KR101869509B1 - Composition and method for detecting a diagnostic marker for sepsis using Testican-1 - Google Patents

Abstract

The present invention relates to a composition for sepsis diagnosis using testican-1 and a method for detecting a diagnostic marker, and more specifically, to a composition for sepsis diagnosis including an agent for measuring the expression level of testican-1 protein or mRNA encoding the testican-1 protein, a kit for diagnosis, and a method for sepsis diagnosis using the same. According to the present invention, the expression level of testican-1 is increased in a sepsis patient and is highly correlated with the severity and survival rate of sepsis. Therefore, the expression level of testican-1 can be analyzed to quickly and accurately diagnose sepsis and determine the severity and prognosis for recovery.

Description

TECHNICAL FIELD The present invention relates to a composition for diagnosing sepsis using testicular-1 and a method for detecting diagnostic markers for sepsis using Testican-1,

TECHNICAL FIELD The present invention relates to a composition for diagnosing sepsis using testican-1 and a method for detecting a diagnostic marker, and more particularly, to a method for detecting the expression level of a testican-1 protein or an mRNA encoding the testican- A diagnostic kit, and a method for diagnosing sepsis using the same.

Rapidly differentiating and diagnosing various systemic inflammatory responses and inflammatory responses caused by pathogenic infections caused by non-infectious causes rather than by pathogen causes infectious inflammation patients to initiate appropriate antibiotic treatment as soon as possible And prevent the use of unnecessary drugs, it is essential for the survival of patients with acute infectious inflammation.

Current diagnostic tools for infection are typically microbial cultures from blood and urine and polymerase chain reaction (PCR) to identify infected organs. However, most of the microbiological culture results in the laboratory are negative, and the PCR method has a time limit. The prohormone of calcitonin (PCT) of calcitonin is elevated within the first three to four hours of infection in most bacterial and fungal infections, but higher levels of change are caused by gram-negative bacteria than other pathogens. It is also unknown whether this level of hormone is elevated by viral infection. Currently, C-reactive protein (CRP) is being used as a common diagnostic marker for a variety of inflammatory diseases. However, infectious inflammation can not be distinguished because the levels of CRP increase in both infectious and noninflammatory inflammatory diseases. Therefore, it is necessary to develop diagnostic reagents that can distinguish inflammation caused by rapid infection.

Sepsis, on the other hand, is a systemic inflammatory reaction caused by an abnormal defensive action of the body against infected microorganisms. The activation of macrophages and thus the excessive production of inflammatory factors are associated with a severe inflammatory response in the whole body. Two or more of the following: fever with a temperature above 38 ° C, hypothermia below 36 ° C, at least 24 breaths per minute (ventilation), at least 90 heartbeats per minute (tachycardia) When symptoms are seen, it is called systemic inflammatory response syndrome (SIRS), and it is called sepsis when the systemic inflammatory response syndrome is caused by microbial infection. Septicemia can potentially lead to septic shock. When septicemia gets worse, the function of various parts of the body (heart, kidney, liver, brain, lungs, etc.) deteriorates and becomes more shocked. A variety of pathogens can cause sepsis, the most common of which is caused by bacteria, but can also be caused by viruses or fungi. Pneumonia causing infection of the lungs, urethral infections causing infection of the bladder and kidneys, skin infections, appendicitis in the abdomen, or meningitis in the brain. For example, if a patient with pneumonia is suffering from sepsis, Severe progression of damage to the heart, liver, lungs, or kidneys occurs. Approximately 20-50% of patients die from septic shock. In addition, post-operative infection may cause sepsis. As a hyperinflammatory response due to infection or postoperative infection, 40 to 90% of deaths are caused by sepsis.

Therefore, rapid diagnosis of early sepsis symptoms before severe sepsis or septic shock may be a very important requirement for lowering the rate of such septic mortality, but the markers capable of quickly and accurately diagnosing it are well known There are many difficulties in diagnosing early sepsis symptoms.

Therefore, the present inventor has made an effort to find a marker capable of rapidly and accurately diagnosing sepsis. As a result, the expression of testican-1 in serum of sepsis animal models and sepsis patients is remarkably increased compared to the normal control, The present inventors have found that the level of expression of ticane-1 is closely related to the severity of disease and the survival rate of the disease.

Accordingly, an object of the present invention is to provide a composition for diagnosing sepsis, which comprises an agent for measuring the expression level of a testican-1 protein or an mRNA encoding the testican-1 protein.

It is another object of the present invention to provide a sepsis diagnosis kit comprising an agent for measuring the level of expression of a testican-1 protein or an mRNA encoding the testican-1 protein.

Another object of the present invention is to provide information necessary for diagnosis of sepsis

(a) providing a sample of the subject; And

(b) measuring the level of expression of the testin-1 protein or the mRNA encoding the testin-1 protein in the sample; And

(c) comparing the expression level of the protein or mRNA with that of a normal person, and determining that the subject having an increased level of expression compared to a normal person has suffered from sepsis.

In order to achieve the object of the present invention, the present invention provides a composition for diagnosing sepsis comprising an agent for measuring the expression level of Testican-1 protein or mRNA encoding the testican-1 protein.

In order to accomplish the object of the present invention, the present invention provides a sepsis diagnosis kit comprising an agent for measuring the expression level of Testican-1 protein or mRNA encoding the testican-1 protein.

According to another aspect of the present invention,

(a) providing a sample of the subject; And

(b) measuring the level of expression of the testin-1 protein or the mRNA encoding the testin-1 protein in the sample; And

(c) comparing the expression level of the protein or mRNA with that of a normal person, and determining that the subject having an increased expression level as compared to a normal person has acquired septicemia.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for diagnosing sepsis comprising an agent for measuring the expression level of a testican-1 protein or an mRNA encoding the testican-1 protein.

Through a series of experiments, the present inventors have found that testicular-1 expression is significantly increased in the serum of sepsis animal models and sepsis patients compared with the normal control, and the expression level of testican-1 is closely correlated with the disease severity and survival rate And that testicular-1 could be used as a diagnostic marker for sepsis.

In the present invention, the testican-1 is a type of proteoglycan, a highly conserved multi-domain proteoglycan that is most prominently expressed in the brain's thalamus and upregulated in activated astrocytes of the brain. Various functions of TESTYCAN-1 are known to inhibit membrane-type MMP, inhibition of cathepsin L, and binding to calcium. Purified proteins have been shown to inhibit cell adhesion and neurite outgrowth in vitro. In vivo, testicular is known to carry not only a significant amount of chondroitin sulfate but also other oligosaccharides, but the precise biological significance is not yet known.

Preferably, in the present invention, the testican-1 protein comprises the amino acid sequence of SEQ ID NO: 1, and the gene encoding the same is also referred to as TESTICAN1 or SPOCK1 and may include the nucleotide sequence of SEQ ID NO: 2 .

In the present invention, the term 'expression' means that a protein or a nucleic acid is produced in a cell. "Protein" is used interchangeably with "polypeptide" or "peptide" and refers to a polymer of amino acid residues as commonly found in natural state proteins. A "polynucleotide" or "nucleic acid" refers to deoxyribonucleotides (DNA) or ribonucleotides (RNA) in the form of single- or double-stranded. Unless otherwise constrained, also includes known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. 'mRNA' is RNA that transfers genetic information (gene-specific nucleotide sequence) to a ribosome in which amino acid sequence is specified from a specific gene during protein synthesis.

In the present invention, the septicemia is caused by inflow of microorganisms symbiotic to the gastrointestinal tract or adjacent tissues of the skin, and the partial infection such as genitourinary tract, bile, lung or gastrointestinal tract is caused by the systemic inflammatory response syndrome (SIRS) It can cause blood infections, and may lead to septicemia by direct blood injection through intravenous injection, and it leads to inflammatory reactions such as high fever, low fever, tachycardia, and tachypnea. Sepsis is a severe sepsis or septic shock or complication that can lead to dysfunction of the lung, kidney, liver, circulatory system, etc., if the cause can not be diagnosed promptly and accurately. (multiple organ dysfunction syndrome, MODS). The sepsis of the present invention encompasses various complications due to such severe sepsis, septic shock, and sepsis.

In the present invention, the severe sepsis refers to sepsis accompanied by at least one sign of organ impairment (for example, hypoxemia, oliguria, metabolic acidosis or cerebral dysfunction) with definite long-term hypoperfusion. In the present invention, the above-mentioned septic shock refers to "severe sepsis" accompanied by a definite hypotension due to the necessity of medication intervention for maintaining the systolic blood pressure <90 mmHg or blood pressure.

In the present invention, complications of the sepsis include multiorgan dysfunction syndrome (MODS), disseminated intravascular coagulation syndrome (DIC), acute respiratory distress syndrome (ARDS), and acute renal failure (AKI). Complications of sepsis are increasingly associated with increased sepsis, severe sepsis, and increased severity of sepsis. For example, "MODS (multiorgan failure syndrome)" is a syndrome in which multiple important organs simultaneously cause dysfunction and are identified in relation to SIRS.

"Sepsis" as used herein includes, but is not limited to, all stages of sepsis, including, but not limited to, the occurrence of sepsis associated with the final stage of sepsis, severe sepsis, septic shock and complications of the sepsis.

When the diagnostic composition of the present invention is to measure the level of expression of the testin-1 protein, the agent may be an antibody that specifically binds to the testin-1 protein.

&Quot; Antibody &quot; means an immunoglobulin that specifically binds to an antigenic site. The TESTYCAN-1 antibody can be produced by cloning the TESTYCAN-1 gene into an expression vector to obtain a protein encoded by the gene and obtaining the protein from the obtained protein according to a conventional method in the art. A testicular-1 protein specific antibody can also be prepared using a fragment of a testican-1 protein comprising a testican-1 antigenic site. The form of the antibody of the present invention is not particularly limited and includes a polyclonal antibody or a monoclonal antibody. Also included in the antibody of the present invention are a part of whole antibodies as long as they have antigen-antibody binding ability, and include all kinds of immunoglobulin antibodies that specifically bind to testkin-1. F (ab '), F (ab '), &lt; / RTI &gt; which have antigen binding functions, as well as complete forms of antibodies with two full length light chains and two full length heavy chains, 2, and Fv. Furthermore, the antibody of the present invention includes a special antibody such as a humanized antibody, a chimeric antibody and a recombinant antibody, as long as it can specifically bind to a testkin-1 protein.

The antibody specifically binding to the testin-1 protein in the present invention is preferably an antibody that specifically binds to a protein having the amino acid sequence shown in SEQ ID NO: 1. The diagnostic composition of the present invention comprising an antibody specific to the testin-1 protein as an agent for measuring the expression level of testin-1 may further comprise a preparation necessary for a method for detecting a known protein, Can be used to determine the level of the testin-1 protein in a subject.

On the other hand, when the diagnostic composition of the present invention is to measure the expression level of mRNA encoding the testin-1 protein, it may be a probe or a primer set that specifically binds to the mRNA.

The mRNA coding for testican-1 may be derived from a mammal, including a human, and preferably comprises the human testin-1 mRNA base sequence shown in SEQ ID NO: 2. The diagnostic composition of the present invention, wherein the testin-1 encoding comprises an mRNA-specific probe or primer set, may further comprise an agent necessary for a method of detecting known RNA. The method of detecting known RNA using this composition can be used without limitation to determine the level of mRNA encoding testican-1 in a subject.

In the present invention, the 'primer' is a short single strand oligonucleotide which serves as a starting point of DNA synthesis. The primer specifically binds to a polynucleotide which is a template under a temperature condition with a suitable buffer, and a DNA polymerase is added to the primer by addition of a nucleoside triphosphate having a base complementary to the template DNA, Is synthesized. The primer is generally composed of 15 to 30 nucleotide sequences, and the melting temperature (Tm) of the primer varies depending on the base structure and length.

The sequence of the primer does not need to have a sequence completely complementary to a part of the nucleotide sequence of the template. It is sufficient if the primer has sufficient complementarity within a range capable of hybridizing with the template and acting as a primer. Thus, in the present invention, the primer for measuring the expression level of mRNA encoding testin-1 does not need to have a perfectly complementary sequence to the testin-1 gene sequence, It suffices that the specific region of the cDNA of the testican-1 is amplified and has a length and complementarity to the purpose of measuring the amount of the mRNA of the testican-1. The primer for the amplification reaction is a set (pair) complementarily binding to the template (or sense) at opposite ends of a specific region of the mRNA of the testan-1 to be amplified and the opposite (antisense) . The primers can be easily designed by those skilled in the art with reference to the mRNA or cDNA base sequence of testican-1.

The primer of the present invention may preferably be a set or a pair which specifically binds to the mRNA nucleotide sequence of testin-1 shown in SEQ ID NO: 2, and most preferably the nucleotide sequence of SEQ ID NO: 3 and SEQ ID NO: 4 Lt; / RTI &gt;

The term "probe" refers to a fragment of a polynucleotide, such as RNA or DNA having a base pair length of several to several hundreds, which can specifically bind to a specific gene mRNA or cDNA (complementary DNA) And it is labeled so that the presence or expression level of mRNA or cDNA to be bound can be confirmed. For the purpose of the present invention, a probe complementary to testicular-1 mRNA can be used for the diagnosis of infectious inflammatory disease by measuring the expression level of testican-1 mRNA by performing hybridization with a test sample have. The selection and hybridization conditions of the probes can be appropriately selected according to techniques known in the art.

The primer or probe of the present invention can be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known methods. In addition, the primer or the probe may be variously modified according to a method known in the art, so long as it does not interfere with the hybridization of the testican-1 with the mRNA. Examples of such modifications include, but are not limited to, methylation, capping, substitution with one or more of the natural nucleotide analogs and modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.) ) Or charged conjugates (eg, phosphorothioate, phosphorodithioate, etc.), and binding of labeling materials using fluorescence or enzymes.

The present invention also provides a sepsis diagnosis kit comprising an agent for measuring an expression level of a testican-1 protein or an mRNA encoding the testican-1 protein.

In the diagnostic kit of the present invention, in order to measure the level of expression of testin-1, an antibody recognizing a testicle-1 protein as a marker or a primer or a probe recognizing a testicle-1 mRNA as a marker, One or more other component compositions, solutions or devices may be included.

In a specific embodiment, the diagnostic kit may be a diagnostic kit comprising essential elements necessary for performing a reverse transcription-polymerase reaction. The RT-PCR kit contains the respective primer pairs specific for the marker gene. A primer is a nucleotide having a sequence specific to a nucleic acid sequence of each marker gene, and has a length of about 7 bp to 50 bp, and more preferably about 10 bp to 30 bp. It may also contain a primer specific for the nucleic acid sequence of the control gene. Other reverse transcription polymerase reaction kits may be used in combination with test tubes or other appropriate containers, reaction buffers (varying in pH and magnesium concentration), deoxynucleotides (dNTPs), enzymes such as Taq polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC DEPC-water, sterile water, and the like.

Another aspect of the present invention is a diagnostic kit characterized in that it includes an essential element necessary for performing a DNA chip. The DNA chip kit may include a substrate to which a cDNA or oligonucleotide corresponding to a gene or a fragment thereof is attached, and reagents, preparations, enzymes, and the like for producing a fluorescent-labeled probe. The substrate may also comprise a cDNA or oligonucleotide corresponding to a control gene or fragment thereof.

Most preferably, it may be a diagnostic kit characterized by comprising essential elements necessary for performing an ELISA. ELISA kits include antibodies specific for the testican-1 marker protein. Antibodies are monoclonal antibodies, polyclonal antibodies or recombinant antibodies with high specificity and affinity for each marker protein and little cross reactivity to other proteins. The ELISA kit may also include antibodies specific for the control protein. Other ELISA kits can be used to detect antibodies that can bind a reagent, such as a labeled secondary antibody, chromophores, an enzyme (in conjugated form with the antibody) and a substrate or antibody capable of detecting the bound antibody Other materials, and the like. In addition, the kit of the present invention may include a washing solution or an eluting solution capable of removing a substrate to be color-developed with the enzyme and unbound proteins and retaining only the bound protein marker.

Samples used for analysis include biological samples that can identify sepsis-specific proteins that can be distinguished from normal conditions, such as blood, serum, urine, leaks, saliva, and the like. Preferably from a biological fluid sample, such as blood, serum, plasma. The sample may be prepared to increase the detection sensitivity of the protein marker. For example, serum samples obtained from a patient may be subjected to anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, Can be pretreated using methods such as sequential extraction or gel electrophoresis.

The present invention also provides a method for diagnosing septicemia,

(a) providing a sample of the subject; And

(b) measuring the level of expression of the testin-1 protein or the mRNA encoding the testin-1 protein in the sample; And

(c) comparing the expression level of the protein or mRNA with that of a normal person, and determining that the subject having an increased expression level as compared to a normal person has acquired septicemia.

The method of the present invention will be described in the following steps.

Step (a) of the method of the present invention is a step of providing a sample of a test object.

The sample of the subject in step (a) can be used without limitation as long as it is collected from a subject to be diagnosed as to whether sepsis is to be diagnosed. Examples thereof include a cell or tissue obtained by biopsy, blood, whole blood, serum, plasma, Cerebrospinal fluid, various secretions, urine, feces, and the like. Preferably blood, plasma, serum, saliva, saliva, sputum, capsular fluid, amniotic fluid, ascites, cervix or vaginal discharge, urine and cerebrospinal fluid. Most preferably blood, plasma, or serum. The sample of the present invention may be derived from a mammal, preferably from a human. A sample of the sample can be obtained by collecting it according to a technique known in the art.

The sample of the subject can also be suitably pretreated according to the method of measuring the level of testin-1 expression as is known in the art. For example, a sample of a test sample may be fixed in a fixative such as formalin, frozen rapidly with liquid nitrogen or the like, and stored frozen at -20 ° C or -70 ° C. Tissue sections may be prepared from frozen or frozen samples and stored frozen.

(B) of the method of the present invention is a step of measuring the expression level of the testican-1 protein or the mRNA encoding the testican-1 protein in the sample provided in the step (a).

The level of expression of testicle-1 mRNA can be determined by amplifying testicle-1 mRNA or cDNA from a test sample using a primer set or a probe specifically binding to testicle-1 mRNA, or by hybridization with a probe ) Reaction can be used to measure the presence and expression level of testican-1 mRNA in the test sample. These primers and probes are as described in the diagnostic composition of the present invention.

Examples of assay methods include reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR (reverse transcription polymerase chain reaction), and the like. (RT-PCR), real-time RT-PCR, RNase protection assay (RPA), northern blotting, DNA microarray chip, RNA base But are not limited to, RNA sequencing, hybridization using nanostrings, in situ hybridization of tissue sections, and the like.

The step (b) may be a step of measuring the expression level of the testin-1 protein.

The level of expression of the testican-1 protein can be detected or measured using an antibody that specifically binds to the testican-1 protein. The antibody is as described in the diagnostic composition of the present invention.

Methods for measuring the expression level of the testican-1 mRNA protein can be used without limitation in the art, including western blotting, dot blotting, enzyme- linked immunosorbent assay, radioimmunoassay (RIA), radial immunodiffusion, Oucheroton immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation, complement fixation, flow cytometry (FACS) or A protein chip method, and the like, but are not limited thereto.

The step (c) is a step of comparing the expression level of the protein or mRNA measured in the step (b) with that of a normal person, and judging that the subject whose expression level is higher than that of a normal person is suffering from sepsis.

The level of expression of testicular-1 in the subject measured by the method of step (b) described above is compared to the level of testicular-1 of normal subjects measured by the same method. It is judged that the testis-1 expression level is higher than that of the healthy normal subjects in the case of sepsis. In addition, the higher the level of testicular-1 expression, the more severe sepsis can be judged. For the degree of testicular-1 expression level increase as a diagnostic criterion, the correlation between the expression level of testican-1 and the severity of sepsis according to the technique known in the art for the method of measuring the selected testicular- May provide an appropriate diagnostic criterion to indicate the severity of sepsis according to the range of levels of testicular-1 expression. In the present invention, the severity of sepsis was closely correlated with the severity of septicemia as the level of testicular-1 expression was increased by using various indicators such as severe sepsis, septic shock, and survival.

According to the present invention, the level of expression of testicular-1 is increased in patients with sepsis and is closely correlated with the severity and survival rate of sepsis. Thus, by analyzing the expression level of testicular-1, it is possible to rapidly and accurately diagnose sepsis, as well as determine the severity and prognosis of sepsis.

Figure 1 shows the results of in vitro tests for the release of Testican-1 by LPS (data are presented as means ± SD of three independent experiments. * P <0.05 vs. vehicle alone, analyzed by one-way ANOVA, by Tukey's test).
Figures 1A and 1B show the amount of testican-1 contained in the medium after incubation of HUVEC cells with LPS (100 ng / mL) (●) or without indicated LPS (O) for the indicated time (ELISA) Results.
1C shows the result of calculating the total testican-1 by adding FIG. 1A and FIG. 1B. FIG.
Figure 1D shows the expression of testican-1 mRNA using qPCR
FIG. 2 shows the results of in vitro test for the release of HMGB1-induced testican-1 (data are presented as means ± SD of three independent experiments. * P <0.05 vs. vehicle alone, analyzed by one-way ANOVA, by Tukey's test).
Figures 2A and 2B show the amount of testican-1 contained in the medium after incubation of the HUVEC cells with HMGB1 (1 [mu] g / mL) (•) or HMGB1 Results.
FIG. 2C shows the result of calculating the total testican-1 by adding FIG. 1A and FIG. 1B.
2D is the result of analysis of testican-1 mRNA expression using qPCR.
Figure 3 shows the results of in vivo observation of testical-1 release (Data are presented as means ± SD of three independent experiments. * P <0.05 vs. vehicle alone, analyzed by one-way ANOVA, by Tukey's test. . Male C57BL / 6 mice were treated with () or without () LPS (A, 10 mg / kg) or HMGB1 (B, 20 mg / kg) And the expression of testican-1 protein was analyzed by ELISA.
Figure 4 shows the results of analysis of testican-1 expression in sepsis or CLP-induced sepsis animal models.
FIG. 4A shows the results of ELISA analysis of testican-1 expression in serum from 21 healthy volunteers and 82 sepsis patients.
FIG. 4B is a graph comparing the results after distinguishing whether the patient survived 15 days after admission or died.
Figure 4C shows the results of ELISA (n = 10, * p &lt; 0.05 vs. sham operated mice) for the concentration of testican-1 after performing CLP-surgery on mice and isolating sera at the indicated time points.
Figure 4D shows the survival rate of sham- and CLP-mice.

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

<Experimental Method>

1. Reagents

Bacterial LPS (lipopolysaccharide, serotype: 0111: B4, L5293) was purchased from Sigma and HMGB1 (high mobility group box1) protein was purchased from Abnova. Testican-1 antibody was purchased from R & D systems and human recombinant testican-1 protein was purchased from Acrobiosystem. Commercially available ELISA (testican-1, testican-2) was purchased from Raybiotech, and testican-3 ELISA was purchased from Mybiosouce.

2. Cell culture

Primary HUVECs (human umbilical vein endothelial cells) were purchased from Cambrex bio science and cultured according to the previously reported method. Passage 3 or 5 HUVECs were used in the experiments.

3. Animal Breeding

Male C57BL / 6 mice (6-7 weeks old, 27 g) were purchased from Orient Bio and given an acclimatization period of 12 days. The mice were housed in polycarbonate cages under the same conditions of temperature, humidity, and light as in the previously reported method. Mice were treated with one dose of LPS 10 mg / kg, HMGB1 20 mg / kg or saline. LPS or HMGB1 was administered every 4 days to recover from weight loss. All animals were managed according to the recommendations of the Animal Ethics Committee of Kyungpook National University.

4. CLP ( Cecal  ligation and puncture

To induce sepsis, male rats were firstly anesthetized with 2% isoflurane (Korea, JW PHARMACEUTICALS, Korea, Inc.) in oxygen carried through a small rodent gas anesthesia machine (RC2, Vetequip, Pleasanton, CA) Forane) was anesthetized. Spontaneous breathing during the operation was allowed. The CLP-induced inflammation model was prepared as previously mentioned. Briefly, a 2 cm center incision was made to expose the intestines adjacent to the appendix. The cecum was tightly bound with 5.0-mm to 3.0-silk structures from cecal tip and punctured using a 22-gauge needle for induction of high-stage inflammation. Squeezed gently to push out a small amount of excrement from the hole location, and started again at the bifurcation. The laparotomy site was sealed with 4.0-silk. In false-control animals, the appendix was unclipped or punctured but revealed and then returned to the abdominal cavity. This method was approved by the Animal Management Committee at Kyungpook National University (IRP No, KNU 2012-13)

5. Patient sample

A healthy volunteer sample was used as a control. Patients were divided into three groups according to the Sepsis Consensus Conference Committee (1992): (1) sepsis, (2) severe sepsis, and (3) septic shock. Clinical records, diagnosis, treatment patterns and blood samples were collected with patient consent. Serum was obtained by centrifugation at 2000 × g for 5 minutes within 48 hours after the blood of the patient was obtained. The serum was stored at -20 ° C. until use for analysis. The experimental protocol was approved by Kyungpook National University Hospital.

6. RNA isolation and quantitative-polymerase chain reaction ( qPCR )

HUVECs were cultured in 6-well plates and cultured with or without treatment with LPS (100 ng / ml) or HMGB1 (1 ug / kg) for the indicated period of time. RNA was isolated using TRI-Reagent (Invitrogen). The isolated RNA was isolated using a PX2 Thermal cycler (Thermo scientific) and a 20-μL reaction reagent (200 U / μL M-MLV reverse transcriptase and 0.5 mg // μL oligo (dT) -adapter primer) Lt; / RTI &gt; Real-time PCR of testican-1 and α-actin was performed using a Mini Opticon Real Time PCR system (Bio-rad) and iQ SYBR Green Supermic (Bio-rad). The sequence information of the primers used in the experiment is as follows:

(1) Testican-1

sense (SEQ ID NO: 3): 5'-GCAAATTGGAGTTCCATGCT-3 '

anti-sense (SEQ ID NO: 4): 5'-TCCTTTCTG CCTTGTGCTTT-3 '

(2) α-actin

sense: 5'-TGAGAGGGAAATCGTGCGTG-3 '

anti-sense: 5'-TTGCTGATCCACATCTGCTGG-3 '

The PCR set conditions for the experiment were as follows:

After the initial denaturation at 95 ° C, the amplification was performed at 95 ° C, 15s and 60 ° C for 35 cycles at 20s, and then the melting curve was cleaved and the temperature was increased from 72 ° C to 98 ° C. Quantitation of gene expression was calculated relative to a-actin.

7. Testican Competitive ELISA of -1

The testican-1 protein contained in 20 mM carbonate / bicarbonate buffer (pH 9.6) containing 0.02% sodium azide was coated on a 96-well plate and cultured overnight at 4 ° C. Plates were washed 3 times with PBS and stored at 4 &lt; 0 &gt; C. Standard testican-1, conditioned culture medium and mouse serum or human serum were pretreated with anti-testeran-1 antibody (1: 500 dilution) at 37 ° C for 90 minutes. The pretreated sample was transferred to a pre-coated plate and incubated at room temperature for 30 minutes. In addition, the plates were washed with PBS-T and reacted with peroxidase-conjugated anti-mouse IgG antibody (1: 2000 dilution) for 90 minutes at room temperature. Plates were washed three times with TBS-T and incubated with 200 μL of substrate solution (100 μg / mL o-phenylenediamine and 0.003% H2O2) for 60 min at room temperature. After stopping the reaction with 50 μL of 8N H2SO4 solution, the absorbance was measured at 490 nm.

&Lt; Example 1 >

Inflammation in vascular endothelial cells or mice In the parameter  by testican -1

LPS is a mediator of the etiology of sepsis that can stimulate inflammatory responses in endothelial cells and mice. ELISA experiments were performed to determine whether LPS induces synthesis and secretion of Testican-1 in HUVEC cells and C57BL / 6 mice.

As a result, the secretion of Testican-1 was significantly increased after 48 hours of LPS administration (Fig. 1A), and a large amount of Testican-1 was synthesized in HUVEC (Fig. 1A-C). As in the ELISA data, test-1 was significantly higher in HUVEC at 36 h after stimulation with 100 ng / mL of LPS than qRT-PCR (Fig. 1D). However, the expression of Testican-2, -3, another subtype of Testican, did not change significantly compared to the control group (results not shown).

To confirm the increased expression of Testican-1 in sepsis, another sepsis mediator, HMGB1, was applied. As a result, as shown in Fig. 2, expression and secretion of Testican-1 in HUVECs were increased by HMGB1 treatment at 1 占 퐂 / mL.

Next, the level of serum Testican-1 was measured after LPS or HMGB1 administration to determine whether testican-1 was systemically released in endotoxemia of mice. Serum Testican-1 was detectable after 36 h of administration of LPS (10 mg / kg) at an intermediate dose (LD50), detectable after 24 h of administration of HMGB1 (20 mg / kg) (Fig. 3A) or HMGB1 (Fig. 3B) was administered intraperitoneally and blood levels remained at 72 hours to 96 hours. These results indicate that inflammatory mediators of sepsis lead to continued synthesis and secretion of Testican-1 in primary cultured human endothelial cells and mice.

&Lt; Example 2 >

Within serum of sepsis patients Testican -1

Next, we examined the concentration of Testican-1 in patients with sepsis. As shown in Figure 4A, the mean value of serum Testican-1 was 7.53 ng / mL (6.97-8.77 ng / mL) in 21 healthy volunteers. Testican-1 was significantly lower in sepsis patients (mean 34.61 ng / mL, range 20.44-63.37 ng / mL, n = 30), severe sepsis patients (mean 69.30 ng / mL, range 41.30-98.69 ng / mL, (115.82 ng / mL, range: 98.10-151.85 ng / mL, n = 30) in patients with hemorrhagic shock. Statistically significant differences were observed between the groups (p <0.00001), confirming that Testican-1 could be used as a marker for the diagnosis and severity of sepsis. Because the data of the present invention showed statistical significance between sepsis and severe sepsis or septic shock and severe sepsis and septic shock patients (Figure 4A). Eight of the 82 patients who participated in the study died within 15 days of admission (2 sepsis, 1 severe sepsis, 5 septic shock). Testican-1 levels were significantly higher in patients who died compared to survivors. : 116.85 ng / mL (60.22-149.31) vs 3.10 ng / mL (25.30-115.45, p <0.000001, FIG. 4B).

ROC curve analysis for Testican-1 to distinguish between sepsis patients (n = 30) and normal controls (n = 20) showed a curvilinear AUC of 1.000 (95% confidence interval [CI] 1.000-1.000) (95% CI 1.000-1.000) for procalcitonin and 1.000 (95% CI 1.000-1.000) for IL-6, respectively. The AUC of testican-1 to distinguish between severe sepsis (n = 22) and sepsis was 0.938 (95% CI 0.905-0.972), 0.692 (95% CI 0.541-0.842) for procalcitonin and 0.712 (95% CI 0.605 - 0.818). In addition, the AUC of testican-1 was 0.986 (95% CI 0.972-1.000) in order to distinguish septic shock (n = 30) from all other severe sepsis patients (n = 52), while for procalcitonin 0.714 % CI 0.596- 0.832) and 0.723 (95% CI 0.602- 0.849) for IL-6.

&Lt; Example 3 >

In an animal model of sepsis testican -1

Because the CLP model is more similar to human sepsis than the endotoxemia model, we used the mouse CLP model of sepsis to measure the serum Testican-1 concentration. After 24 hours of CLP surgery, signs of sepsis, such as tremors, hair straighteness, and sickness, became clear. Serum Testican-1 levels were higher in CLP-induced sepsis mice (Figure 4C). The survival rates of Sham- and CLP-mice are shown in Figure 4d.

As described above, testican-1 can be used not only as a marker for diagnosing sepsis but also as a marker for determining severity of disease.

According to the present invention, the level of expression of testicular-1 is increased in patients with sepsis and is closely correlated with the severity and survival rate of sepsis. Thus, by analyzing the level of expression of testicular-1, it is possible to diagnose sepsis rapidly and accurately, as well as to determine the severity and prognosis of sepsis.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Composition and method for detecting a diagnostic marker for          sepsis using tryptophanyl-tRNA synthetase <130> NP17-0011 <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 439 <212> PRT <213> Artificial Sequence <220> <223> Human testican-1 protein <400> 1 Met Pro Ala Ala Ala Ala   1 5 10 15 Leu Gln Val Glu Ser Arg His Leu Asp Ala Leu Ala Gly Gly Ala Gly              20 25 30 Pro Asn His Gly Asn Phe Leu Asp Asn Asp Gln Trp Leu Ser Thr Val          35 40 45 Ser Gln Tyr Asp Arg Asp Lys Tyr Trp Asn Arg Phe Arg Asp Asp Asp      50 55 60 Tyr Phe Arg Asn Trp Asn Pro Asn Lys Pro Phe Asp Gln Ala Leu Asp  65 70 75 80 Pro Ser Lys Asp Pro Cys Leu Lys Val Lys Cys Ser Pro His Lys Val                  85 90 95 Cys Val Thr Gln Asp Tyr Gln Thr Ala Leu Cys Val Ser Arg Lys His             100 105 110 Leu Leu Pro Arg Gln Lys Lys Gly Asn Val Ala Gln Lys His Trp Val         115 120 125 Gly Pro Ser Asn Leu Val Lys Cys Lys Pro Cys Pro Val Ala Gln Ser     130 135 140 Ala Met Val Cys Gly Ser Asp Gly His Ser Tyr Thr Ser Lys Cys Lys 145 150 155 160 Leu Glu Phe His Ala Cys Ser Thr Gly Lys Ser Leu Ala Thr Leu Cys                 165 170 175 Asp Gly Pro Cys Pro Cys Leu Pro Glu Pro Glu Pro Pro Lys His Lys             180 185 190 Ala Glu Arg Ser Ala Cys Thr Asp Lys Glu Leu Arg Asn Leu Ala Ser         195 200 205 Arg Leu Lys Asp Trp Phe Gly Ala Leu His Glu Asp Ala Asn Arg Val     210 215 220 Ile Lys Pro Thr Ser Ser Asn Thr Ala Gln Gly Arg Phe Asp Thr Ser 225 230 235 240 Ile Leu Pro Ile Cys Lys Asp Ser Leu Gly Trp Met Phe Asn Lys Leu                 245 250 255 Asp Met Asn Tyr Asp Leu Leu Leu Asp Pro Ser Glu Ile Asn Ala Ile             260 265 270 Tyr Leu Asp Lys Tyr Glu Pro Cys Ile Lys Pro Leu Phe Asn Ser Cys         275 280 285 Asp Ser Phe Lys Asp Gly Lys Leu Ser Asn Asn Glu Trp Cys Tyr Cys     290 295 300 Phe Gln Lys Pro Gly Gly Leu Pro Cys Gln Asn Glu Met Asn Arg Ile 305 310 315 320 Gln Lys Leu Ser Lys Gly Lys Ser Leu Leu Gly Ala Phe Ile Pro Arg                 325 330 335 Cys Asn Glu Glu Gly Tyr Tyr Lys Ala Thr Gln Cys His Gly Ser Thr             340 345 350 Gly Gln Cys Trp Cys Val Asp Lys Tyr Gly Asn Glu Leu Ala Gly Ser         355 360 365 Arg Lys Gln Gly Ala Val Ser Cys Glu Glu Glu Gln Glu Thr Ser Gly     370 375 380 Asp Phe Gly Ser Gly Gly Ser Val Val Leu Leu Asp Asp Leu Glu Tyr 385 390 395 400 Glu Arg Glu Leu Gly Pro Lys Asp Lys Glu Gly Lys Leu Arg Val His                 405 410 415 Thr Arg Ala Val Thr Glu Asp Asp Glu Asp Glu Asp Asp Asp Lys Glu             420 425 430 Asp Glu Val Gly Tyr Ile Trp         435 <210> 2 <211> 1320 <212> RNA <213> Artificial Sequence <220> <223> Human testican-1 mRNA <400> 2 atgccggcga tcgcggtgtt ggcggcggcc gccgcggcgt ggtgcttcct ccaagtcgag 60 agccggcacc tggacgcgct cgccggaggc gcgggcccca accacggcaa tttcctagac 120 aatgaccagt ggctgagcac cgtctcccag tacgaccggg acaagtactg gaaccgcttt 180 cgagacgatg attatttcag aaactggaat cccaacaagc cctttgacca agccctggac 240 ccatccaagg acccctgcct gaaggtaaaa tgcagccctc acaaagtgtg tgtgacccag 300 gactaccaga ccgccctgtg tgtcagccgc aagcacctgc tccccaggca aaagaagggg 360 aacgtggccc agaaacactg ggttggacct tcgaatttgg tcaagtgcaa gccctgtccc 420 gtggcacagt cagccatggt ctgcggctca gatggccact cctacacatc caagtgcaaa 480 ttggagttcc atgcttgttc tactggcaaa agcctcgcca ccctctgtga tgggccctgt 540 ccctgtctcc cagagcctga gccaccaaag cacaaggcag aaaggagtgc ctgcacagac 600 aaggagttgc ggaaccttgc ctcccggctg aaggattggt ttggagctct ccacgaggat 660 gcgaacagag tcatcaagcc caccagctcc aacacagccc aaggcaggtt tgacactagc 720 atcctgccca tctgcaagga ctccctgggc tggatgttca acaagttgga catgaactat 780 gacctcctgc ttgacccttc agagatcaat gccatctacc tggataagta cgagccctgt 840 atcaagcctc ttttcaactc gtgtgactcc ttcaaggatg gcaagctttc taacaatgag 900 tggtgctact gcttccagaa gcctggaggt ctcccttgcc agaatgaaat gaacagaatt 960 cagaagctga gtaaggggaa aagcctgttg ggggccttca tacctcggtg taatgaggag 1020 ggctattaca aagccacaca gtgccacggc agcacggggc agtgctggtg tgtggacaaa 1080 tatgggaatg agttggctgg ctccaggaaa cagggtgctg tgagctgtga agaggagcag 1140 gaaacctcag gggattttgg cagtggtggg tccgtggtcc tgctggatga cctagaatat 1200 gaacgggagc tgggaccaaa ggacaaagag gggaagctga gggtgcacac ccgagccgtg 1260 acagaggatg atgaggatga ggatgatgac aaagaggatg aggtcgggta catatggtag 1320                                                                         1320 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Human testican-1 primer forward <400> 3 gcaaattgga gttccatgct 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Human testican-1 primer reverse <400> 4 tcctttctgc cttgtgcttt 20

Claims (9)

1. A composition for diagnosing sepsis comprising an agent for measuring the expression level of a testican-1 protein or an mRNA encoding the testican-1 protein comprising the amino acid sequence represented by SEQ ID NO: 1.
2. The composition of claim 1, wherein the agent is an antibody that specifically binds to a testicle-1 protein.
delete 2. The composition of claim 1, wherein the agent is a probe or primer set that specifically binds to mRNA encoding testin-1.
5. The composition of claim 4, wherein the mRNA comprises the nucleotide sequence of SEQ ID NO: 2.
5. The composition of claim 4, wherein the primer set is represented by SEQ ID NO: 3 and SEQ ID NO:
1. A sepsis diagnosis kit comprising an agent for measuring an expression level of a testican-1 protein or an mRNA encoding the testican-1 protein comprising the amino acid sequence represented by SEQ ID NO: 1.
To provide information necessary for the diagnosis of sepsis
(a) providing a sample of the subject; And
(b) measuring the expression level of a testin-1 protein comprising the amino acid sequence shown in SEQ ID NO: 1 or encoding mRNA thereof in said sample; And
(c) comparing the expression level of the protein or mRNA with that of a normal person, and determining that the subject whose expression level is higher than that of a normal person is septic.
The method according to claim 8, wherein the sample is selected from the group consisting of blood, plasma, serum, saliva, nasal fluid, sputum, capsular fluid, amniotic fluid, ascites, cervix or vaginal discharge, urine and cerebrospinal fluid.

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