KR20210030195A - Compositions for canine pyometra prognosis judgment by measuring the expression procalcitonin - Google Patents

Abstract

The present invention relates to a composition for canine pyometra prognosis judgement by measuring procalcitonin (PCT). In the present invention, when canine with pyometra is treated, PCT is significantly reduced, and PCT exhibits a statistically significant correlation with a tissue damage index, thereby being able to be usefully used to judge the prognosis of canine pyometra through PCT measurement.

Description

Compositions for canine pyometra prognosis judgment by measuring the expression procalcitonin}

The present invention relates to a composition for determining the prognosis of uterine sinusosis 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. Uterine sinusitis (pyometra) in dogs is a representative disease in dogs that causes sepsis. It is asymptomatic before showing clinical symptoms, but if it progresses rapidly to sepsis, it causes serious organ damage.

Dogs with uterine sinusitis often show general symptoms such as vomiting, lethargy, and diarrhea. However, it is necessary to check the symptoms and age of abdominal distension, purulent discharge from the vulva, and whether or not to undergo a neutralization operation, and to confirm specific diagnosis through ultrasound and radiography.

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

Korean Patent Registration No. 17278787

Accordingly, the inventors of the present invention are making efforts to find a method that can accurately diagnose and predict the prognosis of uterine sinusosis in dogs in a simpler way, and use procalcitonin (PCT) as a monitoring index for prognosis and treatment of uterine sinusosis. It was confirmed that the following was suitable 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, including 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, including an agent measuring the level of the mRNA of the procalcitonin (PCT) gene or the protein encoded by the gene.

In addition, the present invention is to provide a kit for predicting the prognosis of uterine sinusitis in dogs, including the composition.

In order to achieve the above object, the present invention is a marker composition for determining the prognosis of canine uterine sinusitis, comprising a procalcitonin (PCT) gene or a protein encoded by the gene, wherein the procalcitonin gene is represented by SEQ ID NO: 1. Characterized in that consisting of a nucleotide sequence, the protein encoded by the procalcitonin gene is made of an amino acid sequence represented by SEQ ID NO: 2, and the prognosis of canine uterine sinusitis is determined after ovarian hysterectomy (OHE), followed by ovarian hysterectomy ( OHE) It provides a marker composition for determining the prognosis of uterine sinusitis in dogs, characterized in that the decrease in the level of procalcitonin is 60 to 70 percent than before.

In addition, the present invention provides a composition for predicting the prognosis of uterine sinusitis in dogs, including an agent measuring the level of the mRNA 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 a sense and antisense primer or a probe that complementarily binds 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, including the composition.

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

In the present invention, when treating a dog suffering from uterine sinusosis, procalcitonin (PCT) was significantly reduced, and procalcitonin (PCT) showed a statistically significant correlation with the tissue damage index, procalcitonin (procalcitonin). It can be usefully used for determining the prognosis of canine uterine sinusosis through PCT) measurement.

1 shows the mean variables for various clinical data in females with uterine sinusitis and healthy females.
2 is a result of confirming that plasma CRP, SAA and cfDNA were increased in females with uterine sinusitis than in healthy females.
3 is a result of a ROC analysis (Receiver operating characteristic analysis) curve comparing diagnostic sensitivity and 1-specificity of each biomarker for detection of a dog with uterine sinusitis.
Figure 4 is a result showing a representative two-dimensional gel electrophoresis (2-DE) map of the uterine tissue of a healthy female (A) and a female with uterine sinusitis (B, C, D).
5 is a result of dividing and confirming the change of the mean variables for the clinical data of females with uterine sinusitis before ovarian hysterectomy (OHE) and after ovarian hysterectomy (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 finding a procalcitonin (PCT) gene marker that can be used for predicting or treating the prognosis of uterine sinusitis as a result of intensive research to discover a genetic marker for diagnosis and prognosis of uterine sinusitis. I did.

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

In addition, the present invention provides a marker composition for predicting the prognosis of dog uterine sinusitis, including the procalcitonin (PCT) gene or a protein encoded by the gene, and a kit for predicting the prognosis of dog uterine sinusitis comprising the composition.

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

The disease targeted by the present invention, “uterine sinusitis”, is a very typical elderly dog disease and commonly occurs in female elderly dogs that have not been neutralized. The diagnosis of uterine sinusosis is made through ultrasound and radiographic examination. If the uterine sinusitis is not treated properly, the mortality rate is very high, so initial diagnosis is very important.

The term "diagnosis" used in the present invention means in a broad sense to judge the condition of a patient's disease in all aspects. The content of the judgment is the name of the disease, etiology, disease type, severity, detailed mode of the bed, and the presence or absence of complications.

In the present invention, the diagnosis is to determine whether the onset of uterine sinusitis and the level of progression stage.

The term "prognosis" as used in the present invention refers to a prediction about the progression and recovery of a condition, and refers to a prospect or a preliminary evaluation. In the present invention, the prognosis refers to recurrence, overall survival, or disease-free survival of uterine sinusosis, but is not limited thereto.

The agent for measuring the mRNA level of the procalcitonin (PCT) gene may be a sense and antisense primer or probe that complementarily binds to the mRNA, but is not limited thereto.

The term “primer” used in the present invention refers to a short gene sequence that serves as the starting point of DNA synthesis, and refers to an oligonucleotide synthesized for use in diagnosis, DNA sequencing, or the like. The primers are usually synthesized and used in a length of 15 to 30 base pairs, but may vary depending on the purpose of use, and may be modified by methylation or capping by a known method.

The term "probe" used in the present invention refers to a nucleic acid capable of specifically binding to an mRNA having a length of several to hundreds of bases produced through enzymatic chemical separation or purification or synthesis. The presence or absence of mRNA can be confirmed 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.

The term “antibody” used in the present invention includes immunoglobulin molecules immunologically reactive with a specific antigen, and includes both monoclonal and polyclonal antibodies. In addition, the antibody includes forms produced by genetic engineering such as chimeric antibodies (eg, humanized murine antibodies) and heterologous antibodies (eg, bispecific antibodies).

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

For example, in order to perform PCR, 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 DNA polymerase, dNTP mixture, PCR buffer solution, and water. It may be a kit including. The PCR buffer solution may contain KCl, Tris-HCl and MgCl2. In addition, constituents necessary for electrophoresis capable of confirming the amplification of the PCR product 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. RT-PCR kits include test tubes or other suitable containers, reaction buffers, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC, in addition to each primer pair specific for the marker gene. -May include DEPC-water, sterilized water, etc. In addition, a primer pair 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 to perform a DNA chip. The DNA chip kit includes a substrate to which cDNA corresponding to a gene or a fragment thereof is attached as a probe, and the substrate may include a cDNA corresponding to a quantitative structural gene or a fragment thereof. In addition, the kit of the present invention may be in the form of a microarray having a substrate on which the marker gene of the present invention is immobilized.

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

The term "antigen-antibody complex" as used herein refers to a combination of a protein encoded by a gene and an antibody specific thereto. The amount of antigen-antibody complex formation can be quantitatively measured through the signal size of a detection label. The detection label may be selected from the group consisting of enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules, and radioactive isotopes, but is not limited thereto.

In addition, the present invention provides an information providing method for predicting 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 tissues, cells, whole blood, blood, saliva, sputum, cerebrospinal fluid, urine, etc., but is not limited thereto.

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

The protein expression level can be determined by conventional methods known in the art: western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme immunoassay (ELISA), immunoprecipitation. , Flow cytometry, immunofluorescence, ouchterlony, complement fixation assay, or protein chip. It can be measured through, but is not limited thereto.

The term "method of providing information for predicting the prognosis of uterine sinusitis" 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 clinical judgment or findings of a doctor do.

Furthermore, the present invention provides a method for diagnosing dog uterine sinusitis, 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 Western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme immunoassay (ELISA), immunoprecipitation. ), flow cytometry, immunofluorescence, ouchterlony, complement fixation assay, and one or more methods selected from the group consisting of protein chips It can be measured through.

Hereinafter, the present invention will be described in more detail by examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill 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 prospective randomized in vitro analysis, it was designed by continuously monitoring blood samples collected before and after surgical treatment (ovariohysterectomy (OHE)). Patient selection, clinical data collection and blood sampling were performed prospectively.

This experiment was performed with the approval of GNU-190218-D0010 of the Institutional Animal Committee (IACUC) of Gyeongsang National University (GNU). Criteria for inclusion in the current study include the diagnosis of pyometra and aliquots of heparin plasma collected twice in a row before and after surgery. In this experiment, dogs with pyometra were reviewed at regional animal hospitals in Seoul, Gwangju, and Jinju during 2016-2019.

The preliminary diagnosis of pyometra was based on signs, case history, and clinical symptoms such as anorexia, multiple sclerosis, polyuria, purulent vaginal discharge, and diagnostic imaging. Confirmation of the diagnosis of pyometra was based on a previously proposed definition as well as an examination of the overall histopathology of the uterine system.

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

Healthy females with their ovaries removed served as control dogs for comparison with pyometra dogs. Before surgery, 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 to healthy dogs

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

<1-2> Uterine tissue collection

Uterine tissue samples from pyometra and healthy dogs were prepared for proteomic analysis to identify the causative biomarkers of inflammation in the uterus. The excised uterine tissue was cut and placed in a 2 ml Eppendorf tube and stored in a deep freezer at -80 °C until analysis. Four randomly selected samples of 3 pyometra and 1 healthy were given 2-DE combined with Matrix-Assisted Laser Desorption / Ionization Time of Flight Tandem (MALDI-TOF / TOF) MS. Tissue protein profiling in samples was based on isoelectric point and molecular weight differences. Researchers analyzing uterine samples were unaware of the patient's results, and a two-dimensional gel analysis was performed to determine the suspected inflammatory protein spot. The corresponding protein spots were applied to MALDI-TOF / TOF MS to obtain a peptide fingerprint, and then confirmed through the NCBI (National Center for Biotechnology Information) database.

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

Blood samples from the treatment group (clinically improved, treated after OHB treatment) were collected before dog surgery (treated group before OHB treatment) and 1-3 days after surgical treatment. Clinical pyometra of intact bitches has provided an 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. A 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 a full blood test and serum biochemical analysis. Exclusion criteria are pregnancy or delivery.

Includes glucose, total protein, albumin, globulin, total bilirubin, alanine aminotransferase (ALT), alkaline phosphatase (ALKP), gamma-glutamyltransferase (GGT), blood urea nitrogen (BUN), creatinine and phosphorus in order to assess the overall clinical status of the organ damage and dogs using a blood gas analyzer (Nova pHOX analyzer, Nova Biomedical, Waltham, MA, USA) with a catalyst Dx ^ⓡ acid-conducting base balance, electrolyte concentrations, and serum biochemistry analysis did. Plasma cholesterol, amylase and lipase were also analyzed immediately after plasma separation in this panel.

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 twice with appropriate controls.

cfDNA.

Measurement of the cfDNA validated for use in dogs in the previous table-top fluorescent measuring instrument (Qubit ^ⓡ dsDNA HS Assay Kit and Qubit ^ⓡ 1.0 fluorescence meter, Life Sciences, Carlsbad, CA, USA) with fluorescence detection method using the relevant reagent for It was carried out using, and it was processed according to the manufacturer's instructions.

Briefly, the reaction volume used per analysis was 200 μl, including 10 μl of plasma samples. 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, the samples were diluted to bring the analysis results into the linear analysis range, and all results were corrected by diluting 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).

Assay, DuoSet ELISA was performed in duplicate 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 with PBS at room temperature. The plate was blocked with a reagent diluent [1% BSA (bovine serum albumin) in PBS (phosphate buffered saline), 300 μl/well, R&D Systems] for 2 hours and washed with a washing buffer (0.05% ^Tween® 20).

Standard and plasma samples were added to each well (100 μl/well) for 2 hours 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 tetra methyl benzidine (TMB); (R&D Systems, Minneapolis, MN, USA) A liquid substrate solution was added, and then each sample was incubated for 20 minutes.

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

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 times prior to the initial measurement, were followed at each analysis step. However, the resulting absorbance values for almost all dogs were above the readable range of the analysis (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 anti-canine-CRP antibodies added to each well. After washing again to remove unbound material, TMB substrate solution was added. SAA was analyzed by sandwich type ELISA using a monoclonal antibody specific for SAA coated on the wells. After 50 μl of sample was added to each well, TMB substrate solution and stop solution were added.

The lower limits of the assay detection were 7.5 ng/mL for CRP and 10 ng/mL for SAA.

HMGB1.

Canine HMGB1 was quantified using a commercial canine ELISA kit (MyBiosource, San Diego, CA, USA) according to the manufacturer's protocol as the sample was not diluted.

Briefly explaining this, 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 at room temperature for 1 hour. After washing, detection was achieved by adding a liquid substrate solution and incubating for 15 minutes under dark conditions. The reaction was stopped by adding an acidic solution, and the lower limit of the assay detection was 6.25 ng/mL.

PCT.

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

Briefly explaining this, 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 fourth wash was added in each washing step and performed using 350 μl of washing buffer per well, then 100 μl of biotin-labeled polyclonal anti-canine procalcitonin was added to each well and 1 hour Reacted for a while. Next, streptavidin-HRP was added and peroxidase activity was quantified using a substrate. The lower limit of detection by PCT analysis was 12.5 pg/mL.

All ELISAs described above ^{use a 450 nm microplate spectrophotometer Versamax TM} (Molecular Devices, Boston, MA, USA) as ^{a reference wavelength to measure the optical density Softmax ®} Pro analysis software (Molecular Devices, Boston, MA, USA). And developed. The wavelength was corrected by subtracting the reading from 540 nm. Readings were made at 450 nm for IL-6, 450 nm for CRP, SAA and PCT, and 450 nm to 630 nm for HMGB1.

4. Proteometics with uterine tissue

<4-1> Sample preparation for 2-DE

Briefly about the experimental method, the uterine tissue was washed twice with PBS to remove residual components, and suspended in lysis buffer ice containing 7M urea, 2M thiourea and 4% (w/v) CHAPS for 1 hour. . Samples were sonicated to confirm destruction and homogenization of all tissue samples. After centrifugation at a temperature of 14,000 rpm and 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 100% acetone cooled with ice, dried with a freeze dryer (SFDSM06, Samwon Freezing Engineering Co., Busan, Korea), and dissolved in 200 µl of sample buffer. Protein concentration was determined according to the manufacturer's protocol using the Pierce™ BCA Protein Assay Kit (Thermo Scientific™ Waltham, MA, USA).

<4-2> 2-DE and image analysis

Isoelectric focusing was performed using GE Healthcare Immobiline™ DryStrip Gels (18cm, pH 4-7, Amersham Biosciences, Uppsala, Sweden), and the strip was 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 in IPG strips. Equipotential focusing is 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) , Slope 10,000V (1 hour), 10,000V (13 hours) and 50V (3 hours).

The strip was first reacted for 15 minutes with 10 mg / ml DTT 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 continuously reacted for 15 minutes.

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

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

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

As processed by the analog of Variations of Analysis, protein spots exceeding 1.5 times and expression changes with P <0.05 were considered to be significantly changed, and the selected spot was visually confirmed and manually on the stained gel. I was restrained.

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

To prepare the gel spots for MALDI-TOF/TOF MS, a bleaching 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, excised protein spots were washed 3 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) for 45 minutes at room temperature, and alkylated. The solution (55 mM iodoacetamide in 0.1 M ammonium bicarbonate) was treated in the dark for 30 minutes. Then, the spots were dehydrated using 0.1M ammonium bicarbonate for 5 minutes and 0.1M ammonium bicarbonate for 15 minutes.

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

Trypsin peptide was extracted using 0.1% trifluoroacetic acid in 66% acetonitrile and dried for 2 hours 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 on the MALDI-TOF / TOF target plate with a pipette. 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 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. Data were used as Mass Standard Kit for 4500 Proteomics Analyzer (calibration Mixture1). The MS/MS spectrum was searched against the NCBI database by Protein Pilot v.3.0 (with MASCOT as the database search engine), and the digestibility and fragment ion mass tolerance was 50 ppm. During the peptide search, carbamidomethylation of cysteine and oxidation of methionine were allowed. The peptide mass tolerance and fragment mass tolerance of the selected protein were set to 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, and was also used to assess 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 associated with pyometra and health-related traits, laboratory conditions, and intervention outcomes were tested using a linear mixed model. Statistical significance was set as 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, 3 pyometra and 1 healthy dog were randomly extracted into uterine tissues to detect biomarkers of infectious agents in the uterus.

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

The varieties of pyometra in 90 cases were as follows; Maltese (24), Shih Tzu (11), Yorkshire Terrier (10), Small Poodle (9), Small Schnauzer (7), Pomeranian (7), Mixed Breed Dog (6), Miniature Pinscher ( 4), Dachshund (2), French Bulldog (2), Jindo (2) and Chow Chow, Caucasian, Golden Retriever, Pointer, Poongsan, and Shiba in each. Characteristics of uterine sinusitis (pyometra) and control healthy dogs 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 pyometra dogs 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 of the 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), pyometra dogs had 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, Each) was significantly higher, and the concentrations of red blood cells, hematocrit, and hemoglobin (6.10 ± 0.12 × 10 ¹² / L, 39.15 ± 0.89%, 14.06 ± 0.52g / dL, respectively) were significantly lower than that of healthy dogs (Fig. 1). See -A,B). However, there were no statistical differences in other variables related to blood cell counts, including lymphocytes and platelets.

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 the healthy control group (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, the ALT and cholesterol concentrations of pyometra (83.29 ± 16.48g / dL, 252.80 ± 11.93mg / dL, respectively) compared to the healthy control group (74.85 ± 8.13 g/dL, 191.83 ± 15.17 mg/dL, respectively). Significantly increased.

Albumin, lipase and chloride concentrations were compared with healthy controls (3.06 ± 0.09 g/dL, 783.83 ± 79.26 U/L, 114.36 ± 0.69 mmol/L) in the pyometra group (2.66 ± 0.05 g/dL, 651.54 ± 159.95). 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. In pyometra, the CRP concentration ranged from 1.8 to 410.4 mg/L, and 6 dogs overlapped the range of healthy dogs (see Fig. 2-A). The difference in CRP concentration was statistically significant ( P = 0.000).

SAA values (range: 26.9 to 21,007.4 mg/L) were recorded variably and widely. Plasma concentrations of SAA in healthy dogs ranged from 26.9 to 725.9 mg/L. SAA concentrations in pyometra ranged from 132.5 to 21,007.4 mg/L. The difference in SAA concentration was 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 concentration was 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, the plasma concentration of IL-6 was below the detection limit. Interestingly, in dogs with pyometra, 30% (11/36) produced similar results with measurable IL-6 in plasma in the range of 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).

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

The usefulness of each biomarker for distinguishing between pyometra dogs and healthy dogs was evaluated using the ROC curve (see Fig. 3). In addition, sensitivity, specificity and blocking values for the 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), 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). The cfDNA concentration was correlated with the number of WBC, neutrophils and globulins (r = 0.46, r = 0.47, r = 0.44, P <0.01). Similarly, there was an appropriate correlation between IL-6 and total protein content (r = 0.88, P = 0.000). In addition, PCT had a significant correlation with hematocrit, monocytes, BUN, total calcium, albumin, globulin and ALKP (see Table 4).

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

Progenesis A total of 44 differentially expressed protein spots were identified using SameSpots image analysis software (version 4.0) (Fold change ≥1.5; P <0.05).

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

Compared to the healthy control, 2 proteins were upregulated and 8 proteins downregulated in pyometra dog #1. In pyometra dog #2, 3 proteins were upregulated and 4 proteins were downregulated. In pyometra dog #3, 2 proteins were upregulated and 10 proteins were downregulated compared to the control. Two proteins were upregulated in all three pyometra dogs.

Among the differentially expressed proteins, proteins related to inflammation that reflect the disease situation were detected. Seven of the 12 known proteins are insulin-like growth factor binding protein 1 (IGFBP1), peroxiredoxin-2 isoform 1 (Prx2), L-lactate dehydrogenase B chain (LDHB), and selenium binding protein 1 ( SELENBP1), vitamin D binding protein isoformX2 (VDBP), alpha -2-HS-glycoprotein (AHSG) and aldehyde dehydrogenase mitochondrial isoform 2 (ALDH2). The rest of the proteins are uncharacteristic for the dog's inflammation.

<Example 5>

Comparison before and after surgery in pyometra dogs

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

Among the analyzed hematological parameters (Table 7), the number of leukocytes and neutrophils were significantly higher after the female neutralization surgery (OHE) in pyometra (P <0.01), and the red blood cells, hematocrit, and hemoglobin were significantly lower than before OHE (P<0.01) (see Figs. 5-A, B).

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

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

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

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, and C).

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

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

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

OHE-dependent parameters were correlated in pyometra. CRP before OHE was correlated with SAA after OHE (r = 0.55, P = 0.007). Similarly, SAA before OHE was correlated with neutrophils (r = 0.54, P = 0.009), and total protein was correlated with IL-6 and CRP (respectively, r = 0.65, P = 0.001, r = 0.53). , P = 0.010). Interestingly, the ionized calcium concentration 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 in order to identify biomarkers capable of determining the diagnosis or prognosis of canine uterine sinusosis, and the following summarizes and interprets the experimental results.

In the present invention, blood test data and plasma proteins of 90 dogs affected by sinusitis and 26 healthy females were compared and analyzed to find a biomarker for diagnosing uterine sinusitis. Among them, three uterine tissues affected by uterine sinusitis and one normal uterine tissue were randomly selected for two-dimensional electrophoresis and mass analysis of inflammatory tissue proteins. Afterwards, in order to find the index of treatment monitoring through inflammation removal, clinical data and plasma proteins of 22 females affected by uterine sinusitis and 9 healthy females were removed before and after the surgical removal of inflammation (ovarian hysterectomy). Comparative analysis was performed.

As a result, in uterine sinusitis, an increase in total white blood cell count, a decrease in red blood cell count, an increase in globulin, a decrease in albumin, 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, 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 tissues, the source of systemic inflammation, confirmed the expression of a total of 32 proteins, including 7 inflammatory proteins and 20 unidentified new proteins. Lastly, in a study to explore treatment monitoring indicators through inflammation removal, interleukin-6 (IL-6), high-mobility group box 1 (HMGB1), and procalcitonin (PCT) were significant along with remarkable decreases in blood globulin, ALKP, and cholesterol levels. A significant decrease was observed. As a result of correlation analysis between clinical data and biomarkers, cfDNA was correlated with WBC (r = 0.46), and PCT was correlated 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.

Taking the above results together, it is believed that cfDNA, CRP and SAA are appropriate as biomarkers for diagnosis of uterine sinusitis, and analysis of PCT, IL-6 and HMGB1 can be used as an indicator of monitoring for treatment of uterine sinusitis. . It is expected that this can be used for fast and accurate diagnosis of sepsis and monitoring of treatment in the future.

So far, the present invention has been looked at around its preferred embodiments. Those of ordinary skill in the art to which the present invention pertains will be able to understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative point of view rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the above 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 uterine sinusitis in dogs, comprising a procalcitonin (PCT) gene or a protein encoded by the gene,
The procalcitonin gene is characterized in that it consists of a nucleotide sequence represented by SEQ ID NO: 1, and the protein encoded by the procalcitonin gene consists of an amino acid sequence represented by SEQ ID NO: 2,
The determination of the prognosis of dog uterine sinusosis is a marker composition for determining the prognosis of uterine sinusosis in dogs, characterized in that procalcitonin decreases to a level of 60 to 70 percent after ovarian hysterectomy (OHE) compared to before ovarian hysterectomy (OHE) .
The method of claim 1,
The prognosis is characterized in that the dog uterine sinusosis does not recur after ovarian hysterectomy (OHE) and is alive or disease-free survival.
A composition for determining the prognosis of uterine sinusitis in dogs, comprising a preparation for measuring the level of a protein encoded by a procalcitonin (PCT) gene,
The gene consists of a nucleotide sequence represented by SEQ ID NO: 1, and the protein consists of an amino acid sequence represented by SEQ ID NO: 2,
The determination of the prognosis of dog uterine sinusosis is characterized in that the procalcitonin decreases by 60 to 70 percent compared to before ovarian hysterectomy (OHE) after ovarian hysterectomy (OHE), a composition for determining prognosis of dog uterine sinusitis.
The method of claim 3,
The composition for determining the prognosis of uterine sinusitis in dogs, characterized in that the agent for measuring the protein level is an antibody that specifically binds to a protein encoded by a gene.
The method of claim 3,
The prognosis is characterized in that the dog uterine sinusosis does not recur and survive or disease-free survival after ovarian hysterectomy (OHE), a composition for determining the prognosis of dog uterine sinusitis.
A kit for determining the prognosis of uterine sinusosis in dogs, comprising the composition of any one of claims 1 to 5.

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