infection.
- WRS can be used as a faster and more accurate diagnostic marker than markers of existing infectious diseases or complications thereof.
- Figure 1 shows a Salmonella WRS changes in accordance vs time (5. typhimurium, 57) the first ( ⁇ 10?, Or from 10 FU abdominal peritoneal exudates (peritoneal lavage fluid of mice injected with) after infection as measured by EUSA.
- the horizontal axis shows the time at which the abdominal effusion was obtained after ST infection (Time after ST innoculat ion (hr)).
- Figure 2 shows the results of ELISA experiments to measure the WRS secretion pattern following virus infection.
- FIG. 3A measures the levels of WRS present in serum of healthy thy control (HC), Severe sepsis, and Septic shock patients. Shows the results of the ELISA experiment. 3B and 3C show the results of ELISA experiments measuring the levels of GRS or KRS in serum of healthy normal (HC) and sepsis patients, respectively (Statistical significance of Dunn's comparison test after ruskal wallis test in FIG. 3A). 3B and 3C were judged by a two-tailed Mann-Whitney test, where *** indicates pO.001 and * indicates p ⁇ 0.05. Ns indicates no statistical significance).
- 4A shows the results of ELISA experiments measuring the levels of WRS present in the serum of healthy normal (H.C) and fungi infected sepsis patients.
- 4B and 3B and 3C show the results of ELISA experiments measuring levels of GRS or KRS present in serum of healthy normal (HC) and sepsis patients, respectively (statistical significance was determined by the two-tailed Mann-Whitney test). *** denotes pO.001 and * denotes pO.05, ns denotes no statistical significance).
- FIG. 5A shows the results of an ELISA experiment measuring the level of WRS present in the serum of Gram-negative bacteria infected patients, Gram-positive bacteria infected patients and Gomwang infected patients.
- Figure 5B shows the results of ELISA experiments to measure the level of WRS present in the serum of single and multi-infected patients (statistical significance was determined by Dunn's comparison test after Kruskal wallis test, *** is ⁇ ⁇ 0 ⁇ 001 , * Indicates p ⁇ 0.05, ns indicates no statistical significance).
- SIRS systemic inflammatory response syndrome
- FIG. 6A asthma (asthma, ASA, FIG. 6B), a disease of chronic inflammatory diseases, and rheumatoid arthritis (RA, FIG. 6C) and the results of comparing WRS levels in patients with Sjogren's syndrome (SS, FIG. 6C) with healthy thy control (HC).
- the experiment was conducted with the permission of the Institutional Review Board (permission number 1502-001-010). Serum samples from healthy controls were collected at the Seoul National University Health Center. Serum samples from 99 sepsis patients were obtained from severe sepsis or septic shock intensive care unit. The patients who provided the serum used in the experiment were admitted to the intensive care unit of a university-affiliated hospital in Seoul. Only patients with confirmed bacterial infection participated in the experiment. Diagnosis of severe sepsis or septic shock was made according to the criteria of the ACCP / SCCM consensus conference (1992). The trial was conducted with informed consent for all patients participating in the trial, in accordance with the review committee's policy. The experiment was also approved by the Review Committee of Seolle Asan Hospital.
- SIRS patients caused by non-infectious diseases
- 166 sepsis patients and 160 septic shock patients were informed of all patients participating in the experiment in accordance with the institution's review committee's policy.
- SIRS systemic inflammatory reaction syndrome
- sepsis or septic shock was confirmed by the ACCP / SCCM consensus conference (1992). It was made according to the standards.
- Serum procalcitonin (RayBiotech, USA Cat No: ELH-PROCALC) and WRS (CUSABI0, China, Cat No: CSB-E11789h) levels were measured using the respective ELISA kits and were measured according to the manufacturer's method. .
- peripheral blood mononuclear cells peripheral blood mononuclear cells
- PBMC peripheral blood mononuclear cells
- CPTTM Cell Preparation Tube
- Bee t onD i ck i nson Cell Preparation Tube
- Salmonella typJiimur iuwi TCC 14028 was obtained from the Korea Microbial Conservation Center (Seoul). Bacteria were commonly cultured using nutrient broth of BD bioscience. The bacteria were incubated overnight before infection and obtained at a density of 1 ⁇ 10 8 CFU. Bacteria density was estimated using an absorbance of 600 nm and a calibration curve. For PBMC or mouse infection experiments, bacteria were washed with PBS and redispersed in medium or serum without PBS. Enzyme—linked immunosorbent assay (ELISA)
- WRS ELISA kit was purchased from CusabioCWuhan, China, catalog number CSB-E11789h), and KRS was purchased from USCN (Wuhan, China, catalog number SED002 Hu). Statistics If the probability value (p-value) is less than 0.05, it was determined that there was statistical significance. All statistical calculations were performed using Graphpad prism 5.0 (GraphPad Software).
- Salmonella typimurium Salmonella typimurium was injected into the mouse abdominal cavity, and the amount of WRS present in the abdominal effusion was checked.
- WRS secreted by intraperitoneal effusion was secreted from the very early stage after Salmonella infection, and the concentration of Salmonella was increased, and the amount of WRS was almost the highest at 1 hour after infection.
- PBMCs peripheral blood monocytes
- RSV respiratory syncytial virus
- PR8 virus an influenza virus
- WRS increased significantly in cell culture medium from 30 minutes after infection.
- FIG. 2A It was confirmed that the level of WRS secreted up to 4 hours after the infection progressed the experiment.
- H.C normal control
- the patients with sepsis who participated in the trial were severe sepsis and septic shock patients who had been identified as having a bacterial infection in the intensive care unit of Asan Medical Center. This (fungi) is as described in Table 1.
- Glycyl-tRNA synthetase Glycyl-tRNA synthetase (GRS)
- ARS secreted aminoacyl tRNA synthetase
- KRS Lysyl-tRNA synthetase
- WRS detected in the serum of systemic inflammatory response syndrome (FIG. 6A), asthma patients (FIG. 6B), rheumatoid arthritis patients (FIG. 6C), and Sjogren's syndrome patients (FIG. 6C) is healthy normal. There was no statistically significant difference compared to the control.
- ROC curve receiver operating characteristics curve
- ⁇ 4-2> Comparison of contents according to survival Patients with sepsis and septic shock were divided into survivors and deaths according to their survival after 28 days.
- the content of WRS and procalcitonin in patient serum of each group was measured quantitatively as in Example ⁇ 4-1>.
- the procalcytonin was not distinguished between the survivors and the dead among the patients, but the WRS was statistically meaningful to confirm that the screening of the patients of death was possible.
- Spearman's correlat ion analys i was performed on sepsis patients using measurements from WRS and procalcitonin in order to correlate the diagnosis results of conventionally used procalcitonin and WRS. As a result, as shown in FIG. 10, it was confirmed that Spearman's rho value (r) was 0.127 and p value was 0.022, indicating an association between WRS and procalcitonin.
- WRS since WRS is increased only in infectious diseases caused by infection, WRS is differentiated from non-infectious diseases and increases rapidly in the early stage of infection. The level of WRS also correlates closely with the severity and prognosis of the disease or complications caused by infection. Thus, WRS can be used as a faster and more accurate diagnostic marker than markers of existing infectious diseases or complications thereof.
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Abstract
The present invention relates to a composition for diagnosing infectious diseases by using a tryptophanyl-tRNA synthetase (WRS) and a method for detecting a diagnostic marker and, more specifically, to: a composition for diagnosing infectious diseases, containing a preparation measuring the WRS protein or mRNA expression level; a diagnostic kit; a method for detecting the WRS for providing information required for the diagnosis of infectious diseases, and a method for determining the infectious disease mortality risk by using the WRS. According to the present invention, the WRS is increased only in infection-induced infectious diseases, differentiating non-infectious diseases therefrom, and is rapidly increased in the early stage of infection. In addition, the level of the WRS is closely correlated with the severity and prognosis of diseases or complications induced by infection. Therefore, the WRS can be used as a marker for more rapid and accurate diagnosis, in comparison to a conventional marker for infectious diseases or complications thereof.
Description
【명세서】 【Specification】
【발명의 명칭】 [Name of invention]
트립토파닐 티알엔에이 합성효소를 이용함 감염 질환 또는 감염 합병증의 진 단용 조성물과 진단 마커 검출 방법 Using Tryptophanyl Thialene Synthesis Enzyme for Diagnosing Infectious Diseases or Complications and Methods for Detecting Diagnostic Markers
【기술분야】 Technical Field
본 출원은 2015년 9월 1일에 출원된 대한 ¾국 특허출원 제 10—2015-0123743 호 및 2016년 3월 2일에 출원된 대한1 ?1국 특허출원 제 10-2016-0025329호를 우선권 으로 주장하고， 상기 명세서 전체는 본 출원의 참고문헌이다. 본 발명은 트립토파닐 티알엔에이 합성효소 ( tryptophanyl-tRNA synthetase , WRS)를 이용한 감염 질환의 진단용 조성물과 진단 마커 검출 방법에 관함 것으로, 보다 상세하게는 트립토파닐 티알엔에이 합성효소 단백질 또는 mRNA의 발현 수준을 측정하는 제제를 포함하는 감염 질환 진단용 조성물, 진단용 키트, 감염 질환의 진 단에 필요한 정보를 제공하기 위한 WRS를 검출하는 방법 , WRS를 이용한 감염 질환 에 대한사망 위험도 판별 방법에 관한 것이다. This application is a priority for ¾ Patent Application No. 10-2015-0123743 and No. 1? For filed on March 2, 2016 1 Patent Application No. 10-2016-0025329, filed on September 1, 2015 And the entirety of the above specification is a reference of the present application. The present invention relates to a composition for diagnosing an infectious disease using tryptophanyl-tRNA synthetase (WRS) and a method for detecting a diagnostic marker, and more particularly, to tryptophanyl thialene synthase protein or mRNA. The present invention relates to a composition for diagnosing an infectious disease, a diagnostic kit, and a method for detecting WRS for providing information necessary for diagnosing an infectious disease, and a method for determining a death risk for an infectious disease using the WRS. .
【배경기술】 Background Art
감염질환은 균， 세균， 바이러스 등의 외래물이 혈액, 체액 및 조직 내에 출 현하여 서식하기 시작하면서 발병하는 질환으로서， 이들의 정확한 동정 및 적절한 치료가 이루어지지 않을 경우, 생명을 잃을 수 있는 질병이다. 위생 수준의 향상에 따라서 감염질환의 유병율은 대체로 감소하는 것으로 보이나， 항생제 투여의 오남 용， 이식에 따른 면역 억제제 사용의 증가， 항암 치료로 인한 면역력 감소， 당뇨, 고혈압 등 기저질환 보유자의 증가 등으로 치명적인 결과를 초래하는 감염질환의 위협은 증가되는 상황이다. 현재 감염에 대한 진단 도구는 환자의 증상을 토대로 하는 임상의사의 경험 적 판단과 더불어 대표적으로 혈액과 소변으로부터의 직접적인 미생물 배양과 감염 된 기관을 확인하기 위한 중합효소연쇄반웅 (polymerase chain react ion, PCR)이 있 다. 그러나 실험실에서의 미생물 배양 결과는 음성인 경우가 대부분이고， PCR 방법 은 시간적인 제한이 있다. 칼시토닌의 프로호르몬 (prohormone of cal ci tonin, PCT) 은 대부분의 세균과 곰광이 감염에서 감염 초기 3-4시간 내에 수치가 증가하지만,
더 높은 수준의 변화는 다른 병원체보다도 그람 음성 세균에 의해 야기된다. 또한 이 호르몬 수준이 바이러스 감염에 의해서도 상승하는지^ 알려져 있지 않다. 이렇 듯, 지난 연구들을 통해 감염질환의 많은 미생물을 검출하는데 상당한 진보가 있어 왔지만 현재 이용되고 있는 진단 방법들은 여전히 많은 노동을 필요로 하고 있고 민감도 및 특이성이 낮은 형편이다. 특히 감염질환은 대부분 감염부위의 염증반웅을 수반하며, 그 중의 일부는 전신 염증반웅을 일으켜 치명적인 결과를 초래하기도 한다. 이러한 전신 염증 반웅 은 그 원인에 따라서 치료법이 상이하여 비감염성 원인으로 유발된 각종 전신 염증 반웅 (systemi c inf lammatory response)과 병원체 감염에 의한 염증 반웅을 신속하 게 구별하여 진단하는 것은 적절한 치료를 위해서 매우 중요하다. 특히 감염질환에 의해 초래되는 감염성 염증 환자는 사망에 이르를 수 있는 등 최대한 빨리 적절한 항생제 치료를 시작하는 것이 중요하기 때문에 이러한 진단은 급성 감염성 염증 환 자의 생존을 위해 필수적이다. Infectious disease is a disease that occurs when foreign organisms such as bacteria, bacteria, and viruses start to live in the blood, body fluids, and tissues, and can cause life loss if they are not correctly identified and treated properly. to be. As the level of hygiene improves, the prevalence of infectious diseases appears to be generally reduced, due to misuse of antibiotics, increased use of immunosuppressants following transplantation, reduced immunity due to chemotherapy, and an increase in the number of underlying diseases such as diabetes and hypertension. The threat of infectious diseases with fatal consequences is increasing. Current diagnostic tools for infections include polymerase chain react ion (PCR) to identify the infected organs and direct microbial culture from blood and urine, as well as empirical judgment by the clinician based on the patient's symptoms. ). However, the results of microbial cultures in the laboratory are mostly negative, and PCR methods have a limited time. Calhortone's prohormone of cal ci tonin (PCT) is elevated in the first 3-4 hours of infection in most bacterial and goiter infections. Higher levels of change are caused by Gram-negative bacteria than other pathogens. It is also unknown whether this hormone level is elevated by viral infection. As such, significant advances have been made in the past studies to detect many microorganisms in infectious diseases, but the current diagnostic methods still require a lot of labor and are low in sensitivity and specificity. Infectious diseases, in particular, are accompanied by inflammatory reactions of the affected areas, and some of them may cause systemic inflammatory reactions and cause fatal consequences. These systemic inflammation reactions differ depending on the cause, and the rapid diagnosis of systemic c inf lammatory response caused by a non-infectious cause and inflammation reaction caused by a pathogen infection is necessary for proper treatment. very important. This diagnosis is essential for the survival of acute infectious inflammatory patients, especially since it is important for patients with infectious inflammatory diseases caused by an infectious disease to begin appropriate antibiotic treatment as soon as possible, such as death.
현재 C-반응 단백질 (C-react ive protein , CRP)이 다양한 염증 질환을 위한 일반적인 진단 마커로 사용되고 있다. 하지만, CRP 수치는 감염과 비감염에 의한 염증 질환에서 모두 수치가 증가하기 때문에 감염성 염증을 구별할 수 없다. 따라 서 빠르게 감염에 의한 염증을 구별할 수 있는 진단용 시약을 개발해야 할 필요가 있다. Currently, C-reactive protein (CRP) is used as a general diagnostic marker for various inflammatory diseases. However, CRP levels are indistinguishable from infectious inflammation because levels are elevated in both inflammatory and non-infectious inflammatory diseases. Therefore, there is a need to develop a diagnostic reagent that can quickly distinguish inflammation caused by infection.
【발명의 상세한 설명】 [Detailed Description of the Invention]
【기술적 과제】 [Technical problem]
이에 본 발명자들은 박테리아， 바이러스 또는 곰광이 (fungi )의 감염에 의한 감염질환 발생시 체내 트립토파닐 티알엔에이 합성효소 (tryptophany卜 tRNA synthetase , TrpRS 또는 WRS, 이하 WRS라 함) 수준이 감염 초기부터 빠르게 증가하 며， 특히 감염성 염증 질환을 수반하는 경우 WRS의 수준이 정상인에 비하여 크게 증가하고, 비감염성 염증 질환의 경우 WRS 수준과 관련이 없는 둥 WRS의 수준은 감 염 질환과 이들의 합병증을 신속하고 정확하게 진단할 수 있는 마커로서 사용될 수 있음을 발견하여 본 발명을 완성하였다. 따라서 본 발명의 목적은 트립토파닐 티알엔에이 합성효소 (tryptophany 1- tRNA synthetase) 단백질 또는 트립토파닐 티알엔에이 합성효소 mRNA의 발현 수준
을 측정하는 제제를 포함하는 감염 질환의 진단용 조성물을 제공하는 것이다. 본 발명의 다른 목적은 트립토파닐 티알엔에이 합성효소 단백질 또는 트립토 과닐 티알엔에이 합성효소 mRNA의 발현 수준을 측정하는 제제를 포함하는 감염 질 환 진단용 키트를 제공하는 것이다. Accordingly, the present inventors have found that the level of tryptophany® tRNA synthetase (TrpRS or WRS, hereinafter WRS) in the body during infection infection caused by bacteria, virus, or fungi infection is rapid from the beginning of infection. In particular, the level of WRS is significantly higher than that of normal people, especially in the case of infectious inflammatory diseases, and in the case of non-infectious inflammatory diseases, the level of WRS, which is not related to WRS levels, can rapidly and rapidly infect infectious diseases and their complications. The present invention has been completed by discovering that it can be used as a marker for accurate diagnosis. Accordingly, an object of the present invention is to provide an expression level of tryptophany 1-tRNA synthetase protein or tryptophanyl thialen synthase mRNA. It is to provide a composition for diagnosis of an infectious disease comprising an agent for measuring the. It is another object of the present invention to provide a kit for diagnosing an infectious disease comprising an agent for measuring the expression level of tryptophanyl thialN synthase protein or tryptophanyl thialA synthase mRNA.
본 발명의 또다른 목적은 감염 질환의 진단에 필요한 정보를 제공하기 위하 여 (a) 피검체의 시료를 제공하는 단계; (b) 상기 시료에서 트립토파닐 tRNA 합성 효소 ( tryptophany tRNA synthetase)의 발현 수준을 측정하는 단계; 및 (c) 상기 트립토파닐 티알엔에이 합성효소의 수준을 정상인과 비교하고 정상인에 비해 트립 토파닐 티알엔에이 합성효소의 발현 수준이 증가한 피검체를 감염 질환에 걸린 것 으로 판정하는 단계를 포함하는 트립토파닐 tRNA 합성효소를 검출하는 방법을 제공 하는 것이다. A further object of the present invention comprises the steps of providing a sample of W (a) the test body in order to provide information necessary for the diagnosis of infectious diseases; (b) measuring the expression level of tryptophany tRNA synthetase in the sample; And (c) comparing the level of the tryptophanyl thiANA synthase to a normal person and determining that the subject has an increased expression level of tryptophanyl thiANA synthase as having an infectious disease. To provide a method for detecting tryptophanyl tRNA synthetase.
본 발명의 또다른 목적은 트립토파닐 티알엔에이 합성효소 단백질 또는 트립 토파닐 티알엔에이 합성효소 mRNA의 발현 수준을 측정하는 제제를 포함하는 감염 질환에 의한 사망 위험도 판별용 조성물을 제공하는 것이다. Still another object of the present invention is to provide a composition for determining the risk of death due to an infectious disease, including an agent for measuring the expression level of tryptophanyl thialN synthase protein or tryptophanyl thialN synthase mRNA.
본 발명의 또다른 목적은 (a) 피검체의 시료를 제공하는 단계; (b) 상기 시 료에서 트립토파닐 tRNA 합성효소 (tryptophanyl-tRNA synthetase)의 발현 수준을 측정하는 단계; 및 (c) 상기 트립토파닐 티알엔에이 합성효소의 수준이 높을 수록 사망 위험도가 높을 것으로 판정하는 단계를 포함하는 감염 질환에 의한 사망 위험 도를 판별하는 방법을 제공하는 것이다. Another object of the present invention is to provide a sample of the subject (a); (b) measuring the expression level of tryptophanyl-tRNA synthetase in the sample; And (c) to provide a method for determining the risk of death due to infectious diseases comprising the step of determining that the higher the level of the tryptophanyl ThialN synthase is higher risk of death.
본 발명의 또다른 목적은 감염 질환의 진단용 제제를 제조하기 위한 트립토 파닐 티알엔에이 합성효소 (tryptophany卜 tRNA synthetase) 단백질 또는 트립토파닐 티알엔에이 합성효소 mRNA의 발현 수준을 측정하는 제제의 용도를 제공하는 것이 다. Another object of the present invention is the use of an agent for measuring the expression level of tryptophany tRNA synthetase protein or tryptophanyl TN synthase mRNA for the preparation of a diagnostic agent for infectious diseases. To provide it.
본 발명의 또다른 목적은 피검체의 시료 내의 트립토파닐 티알엔에이 합성효 소 (tryptophanyl-tRNA synthetase) 단백질 또는 트립토파닐 티알엔에이 합성효소 mRNA의 발현 수준을 측정하는 것을 특징으로 하는 감염 질환을 진단하는 방법을 제 공하는 것이다. Another object of the present invention is to measure the level of expression of tryptophanyl-tRNA synthetase protein or tryptophanyl thial-NA synthase mRNA in a subject's sample. It provides a way to diagnose the problem.
본 발명의 또다른 목적은 감염 질환에 의한 사망 위험도 판별용 제제를 제조 하기 위한 트립토파닐 티알엔에이 합성효소 (tryptophanyl-tRNA synthetase) 단백질 또는 트립토파닐 티알엔에이 합성효소 mRNA의 발현 수준을 측정하는 제제의 용도를 제공하는 것이다.
【기술적 해결방법】 Another object of the present invention is to measure the expression level of tryptophanyl-tRNA synthetase protein or tryptophanyl-thial-NA synthase mRNA for preparing an agent for determining the risk of death due to an infectious disease. It is to provide a use of the formulation. Technical Solution
상기와 같은 목적을 달성하기 위하여， 본 발명은 트립토파닐 티알엔에이 합 성효소 (tryptophanyl-tRNA synthetase) 단백질 또는 트립토파닐 티알엔에이 합성효 소 mRNA의 발현 수준을 측정하는 제제를 포함하는 감염 질환의 진단용 조성물을 제 공한다. In order to achieve the above object, the present invention is an infection comprising an agent for measuring the expression level of tryptophanyl-tRNA synthetase protein or tryptophanyl thial-A synthesis synthetic mRNA It provides a diagnostic composition for the disease.
또한， 본 발명은 트립토파닐 티알엔에이 합성효소 (tryptophanyl-tRNA synthetase) 단백질 또는 트립토파닐 티알엔에이 합성효소 mRNA의 발현 수준을 측 정하는 제제로 구성되는 감염 질환의 진단용 조성물을 제공한다. The present invention also provides a composition for diagnosing an infectious disease consisting of an agent for measuring the expression level of tryptophanyl-tRNA synthetase protein or tryptophanyl thial-A synthase mRNA.
또한， 본 발명은 필수적으로 트립토파닐 티알엔에이 합성효소 ( tryptophany卜 tRNA synthetase) 단백질 또는 트립토파닐 티알엔에이 합성효소 mRNA의 발현 수준 을 측정하는 제제로 구성되는 감염 질환의 진단용 조성물을 제공한다. 본 발명의 다른 목적을 달성하기 위하여 , 본 발명은 트립토파닐 티알엔에이 합성효소 단백질 또는 트립토파닐 티알엔에이 합성효소 mRNA의 발현 수준을 측정하 는 제제를 포함하는 감염 질환 진단용 키트를 제공한다. In addition, the present invention provides a composition for diagnosing an infectious disease consisting essentially of an agent for measuring the expression level of tryptophany tRNA synthetase protein or tryptophanyl thiARNA synthase mRNA. . In order to achieve the other object of the present invention, the present invention provides a kit for diagnosing an infectious disease comprising an agent for measuring the expression level of tryptophanyl thiaL synthase protein or tryptophanyl thiaL synthase mRNA. .
또한， 본 발명은 트립토파닐 티알엔에이 합성효소 단백질 또는 트립토파닐 티알엔에이 합성효소 mRNA의 발현 수준을 측정하는 제제로 구성되는 감염 질환 진 단용 키트를 제공한다. In addition, the present invention provides a kit for diagnosing an infectious disease consisting of an agent for measuring the expression level of tryptophanyl thialN synthase protein or tryptophanyl thialA synthase mRNA.
또한, 본 발명은 필수적으로 트립토과닐 티알엔에이 합성효소 단백질 또는 트립토파닐 티알엔에이 합성효소 mRNA의 발현 수준을 측정하는 제제로 구성되는 감 염 질환 진단용 키트를 제공한다. 본 발명의 또 다른 목적을 달성하기 위하여， 본 발명은 감염 질환의 진단에 필요한 정보를 제공하기 위하여 (a) 피¾체의 시료를 제공하는 단계; (b) 상기 시 료에서 트립토파닐 tRNA 합성효소(^ ^01)11∑1 1 1¾½ synthetase)의 발현 수준을 측정하는 단계; 및 (c) 상기 트립토파닐 티알엔에이 합성효소의 수준을 정상인과 비교하고 정상인에 비해 트립토파닐 티알엔에이 합성효소의 발현 수준이 증가한 피 검체를 감염 질환에 걸린 것으로 판정하는 단계를 포함하는 트립토파닐 tRNA 합성 효소를 검출하는 방법을 제공한다. In addition, the present invention provides a kit for diagnosing an infectious disease, consisting essentially of an agent for measuring the expression level of tryptophanyl thiaL synthase protein or tryptophanyl thiaL synthase mRNA. In order to achieve another object of the present invention, the present invention comprises the steps of (a) providing a sample of the subject to provide information necessary for the diagnosis of an infectious disease; (b) measuring the expression level of tryptophanyl tRNA synthetase (^^ 01) 11∑1 1 1¾½ synthetase in the sample; And (c) comparing the level of the tryptophanyl thiANA synthase with a normal person and determining that a subject having an increased expression level of tryptophanyl thiANA synthase is infected with an infectious disease. Provided are methods for detecting tryptophanyl tRNA synthetase.
또한, 본 발명은 감염 질환의 진단에. 필요한 정보를 제공하기 위하여 (a) 피 검체의 시료를 제공하는 단계; (b) 상기 시료에서 트립토파닐 tRNA 합성효소 ( tryptophanyl-tRNA synthetase)의 발현 수준^ 측정하는 단계; 및 (c) 상기 트립
토파닐 티알엔에이 합성효소의 수준을 정상인과 비교하고 정상인에 비해 트립토파 닐 티알엔에이 합성효소의 발현 수준이 증가한 피검체를 감염 질환에 걸린 것으로 판정하는 단계로 구성되는 트립토파닐 tRNA 합성효소를 감출하는 방법을 제공한다. 또한， 본 발명은 필수적으로 감염 질환의 진단에 필요한 정보를 제공하기 위 하여 (a) 피검체의 시료를 제공하는 단계; (b) 상기 시료에서 트립토파닐 tRNA 합 성효소 ( tryptophany卜 tRNA synthetase)의 발현 수준을 측정하는 단계; 및 (c) 상기 트립토파닐 티알엔에이 합성효소의 수준을 정상인과 비교하고 정상인에 비해 트립 토파닐 티알엔에이 합성효소의 발현 수준이 증가한 피검체를 감염 질환에 걸린 것 으로 판정하는 단계로 구성되는 트립토파닐 tRNA 합성효소를 검출하는 방법을 제공 한다. 본 발명의 또 다른 목적을 달성하기 위하여， 본 발명은 트립토파닐 티알엔에 이 합성효소 단백질 또는 트립토파닐 티알엔에이 합성효소 niRNA의 발현 수준을 측 정하는 제제를 포함하는 감염 질환에 의한사망 위험도 판별용 조성물을 제공한다. 또한， 본 발명은 트립토파닐 티알엔에이 합성효소 단백질 또는 트립토파닐 티알엔에이 합성효소 mRNA의 발현 수준을 측정하는 제제로 구성되는 감염 질환에 의한사망 위험도 판별용 조성물을 제공한다. In addition, the present invention in the diagnosis of infectious diseases. (A) providing a sample of the subject to provide the necessary information; (b) measuring the expression level of tryptophanyl-tRNA synthetase in the sample; And (c) the trip Tryptophanyl tRNA synthetase comprising comparing the level of topanyl thial-A synthase with a normal person and determining that the subject with an increased expression level of tryptophanyl T-A synthase compared to a normal person is infected with an infectious disease Provides a way to hide it. In addition, the present invention essentially provides a sample of a subject to provide information necessary for the diagnosis of an infectious disease; (b) measuring the expression level of tryptophany tRNA synthetase in the sample; And (c) comparing the level of the tryptophanyl thiANA synthase with a normal person and determining that the subject has an increased expression level of tryptophanyl thiANA synthase as having an infectious disease. It provides a method for detecting the tryptophanyl tRNA synthetase. In order to achieve another object of the present invention, the present invention provides a risk of death from an infectious disease comprising an agent for measuring the expression level of tryptophanyl thialene or the expression level of this synthase protein or tryptophanyl thialene synthase niRNA. It provides a composition for determination. In addition, the present invention provides a composition for determining the risk of death by an infectious disease consisting of an agent for measuring the expression level of tryptophanyl thialN synthase protein or tryptophanyl thialN synthase mRNA.
또한， 본 발명은 필수적으로 트립토파닐 티알엔에이 합성효소 단백질 또는 트립토파닐 티알엔에이 합성효소 mRNA의 발현 수준을 측정하는 제제로 구성되는 감 염 질환에 의한 사망 위험도 판별용 조성물을 제공한다. 본 발명의 또 다른 목적을 달성하기 위하여， 본 발명은 (a) 피검체의 시료를 제공하는 단계; (b) 상기 시료에서 트립토파닐 tRNA 합성효소 (tryptophany卜 tRNA synthetase)의 발현 수준을 측정하는 단계; 및 (c) 상기 트립토파닐 티알엔에이 합 성효소의 수준이 높을 수록 사망 위험도가 높을 것으로 판정하는 단계를 포함하는 감염 질환에 의한 사망 위험도를 판별하는 방법을 제공한다. In addition, the present invention provides a composition for determining the risk of death due to an infectious disease consisting essentially of an agent for measuring the expression level of tryptophanyl thialN synthase protein or tryptophanyl thialN synthase mRNA. In order to achieve another object of the present invention, the present invention comprises the steps of (a) providing a sample of the subject; (b) measuring the expression level of tryptophany tRNA synthetase in the sample; And (c) provides a method for determining the risk of death due to infectious diseases comprising the step of determining that the higher the level of the tryptophanyl Thialase synthase is higher risk of death.
또한, 본 발명은 (a) 피검체의 시료를 제공하는 단계; (b) 상기 시료에서 트 립토파닐 tRNA 합성효소 (tryptophanyl— tRNA synthetase)의 발현 수준을 측정하는 단계; 및 (c) 상기 트립토파닐 티알엔에이 합성효소의 수준이 높을 수록 사망 위험 도가 높을 것으로 판정하는 단계로 구성되는 감염 질환에 의한 사망 위험도를 판별 하는 방법을 제공한다. In addition, the present invention comprises the steps of (a) providing a sample of the subject; (b) measuring the expression level of tryptophanyl—tRNA synthetase in the sample; And (c) determining that the higher the level of the tryptophanyl TNA synthase is, the higher the risk of death is.
또한， 본 발명은 필수적으로 (a) 피검체의 시료를 제공하는 ^계; (b) 상기
시료에서 트립토파닐 tRNA 합성효소 ( tryptophany卜 tRNA synthetase)의 발현 수준을 측정하는 단계; 및 (c) 상기 트립토파닐 티알엔에이 합성효소의 수준이 높을 수록 사망 위험도가 높을 것으로 판정하는 단계로 구성되는 감염 질환에 의한 사망 위험 도를 판별하는 방법을 제공한다. 본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 감염 질환의 진단용 제제를 제조하기 위한 트립토파닐 티알엔에이 합성효소 (tryptophanyl-tRNA synthetase) 단백질 또는 트립토파닐 티알엔에이 합성효소 mRNA의 발현 수준을 측 정하는 제제의 용도를 제공한다. 본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 피검체의 시료 내의 트립토파닐 티알엔에이 합성효소 ( tryptophanyl-tRNA synthetase) 단백질 또는 트립 토파닐 티알엔에이 합성효소 mRNA의 발현 수준을 측정하는 것을 특징으로 하는 감 염 질환을 진단하는 방법을 제공한다. 본 발명의 또 다른 목적을 달성하기 위하여 , 본 발명은 감염 질환에 의한 사 망- 위험도 판별용 제제를 제조하기 위한 트립토파닐 티알엔에이 합성효소 ( tryptophanyl-tRNA synthetase) 단백질 또는 트립토파닐 티알엔에이 합성효소 mRNA의 발현 수준을 측정하는 제제의 용도를 제공한다. 이하 본 발명을 상세히 설명한다. 본 발명은 트립토파닐 티알엔에이 합성효소 (tryptophanyl-tRNA synthetase) 단백질 또는 트립토파닐 티알엔에이 합성효소 tnRNA의 발현 수준을 측정하는 제제를 포함하는 감염 질환의 진단용 조성물을 제공한다. 본 발명자들은 WRS가 박테리아， 바이러스또는곰광이 ( fungi )에 의한 감염에 의하여 발현 수준이 감염 초기부터 급속하게 증가하며， 감염 합병증으로 폐렴이나 패혈증 같은 증상이 나타나게 되는 경우 정상인 대조군보다 현저하게 증가함을 처 음으로 확인하였다. 나아가 패혈증 환자의 경우 WRS 발현 수준은 패혈증의 중증도 및 예후와 높은 상관관계를 가지고 있으며, WRS는 감염성 염증에서만 증가하기 때 문에 감염성 염증 질환과 비감염성 염증 질환을 신속하고 정확하게 구분할 수 있
어， 새로운 감염 질환 및 감염 합병증에서의 진단 마커로서의 가치가 매우 높다는 것을 확인하였다. 본 발명자들이 WRS와 감염 질환의 진단과 관련하여 확인한 바는 구체적으로 다음과 같다. In addition, the present invention is essentially (a) ^ system for providing a sample of the subject; (b) above Measuring the expression level of tryptophany tRNA synthetase in the sample; And (c) determining that the higher the level of the tryptophanyl thiA-N synthase is, the higher the risk of death is. To achieve another object of the present invention, the present invention provides an expression of tryptophanyl-tRNA synthetase protein or tryptophanyl thial-A synthase mRNA for the preparation of a diagnostic agent for infectious diseases. Provide the use of the agent to determine the level. In order to achieve another object of the present invention, the present invention provides a method for measuring the expression level of tryptophanyl-tRNA synthetase protein or tryptophanyl thial-A synthase mRNA in a sample of a subject. It provides a method for diagnosing an infectious disease, characterized in that. In order to achieve another object of the present invention, the present invention is a tryptophanyl-tRNA synthetase protein or tryptophanyl thialene for preparing an agent for determining the risk of death by infectious diseases The use of an agent to measure the expression level of A synthase mRNA is provided. Hereinafter, the present invention will be described in detail. The present invention provides a composition for diagnosing an infectious disease comprising an agent for measuring the expression level of tryptophanyl-tRNA synthetase protein or tryptophanyl thial-NA synthase tnRNA. The inventors found that WRS increased rapidly from the beginning of infection due to bacterial, viral or fungi infection, and significantly increased than normal controls when complications such as pneumonia or sepsis appeared as infection complications. It was confirmed for the first time. Furthermore, in patients with sepsis, WRS expression levels are highly correlated with the severity and prognosis of sepsis, and because WRS increases only in infectious inflammation, it is possible to quickly and accurately distinguish between infectious and non-infectious inflammatory diseases. Uh, it was confirmed that the value as a diagnostic marker in new infectious diseases and infectious complications is very high. The inventors have confirmed in connection with the diagnosis of WRS and infectious diseases in detail as follows.
본 발명의 일실시예에서 WRS는 박테리아， 바이러스 또는 곰광이 ( fungi )로 인 한 감염에서 크게 증가함을 확인하였다. 인간 말초혈액 단핵구 세포 (PBMC)가 살모 넬라균 /77∞e//a typhi imirium) 또는 RSV, PR8 등 바이러스에 감염되면 감염 초 기， 늦어도 감염 후 1시간 이내에 이들 PBMC에서 세포 외부로 분비되는 WRS 수준이 급속히 증가한다. 또한 바이러스성 폐렴 환자의 혈청에 존재하는 WRS의 양도 정상 인 대조군에 비하여 증가하였다. In one embodiment of the present invention, WRS was found to increase significantly in infections caused by bacteria, viruses, or fungi. When human peripheral blood mononuclear cells (PBMCs) are infected with viruses such as Salmonella / 77∞e // a typhi imirium or RSV, PR8, WRS secreted out of these cells from these PBMCs early in the infection and at least 1 hour after infection. The level increases rapidly. In addition, the amount of WRS in the sera of patients with viral pneumonia was increased compared to the control group.
본 발명의 다른 일실시예에서는 특히 박테리아 또는 곰광이 (fungi ) 감염으로 인한 패혈증 또는 패혈증 쇼크 환자의 혈청에서 WRS의 양이 건강한 정상인 대조군 의 혈청에 비하여 현저하게 증가하였음을 확인하였다. 세포 외부로 분비되는 다른 종류의 아미노아실 티알엔에이 중합효소 (aminoacyl tRNA synthetase , ARS)인 GRS와 KRS는 패혈증 환자와 정상인 대조군 사이에 차이가 없어， 감염으로 인한 WRS의 증 가는 ARS 일반적인 현상이 아니라 WRS 특이적인 것임을 알 수 있었다. In another embodiment of the present invention, it was confirmed that the amount of WRS was significantly increased in the serum of sepsis or sepsis shock patients due to bacterial or fungi infection, compared to the serum of a healthy control group. GRS and KRS, another type of aminoacyl tRNA synthetase (ARS) that are secreted out of cells, do not differ between sepsis patients and normal controls, and the increase in WRS due to infection is not a general phenomenon. It was found to be WRS specific.
그람-음성균 박테리아, 그람-양성균 박테리아 및 곰광이 감염에 의한 패혈증 환자 각각에서 WRS의 증가 경향에 통계학적으로 유의성 있는 차이가 없는 것으로 보아， 그람-음성균 박테리아， 그람-양성균 박테리아 또는 곰광이 감염에 의한 패혈 증 진단에 모두 유용하게 사용될 수 있음을 확인하였다. 또한， 단일 감염 환자와 다중 감염 환자 사이에서도 혈중 WRS 수치에 유의성 있는 차이가 나타나지 않은 것 으로 보아, 단일 감염 또는 다중 감염으로 인한 패혈증 진단에 모두 유용하게 사용 될 수 있음을 확인하였다. Gram-negative bacteria, Gram-positive bacteria, and Gomstone infections did not appear to be statistically significant in increasing trends in WRS in patients with sepsis due to infection. It was confirmed that both can be usefully used for diagnosing sepsis. In addition, there was no significant difference in blood WRS levels between patients with single infection and patients with multiple infections. Therefore, it could be useful for diagnosing sepsis due to single infection or multiple infections.
특히, 전신성 염증반웅 증후군 (systemi c inf l ammat ion react ive symptom , SIRS) , 비감염성 만성 염증 질환인 천식과 류마티스 관절염, 쇼그렌 증후군 등 자 가면역질환 환자의 혈청에서는 WRS의 양이 정상인 대조군과 비교하여 통계적으로 유의한 차이가 없었다. 따라서 WRS의 발현 수준은 모든 염증 반응에서 증가하는 것 이 아니라， 박테리아， 바이러스 또는 곰광이 ( fungi ) 감염으로 유발된 염증 반웅에 서만 특이적으로 증가하는 것으로 확인되었다. 또한 WRS의 수준은 패혈증 환자에서 보다 패혈성 쇼크 환자에서 더욱 증가하여， WRS의 발현 수준이 패혈증 중증도와도 관련이 있는 것으로 나타났다. 즉, WRS의 발현 수준이 높을수록 패혈증 증상이 심
한 것으로 판단할 수 있다. In particular, in patients with autoimmune diseases such as systemi c inf l ammat ion react ive symptom (SIRS), non-infectious chronic inflammatory diseases, asthma, rheumatoid arthritis, and Sjogren's syndrome, the amount of WRS was higher than that of normal control group. There was no statistically significant difference. Therefore, the expression level of WRS was not increased in all inflammatory reactions, but only specifically in inflammatory reactions caused by bacterial, viral or fungi infection. In addition, the level of WRS was more increased in patients with septic shock than in patients with sepsis, and the expression level of WRS was related to the severity of sepsis. In other words, the higher the expression level of WRS, the more severe sepsis symptoms. You can judge that.
분 발명의 다른 일실시예에서는 WRS의 ROC curve 붐석을 통하여 WRS가 감염 으로 유발된 염증을 진단하는데 있어 민감성 (sensi t ivi ty)과 정확성 (speci f i ci ty) 이 뛰어난 것을 확인하였다. 또한 SOFA score로 표시되는 패혈증의 중증도와의 상 관관계에 있어서도 WRS는 기존의 염증 진단 마커인 CRP보다 더욱 높은 상관관계를 갖는 것으로 분석되었다. 즉, WRS의 발현 수준이 높을수톡 SOFA score가 높아 패혈 증으로 인한 기관 기능상실 (organ fai lure)이 발생할 확를이 높은 것으로 판단할 수 있다. In another embodiment of the invention, it was confirmed that the sensitivity (sensi t ivi ty) and accuracy (speci f i ci ty) were excellent in diagnosing the inflammation caused by the WRS through the ROC curve boom of the WRS. In addition, the correlation between the severity of sepsis expressed by SOFA score and WRS was higher than that of CRP. In other words, the higher the level of expression of WRS and the higher the SOFA score, the higher the probability of organ failure caused by sepsis.
패혈증 진단 28일 후 사망한 패혈증 환자의 WRS 수준은 생존한 패혈증 환자 에서보다 현저하게 높아， WRS가 패혈증 중증도 및 예후와 높은 상관관계가 있음을 더욱 입증하였다. 즉, WRS의 발현 수준이 높을수록 패혈증의 중증도가 높고 예후가 좋지 않은 것으로 예측할 수 있다. WRS levels in sepsis patients who died 28 days after sepsis diagnosis were significantly higher than those in surviving sepsis, further demonstrating that WRS correlates well with sepsis severity and prognosis. In other words, the higher the expression level of WRS, the higher the severity of sepsis and the poorer the prognosis.
또한 본 발명의 일실시예에서는 120명의 정상인, 18명의 (감염질환 이외의 원인에 의한) SIRS 환자， 166명의 패혈증 환자 및 160명의 패혈성 쇼크 환자의 혈 청 ( serum)을 대상으로 연구자 임상실험을 통하여 종래에 사용되는 마커인 프로칼 시토닌 (procalci tonin)과의 비교하여， WRS에 의한 결과가 프로칼시토닌의 결과와 연관성은 있으나, WRS의 경우 감염질환의 합병증에 대해서만 특이적으로 구분되어 검출되며， 사망 우려의 환자의 선별도 가능함을 확인할 수 있었다. In one embodiment of the present invention, the clinical trials were performed on serum of 120 normal persons, 18 SIRS patients (due to infectious diseases), 166 sepsis patients and 160 septic shock patients. Compared with the procal cytonin, which is a marker used in the related art, the results of WRS are related to the results of procalcitonin, but WRS is specifically detected and detected only for complications of infectious diseases. ， It could be confirmed that the screening of death patients is also possible.
앞서 서술한 WRS의 발현 수준과 폐렴 및 패혈증의 중증도 사이의 밀접한 상 관관계를 이용하여 기존의 패혈증 진단 마커보다 더욱 효율적인 패혈증의 진단 마 커로 이용할 수 있음을 알 수 있다. 특히 WRS는 패혈증 특이적으로 증가하고， 감염 초기에 급속하게 증가하기 때문에 감염성 염증 질환인 패혈증과 비감염성 염증 질 환을 신속하게 구분함으로써 염증의 원인을 알지 못하여 발생하는 초기 대웅의 지 연을 방지하고 환자가 가장 적합한 치료를 받도록 할 수 있다. The close correlation between the expression level of WRS and the severity of pneumonia and sepsis can be used as a more efficient marker for sepsis than conventional sepsis diagnostic markers. In particular, WRS increases specifically in sepsis and rapidly increases in the early stages of infection, thereby quickly distinguishing between infectious and non-infectious inflammatory diseases, thereby preventing early Daewoong delays caused by not knowing the cause of inflammation. The patient can be given the most appropriate treatment.
본 발명자들의 발견을 바탕으로 본 발명은 WRS의 발현 수준， 즉 WRS 단백질 또는 WRS mRNA 수준을 측정하는 제제를 포함하는 감염 질환 진단용 조성물을 제공 한다. Based on the findings of the present inventors, the present invention provides a composition for diagnosing an infectious disease, including an agent for measuring the expression level of WRS, that is, WRS protein or WRS mRNA level.
본 발명에서 'WRS ' 는 트립토파닐 티알엔에이 합성효소^^ ！^ ^ ^八 synthetase)를 의미하며， 트립토판 티알엔에이 연결효소 ( t ryptophan-tRNA l igase) , TrpRS, WARS 등으로도 알려져 있다. WRS는 아미노산 트립토판과 tRNA의 아미노아실 레이션 (aminoacyl at ion) 반웅을 매개하는 효소이다. WRS는 인간에서는 WARS 유전자 에 의해 암호화되며， 단백질의 아미노산 서열과 mRNA 염기 서열은 Genbank
accession number NP_004175.2(단백질)， Genbank access i on number NM_004184.3(mRNA 염기서열) 둥으로 공지되어 있다. WRS는 cytoplasmi c form(WARS 또는 tryptophanyl-tRNA synthetase , cytoplasmi c)과 mi tochondr i al form(WARS2 또 는 tryptophanyl-tRNA synthetase , mi tochondr i al )의 두 가지 아형 ( i soform)이 있 4. 본 발명에서의 WRS는 바람직하게는 cytopl asmi c form이다. In the present invention, 'WRS' refers to tryptophanyl thialene synthase ^^！ ^ ^ ^ 八 synthetase, also known as tryptophan thialene ligase (t ryptophan-tRNA l igase), TrpRS, WARS, etc. have. WRS is an enzyme that mediates the aminoacyl at ion reaction of amino acid tryptophan and tRNA. WRS is encoded by the WARS gene in humans, and the amino acid and mRNA nucleotide sequences of the protein accession number NP_004175.2 (protein), Genbank access i on number NM_004184.3 (mRNA sequence). WRS has two subtypes (i soform): cytoplasmic c form (WARS or tryptophanyl-tRNA synthetase, cytoplasmic c) and mi tochondrial form (WARS2 or tryptophanyl-tRNA synthetase, mi tochondrial). WRS in is preferably cytopl asmi c form.
본 발명에서 '발현 (expression) '은 세포에서 단백질 또는 핵산이 생성되는 것을 의미한다. '단백질'은 '폴리펩타이드 (polypept ide) ' 또는 '펩타이드 (pept ide) '와 호환성 있게 사용되며， 예컨대, 자연 상태의 단백질에서 일반적으로 발견되는 바와 같이 아미노산 잔기의 중합체를 말한다. '폴리뉴클레오티드 (polynucleot ide) ' 또는 1핵산'은 단일 -또는 이중-가닥의 형태로 된 데옥시리보뉴 클레오티드 (DNA) 또는 리보뉴클레오티드 (RNA)를 말한다. 다른 제한이 없는 한, 자 연적으로 생성되는 뉴클레오티드와 비슷한 방법으로 핵산에 흔성화되는 자연적 뉴 클레오티드의 공지된 아날로그도 포함된다. 'mRNA'는 단백질 합성 과정에서 특정 유전자로부터 아미노산 서열을 특정하게 되는 리보솜으로 유전 정보 (유전자 특이적 염기 서열)를 전달하는 RNA이다. In the present invention, the expression (expression) means that the protein or nucleic acid is produced in the cell. 'Protein' is used interchangeably with 'polypeptide' or 'peptide' and refers to a polymer of amino acid residues, for example as commonly found in natural proteins. 'Polynucleotide' or ' 1 nucleic acid' refers to deoxyribonucleotides (DNA) or ribonucleotides (RNA) in the form of single- or double-strands. Unless otherwise limited, known analogues of natural nucleotides are also included which are localized to nucleic acids in a manner similar to naturally occurring nucleotides. 'mRNA' is RNA that transfers genetic information (gene-specific nucleotide sequences) to ribosomes that specify amino acid sequences from specific genes during protein synthesis.
'진단 '은 병리 상태의 존재 또는 특징을 확인하는 것을 의미한다, 본 발명에 서의 진단은 WRS 유전자의 발현 수준, 즉 WRS 단백질 또는 WRS mRNA의 수준을 측정 하여 감염 질환의 병리 존재 또는 발병 여부를 확인하는 것이다. 본 발명의 진단용 조성물이 WRS 단백질의 발현 수준을 측정하기 위한 것일 때에는 WRS 단백질 발현 수준을 측정하는 제제는 WRS 단백질에 특이적으로 결합하 는 항체일 수 있다. 'Diagnosis' refers to confirming the presence or characteristics of a pathological condition. The diagnosis in the present invention measures the expression level of the WRS gene, that is, the level of the WRS protein or WRS mRNA, to determine the presence or pathology of an infectious disease. To confirm. When the diagnostic composition of the present invention is for measuring the expression level of the WRS protein, the agent for measuring the expression level of the WRS protein may be an antibody that specifically binds to the WRS protein.
상기 WRS 단백질은 인간을 포함하는 포유류에서 유래한 것일 수 있으며， 바 람직하게는 서열번호 1로 표시되는 인간의 WRS 아미노산 서열을 포함하는 것일 수 있다. The WRS protein may be derived from a mammal including a human, and may preferably include a human WRS amino acid sequence represented by SEQ ID NO: 1.
'항체 (ant ibody) '는 항원성 부위에 특이적으로 결합하는 면역글로불린 ( immunoglobul in)을 의미한다. 본 발명에서의 항체는 WRS 이외에는 다른 종류의 아 미노아실 티알엔에이 합성효소를 포함하는 다른 단백질에는 반웅하지 않고, WRS 단 백질에만 특이적으로 결합하는 항체이다. WRS 항체는 WRS 유전자를 발현백터에 클 로닝하여 상기 유전자에 의해 암호화되는 단백질을 수득하고， 수득한 단백질로부터 당해 기술분야의 통상적인 방법에 따라 제조할 수 있다. WRS 항원성 부위를 포함하 는 WRS 단백질의 단편을 이용하여 WRS 단백질 특이적인 항체를 제조할 수도 있다.
본 발명의 항체의 형태는 특별히 제한되지 않으며, 다클론항체 (polyclonal ant ibody) 또는 단일클론항체 (monoclonal ant ibody)를 포함한다. 또한 항원 -항체 결합성을 갖는 것이면 전체 항체의 일부도 본 발명의 항체에 포함되며， WRS에 특이 적으로 결합하는 모든 종류의 면역글로불린 항체가 포함된다. 예를 들어 2개의 전 체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 갖는 완전한 형태의 항체 뿐 아니라 항체 분자의 기능적인 단편, 즉 항원 결합 기능을 갖는 Fab , F(ab ' ) , F(ab ' )2 및 Fv 등을 포함한다. 나아가 본 발명의 항체에는 WRS 단백질에 특이적으로 결합할 수 있는 것이라면 인간화 항체, 키메릭 항체 등의 특수 항체와 재조합 항체도 포함된 다. 'Antibody' means an immunoglobulin that specifically binds to an antigenic site. The antibody in the present invention is an antibody that specifically binds only to WRS protein without reacting with other proteins including other kinds of aminoacyl thiA-N synthase other than WRS. WRS antibodies can be produced by cloning the WRS gene into an expression vector to obtain a protein encoded by the gene, and from the protein obtained according to conventional methods in the art. Fragments of WRS proteins comprising WRS antigenic sites can also be used to prepare WRS protein specific antibodies. The form of the antibody of the present invention is not particularly limited and includes a polyclonal ant ibody or a monoclonal ant ibody. In addition, as long as it has antigen-antibody binding, a part of the whole antibody is included in the antibody of the present invention, and includes all kinds of immunoglobulin antibodies that specifically bind to WRS. For example, not only full-form antibodies with two full-length light chains and two full-length heavy chains, but also functional fragments of antibody molecules, ie Fab, F (ab '), F (ab' 2) and Fv, and the like. Furthermore, the antibodies of the present invention also include recombinant antibodies and special antibodies such as humanized antibodies and chimeric antibodies as long as they can specifically bind to WRS proteins.
본 발명에서 WRS 단백질은 바람직하게는 서열번호 1로 표시되는 인간의 WRS 아미노산 서열을 포함하는 것으로서, 본 발명에서 WRS 단백질에 특이적으로 결합하 는 항체는, 바람직하게는 서열번호 1로 표시되는 아미노산 서열을 갖는 단백질에 특이적으로 결합하는 항체일 수 있다. 상기 WRS 특이적인 항체를 WRS의 발현 수준 을 측정하는 제제로 포함하는 본 발명의 진단용 조성물은 공지된 단백질을 감지하 는 방법에 필요한 제제를 추가적으로 포함할 수 있으며, 본 조성물을 이용하여 공 지된 단백질을 감지하는 방법을 제한없이 사용하여 피검체에서 WRS 단백질의 수준 을 측정할 수 있다. WRS protein in the present invention preferably comprises a human WRS amino acid sequence represented by SEQ ID NO: 1, the antibody specifically binding to the WRS protein in the present invention, preferably the amino acid represented by SEQ ID NO: 1 It may be an antibody that specifically binds to a protein having a sequence. The diagnostic composition of the present invention comprising the WRS-specific antibody as an agent for measuring the expression level of WRS may further include an agent necessary for a method for detecting a known protein, and using the present composition, a known protein may be used. Any method of detection can be used to determine the level of WRS protein in the subject.
한편 본 발명의 진단용 조성물이 WRS mRNA의 발현 수준을 측정하기 위한 것 일 때에는 WRS mRNA 발현 수준을 측정하는 제제는 WRS mRNA에 특이적으로 결합하는 프로브 또는 프라이머 세트일 수 있다. Meanwhile, when the diagnostic composition of the present invention is for measuring the expression level of WRS mRNA, the agent for measuring the expression level of WRS mRNA may be a probe or primer set that specifically binds to WRS mRNA.
상기 WRS mRNA는 인간을 포함하는 포유류에서 유래한 것일 수 있으며， 바람 직하게는 서열번호 2로 표시되는 인간의 WRS mRNA 염기서열을 포함하는 것일 수 있 다. 상기 WRS mRNA 특이적인 프로브 또는 프라이머 세트를 WRS의 발현 수준을 측정 하는 제제로 포함하는 본 발명의 진단용 조성물은 공지된 RNA를 감지하는 방법에 필요한 제제를 추가로 포함할 수 있다. 본 조성물을 이용하여 공지된 RNA를 감지하 는 방법을 제한없이 사용하여 피검체에서 WRS mRNA의 수준을 측정할 수 있다. The WRS mRNA may be derived from a mammal including a human, and preferably may include a human WRS mRNA nucleotide sequence represented by SEQ ID NO: 2. The diagnostic composition of the present invention comprising the WRS mRNA specific probe or primer set as an agent for measuring the expression level of WRS may further include an agent required for a method for detecting a known RNA. The present composition can be used to measure the level of WRS mRNA in a subject using any known method of detecting RNA.
'프라이머 (pr imer) '는 DNA 합성의 개시점 (start ing point )으로 작용하는 짧 은 단일가닥 올리고뉴클레오티드 (single strand ol igonucl eot ide)이다. 프라이머는 적합한 완층액 (buffer)와 온도 조건에서 주형 (templ ate)인 폴리뉴클레오티드에 특 이적으로 결합하고, DNA 중합효소가 프라이머에 주형 DNA에 상보적인 염기를 갖는 뉴클레오사이드 트리포스페이트를 추가하여 연결함으로써 DNA가 합성된다. 프라이 머는 일반적으로 15 내지 30개의 염기서열로 이루어져 있으며， 염기 구성과 길이에
따라 주형 가닥에 결합하는 온도 (mel t ing temperature , Tm)가 달라진다. The 'primer' is a single stranded oligonucleotide that acts as a starting point for DNA synthesis. The primer specifically binds to a polynucleotide that is a template at suitable buffer and temperature conditions, and the DNA polymerase is linked to the primer by adding nucleoside triphosphate having a base complementary to the template DNA. DNA is synthesized by this. Primers typically consist of 15 to 30 nucleotide sequences, and vary in base composition and length. The temperature at which they bind to the mold strand (melting temperature, Tm) varies.
프라이머의 서열은 주형의 일부 염기 서열과 완전하게 상보적인 서열을 가 질 필요는 없으며, 주형과 흔성화되어 프라이머 고유의 작용을 할 수 있는 범위 내 에서의 층분한 상보성을 가지면 충분하다. 따라서 본 발명에서 WRS mRNA의 발현 수 준을 측정하기 위한 프라이머는 WRS 유전자 서열에 완벽하게 상보적인 서열을 가질 필요는 없으며， DNA 합성을 통해 WRS mRNA 또는 WRS cDNA의 특정 구간을 증폭하여 WRS mRNA의 양을 측정하려는 목적에 맞는 길이와 상보성을 갖는 것이면 충분하다. 상기 증폭 반웅을 위한 프라이머는 증폭하고자 하는 WRS mRNA의 특정 구간의 양쪽 끝부분의 주형 (또는 센스， sense)과 반대편 (안티센스， ant i sense)에 각각 상보적으 로 결합하는 한 세트 (쌍)으로 구성된다. 프라이머는 당업자라면 WRS mRNA 또는 cDNA 염기서열을 참조하여 용이하게 디자인할 수 있다. The sequence of the primer need not have a sequence that is completely complementary to some nucleotide sequences of the template, and it is sufficient to have sufficient complementarity within a range that can be hybridized with the template to perform primer-specific functions. Therefore, the primers for measuring the expression level of the WRS mRNA in the present invention does not need to have a sequence completely complementary to the WRS gene sequence, and the amount of WRS mRNA by amplifying a specific section of the WRS mRNA or WRS cDNA through DNA synthesis. It is sufficient to have a length and complementarity for the purpose to be measured. The primer for amplification reaction consists of a set (pair) of complementary binding to the template (or sense) and the opposite (antisense, ant i sense) at each end of a specific section of the WRS mRNA to be amplified do. Primers can be easily designed by those skilled in the art with reference to WRS mRNA or cDNA sequences.
본 발명의 프라이머는 바람직하게는 서열번호 2로 표시되는 WRS mRNA 염기서 열에 특이적으로 결합하는 한 세트 또는 한 쌍일 수 있으며, 가장 바람직하게는 서 열번호 3과 서열번호 4의 염기서열로 표시되는 프라이머 세트일 수 있다. The primer of the present invention may be preferably a set or a pair specifically binding to the WRS mRNA nucleotide sequence represented by SEQ ID NO: 2, and most preferably represented by the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4 It may be a primer set.
1프로브 (probe) '는 특정 유전자의 mRNA나 cDNA( compl ementary DNA)에 특이적 으로 결합할 수 있는 짧게는 수개 내지 길게는 수백 개의 염기 (base pair ) 길이의 RNA 또는 DNA 등 폴리뉴클레오티드의 단편을 의미하며, 표지 ( l abel ing)되어 있어서 결합하는 대상 mRNA나 cDNA의 존재 유무, 발현양 등을 확인할 수 있다. 본 발명의 목적을 위해서는 WRS mRNA에 상보적인 프로브를 피검체의 시료와 흔성화 반응 (hybr idi zat ion)을 수행하여 WRS mRNA의 발현양을 측정함으로써 감염성 염증 질환 의 진단에 이용할 수 있다. 프로브의 선택 및 흔성화 조건은 당업계에 공지된 기술 에 따라 적절하게 선택할 수 있다. 1 Probe is a fragment of a polynucleotide, such as RNA or DNA, from short to several hundred bases in length that can specifically bind to mRNA or cDNA (complementary DNA) of a specific gene. Meaning, it is labeled (l abel ing) can be confirmed whether the presence of the target mRNA or cDNA to bind, expression amount and the like. For the purposes of the present invention, probes complementary to WRS mRNA can be used to diagnose infectious inflammatory diseases by measuring the expression level of WRS mRNA by performing a hybr idi zat ion with a sample of a subject. The choice and probe conditions of the probe can be appropriately selected according to techniques known in the art.
본 발명의 프라이머 또는 프로브는 포스포아미다이트 (phosphoramidi te) 고체 지지체 합성법이나 기타 널리 공지 된 방법을 이용하여 화학적으로 합성할 수 있 다. 또한 프라이머 또는 프로브는 WRS mRNA와의 흔성화를 방해하지 않는 범위에서 당해 기술분야에 공지된 방법에 따라 다양하게 변형시킬 수 있다. 이러한 변형의 예로는 메틸화， 캡화, 천연 뉴클레오티드 하나 이상의 동족체로의 치환 및 뉴클레 오티드 간의 변형， 예를 들면 하전되지 않은 연결체 (예: 메틸 포스포네이트， 포스 포트리에스테르， 포스포로아미데이트， 카바메이트 등) 또는 하전된 연결체 (예: 포 스포로티오에이트, 포스포로디티오에이트 둥)， 그리고 형광 또는 효소를 이용한 표 지물질 ( l abel ing mater i al )의 결합 등이 있다.
본 발명에서 감염은 하나 또는 두 종류이상의 외인성 박테리아 (세균, 그람 음성균 또는 그람 양성균을 모두 포함한다) , 바이러스, 곰광이 (균)가 몸속에 침입 하여 정착, 증식， 기생상태가 되는 것을 의미하며， 감염 질환은 병원체의 감염와 결과로 생체에서 반응을 일으켜서 발생하는 모든 질환일 수 있다. 감염 질환의 결 과로 나타나는 반응은 염증， 통증, 발열， 피로감, 부종, 혈압 강하 등이 있을 수 있다. 바람직하게는 본 발명의 감염 질환은 살모넬라증 (salmonel losi s) , 식중독， 장티푸스， 파라티푸스， 폐렴， 폐결핵， 결핵, 패혈증 (sepsis) , 패혈성 쇼크 (sept ic shock) , 요로감염， 방광염， 신우신염, 요도염, 전립선염， 상기도감염, 중이염일 수 있으며， 더 바람직하게는 살모넬라증 (salmonel losis) , 식중독, 폐렴, 패혈증, 패혈 성 쇼크일 수 있다. 본 발명에서 상기 패혈증은 감염 질환의 합병증으로 나타나는 전신성 염증 반응 증후군으로 원인을 조기에 신속하고 정확하게 진단하지 못할 경우에는 중증 패혈증 (severe sepsis)이나 패혈성 쇼크 (sept ic shock) , 폐， 신장， 간, 순환기 등 의 기능 장애까지 초래되는 다장기 기능장애 증후군 (mul t iple organ dysfunct ion syndrome, MODS) , 파종성 혈관내 웅고 증후군 (DIC) , 급성 호흡 촉박 증후군 (ARDS) 또는급성 신부전 (AKI)으로진행되어 사망할 수 있는 치명적인 질환이다. 본 명세서에서 사용되는 패혈증은, 패혈증의 최종 단계와 관련된 패혈증， 중 증 패혈증， 패혈증성 쇼크 및 패혈증에 수반하는 다장기 기능장애 증후군 (mul t iple organ dysfunct ion syndrome, MODS) , 파종성 혈관내 웅고 증후군 (DIC) , 급성 호흡 촉박 증후군 (ARDS) 또는 급성 신부전 (AKI )의 발병을 포함하나， 이에 제한되지 않 으며 패혈증의 모든단계를 포함한다. 또한 본 발명은 WRS(tryptophanyl-tRNA synthetase) 단백질 또는 WRS mRNA의 발현 수준을 측정하는 제제를 포함하는 감염 질환 진단용 키트를 제공한다. The primer or probe of the present invention can be synthesized chemically using phosphoramidite solid support synthesis or other well known methods. In addition, primers or probes can be variously modified according to methods known in the art within a range that does not prevent the hybridization with WRS mRNA. Examples of such modifications include methylation, capping, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, for example, uncharged linkages (e.g., methyl phosphonates, phosphoriesters, phosphoramidates, Carbamate, etc.) or charged linkages (eg, phosphorothioate, phosphorodithioate), and binding of a labeling agent (fluorescence or enzyme). In the present invention, the infection means that one or two or more kinds of exogenous bacteria (including bacteria, Gram-negative bacteria or Gram-positive bacteria), viruses, and bearish (bacteria) enter the body and settle, proliferate, and become parasitic. Infectious diseases can be any disease that results from infection of a pathogen and the resulting reaction in vivo. Reactions resulting from infectious diseases may include inflammation, pain, fever, fatigue, swelling, and lowering blood pressure. Preferably, the infectious disease of the present invention is salmonellosis, food poisoning, typhoid fever, paratyphoid, pneumonia, pulmonary tuberculosis, tuberculosis, sepsis, septic shock, urinary tract infection, cystitis, pyelonephritis, It may be urethritis, prostatitis, upper respiratory tract infection, otitis media, more preferably salmonelosis, food poisoning, pneumonia, sepsis, septic shock. In the present invention, the sepsis is a systemic inflammatory syndrome, which is a complication of an infectious disease, and when the cause cannot be diagnosed quickly and accurately early, severe sepsis or septic shock, lung, kidney, liver , Multit organ dysfunct ion syndrome (MODS), disseminated endovascular syndrome (DIC), acute respiratory swelling syndrome (ARDS), or acute renal failure (AKI) It is a fatal disease that can progress and die. Sepsis as used herein includes sepsis, severe sepsis, septic shock and sepsis associated with the final stage of sepsis, mult iple organ dysfunct ion syndrome (MODS), disseminated intravascular arch Syndrome (DIC), Acute Respiratory Tension Syndrome (ARDS) or Acute Renal Failure (AKI), including, but not limited to, all stages of sepsis. The present invention also provides a kit for diagnosing an infectious disease comprising an agent for measuring the expression level of a tryptophanyl-tRNA synthetase (WRS) protein or WRS mRNA.
본 발명의 진단용 키트에는 WRS의 발현 수준을 측정하기 위하여 선택적으로 WRS 단백질을 마커로 인식하는 항체 또는 WRS mRNA를 마커로 인식하는 프라이머， 프로브 뿐만 아니라 분석 방법에 적합한 한 종류 또는 그 이상의 다른 구성 성분 조성물， 용액 또는 장치가포함될 수 있다. In order to measure the expression level of WRS, the diagnostic kit of the present invention includes one or more other component compositions suitable for analytical methods as well as primers or probes that selectively recognize WRS proteins as markers or primers and probes that recognize WRS proteins as markers. Or solutions or devices may be included.
구체적인 양태로서 상기 진단 키트는 역전사 중합효소반웅을 수행하기 위해 필요한 필수 요소를 포함하는 것을 특징으로 하는 진단용 키트일 수 있다. 역전사 중합효소반응 키트는 마커 유전자에 대한 특이적인 각각의 프라이머 쌍을 포함한
다. 프라이머는 각 마커 유전자의 핵산서열에 특이적인 서열을 가지는 뉴클레오타 이드로서, 약 7bp 내지 50bp의 길이， 보다 바람직하게는 약 10bp 내지 30bp의 길이 이다. 또한 대조군 유전자의 핵산 서열에 특이적인 프라이머를 포함할 수 있다. 그 외 역전사 중합효소반웅 키트는 테스트 류브 또는 다른 적절한 컨테이너, 반웅 완 충액 (pH 및 마그네슴 농도는 다양) , 데옥시뉴클레오타이드 (dNTPs) , Taq-폴리머라아 제 및 역전사효소와 같은 효소， DNAse, RNAse 억제제 DEPC-수 (DEPC-water) , 멸균수 등을 포함할 수 있다. In a specific embodiment, the diagnostic kit may be a diagnostic kit comprising essential elements necessary for performing reverse transcription polymerase reaction. The reverse transcription polymerase kit contains individual primer pairs specific for the marker gene. All. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of each marker gene, which is about 7bp to 50bp in length, more preferably about 10bp to 30bp in length. It may also include primers specific for the nucleic acid sequence of the control gene. Other reverse transcriptase polymer reaction kits include test leuub or other suitable containers, reaction buffers (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNAse, RNAse Inhibitor DEPC-water, sterile water, and the like.
또 다른 양태로는 DNA 칩을 수행하기 위해 필요한 필수 요소를 포함하는 것 을 특징으로 하는 진단 키트일 수 있다. DNA 칩 키트는 유전자 또는 그의 단편에 해당하는 cDNA 또는 올리고뉴클레오티드 (ol igonucleot ide)가 부착되어 있는 기판, 및 형광표식 프로브를 제작하기 위한 시약， 제제， 효소 등을 포함할 수 있다. 또한 기판은 대조군 유전자 또는 그의 단편에 해당하는 cDNA 또는 을리고뉴클레오티드를 포함할 수 있다. Another embodiment may be a diagnostic kit characterized by including the necessary elements necessary to carry out the DNA chip. The DNA chip kit may include a substrate on which a cDNA or oligonucleotide corresponding to a gene or a fragment thereof is attached, and a reagent, an agent, an enzyme, or the like for preparing a fluorescent probe. The substrate may also include cDNA or ligonucleotide corresponding to the control gene or fragment thereof.
가장 바람직하게는， ELISA를 수행하기 위해 필요한 필수 요소를 포함하는 갓 을 특징으로 하는 진단 키트일 수 있다. ELISA 키트는 마커 단백질에 대한 특이적 인 항체를 포함한다. 항체는 각 마커 단백질에 대한 특이성 및 친화성이 높고 다른 단백질에 대한 교차 반응성이 거의 없는 항체로, 단클론 항체, 다클론 항체 또는 재조합 항체이다. .또한 ELISA 키트는 대조군 단백질에 특이적인 항체를 포함할 수 있다. 그 외 ELISA 키트는 결합된 항체를 검출할 수 있는 시약， 예를 들면， 표지된 2차 항체， 발색단 (chromophores) , 효소 (항체와 컨주게이트된 형태로서) 및 그의 기 질 또는 항체와 결합할 수 있는 다른 물질 등을 포함할 수 있다. 또한， 본 발명의 키트는 효소와 발색 반웅할 기질 및 결합되지 않은 단백질 등은 제거하고 결합된 단백질 마커 만을 보유할 수 있는 세척액 또는 용리액을 포함할 수 있다. Most preferably, it may be a diagnostic kit characterized by a lampshade containing essential elements necessary to perform an ELISA. ELISA kits contain specific antibodies to the marker protein. Antibodies are antibodies that have high specificity and affinity for each marker protein and have little cross-reactivity to other proteins. They are monoclonal antibodies, polyclonal antibodies, or recombinant antibodies. . The ELISA kit can also include antibodies specific for the control protein. Other ELISA kits may bind to reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (as conjugated to antibodies) and substrates or antibodies thereof. Other substances, and the like. In addition, the kit of the present invention may include a washing solution or an eluent capable of removing the enzyme, the substrate to be reacted with, the unbound protein, and the like, and retaining only the bound protein marker.
분석을 위해 사용되는 시료는 혈액， 혈청， 소변， 누액， 타액 둥 정상적인 상 태와 구별될 수 있는 감염성 염증 질환 특이적 단백질을 확인할 수 있는 생체 시료 를 포함한다. 바람직하게는 생물학적 액체 시료， 예를 들어 혈액, 혈청, 혈장으로 부터 측정될 수 있다. 시료는 단백질 마커의 탐지 감도를 증가시키도록 준비될 수 있는데 예를 들어 환자로부터 수득한 혈청 시료는 음이온 교환 크로마토그래피, 친 화도 크로마토그래피， 크기별 배제 크로마토그래피 (size exclusion chromatography) , 액체 크로마토그래피， 연속추출 (sequent i al extract ion) 또는 젤 전기영동 등의 방법을 이용하여 전처리될 수 있다.
또한 본 발명은 감염 질환의 진단에 필요한 정보를 제공하기 위하여 Samples used for analysis include biological samples capable of identifying infectious inflammatory disease specific proteins that can be distinguished from normal states of blood, serum, urine, tears and saliva. Preferably from a biological liquid sample, for example blood, serum, plasma. Samples may be prepared to increase the detection sensitivity of protein markers, for example serum samples obtained from patients may be anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, continuous It may be pretreated using a method such as extractive extract or gel electrophoresis. In addition, the present invention to provide information necessary for the diagnosis of infectious diseases
(a) 피검체의 시료를 제공하는 단계; (a) providing a sample of the subject;
(b) 상기 시료에서 WRS(tryptophanyl-tRNA synthetase)의 발현 수준을 측정 하는 단계； 및 (b) measuring the expression level of tryptophanyl-tRNA synthetase (WRS) in the sample; and
(c) 상기 WRS의 수준을 정상인과 비교하고 정상인에 비해 WRS의 발현 수준이 증가한 피검체를 감염 질환에 걸린 것으로 판정하는 단계를 포함하는 WRS를 검출하 는 방법을 제공한다. (c) comparing the level of the WRS with a normal person and determining a subject having an increased expression level of the WRS compared to the normal person as having an infectious disease.
본 발명자들은 WRS가 신규한 감염 질환의 마커로서 기능할 수 있음을 처음 발견하여 WRS의 발현 수준을 측정하여 감염 질환의 진단에 필요한 정보를 제공하는 방법을 제공한다. 이하 본 발명의 방법을 단계에 따라 설명한다. We first find that WRS can function as a marker of a novel infectious disease and provide a method of measuring the expression level of WRS to provide information necessary for the diagnosis of an infectious disease. The method of the present invention will be described below step by step.
본 발명의 방법의 (a) 단계는 피검체의 시료를 제공하는 단계이다. Step (a) of the method of the present invention is a step of providing a sample of a subject.
상기 시료는 감염 질환 여부를 진단하고자 하는 피검체에서 채취된 것이면 제한없이 사용할 수 있으며, 예를 들어 생검 등으로 얻어진 세포나 조직, 혈액, 전 혈， 혈청， 혈장, 타액， 뇌척수액, 각종 비물， 소변， 대변 둥일 수 있다. 바람직 하게는 혈액， 혈장， 혈청, 타액， 비액， 객담, 관절낭액, 양수, 복수， 자궁경부 또 는 질 분비물， 소변 및 뇌척수액일 수 있다. 가장 바람직하게는 혈액， 혈장， 또는 혈청일 수 있다. The sample may be used without limitation as long as it is collected from a subject to diagnose an infectious disease, for example, cells or tissues obtained by biopsy, blood, whole blood, serum, plasma, saliva, cerebrospinal fluid, various rainwater, urine. Or can be stool. Preferably blood, plasma, serum, saliva, nasal fluid, sputum, joint salivary fluid, amniotic fluid, ascites, cervical or vaginal discharge, urine and cerebrospinal fluid. Most preferably, blood, plasma, or serum.
본 발명의 방법의 (b) 단계는 (a) 단계에서 제공한 시료에서 WRS의 발현 수 준을 측정하는 단계이다. 상기 WRS의 발현 수준은 WRS 단백질 또는 WRS mRNA의 발 현 수준일 수 있다. Step (b) of the method of the present invention is a step of measuring the expression level of WRS in the sample provided in step (a). The expression level of the WRS may be the expression level of the WRS protein or WRS mRNA.
WRS 단백질의 수준은 WRS 단백질에 특이적으로 결합하는 항체를 이용하여 검 출하거나 측정할 수 있다. WRS 단백질 특이적인 항체는 본 발명의 진단용 조성물에 서술한 바와 같다. WRS 단백질의 발현 수준을 측정하는 방법은 당업계에서 공지되 어 있는 방법은 제한 없이 사용할 수 있으며， 그 예로 웨스턴 블랏팅 (western blott ing) , 닷 블랏팅 (dot blott ing) , 효소면역분석법 (enzyme- 1 inked immunosorbent assay, ELISA) , 방사능 면역분석법 (RIA) , 방사면역확산법, 오우크테 로니 면역 확산법， 로케트 면역 전기영동， 면역조직화학염색， 면역침전법 ( immunoprecipi tat i on) , 보체 고정 분석법, 유세포 분석법 (FACS) 또는 단백질 칩 (chip) 방법 등이 있으나， 이들에 한정되는 것은 아니다. 바람직하게는 ELISA 방법 을 이용할 수 있다. Levels of WRS proteins can be detected or measured using antibodies that specifically bind to WRS proteins. WRS protein specific antibodies are as described in the diagnostic composition of the present invention. Methods for measuring the expression level of the WRS protein can be used without limitation, methods known in the art, such as Western blotting, dot blotting, enzyme immunoassay (enzyme) 1 inked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunoassay, oukteroni immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipi tat i on, complement fixation assay, Flow cytometry (FACS) or protein chip methods, but are not limited to these. Preferably, an ELISA method can be used.
WRS mRNA 수준은 WRS mRNA에 특이적으로 결합하는 프라이머 세트나 프로브를 이용하여 피검체의 시료로부터 WRS의 mRNA나 cDNA를 증폭하거나, 프로브와 흔성화
반웅 (hybridizat ion)을 이용하여 피검체 시료 내 WRS mRNA의 존재와 발현량을 측정 할 수 있다. WRS의 프라이머와 프로브는 본 발명의 진단용 조성물에서 서술한 바와 같다. WRS mRNA 발현 수준의 측정은 당업계에서 통상적인 발현 수준 확인 방법을 제한 없이 사용할 수 있으며, 분석 방법의 예로 역전사중합체연쇄반웅 (reverse transcript ion polymerase chain react ion, RT-PCR) , 경쟁적 RTᅳ PCR( compet i t ive RT-PCR) , 실시간 RT-PCR(real-t ime RT-PCR) , RNase 보호 분석법 (RPA:RNase protect ion assay) , 노던 블랏팅 (northern blott ing) , DNA 마이크로어레이 칩 (microarray chip) , RNA 염기서열분석 (RNA sequencing) , 나노스트링 (nanostr ing)을 이용한 혼성화 방법， 조직 절편의 인시투 혼성화 방법 ( in si tu hybridizat ion) 등 이 있으나， 이들에 한정되는 것은 아니다. WRS mRNA levels can be expressed by amplifying WRS mRNA or cDNA from a sample of a subject using a primer set or probe that specifically binds to WRS mRNA, or by localizing with a probe. The presence and expression of WRS mRNA in the subject sample can be measured using a hybridization ion. Primers and probes of the WRS are as described in the diagnostic composition of the present invention. Determination of WRS mRNA expression level can be used without limitation the conventional expression level confirmation method in the art, and examples of the analysis method is reverse transcript ion polymerase chain react ion (RT-PCR), competitive RT ᅳ PCR ( compet it ive RT-PCR, real-time RT-PCR, RNase protect ion assay (RPA), northern blotting, DNA microarray chip ), RNA sequencing, hybridization using nanostrings, in situ hybridization of tissue sections, and the like, but are not limited thereto.
본 발명의 방법의 (c) 단계는 (b) 단계에서 측정한 피검체 시료의 WRS의 수 준을 정상인과 비교하고 정상인에 비해 WRS의 발현 수준이 증가한 피검체를 감염성 질환에 걸린 것으로 판정하는 단계이다. Step (c) of the method of the present invention is to compare the level of the WRS of the subject sample measured in step (b) with a normal person, and to determine that the subject has an increased infectious disease level compared to the normal person as having an infectious disease. to be.
상술한 (b) 단계의 방법으로 측정한 피검체의 WRS의 발현 수준을， 동일한 방 법으로 측정한 정상인의 WRS 수준과 비교한다. WRS의 발현 수준이 건강한 정상인에 비하여 증가한 피검체를 감염성 질환에 걸린 것으로 판정한다. 또한 감염성 질환이 패혈증인 경우, WRS의 발현 수준이 높을수록 패혈증이 중증인 것으로 판단할 수 있 다. 진단의 기준이 되는 WRS 발현 수준 증가의 정도에 대하여서는ᅳ 선택한 WRS 발 현 수준 측정 방법에 맞게 당업계에 공지된 기술에 따라 WRS의 발현 수준과 패혈증 중증도 사이의 상관성을 분석하여 WRS 발현 수준의 범위에 따라 패혈증의 중증도를 나타내도록 적절한 진단의 기준을 제공할 수도 있다. 본 발명에서는 패혈증의 증증 도로서 중증 패혈증과 패혈성 쇼크, SOFA score , 패혈증 진단 후 28일째 생존 여부 등 다양한 지표를 이용하여 WRS의 발현 수준이 높을수록 패혈증 중증도도 높아지는 밀접한 상관관계가 있음을 보인 바 있다. 한편， 본 발명의 일실시예에서는 연구자 임상실험을 통하여 사망 환자의 혈 청에서의 WRS의 발현양이 생존 환자에 비해서 유의하게 증가되어 있음을 확인하였 고， 이는 종래에 사용되는 마커인 프로칼시토닌 (procalci tonin)에서는 구분할 수 없는 것임을 확인하였다. The expression level of the WRS of the subject measured by the method of step (b) described above is compared with the WRS level of the normal person measured by the same method. Subjects with increased levels of expression of WRS compared to healthy normal subjects are determined to have an infectious disease. In addition, when the infectious disease is sepsis, the higher the expression level of WRS, it can be determined that sepsis is more severe. The extent of WRS expression level is analyzed by analyzing the correlation between WRS expression level and sepsis severity according to techniques known in the art for the selected method of measuring WRS expression level. In some cases, an appropriate diagnosis may be provided to indicate the severity of sepsis. In the present invention, the higher the expression level of WRS using various indicators such as severe sepsis and septic shock, SOFA score, and survival at 28 days after sepsis diagnosis, the higher the sepsis severity was. have. On the other hand, in one embodiment of the present invention it was confirmed that the expression of WRS in the serum of the deceased patient significantly increased compared to the surviving patient through a clinical trial of the investigator, which is a procalcitonin (marker used conventionally procalci tonin) was indistinguishable.
따라서, 본 발명은 트립토파닐 티알엔에이 합성효소 (tryptophanyl-tRNA synthetase) 단백질 또는 트립토파닐 티알엔에이 합성효소 mRNA의 발현 수준을 측 정하는 제제를 포함하는 감염 질환에 의한사망 위험도 판별용 조성물을 제공한다.
본 발명에서 사망 위험도는 감염 질환에 의한 사망 위험도를 의미하며 , 감염 에 의한 사망은 염증반응， 고열 통증， 호흡곤란, 저체온， 혈압강하 등의 일부 또 는 전부의 증상을 보이다， 쇼크， 일부 또는 다발성의 장기 부전으로 사망에 이르게 되는 것을 의미한다. Accordingly, the present invention provides a composition for determining the risk of death by an infectious disease comprising an agent for measuring the expression level of tryptophanyl-tRNA synthetase protein or tryptophanyl thial-NA synthase mRNA to provide. In the present invention, the risk of death refers to the risk of death due to an infectious disease, and the death due to infection shows some or all symptoms of inflammatory reaction, high fever pain, difficulty breathing, hypothermia, lowering blood pressure, shock, partial or multiple symptoms. This means death of organs due to organ failure.
아울러， 본 발명은 In addition, the present invention
(a) 피검체의 시료를 제공하는 단계; 및 (a) providing a sample of the subject; And
(b) 상기 시료에서 트립토파닐 tRNA 합성효소 (tryptophanyl-tRNA synthetase)의 발현 수준을 측정하는 단계; 및 (b) measuring the expression level of tryptophanyl-tRNA synthetase in the sample; And
(c) 상기 트립토파닐 티알엔에이 합성효소의 수준이 높을 수록 사망 위험도 가 높을 것으로 판정하는 단계를 포함하는 감염 질환에 의한 사망 위험도를 관별하 는 방법을 제공한다. (C) provides a method for identifying the risk of death due to an infectious disease comprising the step of determining that the higher the level of the tryptophanyl TNA synthase is higher risk of death.
본 발명의 판별 방법에서의 시료 등에 대해서는 상기한 바와 같다. 본 발명은 감염 질환의 진단용 제제를 제조하기 위한 트립토파닐 티알엔에이 합성효소 (tryptophany卜 tRNA synthetase) 단백질 또는 트립토과닐 티알엔에이 합성 효소 mRNA의 발현 수준을 측정하는 제제의 용도를 제공한다. 본 발명은 피검체의 시료 내의 트립토파닐 티알엔에이 합성효소 ( tryptophanyl-tRNA synthetase) 단백질 또는 트립토파닐 티알엔에이 합성효소 mRNA의 발현 수준을 측정하는 것을 특징으로 하는 감염 질환을 진단하는 방법을 제 공한다. 본 발명은 감염 질환에 의한사망 위험도 판별용 제제를 제조하기 위한 트립 토파닐 티알엔에이 합성효소 (tryptophanyl-tRNA synthetase) 단백질 또는 트립토파 닐 티알엔에이 합성효소 mRNA의 발현 수준을 측정하는 제제의 용도를 제공한다. 본 발명의 상기피검체란 동물， 바람직하게는 포유동물， 특히 인간을 포함하 는 동물일 수 있으며， 더 바람직하게는 진단이 필요한 인간 또는 환자 (pat i ent ) 일 수 있다. 본 발명에서 상기 감옆 질환을 진단하는 방법의 진단은 측정한 피검체 시료 의 WRS의 수준을 정상인과 비교하고 정상인에 비해 WRS의 단백질 또는 mRNA 발현
수준이 증가한 피검체를 감염성 질환에 걸린 것으로 진단하는 단계이다. 이와 같은 정상인과의 비교는 상술한 바와 같다. 본 발명의 용어 '-을 포함하는 (comprising)'이란 '함유하는' 또는 '특징으로 하는'과 동일하게 사용되며， 조성물 또는 방법에 있어서, 언급되지 않은 추가적인 성분 요소 또는 방법 단계 등을 배제하지 않는다. 용어 '-로 구성되는 (consisting of)'이란 별도로 기재되지 않은 추가적인 요소, 단계 또는 성분 등을 제외한다. 용 어 '필수적으로 -로 구성되는 (essentially consisting of)'이란 조성물 또는 방법 의 범위에 있어서, 기재된 물질 또는 단계와 더불어 이의 기본적인 특성에 실질적 으로 영향을 미치지 않는 물질 또는 단계 등을 포함하는 것을 의미한다. The sample and the like in the determination method of the present invention are as described above. The present invention provides a use of an agent for measuring the expression level of tryptophany® tRNA synthetase protein or tryptophanyl thialene synthase mRNA for the preparation of a diagnostic agent for infectious disease. The present invention provides a method for diagnosing an infectious disease characterized by measuring the expression level of tryptophanyl-tRNA synthetase protein or tryptophanyl thial-NA synthase mRNA in a sample of a subject. to provide. The present invention is the use of an agent for measuring the expression level of tryptophanyl-tRNA synthetase protein or tryptophanyl thial-A synthase mRNA for the preparation of the agent for determining the risk of death by infectious diseases To provide. The subject of the present invention may be an animal, preferably a mammal, particularly an animal including a human, more preferably a human or a patient (pat i ent) in need of diagnosis. In the present invention, the method of diagnosing the perioral disease is to compare the WRS level of the measured subject sample with a normal person and express the protein or mRNA of the WRS compared to the normal person. Increasing levels of a subject are diagnosed as having an infectious disease. The comparison with the normal person is as described above. The term 'comprising' of the present invention is used in the same way as 'containing' or 'featured' and does not exclude additional component elements or method steps that are not mentioned in the composition or method. . The term 'consisting of' excludes additional elements, steps or components, etc., unless otherwise noted. The term 'essentially consisting of' means, in the scope of the composition or method, that the substance or step is included in addition to the substance or step described and does not substantially affect its basic properties. .
【유리한 효과】 Advantageous Effects
따라서， 본 발명에 따르면 WRS는 감염으로 유발되는 감염 질환에서만 증가되 기 때문에 비감염성 질환과 차별되며, 감염 초기에 급속하게 증가한다. 또한 WRS의 수준은 감염으로 유발되는 질환 또는 합병증의 증증도 및 예후와 밀접한 상관관계 를 가지고 있다. 따라서 WRS는 기존의 감염 질환 또는 이의 합병증의 마커보다 더 욱 신속하고 정확한 진단 마커로 사용될 수 있다. Therefore, according to the present invention, WRS is increased only in an infectious disease caused by an infection, and thus is distinguished from a non-infectious disease and rapidly increases in the early stage of infection. Levels of WRS also correlate closely with the severity and prognosis of diseases or complications caused by infection. Thus, WRS can be used as a faster and more accurate diagnostic marker than markers of existing infectious diseases or complications thereof.
【도면의 간단한 설명】 [Brief Description of Drawings]
도 1은 살모넬라균 (5. typhimurium, 57)을 1(Λ 10? 또는 10 FU로 복강 주사 한 마우스의 복강삼출액 (peritoneal lavage fluid)에서 EUSA로 측정한 감염 후 시 간에 따른 WRS의 변화를 나타낸다. 가로축은 ST 감염 후 복강 삼출액을 수득한 시 간을 나타낸다 (Time after ST innoculat ion(hr)) . Figure 1 shows a Salmonella WRS changes in accordance vs time (5. typhimurium, 57) the first (Λ 10?, Or from 10 FU abdominal peritoneal exudates (peritoneal lavage fluid of mice injected with) after infection as measured by EUSA. The horizontal axis shows the time at which the abdominal effusion was obtained after ST infection (Time after ST innoculat ion (hr)).
도 2는 바이러스 감염에 따른 WRS 분비 양상을 측정하는 ELISA 실험 결과를 나타낸다. Figure 2 shows the results of ELISA experiments to measure the WRS secretion pattern following virus infection.
도 2의 A는 RSV(M0I=2) 또는 PR8 바이러스 (M0I=2)로 감염한 말초혈액 단핵구 세포 (PBMC)의 배양액에 존재하는 WRS 수준의 시간에 따른 변화를 나타낸다. 도 2의 B는 정상인 (H.C, n=20), 바이러스성 폐렴 (viral pneumonia, n=5) 환자의 혈청에 존 재하는 WRS의 수준을 측정하는 ELISA 실험 결과를 나타낸다. 통계적 유의성은 Mann Whitney test를 이용하였으며 * 표시한 p 값은 0.04이다. 2A shows the change over time of the WRS level present in the culture of peripheral blood mononuclear cells (PBMC) infected with RSV (M0I = 2) or PR8 virus (M0I = 2). 2B shows the results of ELISA experiments measuring the levels of WRS present in the serum of normal (H.C, n = 20), viral pneumonia (n = 5) patients. Statistical significance was obtained using the Mann Whitney test. * P value is 0.04.
도 3A는 건강한 정상인 (heal thy control, H.C), 중증 패혈증 (Severe sepsis), 패혈성 쇼크 (Septic shock) 환자의 혈청에 존재하는 WRS의 수준을 측정하
는 ELISA 실험 결과를 나타낸다. 도 3B 및 도 3C는 각각 건강한 정상인 (H.C)과 패 혈증 환자의 혈청에 존재하는 GRS 또는 KRS의 수준을 측정하는 ELISA 실험 결과를 나타낸 것이다 (통계적 유의성 도 3A의 경우 Dunn's comparison test after ruskal wallis test로 판단하였으며 도 3B 및 도 3C는 two-tailed Mann-Whitney test로 판 단하였다 ， ***는 pO.001, *는 p<0.05를 나타낸다. n.s는 통계적으로 유의하지 않음을 나타낸다). FIG. 3A measures the levels of WRS present in serum of healthy thy control (HC), Severe sepsis, and Septic shock patients. Shows the results of the ELISA experiment. 3B and 3C show the results of ELISA experiments measuring the levels of GRS or KRS in serum of healthy normal (HC) and sepsis patients, respectively (Statistical significance of Dunn's comparison test after ruskal wallis test in FIG. 3A). 3B and 3C were judged by a two-tailed Mann-Whitney test, where *** indicates pO.001 and * indicates p <0.05. Ns indicates no statistical significance).
도 4A는 건강한 정상인 (H.C)과 곰광이 (fungi) 감염 패혈증 환자의 혈청에 존 재하는 WRS의 수준을 측정하는 ELISA실험 결과를 나타낸다. 도 4B 및 도 3B 및 도 3C는 각각 건강한 정상인 (H.C)과 패혈증 환자의 혈청에 존재하는 GRS 또는 KRS의 수준을 측정하는 ELISA 실험 결과를 나타낸 것이다 (통계적 유의성은 two-tailed Mann-Whitney test로 판단하였으며, ***는 pO.001, *는 pO.05를 나타낸다. n.s 는 통계적으로 유의하지 않음을 나타낸다). 4A shows the results of ELISA experiments measuring the levels of WRS present in the serum of healthy normal (H.C) and fungi infected sepsis patients. 4B and 3B and 3C show the results of ELISA experiments measuring levels of GRS or KRS present in serum of healthy normal (HC) and sepsis patients, respectively (statistical significance was determined by the two-tailed Mann-Whitney test). *** denotes pO.001 and * denotes pO.05, ns denotes no statistical significance).
도 5A는 그람-음성균 감염 환자， 그람-양성균 감염 환자 및 곰광이 감염 환 자의 혈청에 존재하는 WRS의 수준을 측정하는 ELISA 실험 결과를 나타낸다. 도 5B 는 단일 감염 환자 및 다중 감염 환자의 혈청에 존재하는 WRS의 수준을 측정하는 ELISA 실험 결과를 나타낸다 (통계적 유의성은 Dunn's comparison test after Kruskal wallis test로 판단하였으며, ***는 ρ<0·001, *는 p<0.05를 나타낸다. n.s는 통계적으로 유의하지 않음을 나타낸다). FIG. 5A shows the results of an ELISA experiment measuring the level of WRS present in the serum of Gram-negative bacteria infected patients, Gram-positive bacteria infected patients and Gomwang infected patients. Figure 5B shows the results of ELISA experiments to measure the level of WRS present in the serum of single and multi-infected patients (statistical significance was determined by Dunn's comparison test after Kruskal wallis test, *** is ρ <0 · 001 , * Indicates p <0.05, ns indicates no statistical significance).
도 6은 전신선 염증반응 증후군 (systemic inflammatory response syndrome, SIRS, 도 6A), 만성 무균성 염증 (sterile chronic inflammatory diseases) 질환인 천식 (asthma, ASA, 도 6B)， 류마티스 관절염 (rheumatoid arthritis, RA, 도 6C) 그 리고 쇼그렌 증후군 (Sjogren's syndrome, SS, 도 6C) 환자의 WRS의 수준을 건강한 정상인 대조군 (heal thy control, HC)과 비교한 결과를 나타낸 것이다. 6 is a systemic inflammatory response syndrome (SIRS, FIG. 6A), asthma (asthma, ASA, FIG. 6B), a disease of chronic inflammatory diseases, and rheumatoid arthritis (RA, FIG. 6C) and the results of comparing WRS levels in patients with Sjogren's syndrome (SS, FIG. 6C) with healthy thy control (HC).
도 7은 WRS의 ROC curve를 나타낸다. 7 shows the ROC curve of the WRS.
도 8은 WRS와 CRP의 SOFA score와의 상관성을 보여주는 그래프를 나타낸다. r은 피어슨 상관계수， p는 확률값을 나타낸다. 8 shows a graph showing the correlation between the WRS and the SOFA score of the CRP. r is Pearson's correlation coefficient and p is a probability value.
도 9는 패혈증 진단 28일 후 생존한 환자와 사망한 환자의 WRS수준을 나타 낸다. 통계적 유의성은 Mann Whitney test를 이용하였으며， *** 표시한 p 값은 0.0007이다. 9 shows the WRS levels of surviving patients and deceased patients 28 days after sepsis diagnosis. Statistical significance was obtained using the Mann Whitney test, and *** p value was 0.0007.
도 10은 혈청에서의 WRS와 프로칼시토닌 (procalcitonin) 수준을 스피어맨스 연관 분석을 통해 확인한 것이다.
【발명의 실시를 위한 형태】 10 confirms the WRS and procalcitonin levels in serum through Spearman's correlation analysis. [Form for implementation of invention]
이하 본 발명을 상세히 설명한다. Hereinafter, the present invention will be described in detail.
단， 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실 시예에 한정되는 것은 아니다. 임상실험 However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples. Clinical trial
실험 방법은 서을대학교 검토 위원회 (institutional review board)의 허가를 받고 진행하였다 (허가번호 1502-001-010). 건강한 대조군의 혈청 (serum) 샘플은 서 울대학교 건강 센터에서 수집하였다. 99명의 패혈증 환자의 혈청 샘플은 중증 패혈 증이나 패혈성 쇼크 중환자실을 통해 구하였다. 실험에 사용한 혈청을 제공한 환자 들은 서울의 대학 연계 병원의 중환자실에 입원했던 환자들로， 박테리아 감염이 확 인된 환자들만 실험에 참여하였다. 중증 패혈증이나 패혈성 쇼크의 진단은 ACCP/SCCM consensus conference(1992년)의 기준에 따라 이루어졌다. 실험은 검토 위원회의 방침에 따라 실험에 참여한 모든 환자들에 대하여 고지에 입각한 동의를 얻고 진행되었다. 실험은 또한 서을아산병원의 검토 위원회의 허가를 받았다. 2014 년 1월과 2015년 7월 사이에 서을 세브란스 병원 알레르기 -천식 클리닉에 입원한 총 35명의 환자가 실험에 참여하였다. 실험은 26명의 건강한 대조군과, 알레르기 전문의에 의해 증상과 폐기능 검사 결과 (기관지 확장제 (bronchodilator) 사용 후 1 초간 강제된 내쉬는 호흡 부피 (forced expiratory volume for 1 second, FEV1)의 증가율이 12% 초과)를 바탕으로 천식으로 진단받은 35명의 안정된 천식 환자들을 포함하였다. 상기 안정된 천식은 최근 1개월간 약물 투여랑의 증가 없이 통상적인 약물 투여량을 유지해 온 천식 환자들로 정의하였다ᅳ 임상실험은 세브란스 병원과 연세대학교 건강 시스템의 허가를 받았다 (허가번호 4-2013-0397). 42명의 원발성 쇼그렌 증후군 환자 (primary Sjogren's syndrome, pSS), 35명의 류마티스 관절염 (rheumatoid arthritis, RA) 환자， 그리고 20명의 건강한 대조군의 혈청을 수집하 였다. 원발성 쇼그렌 증早군은 American-European Consensus Group criteria for pSS또는 2012 American College of Rheumatology criteria에 따라 진단되었다. 류 마티스 관절염은 1987 revised criteria for the classified ion of rheumatoid arthritis 또는 2010 rheumatoid arthritis classification criteria에 따라 진단 되었다. 헬싱키 선언의 원칙에 따라 실험에 참여한 모든 환자와 건강한 대조군으로 부터 실험에 대한 고지에 입각한 동의를 받았다. 상기 실험은 서울 성모 병원 검토 위원회의 허가를 받았다 (KC130NMI0646) .
임상실험 -프로칼시토닌과의 비교실험 The experiment was conducted with the permission of the Institutional Review Board (permission number 1502-001-010). Serum samples from healthy controls were collected at the Seoul National University Health Center. Serum samples from 99 sepsis patients were obtained from severe sepsis or septic shock intensive care unit. The patients who provided the serum used in the experiment were admitted to the intensive care unit of a university-affiliated hospital in Seoul. Only patients with confirmed bacterial infection participated in the experiment. Diagnosis of severe sepsis or septic shock was made according to the criteria of the ACCP / SCCM consensus conference (1992). The trial was conducted with informed consent for all patients participating in the trial, in accordance with the review committee's policy. The experiment was also approved by the Review Committee of Seolle Asan Hospital. Between January 2014 and July 2015, a total of 35 patients were admitted to the Severance Hospital Allergy-Asthma Clinic. Experiments included 26 healthy controls and allergy specialists for symptom and pulmonary function tests (more than 12% increase in forced expiratory volume for 1 second (FEV1) for 1 second after bronchodilator) The study included 35 stable asthmatic patients diagnosed with asthma. The stable asthma was defined as patients with asthma who had maintained their usual drug dose without increasing drug dosage in the last month. The clinical trial was licensed by Severance Hospital and Yonsei University Health System (Permission No. 4-2013-0397). ). Serum from 42 patients with primary Sjogren's syndrome (pSS), 35 patients with rheumatoid arthritis (RA), and 20 healthy controls were collected. Primary Sjogren's syndrome was diagnosed according to the American-European Consensus Group criteria for pSS or the 2012 American College of Rheumatology criteria. Rheumatoid arthritis was diagnosed according to the 1987 revised criteria for the classified ion of rheumatoid arthritis or 2010 rheumatoid arthritis classification criteria. In accordance with the principles of the Helsinki Declaration, informed consent was obtained from all patients and healthy controls. The experiment was approved by Seoul St. Mary's Hospital Review Committee (KC130NMI0646). Clinical Trial-Comparison with Procalcitonin
120명의 정상인， .18명의 (감염질환 이외의 원인에 의한) SIRS 환자 , 166명 의 패혈증 환자 및 160명의 패혈성 쇼크 환자를 대상으로 각 기관의 검토 위원회의 방침에 따라 실험에 참여한 모든 환자들에 대하여 고지에 입각한 동의를 얻고 진행 되었다ᅳ 환자의 경우 서울의 대학병원 중환자실에 입원했던 환자들로부터 얻은 것 으로， 전신성 염증반웅 증후군 (SIRS), 패혈증이나 패혈성 쇼크의 진단은 ACCP/SCCM consensus conference(1992년)의 기준에 따라 이루어졌다. 120 normal subjects. 18 SIRS patients (caused by non-infectious diseases), 166 sepsis patients and 160 septic shock patients were informed of all patients participating in the experiment in accordance with the institution's review committee's policy. In the case of patients who were admitted to the intensive care unit of a university hospital in Seoul, the diagnosis of systemic inflammatory reaction syndrome (SIRS), sepsis or septic shock was confirmed by the ACCP / SCCM consensus conference (1992). It was made according to the standards.
혈청의 프로칼시토닌 (RayBiotech, USA Cat No ： ELH-PROCALC)과 WRS (CUSABI0, China, Cat No: CSB-E11789h)수준은 각각의 ELISA kit 를 이용하여 측정 한 것으로 제조사가 제시한 방법에 따라 측정하였다. 세포 배양 Serum procalcitonin (RayBiotech, USA Cat No: ELH-PROCALC) and WRS (CUSABI0, China, Cat No: CSB-E11789h) levels were measured using the respective ELISA kits and were measured according to the manufacturer's method. . Cell culture
인간 말초혈액 단핵구 세포 (peripheral blood mononuclear cells, PBMC)는 sodium citrate를 포함하는 Cell Preparation Tube ( CPTTM , Bee t onD i ck i nson )■§· 이용' 하여 분리하였다. 박테리아 종류 (strain)와 감 Human peripheral blood mononuclear cells (peripheral blood mononuclear cells, PBMC) are Cell Preparation Tube (CPTTM, Bee t onD i ck i nson) containing sodium citrate was isolated by ■ § · used. Bacteria strain and persimmon
Salmonella typJiimur iuwi TCC 14028)은 한국 미생물 보존 센터 (서울)에서 구 하였다. 박테리아는 BD bioscience의 영양 배지 (nutrient broth)를 이용하여 통상 적으로 배양하였다. 감염 전에 박테리아는 하룻밤 동안 배양하고， 1X108CFU의 밀도 로 수득하였다. 박테리아 밀도는 600nm의 흡광도와 검정선 (calibration curve)를 이용하여 추산하였다. PBMC나 마우스 감염 실험을 위해서 박테리아는 PBS로 세척하 고 혈청을 포함하지 않는 배지나 PBS에 재분산하였다. 효소면역즉정법 (Enzyme— linked immunosorbent assay. ELISA) Salmonella typJiimur iuwi TCC 14028) was obtained from the Korea Microbial Conservation Center (Seoul). Bacteria were commonly cultured using nutrient broth of BD bioscience. The bacteria were incubated overnight before infection and obtained at a density of 1 × 10 8 CFU. Bacteria density was estimated using an absorbance of 600 nm and a calibration curve. For PBMC or mouse infection experiments, bacteria were washed with PBS and redispersed in medium or serum without PBS. Enzyme—linked immunosorbent assay (ELISA)
마우스와 인간의 WRS, KRS의 정량적 분석을 위하여 배양 배지나 혈청에 분비 된 단백질의 양을 ELISA 키트를 이용하여 측정하였다. WRS ELISA kit는 CusabioCWuhan, China, 카탈로그 번호 CSB-E11789h)로부터 구입하였으며, KRS는 USCN( Wuhan, China, 카탈로그 번호 SED002 Hu)에서 구입하여서 측정하였다. 통계 (Statistics)
확률값 (provability value, p-value)이 0.05 미만인 경우 통계적 유의성이 있는 것으로 판단하였다. 모든 통계적 계산은 Graphpad prism 5.0(GraphPad Software)을 이용하여 실시하였다. For quantitative analysis of mouse and human WRS and KRS, the amount of protein secreted in culture medium or serum was measured using an ELISA kit. WRS ELISA kit was purchased from CusabioCWuhan, China, catalog number CSB-E11789h), and KRS was purchased from USCN (Wuhan, China, catalog number SED002 Hu). Statistics If the probability value (p-value) is less than 0.05, it was determined that there was statistical significance. All statistical calculations were performed using Graphpad prism 5.0 (GraphPad Software).
<실시예 1> <Example 1>
박테리아 또는 바이러스 감염으로 ᅵ한 WRS수준 증가 <1-1>살모넬라균 감염에 의한 WRS 분비 Increased WRS levels caused by bacterial or viral infections <1-1> WRS secretion by Salmonella infection
마우스 복강에 살모넬라균 typimurium)을 주입하고, 복강삼출액 에 존재하는 WRS의 양을 확인하였다. Salmonella typimurium) was injected into the mouse abdominal cavity, and the amount of WRS present in the abdominal effusion was checked.
9~10주령 C57BL/6 암컷에 1C)6, IQ 또는 10¾ 의 살모넬라균을 복강주사하 고， 박테리아 감염 후 네 시간까지 한 시간마다 5~11마리의 마우스에서 복강삼출액 을 수득하여 복강삼출액에 존재하는 WRS의 양을 ELISA로 측정하였다 (도 1). Intraperitoneal injection of 1C) 6 , IQ or 10¾ Salmonella in 9 to 10 week old C57BL / 6 females, and obtained peritoneal effusion from 5 to 11 mice every hour up to 4 hours after bacterial infection. The amount of WRS was measured by ELISA (FIG. 1).
복강 삼출액으로 분비된 WRS는 살모넬라균 감염 후 매우 초기 단계에서부터 분비되어 살모넬라균 농도의존적으로 증가하였으며， 감염 후 1시간째 WRS의 양은 거의 최고치에 달하였다. WRS secreted by intraperitoneal effusion was secreted from the very early stage after Salmonella infection, and the concentration of Salmonella was increased, and the amount of WRS was almost the highest at 1 hour after infection.
<1-2> 바이러스 감염에 의한 WRS 분비 <1-2> WRS secretion by virus infection
박테리아 외에 바이러스 감염에 의하여 WRS의 분비가 일어나는지를 인간 말 초혈액 단핵구 세포와 바이러스성 폐렴 환자의 혈청을 이용하여 알아보았다. In addition to bacteria, the secretion of WRS by viral infection was investigated using human peripheral blood mononuclear cells and serum from patients with viral pneumonia.
인간 말초혈액 단핵구 세포 (PBMC)들을 호흡기 세포융합 바이러스 (respiratory syncytial virus, RSV)와 인플루엔자 바이러스 (influenza virus)인 PR8 바이러스로 감염하였을 때, 세포 배양 배지에서는 감염 후 30분부터 WRS가 크 게 증가하였으며， 실험을 진행한 감염 후 4시간까지 분비된 WRS의 수준이 유지되는 것을 확인하였다 (도 2의 A). 또한 바이러스성 폐렴 (viral pneumonia) 환자의 혈청 에서 정상인 대조군 (H.C)과 비교하여 WRS의 양이 현저하게 증가한 것을 확인하였다 (도 2의 B). When human peripheral blood monocytes (PBMCs) were infected with respiratory syncytial virus (RSV) and PR8 virus, an influenza virus, WRS increased significantly in cell culture medium from 30 minutes after infection. , It was confirmed that the level of WRS secreted up to 4 hours after the infection progressed the experiment (FIG. 2A). In addition, it was confirmed that the amount of WRS was significantly increased in the sera of patients with viral pneumonia compared to the normal control (H.C) (FIG. 2B).
이상의 실험 결과는 바이러스 감염에 의하여 WRS의 수준이 증가함을 보여준 다. The above experimental results show that the WRS level is increased by viral infection.
<실시예 2> <Example 2>
감염성 염중 화에서의 WRS특이적 분비
<2-l> 패혈증과 패혈성 쇼크 환자에서의 WRS의 증가 WRS specific secretion in infectious salt neutralization <2-l> Increase in WRS in Patients with Sepsis and Septic Shock
감염으로 인한 합병증인 패혈증과 패혈성 쇼크 환자의 WRS의 수준을 정상인 대조군과 비교하였다. Levels of WRS in sepsis and septic shock, complications due to infection, were compared with normal controls.
본 실험에 참여한 패혈증 환자들은 서울아산병원의 중환자실에 입원한 박테 리아 감염이 확인된 중증 패혈증 환자 (severe sepsis)와 패혈성 쇼크 (sept i c shock) 환자들로， 환자들에서 검출된 박테리아 및 곰광이 (fungi )는 표 1에 기재한 바와 같다. The patients with sepsis who participated in the trial were severe sepsis and septic shock patients who had been identified as having a bacterial infection in the intensive care unit of Asan Medical Center. This (fungi) is as described in Table 1.
중증 패혈증 환자와 패혈성 쇼크 환자의 혈청에서는 건강한 정상인 (heal thy control , H.C)에 비하여 WRS의 수준이 크게 증가한 것을 확인하였다 (도 3A) . 측정 된 WRS 수치는 건강한 정상인에서 0.18±0.06 ng/ml (n=20)인데 비하여 패혈증 환자 에서는 2.63土 0.62 ng/ml (n=37)로, 약 20배 증가하였다. 또한 패혈증 쇼크 환자의 WRS 수치는 6.35±0.90 ng/ml (n=63)으로， 정상인 대조군에 비해서는 약 50배 증가 하였다. 패혈증 쇼크 환자의 WRS 수치는 중증 패혈증 환자에서보다 더욱 증가하여 WRS의 수준이 패혈증 중증도 (sever i ty)와도 밀접한 관련이 있음을 알 수 있다. 패혈증 환자에서 WRS 수준이 정상인보다 크게 증가한 것과는 대조적으로 다 른 종류의 분비되는 아미노아실 티알엔에이 합성효소 (aminoacyl tRNA synthetase, ARS)인 글라이실 티알엔에이 합성효소 (glycyl-tRNA synthetase, GRS) 및 라이실 티 알엔에이 합성효소 ( lysyl-tRNA synthetase , KRS)는 유의한 변화를 보이지 않았다 ( 도 3B 및 도 3C) .
In the serum of patients with severe sepsis and septic shock, it was confirmed that the level of WRS was significantly increased compared to healthy thy control (HC) (FIG. 3A). The measured WRS level was 0.18 ± 0.06 ng / ml (n = 20) in healthy healthy subjects, compared to 2.63 土 0.62 ng / ml (n = 37) in sepsis patients. In addition, the WRS level in patients with sepsis shock was 6.35 ± 0.90 ng / ml (n = 63), which was about 50-fold higher than that of the normal control group. WRS levels in sepsis shock patients were increased more than in severe sepsis patients, indicating that WRS levels are closely related to sever i ty. Glycyl-tRNA synthetase (GRS), another type of secreted aminoacyl tRNA synthetase (ARS), in contrast to significantly increased WRS levels in sepsis patients and Lysyl-tRNA synthetase (KRS) showed no significant change (FIGS. 3B and 3C).
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한편， 도 4에 나타낸 바와 같이 , 박테리아 감염뿐만 아니라 곰팡이 (fungi) 패혈증 환자의 혈청에서도 WRS의 수치는 증가되어 있는 것으로 확인되으며 (n=8, 4.52土 1.83 ng/mL, Mann-Whitney test), GRS 및 KRS의 혈청 수치에서는 유의한 변 화가 나타나지 않았다. On the other hand, as shown in Figure 4, not only the bacterial infection but also fungi (fungi) sepsis patients serum WRS was found to be increased (n = 8, 4.52 土 1.83 ng / mL, Mann-Whitney test) , Serum levels of GRS and KRS did not show significant changes.
상기 패혈증 환자 중 그람-음성균 박테리아 감염 환자 (n=62, 5.98土 0.93ng/mL) 및 그람-양성균 박테리아 감염 환자 (n=25, 2.87±0.72ng/mL)ᅳ 곰광이 감염 환자 (n=8, 4.52±1.83) 사이에서는 유의적인 차이가 관찰되지 않았으며， 다중
병원균 감염 환자 (n=ll, 3.53±1.22ng/mL) 및 단일 병원균 감염 환자 (n=94, 5.01土 0.67ng/mL) 사이에서도 유의적인 차이가 관찰되지는 않았다 (도 5). Gram-negative bacterial infection patients (n = 62, 5.9862 0.93ng / mL) and Gram-positive bacteria bacterial infection patients (n = 25, 2.87 ± 0.72ng / mL) , 4.52 ± 1.83), no significant difference was observed. No significant difference was observed between pathogen infected patients (n = ll, 3.53 ± 1.22 ng / mL) and single pathogen infected patients (n = 94, 5.01 土 0.67 ng / mL) (FIG. 5).
<2-2> 비감염성 염중 질환에서의 WRS수준 <2-2> WRS Levels in Diseases of Non-Infectious Salts
감염에 의하지 않은 전신선 염증반응 증후군 (systemic inflammatory response syndrome, SIRS) , 만성 무균성 염증 (sterile chronic inflammatory diseases) 질환인 천식 (asthma), 류마티스 관절염 (rheumatoid arthritis) 그리고 쇼그렌 증후군 (Sjogren's syndrome) 환자의 WRS의 수준을 건강한 정상인 대조군과 비교하였다. WRS in patients with systemic inflammatory response syndrome (SIRS) without infection, asthma, sterile chronic inflammatory diseases, rheumatoid arthritis and Sjogren's syndrome The levels of were compared with healthy healthy controls.
이들 비감염성 염증 질환 환자의 혈청에 존재하는 WRS의 수춘은 건강한 정상 인 대조군과 크게 다르지 않음을 관찰하였다 (도 6). 전신성 염증반응 증후군 (도 6 의 A)， 천식 환자 (도 6의 B), 류마티스 관절염 환자 (도 6의 C), 그리고 쇼그렌 증 후군 환자 (도 6의 C)의 혈청에서 감지되는 WRS는 건강한 정상인 대조군과 비교하여 통계적으로 유의한 차이를 나타내지 않았다. It was observed that the water level of WRS present in the serum of these patients with non-infectious inflammatory disease was not significantly different from healthy normal controls (FIG. 6). WRS detected in the serum of systemic inflammatory response syndrome (FIG. 6A), asthma patients (FIG. 6B), rheumatoid arthritis patients (FIG. 6C), and Sjogren's syndrome patients (FIG. 6C) is healthy normal. There was no statistically significant difference compared to the control.
<실시예 3> <Example 3>
감엮성 중 짐화의 ?1다 마커로서의 WRS의 효음성 <3-1> 감염성 염증 질환마커로서 WRS의 성능 Jimhwa the sense of yeokseong? Efficacy of WRS as a Multivalent Marker Performance of WRS as an Infectious Inflammatory Disease Marker
감염성 염증 질환 마커로서의 WRS의 성능을 확인하기 위하여 수용자 작용 특 징 곡선 (receiver operating characteristics curve , ROC curve)를 작성하였다. 박테리아 감염 패혈증 환자 100명의 데이터로 산출한 ROC curve에 나타난 WRS의 AUC는 0.90(p<0.0001), cut off 수치는 0.28ng/mL, 민감성 (sensitivity)은 82% 그리고 정확성 (specificity)은 80%인 것으로 나타났다 (도 7). In order to confirm the performance of WRS as an infectious inflammatory disease marker, a receiver operating characteristics curve (ROC curve) was prepared. The AUC of WRS on the ROC curve of 100 patients with bacterial infection sepsis was 0.90 (p <0.0001), 0.28 ng / mL cut off, 82% sensitivity and 80% specificity. (FIG. 7).
<3-2> WRS의 수준과 감염성 염증 질환 중증도 및 예후사이의 상관 감옇성 염증 질환 마커로서의 WRS의 성능을 더욱 확인하기 위하여 WRS의 수 준과 패혈증 중증도와 패혈증 예후의 상관성을 알아보았다. <3-2> Correlation between WRS Level, Infectious Inflammation Disease Severity and Prognosis In order to further confirm the performance of WRS as a marker of susceptibility to inflammatory disease, the correlation between WRS level, sepsis severity and sepsis prognosis was examined.
패혈증 환자의 예후를 나타내는 순차기관 기능상실 평가 스코어 (sequential organ failure assessment score, SOFA score)와 환자의 혈청에서 감지되는 WRS 또는 CRP의 수준을 그래프로 나타내고 피어슨 상관계수 (Pearson correlation coefficient)를 계산하여 상관성 여부를 판단하였다 (도 8). WRS의 피어슨 상관계수 는 0.43으로, CRP의 0.15보다 현저히 높았으며, 통계적으로도 유의한 상관성을 나
타내었다. 즉， WRS의 수준은 기존의 염증 지표인 CRP보다 패혈증 환자의 SOFA score와 더욱 밀접한 상관관계를 가지고 있음을 알 수 있다. Graph the sequential organ failure assessment score (SOFA score) that indicates the prognosis of the sepsis patient and the level of WRS or CRP detected in the patient's serum, and calculate the correlation by calculating the Pearson correlation coefficient. It was judged whether or not (FIG. 8). Pearson's correlation coefficient of WRS was 0.43, which was significantly higher than 0.15 of CRP. Burned out. In other words, the level of WRS is more closely correlated with the SOFA score of sepsis patients than CRP, which is an existing inflammatory marker.
또한 패혈증 환자의 예후를 나타내는 다른 지표로서 패혈증 진단 후 28일의 생존 여부와 WRS의 수준을 확인하고 만 휘트니 테스트로 통계적 유의성을 판단하였 다 (도 9) . 패혈증 진단 후 28일에 사망한 환자들에서는 생존한 환자에서보다 WRS 수준이 통계적으로도 유의한 큰 차이를 보였다 (사망， n=27 , WRS 9. 14+1.63ng/ml ;생 존, n=72 , WRS 3.36+0.49ng/ml ) . 즉， WRS 수준이 높을수록 패혈증 생존 예후가 좋 지 않음을 알 수 있다. In addition, as another index indicating the prognosis of the sepsis patient, the survival and the level of WRS were checked for 28 days after sepsis diagnosis, and the statistical significance was judged only by the Whitney test (FIG. 9). In patients who died 28 days after sepsis diagnosis, WRS levels were significantly higher than those in survivors (death, n = 27, WRS 9.14 + 1.63 ng / ml; survival, n = 72, WRS 3.36 + 0.49 ng / ml). In other words, the higher the WRS level, the better the prognosis of sepsis survival.
<실시예 4> <Example 4>
프로 ·시토닌 (Drocalcitonin)과의 비교 <4-1> 혈청내의 함량 비교 Comparison with pro-cytonin (Drocalcitonin) <4-1> Comparison of contents in serum
120명의 정상인， 18명의 (감염질환 이외의 원인에 의한) SIRS 환자 ， 166명 의 패혈증 환자 및 160명의 패혈성 쇼크 환자의 혈청 (serum)을 대상으로 이들 각 환자에서의 WRS (CUSABIO, China, Cat No : CSB-E11789h)와 주요 염증 마커로 알려 져 있는 프로칼시토닌 (RayBiotech , USA Cat No : ELH-PROCALC)을 대상으로 이들의 양을 제조사의 지침에 따라 ELISA방법으로 정량적으로 측정하였다. 그 결과， 하기 표 2에서 보듯이 프로칼시토닌의 경우 정상인에 대비해서 비 감염성 전신성 염증 증상인 SIRS가 구분되고， 감염질환의 합병증인 패혈증이나 패 혈성 쇼크에서는 더 많은 양이 검출되는 것에 비해서, WRS의 경우 감염질환의 합병 증에 대해서만 특이적으로 구분됨을 알 수 있었다. WRS in each of these patients (CUSABIO, China, Cat) in 120 normal subjects, 18 SIRS patients (due to infectious diseases), 166 sepsis patients and 160 septic shock patients No: CSB-E11789h) and procalcitonin (RayBiotech, USA Cat No: ELH-PROCALC), known as major inflammatory markers, were measured quantitatively by ELISA according to the manufacturer's instructions. As a result, as shown in Table 2 below, SIRC, which is a non-infectious systemic inflammatory symptom, is distinguished from procalcitonin in comparison with a normal person, and a greater amount is detected in sepsis or septic shock, which is a complication of an infectious disease. In this case, only the complications of infectious diseases were identified.
【표 2】 Table 2
정상 대조군과 환자에서의 WRS와 프로칼시토닌의 비교 Comparison of WRS and Procalcitonin in Normal Controls and Patients
<4-2> 생존여부에 따른 함량의 비교
패혈증， 패혈성 쇼크 환자에 대해서 28일 경과시의 생존여부에 따라서 생존 자와 사망자의 군을 나누었다. 각 군의 환자 혈청에서의 WRS와 프로칼시토닌의 함 량을 상기 실시예 <4-1>에서와 같이 정량적으로 측정하였다. 그 결과 하기 표 3에서 보듯이， 프로칼시토닌의 경우 환자 중 생존자와 사망 자간의 구분이 되지 않았으나, WRS의 경우 통계적으로 의의가 있도록 구분되어 사 망 우려의 환자의 선별이 가능함을 확인할 수 있었다. <4-2> Comparison of contents according to survival Patients with sepsis and septic shock were divided into survivors and deaths according to their survival after 28 days. The content of WRS and procalcitonin in patient serum of each group was measured quantitatively as in Example <4-1>. As a result, as shown in Table 3 below, the procalcytonin was not distinguished between the survivors and the dead among the patients, but the WRS was statistically meaningful to confirm that the screening of the patients of death was possible.
【표 3】 Table 3
생존 환자와 사망 환자에서의 WRS와프로칼시토닌의 비교
Comparison of WRS and Procalcitonin in Survival and Death Patients
<4-3>프로칼시토닌과의 연관 분석 <4-3> Analysis of association with procalcitonin
종래에 사용되는 프로칼시토닌과 WRS의 진단 결과의 상호 연관성을 확인하기 위하여 패혈증 환자에 대해서 WRS와 프로칼시토닌에서의 측정값을 이용하여 스피어 맨스 연관 분석 (Spearman ' s correlat ion analys i s)를 수행하였다. 그 결과， 도 10에서 보듯이, WRS와 프로칼시토닌간에 Spearman ' s rho 값 (r) 이 0.127 , p값이 0.022로 나타나 연관성을 나타냄을 확인할 수 있었다. Spearman's correlat ion analys i was performed on sepsis patients using measurements from WRS and procalcitonin in order to correlate the diagnosis results of conventionally used procalcitonin and WRS. As a result, as shown in FIG. 10, it was confirmed that Spearman's rho value (r) was 0.127 and p value was 0.022, indicating an association between WRS and procalcitonin.
【산업상 이용가능성】 Industrial Applicability
이상 살펴본 바와 같이 , WRS는 감염으로 유발되는 감염 질환에서만 증가되기 때문에 비감염성 질환과 차별되며， 감염 초기에 급속하게 증가한다. 또한 WRS의 수 준은 감염으로 유발되는 질환 또는 합병증의 중증도 및 예후와 밀접한 상관관계를 가지고 있다. 따라서 WRS는 기존의 감염 질환 또는 이의 합병증의 마커보다 더욱 신속하고 정확한 진단 마커로 사용될 수 있다.
As described above, since WRS is increased only in infectious diseases caused by infection, WRS is differentiated from non-infectious diseases and increases rapidly in the early stage of infection. The level of WRS also correlates closely with the severity and prognosis of the disease or complications caused by infection. Thus, WRS can be used as a faster and more accurate diagnostic marker than markers of existing infectious diseases or complications thereof.
Claims
【청구항 1】 [Claim 1]
트립토파닐 티알엔에이 합성효소 (tryptophanyl -tRNA synthetase) 단백질 또 는 트립토파닐 티알엔에이 합성효소 mRNA의 발현 수준을 측정하는 제제를 포함하는 감염 질환의 진단용 조성물. A composition for diagnosing an infectious disease comprising an agent for measuring the expression level of tryptophanyl-tRNA synthetase protein or tryptophanyl thial-NA synthase mRNA.
【청구항 2] [Claim 2]
제 1항에 있어서， 상기 감염은 바이러스, 박테리아 및 곰광이 ( fungi )로 이루 어진 군에서 선택된 하나 이상에 의한 감염인 것을 특징으로 하는 조성물. The composition of claim 1, wherein the infection is an infection caused by one or more selected from the group consisting of viruses, bacteria and fungi.
【청구항 3】 [Claim 3]
제 2항에 있어서， 상기 박테리아는 그람-음성균 또는 그람-양성균인 것을 특 징으로 하는 조성물. The composition of claim 2, wherein the bacterium is Gram-negative or Gram-positive.
【청구항 4】 [Claim 4]
제 1항에 있어서, 상기 제제는 트립토파닐 티알엔에이 합성효소 단백질에 특 이적으로 결합하는 항체인 것을 특징으로 하는 조성물. The composition of claim 1, wherein the agent is an antibody that specifically binds to tryptophanyl thialA synthase protein.
【청구항 5】 [Claim 5]
제 4항에 있어서， 상기 트립토파닐 티알엔에이 합성효소 단백질은 서열번호 1 로 표시되는 아미노산 서열을 포함하는 것을 특징으로 하는 조성물. The composition according to claim 4, wherein the tryptophanyl thialN synthase protein comprises an amino acid sequence represented by SEQ ID NO: 1.
【청구항 6】 [Claim 6]
제 1항에 '있어서， 상기 제제는 트립토파닐 티알엔에이 합성효소 mRNA에 특이 적으로 결합하는 프로브 또는 프라이머 세트인 것을 특징으로 하는 조성물. The composition of claim 1, wherein the agent is a set of probes or primers that specifically bind to tryptophanyl thiANA synthase mRNA.
【청구항 7】 [Claim 7]
제 6항에 있어서, 상기 트립토파닐 티알엔에이 합성효소 mRNA는 서열번호 2로 표시되는 염기서열을 포함하는 것을 특징으로 하는 조성물. 7. The composition of claim 6, wherein the tryptophanyl thiAlN synthase mRNA comprises a nucleotide sequence represented by SEQ ID NO: 2.
【청구항 8】 [Claim 8]
제 6항에 있어서, 상기 프라이머 세트는 서열번호 3 및 서열번호 4로 표시되
는 것을 특징으로 하는 조성물. The method of claim 6, wherein the primer set is represented by SEQ ID NO: 3 and SEQ ID NO: 4 Composition characterized in that.
【청구항 9】 [Claim 9]
제 1항에 있어서， 상기 감염질환은 살모넬라증 (salmonel losis) , 식중독， 장티 푸스， 파라티푸스， 폐렴， 폐결핵， 결핵， 패혈증 (sepsis) , 패혈성 쇼크 (sept i c shock) , 요로감염， 방광염， 신우신염， 요도염， 전립선염， 상기도감염, 중이염으로 이루어진 군에서 선택된 것임을 특징으로 하는 조성물. The method of claim 1, wherein the infectious disease is salmonel losis, food poisoning, typhoid fever, paratyphoid, pneumonia, pulmonary tuberculosis, tuberculosis, sepsis, septic shock, urinary tract infection, cystitis, pyelonephritis , Urethritis, prostatitis, upper respiratory tract infection, otitis media, the composition characterized in that it is selected from the group consisting of.
【청구항 10] [Claim 10]
트립토파닐 티알엔에이 합성효소 (tryptophanyl-tRNA synthetase) 단백질 또 는 트립토파닐 티알엔에이 합성효소 mRNA의 발현 수준을 측정하는 제제를 포함하는 감염 질환 진단용 키트. A kit for diagnosing an infectious disease comprising an agent for measuring the expression level of tryptophanyl-tRNA synthetase protein or tryptophanyl thial-NA synthase mRNA.
【청구항 11】 [Claim 11]
감염 질환의 진단에 필요한 정보를 제공하기 위하여 To provide you with the information you need to diagnose infectious diseases
(a) 피검체의 시료를 제공하는 단계; (a) providing a sample of the subject;
(b) 상기 시료에서 트립토파닐 tRNA 합성효소 (tryptophanyl— tRNA synthetase)의 발현 수준을 측정하는 단계; 및 (b) measuring the expression level of tryptophanyl—tRNA synthetase in the sample; And
(c) 상기 트립토파닐 티알엔에이 합성효소의 수준을 정상인과 비교하고 정상 인에 비해 트립토파닐 티알엔에이 합성효소의 발현 수준이 증가한 피검체를 감염 질환에 걸린 것으로 판정하는 단계를 포함하는 트립토파닐 tRNA 합성효소를 검출하 는 방법ᅳ (c) comparing the level of the tryptophanyl thial-A synthase with a normal person and determining a subject having an infectious disease that has an increased expression level of tryptophanyl thial-A synthase compared to a normal person Method for detecting tryptophanyl tRNA synthetase ᅳ
【청구항 12】 [Claim 12]
제 10항에 있어서, 상기 시료는 혈액， 혈장， 혈청, 타액， 비액， 객담, 관절낭 액， 양수， 복수， 자궁경부 또는 질 분비물， 소변 및 뇌척수액으로 이루어진 군에서 선택되는 것을 특징으로 하는 방법. The method of claim 10, wherein the sample is selected from the group consisting of blood, plasma, serum, saliva, nasal fluid, sputum, articular sac, amniotic fluid, ascites, cervical or vaginal discharge, urine and cerebrospinal fluid.
【청구항 13] [Claim 13]
제 10항에 있어서, 상기 트립토파닐 티알엔에이 합성효소의 발현 수준을 측정 하는 단계는 트립토파닐 티알엔에이 합성효소의 단백질 또는 트립토파닐 티알엔에 이 합성효소 mRNA의 발현 수준을 측정하는 것을 특징으로 하는 방법 .
The method of claim 10, wherein the step of measuring the expression level of tryptophanyl thiAen synthase is to measure the expression level of this synthase mRNA in tryptophanyl thiAN synthase protein Characterized in that the method.
【청구항 14] [Claim 14]
트립토파닐 티알엔에아 합성효소 (tryptophanyl-tRNA synthetase) 단백질 또 는 트립토파닐 티알엔에이 합성효소 mRNA의 발현 수준을 측정하는 제제를 포함하는 감염 질환에 의한 사망 위험도 판별용 조성물. A composition for determining the risk of death due to an infectious disease comprising an agent for measuring the expression level of tryptophanyl-tRNA synthetase protein or tryptophanyl thialene synthase mRNA.
【청구항 15】 [Claim 15]
(a) 피검체의 시료를 제공하는 단계; (a) providing a sample of the subject;
(b) 상기 시료에서 트립토파닐 tRNA 합성효소 ( tryptophany卜 tRNA synthetase)의 발현 수준을 측정하는 단계; 및 (b) measuring the expression level of tryptophany tRNA synthetase in the sample; And
(c) 상기 트립토파닐 티알엔에이 합성효소의 수준이 높을 수록 사망 위험도 가 높을 것으로 판정하는 단계를 포함하는 감염 질환에 의한 사망 위험도를 판별하 는 방법 . (c) determining the risk of death due to an infectious disease, comprising determining that the higher the level of tryptophanyl TNA is, the higher the risk of death.
【청구항 16】 [Claim 16]
감염 질환의 진단용 제제를 제조하기 위한 트립토파닐 티알엔에이 합성효소 Tryptophanyl Thialene Synthase for the Preparation of Diagnostic Agents for Infectious Diseases
( tryptophanyl-tRNA synthetase) 단백질 또는 트립토파닐 티알엔에이 합성효소 mRNA의 발현 수준을 측정하는 제제의 용도. tryptophanyl-tRNA synthetase Use of an agent to measure the expression level of a protein or tryptophanyl thialene synthase mRNA.
【청구항 17】 [Claim 17]
피검체의 시료 내의 트립토파닐 티알엔에이 합성효소 ( tryptophany卜 tRNA synthetase) 단백질 또는 트립토파닐 티알엔에이 합성효소 mRNA의 발현 수준을 측 정하는 것을 특징으로 하는 감염 질환을 진단하는 방법 . A method for diagnosing an infectious disease, characterized by measuring the expression level of tryptophany® tRNA synthetase protein or tryptophanyl thialen synthase mRNA in a sample of a subject.
【청구항 18】 [Claim 18]
감염 질환에 의한 사망 위험도 판별용 제제를 제조하기 위한 트립토파닐 티 알엔에이 합성효소 (tryptophany卜 tRNA synthetase) 단백질 또는 트립토파닐 티알엔 에이 합성효소 mRNA의 발현 수준을 측정하는 제제의 용도.
Use of an agent to measure the expression level of tryptophany tRNA synthetase protein or tryptophanyl thialen synthase mRNA to prepare an agent for determining the risk of death from an infectious disease.
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| PL16842325T PL3346270T3 (en) | 2015-09-01 | 2016-09-01 | Composition for diagnosing infectious diseases or infectious complications by using tryptophanyl-trna synthetase and method for detecting diagnostic marker |
| EP16842325.9A EP3346270B1 (en) | 2015-09-01 | 2016-09-01 | Composition for diagnosing infectious diseases or infectious complications by using tryptophanyl-trna synthetase and method for detecting diagnostic marker |
| ES16842325T ES2911270T3 (en) | 2015-09-01 | 2016-09-01 | Composition for diagnosing infectious diseases or infectious complications by using tryptophanil-tRNA synthetase and method for detecting the diagnostic marker |
| JP2018530462A JP6732914B2 (en) | 2015-09-01 | 2016-09-01 | Composition for diagnosing infectious disease or infectious complication using tryptophanyl tRNA synthetase and method for detecting diagnostic marker |
| CN201680050793.5A CN108449999B (en) | 2015-09-01 | 2016-09-01 | Compositions for diagnosing infectious diseases or complications thereof using tryptophamide-tRNA synthetase and methods for detecting diagnostic markers |
| US15/908,568 US10788493B2 (en) | 2015-09-01 | 2018-02-28 | Composition for diagnosing infectious diseases or infectious complications by using tryptophanyl-tRNA synthetase and method for detecting diagnostic marker |
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| CN113574075B (en) | 2019-03-14 | 2023-01-10 | 高丽大学校产学协力团 | Carrageenan derivative, probe for marking macrophage and method for preparing the same |
| CN114502732A (en) | 2019-07-18 | 2022-05-13 | Jw生物科学股份有限公司 | Antibodies that specifically bind WRS proteins and uses thereof |
| EP4238995A4 (en) | 2020-10-27 | 2024-08-14 | Mirimgene Co Ltd | Wars-neutralizing antibody and use thereof |
| KR20220055856A (en) | 2020-10-27 | 2022-05-04 | 주식회사 미림진 | Methods for determining the risk of death from infectious inflammatory diseases based on WARS and cytokine concentrations |
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