KR102317330B1 - Compositions for canine pyometra prognosis judgment by measuring the expression procalcitonin - Google Patents

Abstract

The present invention relates to a composition for determining the prognosis of uterine sinusitis in dogs by measuring procalcitonin. In the present invention, when a dog with uterine sinusitis was treated, procalcitonin (PCT) was significantly reduced, and procalcitonin (PCT) showed a statistically significant correlation with the tissue damage index, procalcitonin (procalcitonin) ; PCT) measurement can be usefully used to determine the prognosis of canine uterine sinusitis.

Description

Compositions for judging the prognosis of canine uterine sinusitis by measuring procalcitonin {Compositions for canine pyometra prognosis judgment by measuring the expression procalcitonin}

The present invention relates to a composition for determining the prognosis of uterine sinusitis in dogs by measuring procalcitonin.

In veterinary medicine, sepsis is as high as 20-70%, but there is still no fast and accurate diagnostic method for it. Pyometra in dogs is a representative disease in dogs that causes sepsis. It is asymptomatic before showing clinical symptoms, but when it progresses rapidly to sepsis, it causes serious organ damage.

Dogs with uterine sinusitis often show general symptoms such as vomiting, lethargy, and diarrhea in the early stages. However, it is necessary to check the symptoms of abdominal distension, purulent discharge from the vulva, age, and whether or not neutralization has been performed, and to confirm a specific diagnosis through ultrasound and radiographic examination.

Accordingly, there is a need for a method capable of accurately diagnosing uterine sinusitis in dogs and predicting its prognosis in a simpler way, but the research is still insignificant.

Korean Patent No. 1732787

Accordingly, the present inventors are making diligent efforts to find a method for accurately diagnosing uterine sinusitis in dogs and predicting its prognosis in a more convenient way, and using procalcitonin (PCT) as a monitoring indicator for prognosis and treatment It was confirmed that the following suitability was completed, and the present invention was completed.

Accordingly, an object of the present invention is to provide a marker composition for predicting the prognosis of uterine sinusitis in dogs, comprising a procalcitonin (PCT) gene or a protein encoded by the gene.

In addition, the present invention is to provide a composition for predicting the prognosis of uterine sinusitis in dogs, comprising an agent for measuring the mRNA level of the procalcitonin (PCT) gene or the protein encoded by the gene.

In addition, the present invention provides a kit for predicting the prognosis of uterine sinusitis in dogs, comprising the composition.

In order to achieve the above object, the present invention provides a marker composition for determining the prognosis of uterine sinusitis in dogs, comprising a procalcitonin (PCT) gene or a protein encoded by the gene, wherein the procalcitonin gene is represented by SEQ ID NO: 1 It is characterized in that it consists of a nucleotide sequence, and the protein encoded by the procalcitonin gene consists of the amino acid sequence represented by SEQ ID NO: 2, and the prognosis of canine uterine sinusitis is determined after ovariectomy (OHE) after ovariectomy ( OHE) provides a marker composition for determining the prognosis of uterine sinusitis in dogs, characterized in that it confirms a decrease in procalcitonin to a value of 60 to 70 percent compared to before.

In addition, the present invention provides a composition for predicting the prognosis of uterine sinusitis in dogs, comprising an agent for measuring the mRNA level of the procalcitonin (PCT) gene or the protein encoded by the gene.

In one embodiment of the present invention, the agent for measuring the mRNA level may be sense and antisense primers or probes complementary to the mRNA of a gene.

In one embodiment of the present invention, the agent for measuring the protein level may be an antibody that specifically binds to a protein encoded by a gene.

In addition, the present invention provides a kit for predicting the prognosis of uterine sinusitis in dogs, comprising the composition.

In one embodiment of the present invention, the prognosis may be survival or disease-free survival without recurrence of uterine sinusitis in dogs after ovarian hysterectomy (OHE).

In the present invention, when a dog with uterine sinusitis was treated, procalcitonin (PCT) was significantly reduced, and procalcitonin (PCT) showed a statistically significant correlation with the tissue damage index, procalcitonin (procalcitonin) ; PCT) measurement can be usefully used to determine the prognosis of canine uterine sinusitis.

1 shows the mean parameters for various clinical data of females with and healthy females with uterine sinusitis.
2 is a result confirming that plasma CRP, SAA and cfDNA increased in females with uterine sinusitis than in healthy females.
3 is a result showing a ROC analysis (Receiver operating characteristic analysis) curve comparing the diagnostic sensitivity and 1-specificity of each biomarker for detection of a dog with uterine sinusitis.
4 is a result showing representative two-dimensional gel electrophoresis (2-DE) maps of uterine tissues of healthy females (A) and females with uterine sinusitis (B, C, D).
Figure 5 is the result of confirming the change of the average variable for the clinical data of females with uterine sinusitis divided into before oophorectomy (OHE) and after oophorectomy (OHE).
6 is a result of confirming the average concentration of inflammatory parameters in healthy females and females with uterine sinusitis.

The present inventors completed the present invention by discovering procalcitonin (PCT) gene markers that can be used for prognosis prediction or treatment of uterine sinusitis as a result of intensive research to discover genetic markers for diagnosis and prognosis of uterine sinusitis. did.

Accordingly, the present invention provides a marker composition for predicting the prognosis of uterine sinusitis in dogs, comprising a procalcitonin (PCT) gene or a protein encoded by the gene.

The present invention also provides a marker composition for predicting the prognosis of uterine sinusitis in dogs, comprising a procalcitonin (PCT) gene or a protein encoded by the gene, and a kit for predicting the prognosis of uterine sinusitis in dogs comprising the composition.

The procalcitonin (PCT) gene of the present invention may consist of the nucleotide sequence of SEQ ID NO: 1, and the protein encoding the procalcitonin (PCT) gene may consist of the amino acid sequence of SEQ ID NO: 2 .

"Uterine sinusitis", a disease targeted by the present invention, is a very representative disease of elderly dogs and commonly occurs in elderly female dogs that have not been neutered. The diagnosis of uterine sinusitis is made through ultrasound and radiographic examination. Since uterine sinusitis has a very high fatality rate if not treated properly, early diagnosis is very important.

As used herein, the term “diagnosis” in a broad sense means judging the actual condition of a patient's disease in all aspects. The content of the judgment is the disease name, etiology, disease type, severity, detailed mode of the disease, and the presence or absence of complications.

Diagnosis in the present invention is to determine whether or not the onset of uterine sinusitis and the level of progression.

As used herein, the term “prognosis” refers to prediction of disease progression and recovery, and refers to a prospect or a preliminary evaluation. In the present invention, the prognosis means, but is not limited to, recurrence of uterine sinusitis, overall survival, or disease-free survival.

The agent for measuring the mRNA level of the procalcitonin (PCT) gene may be sense and antisense primers or probes complementary to mRNA, but is not limited thereto.

As used herein, the term “primer” refers to an oligonucleotide synthesized for use in diagnosis, DNA sequencing, etc. as a short gene sequence serving as a starting point of DNA synthesis. The primers may be synthesized and used with a length of typically 15 to 30 base pairs, but may vary depending on the purpose of use, and may be modified by methylation, capping, etc. by a known method.

As used herein, the term “probe” refers to a nucleic acid capable of specifically binding to mRNA having a length of several bases to several hundreds of bases produced through enzymatic chemical separation and purification or synthesis. The presence or absence of mRNA can be checked by labeling radioactive isotopes or enzymes, and it can be designed and modified by a known method.

The agent for measuring the protein level may be an antibody that specifically binds to a protein encoded by a gene, but is not limited thereto.

As used herein, the term “antibody” includes immunoglobulin molecules having immunological reactivity with a specific antigen, and includes both monoclonal and polyclonal antibodies. The antibody also includes forms produced by genetic engineering such as chimeric antibodies (eg, humanized murine antibodies) and heterologous antibodies (eg, bispecific antibodies).

The prognosis prediction kit of the present invention consists of one or more other component compositions, solutions or devices suitable for the analysis method.

For example, the kit of the present invention contains genomic DNA derived from a sample to be analyzed, a primer set specific for the marker gene of the present invention, an appropriate amount of a DNA polymerase, a dNTP mixture, a PCR buffer, and water to perform PCR. It may be a kit comprising The PCR buffer may contain KCl, Tris-HCl and MgCl2. In addition, components necessary for performing electrophoresis that can confirm whether the PCR product is amplified may be additionally included in the kit of the present invention.

In addition, the kit of the present invention may be a kit including essential elements necessary for performing RT-PCR. In addition to each primer pair specific for a marker gene, the RT-PCR kit includes a test tube or other suitable container, reaction buffer, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC -Water (DEPC-water), sterile water, etc. may be included. In addition, a pair of primers specific to a gene used as a quantitative control may be included.

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

In addition, the kit of the present invention may be a kit characterized in that it includes essential elements necessary for performing ELISA. The ELISA kit includes an antibody specific for a marker protein, and an agent for measuring the protein level. The ELISA kit may include a reagent capable of detecting an antibody that has formed an “antigen-antibody complex”, for example, a labeled secondary antibody, chromopores, an enzyme, and a substrate thereof. In addition, an antibody specific for the quantitative control protein may be included.

As used herein, the term “antigen-antibody complex” refers to a combination of a protein encoded by a gene and an antibody specific thereto. The amount of formation of the antigen-antibody complex can be quantitatively measured through the magnitude of the signal of the detection label. The detection label may be selected from the group consisting of an enzyme, a fluorescent substance, a ligand, a luminescent substance, a microparticle, a redox molecule, and a radioisotope, but is not limited thereto.

In addition, the present invention provides an information providing method for predicting the prognosis of uterine sinusitis, comprising measuring the expression level of the mRNA of the procalcitonin (PCT) gene or the protein encoded by the gene in a biological sample derived from a subject. to provide.

The biological sample derived from the subject may include, but is not limited to, tissues, cells, whole blood, blood, saliva, sputum, cerebrospinal fluid, and urine.

The expression level of the mRNA is polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time PCR), RNase protection assay (RNase) by a conventional method known in the art. Protection assay; RPA), microarray (microarray), or can be measured through one or more methods selected from the group consisting of northern blotting (northern blotting), but is not limited thereto.

The protein expression level is determined by a conventional method known in the art, such as western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme immunoassay (ELISA), immunoprecipitation. , flow cytometry, immunofluorescence, ouchterlony, complement fixation assay, or at least one method selected from the group consisting of a protein chip It can be measured through, but is not limited thereto.

The term "information providing method for predicting the prognosis of uterine sinusitis in dogs" as used in the present invention is a preliminary step for predicting the prognosis and provides objective basic information necessary for predicting the prognosis of uterine sinusitis, excluding the clinical judgment or opinion of the doctor. do.

Furthermore, the present invention provides a method for diagnosing uterine sinusitis in dogs, comprising measuring the expression level of procalcitonin (PCT) in a biological sample derived from a subject.

In the present invention, the expression level of procalcitonin (PCT) is determined by western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme immunoassay (ELISA), immunoprecipitation. ), flow cytometry, immunofluorescence, ouchterlony, complement fixation assay, and at least one method selected from the group consisting of a protein chip can be measured through

Hereinafter, the present invention will be described in more detail by way of Examples. These examples are merely for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

<Example 1>

Experimental materials and methods

1. Experimental design

As a result of analyzing several biomarkers of inflammation through a prospective randomized in vitro analysis, blood samples collected before and after surgical treatment (ovariohysterectomy (OHE)) were continuously monitored and designed. Patient selection, clinical data collection and blood sampling were performed prospectively.

This experiment was performed with the approval of the Institutional Animal Committee (IACUC) GNU-190218-D0010 of Gyeongsang National University (GNU). Criteria for inclusion in the current study included the diagnosis of pyometra and aliquots of heparinized plasma collected two consecutive times before and after surgery. In this experiment, dogs with pyometra from regional veterinary hospitals in Seoul, Gwangju, and Jinju were reviewed during 2016-2019.

A preliminary diagnosis of pyometra was made based on signs, case history, and clinical symptoms such as anorexia, multiple sclerosis, polyuria, purulent vaginal discharge, and diagnostic images. Confirmation of the diagnosis of pyometra was based on a general histopathological examination of the uterine system as well as on previously proposed definitions.

Medical records, including medical records, signs, physical examination, clinical signs, routine blood analysis and diagnostic images of pyometra were reviewed.

Healthy bitches with ovaries removed served as control dogs for comparison with dogs with pyometra. Preoperative dogs were classified as healthy based on historical undetectable results, physical examination, complete blood count, and serum biochemical analysis.

<1-1> Collection of blood samples to compare pyometra and healthy dogs

Samples were collected via venipuncture and stored in EDTA tubes or lithium heparin tubes. EDTA blood samples were analyzed using an automated hematology analyzer. Heparinized plasma was separated from the heparin tube by centrifugation at 3,000 × g for 10 minutes. Plasma was stored in plastic cryotubes and frozen at -20 °C until shipped to Kyungpook National University Veterinary Teaching Hospital for periodic analysis of inflammatory biomarkers. Samples from the local veterinary hospital were stored on dry ice and transported to Kyungpook National University. Upon arrival, all samples were frozen and the plasma stored at -80°C in a deep freezer until needed for analysis of inflammatory biomarkers. Freezing and thawing were not repeated, and heparinized plasma samples were thawed at room temperature (RT) and centrifuged immediately prior to analysis.

<1-2> Uterine tissue collection

Uterine tissue samples from healthy dogs with pyometra were prepared for proteomic analysis to identify causative biomarkers of inflammation in the uterus. The excised uterine tissue was excised, placed in 2 ml eppendorf tubes, and stored in a -80 °C deep freezer until analysis. Four randomly selected samples, three with pyometra and one healthy, underwent 2-DE combined with Matrix-Assisted Laser Desorption / Ionization Time of Flight Tandem (MALDI-TOF / TOF) MS. Tissue protein profiling in the samples was based on isoelectric point and molecular weight differences. Researchers analyzing the uterine samples were unaware of the patient's findings and performed a two-dimensional gel analysis to determine suspected inflammatory protein spots. The corresponding protein spots were subjected to MALDI-TOF / TOF MS to obtain peptide fingerprints, which were then verified through the National Center for Biotechnology Information (NCBI) database.

<1-3> Comparison of blood analysis between pyometra and healthy dogs after surgical resection

The initial blood samples were collected from the dogs before the procedure (treatment group before the OHB procedure) and from the treatment group 1-3 days after the surgical treatment (clinically improved, the treatment group after the OHB procedure). Clinical pyometra in intact bitches provided the opportunity to detect dynamic changes occurring in inflammatory biomarkers from the appearance of inflammatory stimuli to the elimination of inflammation 1-3 days after OHE. Comparison of organ dysfunction between pyometra and healthy controls is also possible.

2. Comparison of clinical parameters of pyometra

Data collected from medical records includes evaluation of all clinical data including breed, age, weight, case history, physical examination results, at least complete blood tests and serum biochemical analysis. Exclusion criteria were pregnancy or delivery.

Contains glucose, total protein, albumin, globulin, total bilirubin, alanine aminotransferase (ALT), alkaline phosphatase (ALKP), gamma-glutamyltransferase (GGT), blood urea nitrogen (BUN), creatinine and phosphorus Acid-base balance, electrolyte concentration, and serum biochemical analyzes were performed using a blood gas analyzer (Nova pHOX analyzer, Nova Biomedical, Waltham, MA, USA) and Catalyst Dx ^{ⓡ to evaluate the overall clinical status of dogs.} did. Plasma cholesterol, amylase and lipase were also analyzed in this panel immediately after plasma separation.

3. Comparison of inflammatory parameters in pyometra

Frozen plasma samples, once thawed at room temperature, were centrifuged, carefully mixed and pipetted for analysis to minimize the risk of disturbing the buffy coat. All biomarker measurements were performed in duplicate with appropriate controls.

cfDNA.

Measurement of cfDNA previously validated for use in dogs was performed using a benchtop fluorometer (Qubit ^ⓡ dsDNA HS Assay kit and Qubit ^ⓡ 1.0 fluorometer, Life Sciences, Carlsbad, CA, USA) through a fluorescence detection method using relevant reagents. was used and processed according to the manufacturer's instructions.

Briefly, the reaction volume used per assay was 200 μl including 10 μl of plasma sample. The fluorometer was calibrated using standard measurements prior to analysis of each batch. The concentration of cfDNA was ^{determined using the dilution algorithm Qubit ⓡ} 1.0 (Life Sciences, Carlsbad, CA, USA). If necessary, samples were diluted to bring the analysis results into the linear analysis range, and all results were corrected by dilution by 2 times.

IL-6.

Plasma IL-6 was measured by ELISA using a commercially available matched canine antibody pair (DuoSet ELISA Development Kit, R&D Systems, Minneapolis, MN, USA).

The assay was performed in duplicate using DuoSet ELISA using DuoSet Ancillary Reagent Kit2 (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

Briefly, wells of 96-well plates were coated with goat anti-canine IL-6 capture antibodies and diluted overnight in PBS at room temperature. The plate was blocked with a reagent diluent [1% bovine serum albumin (BSA) in PBS (phosphate buffered saline), 300 μl/well, R&D Systems] for 2 hours and washed with a wash buffer (0.05% Tween ^ⓡ 20).

Standard and plasma samples were added to each well (100 μl/well) for 2 h at room temperature. After washing, a detection antibody (biotinylated goat anti-canine IL-6 antibody) was added to each sample, and streptavidin-horseradish peroxidase (HRP); (1:200 in reagent diluent, 100 μl/well, R&D Systems) was added. detection was achieved; and tetramethyl benzidine (TMB); (R&D Systems, Minneapolis, MN, USA) Liquid substrate solution was added and then each sample was incubated for 20 min.

The reaction was then stopped using a stop solution (2N H ₂ SO ₄ , 50 μl/well, R&D Systems). The limit of detection of IL-6 was 62.5 pg/ml. Plasma samples from all dogs were used as the basis for the spiking and recovery experiments, and the spiking level was determined based on the detection limit of the assay. A recovery of 75% to 125% was considered acceptable in the present invention.

CRP and SAA.

CRP and SAA were analyzed using a commercially available ELISA kit (Phase SAA, CRP kit, Tridelta Development Limited, County Kildare, Ireland).

The manufacturer's instructions, such as diluting the sample 500-fold prior to the initial measurement, were followed for each assay step. However, the resulting absorbance values for almost all dogs were above the readable range of the assay (data not shown). All subsequent assays were performed using a 200,000-fold dilution for SAA and a 2,000-fold dilution for CRP. Analysis of canine CRP was performed using an anti-canine-CRP antibody added to each well. After washing again to remove unbound material, TMB substrate solution was added. SAA was assayed via sandwich-type ELISA using monoclonal antibodies specific for SAA coated in wells. After adding 50 μl of sample to each well, TMB substrate solution and stop solution were added.

The lower limit of assay detection was 7.5 ng/mL for CRP and 10 ng/mL for SAA.

HMGB1.

Canine HMGB1 was quantified using a commercially available canine ELISA kit (MyBiosource, San Diego, CA, USA) following the manufacturer's protocol such as undiluted samples.

Briefly, standard and plasma samples were added to each strip plate well (50 μl/well). Then, 100 μl of HRP-conjugate reagent was added to each well and reacted for 1 hour at room temperature. After washing, detection was achieved by adding liquid substrate solution and incubating for 15 min under dark conditions. The reaction was stopped by addition of acidic solution, and the lower limit of assay detection was 6.25 ng/mL.

PCT.

PCT was measured using a commercially available canine ELISA kit (Biovendor LLC, Asheville, NC, USA) according to the manufacturer's instructions.

Briefly, standard and plasma samples were pipetted into each well of a 96-well plate (100 μl/well) and incubated for 1 hour at room temperature.

After washing according to the manual, a 4th wash in each wash step was added using 350 μl of wash buffer per well, then 100 μl of biotin-labeled polyclonal anti-canine procalcitonin was added to each well and 1 h reacted while Next, streptavidin-HRP was added and the substrate was used to quantify peroxidase activity. The lower limit of detection by PCT assay was 12.5 pg/mL.

All ELISAs described above ^{were performed using a microplate spectrophotometer Versamax TM} (Molecular Devices, Boston, MA, USA) with ^{a reference wavelength of 450 nm for measuring optical density Softmax ⓡ} Pro analysis software (Molecular Devices, Boston, MA, USA). was developed. The wavelength was corrected by subtracting the reading from 540 nm. The readings were at 450 nm for IL-6, 450 nm for CRP, SAA and PCT, and 450 nm to 630 nm for HMGB1.

4. Proteomics with Uterine Tissue

<4-1> Sample preparation for 2-DE

Briefly about the experimental method, the uterine tissues were washed twice with PBS to remove residual components, and lysis buffer containing 7M urea, 2M thiourea and 4% (w/v) CHAPS was suspended on ice for 1 hour. . The samples were sonicated to confirm destruction and homogenization of all tissue samples. After centrifugation at 14,000 rpm and a temperature of 4° C. for 15 minutes, the supernatant was separated and placed in an Eppendorf tube. In order to precipitate the protein, the protein pellet was resuspended in 20% trichloroacetic acid (TCA) at a temperature of 4° C. for 30 minutes and crushed using a homogenizer. The resulting pellet was washed twice with ice-cooled 100% acetone, dried with a freeze dryer (SFDSM06, Samwon Freezing Engineering Co., Busan, Korea), and dissolved in 200 μl of sample buffer. Protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Scientific™ Waltham, MA, USA) according to the manufacturer's protocol.

<4-2> 2-DE and image analysis

Isoelectric focusing was performed using GE Healthcare Immobiline™ DryStrip Gels (18 cm, pH 4-7, Amersham Biosciences, Uppsala, Sweden), the strips were 7M urea, 2% CHAPRS, 20mM DTT, 0.5 IPG buffer, bromophenol blue. was added and incubated at room temperature. Equal amounts (500 μg) of each protein sample were rehydrated on IPG strips. Equipotential focusing was performed under the following conditions of Ettan IPGphor 3 (GE Healthcare, Uppasala, Sweden): 50V (1 hour), 200V (1 hour), 500V (30 minutes), slope 4,000V (30 minutes), 4,000V (1 hour) , slopes of 10,000V (1 hour), 10,000V (13 hours) and 50V (3 hours).

The strips were first reacted with 10 mg/ml DTT for 15 min in equilibration buffer [6 M urea, 30% glycerol, 2% sodium dodecyl sulfate (SDS), 50 mM Tris-HCl], followed by 40 mg/ml iodoacetamide (IAA) ) and were continuously reacted for 15 min.

Following the first dimension, a second dimension using 12% SDS-PAGE was performed on the Ettan DALT II system at 20 °C with a constant current of 25 mA/gel. 2-DE was performed using a Protein-II XI electrophoresis instrument (Bio-Rad) at room temperature until the dye reached the bottom of the gel.

After electrophoresis, it was stained with silver nitrate along with fertilization. A total of 4 independent gels were used in triplicate. Briefly, the gel was incubated in a fixative solution (5% acetic acid in 50% ethanol) for 3 h, washed once with 30 min ethanol for 15 min and rinsed 3 times with distilled water for 5 min each. After washing, the gel was sensitized with 0.02% sodium thiosulfate for 10 minutes, and rinsed 3 times with distilled water for 1 minute each time. The gel was stained with silver staining solution (0.3% silver nitrate) for 20 min at room temperature, washed 3 times with distilled water for 1 min each time, and protein spots with a solution containing 3% sodium carbonate, 0.02% sodium thiosulfate and 0.05% formalin This was developed until it could be sufficiently visualized. The reaction was then stopped using a stop solution (6% acetic acid).

The silver-stained gels were scanned with a BioRad GS-800 densitometer and ^{image analysis was performed through Progenesis SameSpots TM} 2D software (Nonlinear Dynamics, Newcastle, UK).

Protein spots greater than 1.5 fold and expression changes with P < 0.05 as processed by analogs of Variations of Analysis were considered to be significantly changed, and the selected spots were visually confirmed and manually in the stained gel. was isolated.

<4-3> Protein identification by MALDI-TOF / TOF MS analysis

To prepare the gel spots for MALDI-TOF/TOF MS, a decolorizing solution (30 mM potassium hexacyanoferrate, 100 mM sodium thiosulfate) was added to all spots to remove silver staining.

Protein digestion was performed with slight modifications from the existing method. Briefly, the excised protein spots were washed three times with 10% acetic acid in 50% methanol. After washing, the gel spots were treated with 100 μl of acetonitrile and 100 μl of 100 mM ammonium bicarbonate for 5 minutes each. Then, the excised gel spot was dried using a vacuum freeze dryer (SFDSM06, Samwon Freezing Engineering Co., Busan, Korea), incubated in a reducing solution (10 mM DTT in 0.1 M ammonium bicarbonate) at room temperature for 45 min, and alkylated solution (55 mM iodoacetamide in 0.1 M ammonium bicarbonate) for 30 min in the dark. The spots were then dehydrated using 0.1M ammonium bicarbonate for 5 minutes and 0.1M ammonium bicarbonate for 15 minutes.

Finally, gel spots were proteolyzed using digestion buffer containing 20 ng/μl trypsin (Promega Corporation, Madison, WI, USA) and 0.1% octyl beta-D glucopyranoside in ice-cooled 25 mM ammonium bicarbonate. After 45 min, the solution _{was replaced with 30 μl of 50 mM NH 4} HCO ₃ without trypsin, and proteolysis was performed at 37° C. overnight.

The trypsin peptide was extracted using 0.1% trifluoroacetic acid in 66% acetonitrile and dried for 2 h using a vacuum freeze dryer.

MALDI-TOF/TOF MS analysis was performed as described above. Briefly, the digested peptide solution (in 50% ACN, 0.1% TFA) was spotted with a pipette onto a MALDI-TOF/TOF target plate. MS/MS analysis was performed on an ABI 4800 Plus TOF-TOF mass spectrometer (Applied Biosystems, Framingham, MA, USA) using a 200 Hz ND:YAG laser operating at 355 nm. The 10 strongest ions per MALDI spot with a signal/noise ratio >25 were selected for subsequent MS/MS analysis in 1 KV mode using 800–1,000 consecutive laser shots. Air was used as the collision gas during MS/MS analysis. The data were used as a Mass Standard Kit for the 4500 Proteomics Analyzer (calibration Mixture1). MS/MS spectra were searched against the NCBI database by Protein Pilot v.3.0 (with MASCOT as the database search engine), digestibility and fragment ion mass tolerances were 50 ppm. Carbamidomethylation of cysteine and oxidation of methionine were allowed during peptide screening. The peptide mass tolerance and fragment mass tolerance of the selected protein were set at 50 ppm. Individual peptide ion scores were investigated using a statistically significant threshold of P = 0.05.

5. Statistical Analysis

Mean, standard deviation and significant difference were calculated. Comparison of differences between pyometra and healthy animals was performed using the Mann-Whitney U test, which was also used to evaluate differences in inflammatory parameters between subgroups of pyometra dogs showing different clinical data. The linear correlation between the two variables was tested through Pearson's correlation coefficient. For continuous variables, the Wilcoxon 2 sample test was used to test differences between pyometra and healthy dogs and before and after OHE. In addition, dogs with pyometra and health-related traits, laboratory conditions and intervention outcomes were tested using a linear mixed model. Statistical significance was set at P <0.05 and P <0.01. All statistical evaluations were performed using the statistical software package SPSS (SPSS 25.0, SPSS Inc, Chicago, IL, USA). All graphs were plotted using GraphPad Prism 8.0 (GraphPad Software, La Jolla, CA, USA). In addition, all results were expressed as mean ± standard deviation (SD).

<Example 2>

Classification and Characteristics of Dogs

In the present invention, 116 female dogs of various breeds, weights and ages were included in the study. These animals included 90 cases of pyometra and 26 cases of healthy control dogs used for clinical data analysis. Among them, three pyometra and one healthy dog were randomly selected as uterine tissues to detect biomarkers of intrauterine infectious agents.

Postoperatively, 22 pyometra and 9 healthy control samples were used for treatment monitoring index analysis. Of the 90 cases of pyometra dogs, 54 were analyzed for pre-OHE clinical data only, and 14 cases were used for laboratory analysis of data during the pre-OHE period due to lack of plasma samples after OHE. Similarly, 15 dogs out of 26 healthy controls were analyzed for pre-OHE clinical data only.

The cultivar of pyometra in 90 cases were as follows; Maltese (24), Shih Tzu (11), Yorkshire Terrier (10), Miniature Poodle (9), Miniature Schnauzer (7), Pomeranian (7), Mixed Breed Dog (6), Miniature Pinscher ( 4), Dachshund (2), French Bulldog (2), Jindo (2) and Chow Chow, Cocker Spaniel, Golden Retriever, Pointer, Poongsan, and Shiba Inu. Characteristics of healthy dogs with pyometra and control are tabulated (see Table 1).

All healthy dogs used as controls consisted of healthy female dogs with a median age of 5 years (range 1-10). Most healthy dog breeds were mixed breeds. The age of dogs with pyometra was significantly higher than that of healthy dogs used as controls (mean 9 years for pyometra dogs versus 5 years for healthy controls, P = 0.000).

<Example 3>

Comparison of pyometra and healthy female dogs

<3-1> Clinical data evaluation result

In the present invention, clinical data including blood and biochemical data were analyzed for most subjects. Analysis of most clinical data showed significant differences between dogs with pyometra and healthy controls. Hematological data for all 116 dogs used in this experiment were analyzed.

As a result, among the analyzed hematological parameters (see Table 2), the number of pyometra dogs was the number of leukocytes, neutrophils and monocytes (24.29 ± 2.08 × 10 ⁹ , 16.91 ± 1.67 × 10 ⁹ , 1.95 ± 0.29 × 10 ⁹ / L, respectively), and the red blood cell, hematocrit, and hemoglobin concentrations (6.10 ± 0.12 × 10 ¹² / L, 39.15 ± 0.89%, 14.06 ± 0.52 g / dL, respectively) were significantly lower than that of healthy dogs (Fig. 1). - see A, B). However, other variables related to blood count, including lymphocytes and platelets, were not statistically different.

Total protein, globulin, ALKP, and amylase concentrations were significantly increased in pyometra dogs (8.07 ± 0.82 g / dL, 4.66 ± 0.13 g / dL, 497.85 ± 81.59 U / L, 1334.57 ± 354.85 U / L). , each); (see Figures 1-C, D), compared to healthy controls (6.34 ± 0.15 g/dL, 3.29 ± 0.11 g/dL, 126.00 ± 18.61 U/L, 561.35 ± 48.85 U/L, respectively); (P < 0.01). In addition, ALT and cholesterol concentrations in pyometra (83.29 ± 16.48 g/dL, 252.80 ± 11.93 mg/dL, respectively) were higher than in healthy controls (74.85 ± 8.13 g/dL, 191.83 ± 15.17 mg/dL, respectively). significantly increased.

Albumin, lipase and chloride concentrations were higher in the pyometra group (2.66 ± 0.05 g/dL, 651.54 ± 159.95) compared to healthy controls (3.06 ± 0.09 g/dL, 783.83 ± 79.26 U/L, 114.36 ± 0.69 mmol/L). U/L, 107.07 ± 1.968 mmol/L, respectively).

<3-2> Assessment of inflammatory parameters

Figure 2 shows the concentration of inflammatory parameters in the pyometra group and healthy dogs.

Plasma concentrations of CRP in healthy dogs ranged from 0.2 to 24.0 mg/L. CRP concentrations in pyometra ranged from 1.8 to 410.4 mg/L, and 6 dogs overlapped the range in healthy dogs (see Fig. 2-A). The difference in CRP concentration was statistically significant ( P = 0.000).

SAA levels (range: 26.9 to 21,007.4 mg/L) were reported to be variable and wide. Plasma concentrations of SAA in healthy dogs ranged from 26.9 to 725.9 mg/L. In pyometra, SAA concentrations ranged from 132.5 to 21,007.4 mg/L. The differences in SAA concentrations were significant ( P = 0.001; Mann-Whitney U test); (See Figure 2-B).

Plasma concentrations of cfDNA in healthy dogs ranged from 554.5 to 1,015 µg/L. In pyometra, cfDNA ranged from 638.5 to 3,860 μg/L. Similar to SAA, cfDNA concentrations were variable. The difference in cfDNA concentration was significant ( P = 0.001; Mann-Whitney U test); (See Figure 2-C).

In 78% (7/9) of healthy dogs, plasma concentrations of IL-6 were below the detection limit. Interestingly, pyometra dogs yielded similar results, with 30% (11/36) having measurable IL-6 in plasma ranging from 0.16 to 106.63 ng/mL. There was no significant difference between plasma IL-6 concentrations ( P = 0.65, Mann-Whitney U test); (See Figure 2-D).

Plasma concentrations of PCT in healthy dogs ranged from 50 to 297 pg/mL. In pyometra, PCT ranged from 5 to 1,420 μg/L. There was no significant difference between plasma PCT concentrations ( P = 0.35, Mann-Whitney U test); (See Figure 2-E).

Plasma concentrations of HMGB1 in healthy dogs (n = 3) ranged from 42.6 to 56.4 μg/L. In pyometra (n = 34), HMGB1 concentrations ranged from 6.25 to 190.8 μg/L. Mean values of healthy dogs were higher than those of pyometra, but there was no significant difference in plasma HMGB1 concentrations ( P = 0.24, Mann-Whitney U test); (See Figure 2-F).

The usefulness of each biomarker for differentiating pyometra and healthy dogs was evaluated using ROC curves (see FIG. 3 ). In addition, the sensitivity, specificity, and blocking values for detection of pyometra in dogs were evaluated (see Table 3). Plasma cfDNA showed the best sensitivity (97%) and specificity (80%) for differentiated dogs with pyometra (cut-off value 682.75 μg/L), with a combined sensitivity of 88% and specificity. The 100% cut-off value is 13.17 mg/L.

There was a significant correlation between SAA and CRP in dogs with pyometra (r = 0.68, P = 0.000). cfDNA concentrations were correlated with the WBC, neutrophil and can globulin (r = 0.46, r = 0.47 , r = 0.44, P <0.01). Similarly, there was a moderate correlation between IL-6 and total protein content (r = 0.88, P = 0.000). In addition, PCT was significantly correlated with hematocrit, monocytes, BUN, total calcium, albumin, globulin and ALKP (see Table 4).

Some independent categories were also evaluated to obtain details on the differences in inflammatory parameters between subgroups of pyometra dogs (clinically bad dogs) as determined from clinical data (see Table 5). Differences in inflammatory parameters were found in cfDNA and CRP, and the two biomarkers were associated with several important clinical parameters.

<Example 4>

Results of profiling of uterine proteins by 2-DE

The present inventors performed 2-DE analysis by confirming a representative 2-DE pattern of healthy control and pyometra as shown in FIGS. 4-A (control) and 4-B, 4-C, and 4-D (pyometra). did.

Progenesis SameSpots image analysis software (version 4.0) was confirmed by a protein spot with a total of 44 differentially expressed using (Fold change ≥1.5; P <0.05 ).

A total of 32 proteins were successfully identified by MALDI-TOF/TOF MS analysis. Differentially expressed proteins were functionally classified using the NCBI and Universal Protein Resource Knowledgebase (UniProtKB) databases. Repeat proteins were deleted and 12 proteins were finalized according to data availability in the Uniprot protein database. A description of all identified proteins with analyzed molecular weight, assay isoelectric point, sequence range and number of peptide matches and MOWSE score and fold change is presented (Table 6).

Compared to healthy controls, 2 proteins were upregulated and 8 proteins were downregulated in pyometra dog #1. Three proteins were upregulated and four proteins were downregulated in pyometra dog #2. In pyometra dog #3, 2 proteins were upregulated and 10 proteins were downregulated compared to the control group. Two proteins were upregulated in all three pyometra dogs.

Among the differentially expressed proteins, proteins related to inflammation reflecting the disease state were detected. 7 out of 12 known proteins are insulin-like growth factor binding protein 1 (IGFBP1), peroxyredoxin-2 isoform 1 (Prx2), L-lactate dehydrogenase B chain (LDHB), selenium binding protein 1 ( SELENBP1), vitamin D binding protein isoformX2 (VDBP), alpha-2-HS-glycoprotein (AHSG), and aldehyde dehydrogenase mitochondrial isoform 2 (ALDH2). The remaining proteins are uncharacteristic of canine inflammation.

<Example 5>

Comparison before and after surgery in dogs with pyometra

<5-1> Evaluation of clinical data according to surgical treatment

Among the hematological parameters analyzed (Table 7), the number of leukocytes and neutrophils after neutering operation (OHE) in females in pyometra were significantly higher (P <0.01), and erythrocytes, hematocrit and hemoglobin were significantly lower than before OHE. (P < 0.01) (see FIGS. 5-A, B).

Likewise, total protein, globulin, cholesterol, pH and potassium concentrations in the biochemical panel were significantly lower (P <0.01) for glucose, BUN, creatinine, albumin, ALKP and ionized calcium concentrations (P < 0.05). (Fig. 5-C , see D). After OHE, only sodium and chloride concentrations increased significantly (P <0.01). Increased leukocyte and neutrophil counts after OHE were also statistically significant in healthy dogs after OHE (data not shown).

Interestingly, differences before and after OHE between dogs with pyometra and healthy dogs were found only for globulin (P = 0.001) and sodium (P = 0.027).

<5-2> Evaluation of inflammatory parameters according to surgical procedures

There was no difference in the values of CRP, SAA and cfDNA before or after OHE in pyometra dogs (P = 0.095, 0.052 and 0.168, respectively) (see FIGS. 6-A, B, C).

IL-6 concentrations before OHE (8.46 ng/ml, 0.063-106.63) were significantly higher than those after OHE (4.71 ng/ml, 0.063-48.58) (P = 0.025, Wilcoxon's signed ranks test); (See Figure 6-D).

PCT concentration before OHE (212.41 ng/ml, 5-1420) was significantly higher than PCT concentration after OHE (137.77 ng/ml, 5-828) (P = 0.027, Wilcoxon's signed ranks test); (See Figure 6-E).

HMGB1 concentration before OHE (50.87 ng/ml, 6.25-190.88) was significantly higher than HMGB1 concentration after OHE (43.05 ng/ml, 6.25-149.5) (P = 0.034, Wilcoxon's signed ranks test); (See Figure 6-F).

OHE-dependent parameters were correlated in pyometra. CRP before OHE correlated with SAA after OHE (r = 0.55, P = 0.007). Similarly, SAA prior to OHE correlated with neutrophils (r = 0.54, P = 0.009) and total protein correlated with IL-6 and CRP (r = 0.65, P = 0.001, r = 0.53, respectively, respectively). , P = 0.010). Interestingly, ionized calcium concentrations before OHE correlated with IL-6 after OHE (r = 0.71, P = 0.000).

<Example 6>

Summary and Interpretation of Experimental Results

The present inventors obtained the same experimental results as in Examples 2 to 5 above in order to identify biomarkers capable of determining the diagnosis or prognosis of canine uterine sinusitis, and a summary and interpretation of the experimental results are provided below.

In the present invention, blood test data and plasma proteins of 90 dogs with sinusitis and 26 healthy bitches were compared and analyzed to find biomarkers for uterine sinusitis diagnosis. In addition, three uterine tissues affected with uterine sinusitis and one normal uterine tissue were randomly selected and subjected to two-dimensional electrophoresis and mass spectrometry of inflammatory tissue proteins. In order to find the therapeutic monitoring index through the subsequent removal of inflammation, clinical data and plasma proteins of 22 females with uterine sinusitis and 9 healthy females were analyzed before and after surgical removal of inflammation (hysterectomy). Comparative analysis was performed.

As a result, in uterine sinusitis, an increase in the total number of white blood cells, a decrease in the number of red blood cells, an increase in globulin, a decrease in albumin, and an increase in ALT, ALKP, cholesterol, and amylase were observed. SAA), a significant increase in cellfree DNA (cfDNA) was additionally observed. In particular, as a result of analyzing the receiver operating characteristic curve among them, cfDNA (AUC = 0.959) was identified as a more useful biomarker for diagnosing uterine sinusitis than CRP and SAA. Two-dimensional electrophoresis and mass spectrometry of proteins in uterine tissue, the source of systemic inflammation, confirmed the expression of a total of 32 proteins, 7 inflammatory proteins and 20 new unidentified proteins. Finally, in a study to explore treatment monitoring indicators by eliminating inflammation, interleukin-6 (IL-6), high-mobility group box 1 (HMGB1), and procalcitonin (PCT) were significantly reduced in blood globulin, ALKP, and cholesterol levels. A significant decrease was confirmed. As a result of correlation analysis of clinical data and biomarkers, cfDNA showed a correlation with WBC (r = 0.46), and PCT showed a correlation with BUN (r = 0.41), albumin (r = -0.37), globulin (r = 0.33), ALKP (r = 0.34), it was confirmed that the correlation between the values was significant.

Considering the above results, cfDNA, CRP, and SAA are suitable as biomarkers for diagnosing uterine sinusitis, and the analysis of PCT, IL-6 and HMGB1 is considered to be useful as an indicator for monitoring the treatment of uterine sinusitis. . It is expected that this will be utilized for the rapid and accurate diagnosis and monitoring of treatment of sepsis in the future.

So far, with respect to the present invention, the preferred embodiments have been looked at. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY KONGJU NATIONAL UNIVERSITY INDUSTRY-UNIVERSITY COOPERATION FOUNDATION <120> Compositions for canine pyometra prognosis prediction by measuring the expression procalcitonin <130> PN2007-292 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 488 <212> DNA <213> Artificial Sequence <220> <223> canine procalcitonin's base sequence <400> 1 caggagcagg agcaggagca ggagactgag ggctccagcc tggacagctc cagagctaag 60 cggtgcagta atctgagtac ctgtgtgctg ggcacatact cgaaggacct gaacaacttt 120 catacattct ctggcatcgg cttcggggct gaaacacctg gcaagaaaag ggacatagcc 180 agcggcttgg agaggggccg ctaacctcgc tttggggtgc cccaggatgc caactgatct 240 ccttccactc cttcctgact tcccttcttg ctcccattga tgaatttaat gcatgcagat 300 ttcacttgga ttgctcttcc agctggcatt ggtagctttg cttgcgagag agaatattgg 360 ggatctcagg atggaaggga agtgagcagc actcatagac caggtatgag gtctttttgc 420 tcctttagag gttagaggta agagtgaggg aagcctcttg agattccaga ggatttcgag 480 gtagagtc 488 <210> 2 <211> 130 <212> PRT <213> Artificial Sequence <220> <223> canine procalcitonin's amino acid sequence <400> 2 Met Gly Leu Trp Lys Ser Ser Pro Phe Leu Ala Phe Ser Ile Leu Val 1 5 10 15 Leu Cys Gln Ala Gly Gly Leu Gln Ala Ala Pro Phe Arg Ser Ala Leu 20 25 30 Glu Gly Leu Pro Asp Pro Thr Ala Leu Ser Glu Lys Glu Gly Arg Leu 35 40 45 Leu Leu Ala Ala Leu Val Lys Ala Tyr Val Gln Arg Lys Asn Glu Leu 50 55 60 Glu Gln Glu Gln Glu Gln Glu Thr Glu Gly Ser Ser Leu Asp Ser Ser 65 70 75 80 Arg Ala Lys Arg Cys Ser Asn Leu Ser Thr Cys Val Leu Gly Thr Tyr 85 90 95 Ser Lys Asp Leu Asn Asn Phe His Thr Phe Ser Gly Ile Gly Phe Gly 100 105 110 Ala Glu Thr Pro Gly Lys Lys Arg Asp Ile Ala Ser Gly Leu Glu Arg 115 120 125 Gly Arg 130

Claims (6)

A marker composition for determining the prognosis of obstructive uterine sinusitis in dogs after oophorectomy , comprising a procalcitonin (PCT) gene or a protein encoded by the gene,
The procalcitonin gene is characterized in that it consists of the nucleotide sequence shown in SEQ ID NO: 1, the protein encoded by the procalcitonin gene consists of the amino acid sequence shown in SEQ ID NO: 2, and the prognosis of the obstructive dog uterine sinusitis is determined A marker composition for determining the prognosis of obstructive canine uterine sinusitis after oophorectomy , characterized in that it confirms a decrease in procalcitonin by 60 to 70 percent compared to before oophorectomy (OHE) after oophorectomy (OHE) .
According to claim 1,
The prognosis is a marker composition for determining the prognosis of obstructive uterine sinusitis in dogs after oophorectomy , characterized in that the prognosis is survival or disease-free survival without recurrence of obstructive uterine sinusitis after oophorectomy (OHE).
A composition for judging the prognosis of obstructive uterine sinusitis after ovariectomy , comprising an agent for measuring the protein level encoded by the procalcitonin (PCT) gene, wherein the gene consists of the nucleotide sequence represented by SEQ ID NO: 1 and the protein consists of the amino acid sequence represented by SEQ ID NO: 2, and the prognosis of dog uterine sinusitis is 60 to 70 percent of procalcitonin after oophorectomy (OHE) compared to before oophorectomy (OHE). A composition for judging the prognosis of obstructive uterine sinusitis in dogs after oophorectomy , characterized in that it confirms a decrease.
4. The method of claim 3,
The agent for measuring the protein level is a composition for determining the prognosis of obstructive uterine sinusitis in dogs after oophorectomy , characterized in that the antibody specifically binds to the protein encoded by the gene.
4. The method of claim 3,
Wherein the prognosis is survival or disease-free survival without recurrence of canine uterine sinusitis after oophorectomy (OHE) .
A kit for judging the prognosis of obstructive uterine sinusitis in dogs after ovarian hysterectomy , comprising the composition of any one of claims 1 to 5.

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