KR102562614B1 - Kit for periodontal disease diagnosis using non-invasive methods - Google Patents

Abstract

The present invention relates to a kit for diagnosing periodontal disease using a non-invasive method, and relates to a composition capable of diagnosing periodontal disease and a diagnostic kit comprising the same.
The composition for diagnosing periodontal disease according to the present invention can diagnose periodontal disease very accurately and simply and quickly in a non-invasive manner. In addition, by enabling early diagnosis of periodontal disease, it can be used for early detection and treatment of periodontal disease.

Description

Kit for periodontal disease diagnosis using non-invasive methods}

The present invention relates to a kit for diagnosing periodontal disease using a non-invasive method, and relates to a composition capable of diagnosing periodontal disease and a diagnostic kit comprising the same.

Periodontal disease includes gingivitis occurring in the gums and periodontitis occurring in the alveolar bone, and belongs to chronic inflammatory diseases as a disease in which the surrounding tissue supporting the teeth is destroyed, not a disease in which the teeth themselves are damaged. In many cases, patients do not recognize the onset of periodontal disease until the inflammation gradually progresses and the alveolar bone around the teeth is destroyed, and the teeth must be extracted. In order to prevent this, it is necessary to have a technology that can diagnose periodontal disease at an early stage.

Until now, various studies related to periodontal disease are in progress worldwide, but research is only focused on treating periodontal disease after it has progressed to a certain extent. The reality is that it is limited to qualitative diagnosis through Currently, the diagnosis of periodontal disease relies on determining the degree of periodontitis progression or analyzing the degree of bacterial infection by measuring the depth of the periodontal pocket and the degree of gum recession using a periodontal pocket probe after subjective symptoms and radiographs of the patient.

However, in order to effectively treat periodontal disease, it is necessary to develop periodontal disease diagnostic factors capable of early diagnosis and/or simple examination, but it is insufficient.

Accordingly, while developing a highly reliable biomarker for diagnosing periodontal disease, the present inventors performed proteomics analysis on proteins in samples obtained from tissues and gingival crevicular fluid (GCF) of patients with periodontitis and normal subjects to perform DEP (Differentially expressed protein) was found, and as a result, the present invention was completed by confirming a group of biomarkers effective for diagnosing periodontal disease.

One object of the present invention is to provide a composition or kit that can easily and accurately diagnose periodontal disease.

Another object of the present invention is to provide a method for providing information for diagnosing periodontal disease.

Another object of the present invention is to provide a device for diagnosing periodontal disease.

Another object of the present invention is to provide a marker composition for diagnosing periodontal disease.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention provides a composition for diagnosing periodontal disease, including an agent for measuring the expression level of at least one protein selected from the group consisting of P01622, A0A087X0N5, E9PH25, A0A0B4J1V0, HOYI36, Q9NZT2, and P07305 or a gene encoding the same.

At least one protein selected from the group consisting of P01622, A0A0B4J1V0, H0YI36, and Q9NZT2 may be overexpressed in a sample of a patient with periodontal disease.

In addition, at least one protein selected from the group consisting of A0A087X0N5, E9PH25, and P07305 may be low-expressed in a sample of a periodontal disease patient.

The periodontal disease is a group consisting of gingivitis, periodontal inflammation, gingival bleeding, gingival retraction, periodontal pocket, pericoronitis and periodontal abscess. It may be one or more selected from.

The agent for measuring the protein expression is an oligopeptide, monoclonal antibody, polyclonal antibody, chimeric antibody, ligand, PNA (peptide nucleic acid) or aptamer that specifically binds to the protein can be

An agent for measuring the expression of the nucleic acid may be a primer, a probe, or an antisense nucleotide that specifically binds to the nucleic acid.

In addition, the present invention provides a kit for diagnosing periodontal disease comprising the composition.

The kit may be a RT-PCR kit, a DNA chip kit, or a protein chip kit.

In addition, the present invention is a method for diagnosing periodontal disease, comprising measuring the expression level of at least one protein selected from the group consisting of P01622, A0A087X0N5, E9PH25, A0A0B4J1V0, H0YI36, Q9NZT2, and P07305 from a biological sample or a gene encoding the same. Provides a method for providing information about

The present invention relates to data including the expression level of at least one protein selected from the group consisting of P01622, A0A087X0N5, E9PH25, A0A0B4J1V0, H0YI36, Q9NZT2, and P07305 measured in a biological sample of an individual or a gene encoding the same, periodontal disease Provided is a periodontal disease diagnosis device including a diagnosis unit for determining diagnosis information.

The present invention provides a biomarker composition for diagnosing periodontal disease comprising at least one protein selected from the group consisting of P01622, A0A087X0N5, E9PH25, A0A0B4J1V0, HOYI36, Q9NZT2, and P07305 or a gene encoding the same.

The composition for diagnosing periodontal disease according to the present invention can diagnose periodontal disease very accurately and simply and quickly in a non-invasive manner.

In addition, by enabling early diagnosis of periodontal disease, it can be used for early detection and treatment of periodontal disease.

1 is a diagram showing the BCA protein assay results.
2a and 2b are diagrams showing the results of Label-free quantification using LC-MS/MS (Q-Exactive) in periodontal tissue.
3 is a diagram showing the results of Volcano plots analysis of LC-MS/MS data of patients with periodontal disease and normal patients.
4 is a diagram showing the results of principal component analysis (PCA) analysis of LC-MS/MS data of a periodontal disease patient group and a normal patient group.
5 is a diagram showing heat map analysis results based on the protein analysis results of periodontal patients and normal groups.
6A is a BCA standard curve of a GCF sample from a patient with periodontitis, and FIG. 6B is a diagram showing a BCA standard curve of a GCF sample from a normal person.
7a and 7b are diagrams showing the results of label-free quantification using LC-MS/MS (Q-Exactive) in GCF.
8 is a diagram showing the results of Volcano plots analysis of LC-MS/MS data of a periodontal disease patient group and a normal patient group.
9 is a diagram showing PCA analysis results of LC-MS/MS data of patients in a periodontal disease patient group and a normal group.
10 is a diagram showing Heatmap analysis results based on protein analysis results of periodontal patients and normal groups.
11 is a diagram showing the results of DEP analysis on the expression patterns of proteins identified in tissues and GCF samples of patients with periodontitis and normal subjects.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in this application, it should not be interpreted in an ideal or excessively formal meaning. don't In addition, in describing the present invention, if it is determined that a detailed description of a related known technology may unnecessarily obscure the subject matter of the present invention, the detailed description will be omitted.

While developing a highly reliable biomarker for diagnosing periodontal disease, the present inventors analyzed differentially expressed protein (DEP) proteomics for proteins in samples obtained from tissues and gingival crevicular fluid (GCF) of patients with periodontitis and normal subjects. was carried out, and as a result, a novel biomarker was identified.

Accordingly, according to one embodiment of the present invention, the present invention includes an agent for measuring the expression level of at least one protein selected from the group consisting of P01622, A0A087X0N5, E9PH25, A0A0B4J1V0, HOYI36, Q9NZT2, and P07305 or a gene encoding the same. It relates to a composition for diagnosing periodontal disease.

The P01622 is an IGKV3-20 (Ig kappa chain V-III region Ti) protein, which may be the amino acid sequence of SEQ ID NO: 1 or a variant thereof.

The A0A087X0N5 is an IGKV1-17 (Immunoglobulin Kappa Variable 1-17) protein, and may be the amino acid sequence of SEQ ID NO: 2 or a variant thereof, but is not limited thereto.

The E9PH25 is a GYPA (Glycophorin A) protein, which is known as a cell membrane component, and may be the amino acid sequence of SEQ ID NO: 3 or a variant thereof, but is not limited thereto.

The A0A0B4J1V0 is IGHV3-15 (Immunoglobulin Heavy Variable 3-15), which is a component of the plasma membrane and plays a role in receptor binding in signal transduction. The A0A0B4J1V0 may be the amino acid sequence of SEQ ID NO: 4 or a variant thereof, but is not limited thereto.

The H0YI36 is ARHGAP9 (Rho GTPase Activating Protein 9) involved in signal transduction, and may be the amino acid sequence of SEQ ID NO: 5 or a variant thereof, but is not limited thereto.

The Q9NZT2 is involved in signal transduction with OGFR (opioid growth factor receptor), is a component of membranes, nuclei, or cells, and may be the amino acid sequence of SEQ ID NO: 6 or a variant thereof, but is not limited thereto.

The P07305 is H1-0 (histone H 1.0), plays a role in cell organization and biogenesis, is an ER/Golgi, nucleus, and cell component, and is an amino acid sequence of SEQ ID NO: 7 or a variant thereof It may be, but is not limited thereto.

The expression level of one or more proteins selected from the group consisting of P01622, A0A0B4J1V0, HOYI36, and Q9NZT2 or a gene encoding the same may be overexpressed in a sample of a patient with periodontal disease compared to a sample of a normal person.

In addition, the expression level of at least one protein selected from the group consisting of A0A087X0N5, E9PH25, and P07305 or a gene encoding the same may be lower in a sample of a patient with periodontal disease than in a sample of a normal person.

In the present invention, “periodontal disease” refers to symptoms related to inflammatory diseases that destroy soft tissues such as periodontal ligaments and gingiva surrounding teeth and hard tissues such as alveolar bone.

The periodontal disease may be gingivitis, periodontal inflammation, gingival bleeding, gingival retraction, periodontal pocket, pericoronitis, periodontal abscess, and the like. but not limited thereto.

In the present invention, "diagnosis" means confirming the presence or characteristics of a pathological state, determining the susceptibility of a subject to a specific disease or disorder, or determining whether an object currently has a specific disease or disorder determining whether or not, determining the prognosis of a subject suffering from a particular disease or disorder, or therametrics. For the purposes of the present invention, the specific disease or condition may be for periodontal disease.

In the present invention, "biomarker for diagnosing periodontal disease" is a substance that can be diagnosed by distinguishing a biological sample related to periodontal disease from a normal sample, and a polypeptide or organic biomolecules such as nucleic acids (eg mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.); and the like. For the purpose of the present invention, it is a gene or protein whose expression is increased or decreased in bodily fluids such as tracheal disease tissues, cells, or gingival crevicular fluid (GCF).

The selection and application of significant diagnostic markers determines the reliability of diagnostic results. A significant diagnostic marker is accurate in terms of diagnostic results and has high validity and reliability to show consistent results even during repeated measurements. ) indicates a high marker. The periodontal disease diagnostic marker of the present invention is a protein or gene expression whose expression is changed compared to a normal sample as a direct or indirect factor with the onset of periodontal disease, and can show the same results even in repeated experiments. Accordingly, the diagnosis result based on the result obtained by measuring the expression level of the significant diagnostic marker of the present invention can be reasonably reliable.

In the present invention, "an agent for measuring the expression level" is an agent capable of confirming the presence and expression level of mRNA of a protein or marker gene expressed in a biological sample for the diagnosis of periodontal disease.

The agent for measuring the protein expression is an oligopeptide, monoclonal antibody, polyclonal antibody, chimeric antibody, ligand, PNA (peptide nucleic acid) or aptamer that specifically binds to the protein It may be, but is not limited thereto.

In the present invention, the "antibody" refers to a substance that specifically binds to an antigen and causes an antigen-antibody reaction. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to the biomarker protein. Antibodies of the present invention include both polyclonal antibodies, monoclonal antibodies and recombinant antibodies. Such antibodies can be readily prepared using techniques well known in the art. For example, polyclonal antibodies can be produced by a method well known in the art, including a process of injecting an antigen of the protein into an animal and collecting blood from the animal to obtain serum containing the antibody. Such polyclonal antibodies can be prepared from any animal, such as goat, rabbit, sheep, monkey, horse, pig, cow, dog, and the like. In addition, monoclonal antibodies can be prepared using the hybridoma method well known in the art (see Kohler and Milstein (1976) European Journal of Immunology 6:511-519), or the phage antibody library technique (Clackson et al, Nature, 352). :624-628, 1991; Marks et al, J. Mol. Biol., 222:58, 1-597, 1991). Antibodies prepared by the above methods may be separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography. In addition, the antibodies of the present invention include functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule means a fragment having at least an antigen-binding function, and includes Fab, F(ab'), F(ab')2, and Fv.

In the present invention, the "PNA (Peptide Nucleic Acid)" refers to an artificially synthesized polymer similar to DNA or RNA, and was first introduced in 1991 by Professors Nielsen, Egholm, Berg and Buchardt of the University of Copenhagen, Denmark. Whereas DNA has a phosphate-ribose backbone, PNA has a repeated N-(2-aminoethyl)-glycine backbone linked by peptide bonds, which greatly increases the binding force and stability to DNA or RNA, and is thus used in molecular biology. , diagnostic assays and antisense therapies. PNA is described by Nielsen PE, Egholm M, Berg RH, Buchardt O (December 1991). "Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide". Science 254(5037): 1497-1500.

In the present invention, the "aptamer" is an oligonucleic acid or peptide molecule, and a general description of the aptamer is described in the literature [Bock LC et al., Nature 355(6360):5646(1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78(8):42630 (2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95(24): 142727 (1998).

Analysis methods for confirming the amount of protein include western blotting, tissue immunostaining, immunoprecipitation assay, complete fixation assay, FACS, and protein chip. However, it is not limited thereto.

The protein expression level can be measured using an ELISA method (direct ELISA, indirect ELISA or sandwich ELISA, etc.), Western blot or protein chip. The protein chip hybridizes a protein in which one or more antibodies to a marker are arranged at a predetermined position on a substrate and immobilized at a high density with the protein chip to form an antigen-antibody complex, and reads the protein to determine the presence or expression level of the protein. You can check. In the present invention, through this method, marker protein expression levels in normal control subjects and subjects suspected of periodontal disease can be compared, and when overexpression or underexpression of the marker protein is confirmed, the target patient can be diagnosed as having periodontal disease. .

Agents for measuring gene expression levels may be primers, probes, or antisense nucleotides that specifically bind to the gene or nucleic acid.

Analysis methods for confirming the amount of mRNA of the marker gene include RT-PCR, competitive RT-PCR, real-time RT-PCR, and RNase protection assay (RPA; RNase protection assay), northern blotting, or a DNA microarray chip, but is not limited thereto. In the present invention, through this detection method, mRNA expression levels in normal control subjects and subjects suspected of periodontal disease can be compared.

The "primer" is a nucleic acid sequence having a short free 3'hydroxyl group, which can form a base pair with a complementary template and serves as a starting point for copying the template. Refers to a short nucleic acid sequence. Primers can initiate DNA synthesis in the presence of a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates in an appropriate buffer and temperature. In the present invention, PCR amplification is performed using the sense and antisense primers of the marker polynucleotide of the present invention, and periodontal disease onset can be diagnosed or prognosis can be predicted through whether or not a desired product is produced. PCR conditions and lengths of sense and antisense primers can be modified based on those known in the art.

The "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to as short as several bases to as long as hundreds of bases capable of specific binding to mRNA, and is labeled, so that the presence or absence of a specific mRNA can be confirmed. there is. The probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like. In the present invention, diagnosis or prognosis of periodontal disease can be predicted through hybridization by using the marker polynucleotide of the present invention and a complementary probe. Selection of suitable probes and hybridization conditions can be modified based on those known in the art.

Such nucleic acid sequences can also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution of one or more natural nucleotides with homologues, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phosphotriesters, phosphoro amidates, carbamates, etc.) or to charged linkages (eg phosphorothioates, phosphorodithioates, etc.).

In the present invention, the "antisense" refers to a sequence of nucleotide bases in which an antisense oligomer is hybridized with a target sequence in RNA by Watson-Crick base pairing, allowing formation of a typical mRNA and RNA: oligomer heteroduplex in the target sequence and an oligomer having an inter-subunit backbone. Oligomers may have exact sequence complementarity or near complementarity to the target sequence.

In another aspect, the present invention relates to a kit for diagnosing periodontal disease comprising the composition for diagnosis. Specifically, the kit for diagnosing periodontal disease may be a reverse transcription polymerase-PCR (RT-PCR) kit, a DNA chip kit, or a protein chip kit.

The kit for diagnosing periodontal disease may further include one or more other component compositions, solutions, or devices suitable for the analysis method. The diagnostic kit may be a diagnostic kit characterized in that it includes essential elements necessary for carrying out a reverse transcription polymerase reaction. The reverse transcription polymerase reaction kit includes each pair of primers specific for a marker gene. The primer is a nucleotide having a sequence specific to the nucleic acid sequence of each marker gene, and is about 7 bp to 50 bp in length, more preferably about 10 bp to 30 bp in length. In addition, a primer specific for the nucleic acid sequence of the control gene may be included. Other reverse transcription polymerase reaction kits include test tubes or other suitable container, reaction buffer (with varying pH and magnesium concentration), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC -Water (DEPC-water), sterilized water, etc. may be included.

In addition, it may be a diagnostic kit characterized in that it includes essential elements required to perform a DNA chip. The DNA chip kit may include a substrate to which cDNA or oligonucleotides corresponding to genes or fragments thereof are attached, and reagents, reagents, enzymes, and the like for producing fluorescently labeled probes. In addition, the substrate may include a cDNA or oligonucleotide corresponding to a control gene or a fragment thereof.

In addition, it may be a diagnostic kit characterized in that it includes essential elements required to perform a protein chip or ELISA. Protein chip kits or ELISA kits contain antibodies specific for marker proteins. An antibody is an antibody that has high specificity and affinity for each marker protein and little cross-reactivity to other proteins, and is a monoclonal antibody, polyclonal antibody, or recombinant antibody. ELISA kits may also include antibodies specific for a control protein. Other ELISA kits include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (eg, conjugated with antibodies) and substrates thereof or those capable of binding the antibody. may contain other substances and the like.

In another aspect, the present invention provides a periodontal method comprising measuring the expression level of at least one protein selected from the group consisting of P01622, A0A087X0N5, E9PH25, A0A0B4J1V0, H0YI36, Q9NZT2, and P07305 from a biological sample or a gene encoding the same. It relates to a method of providing information on disease diagnosis.

The protein or the gene encoding it is as described above.

In the method for providing information for diagnosing periodontal disease of the present invention, it is possible to detect the mRNA expression level of the fusion gene encoding the MFSD7-ATP5I fusion protein or the level of the fusion protein expressed therefrom, and biological samples isolated from patients The expression level can be detected through a known process by isolating mRNA or protein. Here, the biological sample isolated from the patient is a tissue, for example, a cell, tissue, organ, body fluid (blood, lymph, GCF, etc. ), digestive juices, sputum, alveolar-bronchial lavage fluid, urine, feces, as well as nucleic acid extracts (genomic extracts, transcript extracts, cDNA preparations or aRNA preparations prepared from transcript extracts, etc.) or proteins obtained from these biological samples including, but not limited to, extracts as well. Further, the sample may be subjected to formalin fixation treatment, alcohol fixation treatment, freezing treatment or paraffin embedding treatment. In addition, genes, transcription products, cDNAs, or proteins can be prepared by selecting a known method suitable for the type and condition of the sample.

In the present invention, the expression level of at least one protein selected from the group consisting of P01622, A0A087X0N5, E9PH25, A0A0B4J1V0, H0YI36, Q9NZT2, and P07305 measured in a biological sample of an individual or a gene encoding the same is increased compared to a normal control group, or , If the periodontal disease is reduced, it may further include the step of predicting that periodontal disease has occurred or the possibility of developing periodontal disease is high.

When the expression level of at least one protein selected from the group consisting of P01622, A0A0B4J1V0, H0YI36, and Q9NZT2 or a gene encoding the same is overexpressed in a sample of a patient with periodontal disease compared to a sample of a normal person, periodontal disease has occurred or the possibility of developing periodontal disease can be predicted to be high. 1.2 to 20 fold or more, for example, 1.2 fold or more, 2 fold or more, 3 fold or more, 4 fold or more, 5 fold or more, 6 If it is more than twice, more than seven times, or more than eight times, it can be predicted that the possibility of developing periodontal disease is high.

In addition, when the expression level of at least one protein selected from the group consisting of A0A087X0N5, E9PH25, and P07305 or a gene encoding the same is lower in a sample of a patient with periodontal disease than in a sample of a normal person, periodontal disease has occurred or periodontal disease has occurred. It can be predicted with high probability. The expression level of the measured protein or encoding gene is 0.9 to 0.00001 fold or more, for example, 0.9 fold or more, 0.8 fold or more, 0.7 fold or more, 0.6 fold or more, 0.5 fold or more, 0.1 fold or more compared to the normal control group If it is more than twice, more than 0.01 times, or more than 0.001 times, 0.0001, 0.00001, it can be predicted that the possibility of developing periodontal disease is high.

Furthermore, in the present invention, when predicting or diagnosing a high possibility of developing periodontal disease by measuring the expression level of the protein or the gene encoding the protein measured for the biological sample of the subject as described above, the disease for the subject It may further include a step of performing an appropriate treatment such as drug administration, physical therapy, gene therapy, radiation therapy, or immunotherapy.

According to another embodiment of the present invention, the expression level of one or more proteins selected from the group consisting of P01622, A0A087X0N5, E9PH25, A0A0B4J1V0, H0YI36, Q9NZT2, and P07305 measured with respect to a biological sample of an individual or a gene encoding the same It relates to a periodontal disease diagnosis apparatus including a diagnosis unit for determining periodontal disease diagnosis information with respect to data included therein.

The apparatus for diagnosing periodontal disease of the present invention may further include a sample receiving unit for receiving a biological sample of an individual.

In the present invention, the biological sample is any material obtained from or derived from an individual, for example, a liquid biopsy, and may be, for example, blood, serum, plasma, GCF, or saliva.

The periodontal disease diagnosis device of the present invention measures the expression of at least one protein selected from the group consisting of P01622, A0A087X0N5, E9PH25, A0A0B4J1V0, H0YI36, Q9NZT2, and P07305, or a gene encoding the same, measured with respect to the biological sample accommodated in the sample receiving unit. An input unit for inputting a level (data to be diagnosed) may be further included. In the present invention, the input unit is the data to be diagnosed, for example, P01622, A0A087X0N5, E9PH25, A0A0B4J1V0, H0YI36, Q9NZT2, and P07305 for standardized housekeeping proteins or genes measured for a biological sample of an individual. A ΔCt value, which is a value of the relative expression level of at least one protein selected from the group consisting of or a gene encoding the same, may be input.

In addition, in the present invention, the input unit may perform preprocessing such as sorting, normalization, and/or scaling on the data to be diagnosed, or the data to be diagnosed may be input to the input unit after preprocessing.

In the present invention, multiple data to be diagnosed can be input to the input unit for one individual.

In particular, in the input unit of the periodontal disease diagnosis device of the present invention, to input the expression level of at least one protein selected from the group consisting of P01622, A0A087X0N5, E9PH25, A0A0B4J1V0, H0YI36, Q9NZT2, and P07305 or a gene encoding the same. The formulation and method for measuring the expression level overlap with those described in the method for providing information for diagnosing periodontal disease, so that detailed descriptions thereof are omitted below to prevent excessive complexity of the specification.

The apparatus for diagnosing periodontal disease of the present invention may include a diagnosis unit that determines periodontal disease diagnosis information with respect to data to be diagnosed input through the input unit.

In the present invention, the diagnosis unit may determine whether periodontal disease is positive or negative based on the possibility of periodontal disease or whether or not periodontal disease has occurred with respect to diagnosis subject data.

In the present invention, the diagnostic unit is one or more proteins selected from the group consisting of P01622, A0A087X0N5, E9PH25, A0A0B4J1V0, H0YI36, Q9NZT2, and P07305 measured with respect to the biological sample of the subject, as the diagnosis target data input to the input unit, or When the expression level of the coding gene is increased or decreased compared to the normal control group, the possibility of developing periodontal disease is high, or it can be determined as being positive for periodontal disease.

The present invention will be described in more detail through the following examples. These examples are only intended to illustrate the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

<Example 1> Analysis of expression markers in periodontal tissue

Example 1-1: Periodontal tissue analysis method

Periodontal tissues of the normal group (HT1-HT5) and the periodontitis patient group (IT1-IT18) were prepared.

In order to analyze the periodontal tissue, the analysis was performed in the following steps.

1) A protein mixture was obtained by tissue pulverization using a Covaris grinder under -80°C conditions.

2) The protein mixture was treated with 8M urea and lysed through high frequency sonication for 20 minutes, thereby obtaining a lysed protein mixture.

3) Protein was quantified through BCA protein assay with respect to the obtained protein mixture.

4) 5mM TCEP (tris(2-carboxyethyl)phosphine) was treated as a reducing agent, and incubation was performed at 37°C for 30 minutes.

5) 15mM IAA (Iodoacetamide) was treated and incubation was performed at 25° C. for 30 minutes in a dark environment.

6) 3 times the volume of the sample was treated with 1M Tris, and pH 8-9 was confirmed.

7) Trypsin was treated at a ratio of 1:50 to the amount of protein and incubated at 37 °C for 16 hours.

8) Formic acid was treated to confirm pH 2-3.

9) A desalting process was performed on the sample.

10) Protein was analyzed using LC-MS/MS (Q-Exactive).

11) Data derived through Thermo Scientific™ Proteome Discoverer 2.5.0.400 were analyzed.

Example 1-2: Protein quantification method (BCA protein assay)

1 shows the BCA protein analysis results for the samples of the normal group (HT1-HT5) and the periodontitis patient group (IT1-IT18).

In addition, the amount of protein for each sample through the Bicinchoninic acid assay (BCA protein assay) is shown in Table 1 below.

[Table 1]

Table 1 shows the results of quantitative analysis of protein through quality control (QC) of the sample using the BCA protein quantitative analysis method. Equal amounts of protein were processed for Trypsin digestion. The same amount of protein was subjected to Trypsin digestion, and then the same amount of peptide was subjected to LC-MS/MS analysis.

Example 1-3: Derivation of expressed protein

Label-free quantification using LC-MS/MS (Q-Exactive) was performed to analyze the protein. Chromatograms of protein analysis for each sample are shown in FIGS. 2a and 2b.

2a and 2b show base peak chromatograms of protein LC-MS/MS analysis results for each tissue sample of a normal group and a periodontitis patient group. The analysis time is 180 minutes, and the peptides separated by the gradient of the solvent and eluted are analyzed by MS/MS and appear as peaks. Label-free quantification analysis of tissue proteins was performed using the peak intensity of peptides. The conditions are as follows.

[Table 2]

In addition, the number of proteins for each sample derived through label-free quantification using LC-MS/MS (Q-Exactive) is shown in Table 3 below.

[Table 3]

Table 3 is The results show the number of proteins quantitatively and qualitatively analyzed in the periodontal tissue samples of the normal group and the periodontitis patient group through Thermo Scientific™ Proteome Discoverer 2.5.0.400. Among the quantitatively analyzed proteins, 262 over-expressed proteins and 869 under-expressed proteins were found in the periodontitis patient group. It can be seen that the number of proteins quantitatively and qualitatively analyzed is higher in the periodontitis patient group. In addition, more low-expressed proteins were identified in the periodontitis patient group.

Example 1-4: Proteomics analysis

Based on the LC-MS/MS analysis results, proteomics analysis was performed using Thermo Scientific™ Proteome Discoverer 2.5.0.400.

First, the LC-MS / MS analysis results of the periodontal disease patient group and the normal group were compared by performing Volcano plot analysis, and the results are shown in FIG. 3. (P-value = 0.05, Log2 fold change = 1)

Figure 3 shows the DEP of P-value < 0.05 and Log2 ratio > 1 or < -1 by statistical analysis (t-test) of tissue samples from the normal group and periodontitis patient group using Volcano plot. The x-axis represents Log2 ratio and the y-axis represents -Log10 (P-value). Among the overexpressed proteins in the periodontitis patient group, there were 1886 proteins with a Log2 ratio > 1 and 869 proteins with a P-value < 0.05. Among the low-expression proteins in the periodontitis patient group, there were 444 proteins with Log2 ratio < -1 and 262 proteins with P-value < 0.05. A total of 1131 different DEPs in the tissue samples of the normal group and the periodontitis patient group were determined as biomarker candidates.

In addition, principal component analysis (PCA) was performed on the LC-MS/MS analysis results of the periodontal disease patient group and the normal group (X data = PC1, Y data = PC2). The results are shown in FIG. 4 .

Figure 4 shows the difference between each group through PCA results by statistically analyzing the protein quantification of the tissue samples of the normal group and the periodontitis patient group with the intensity value of each peak of the protein. PCA shows an overall understanding of DEP and how the quantification of proteins differs between groups.

In addition, Heatmap analysis was performed based on the protein analysis results of periodontal patients and normal groups, and the results are shown in FIG. 5.

Figure 5 shows the statistical analysis of the protein quantification of the tissue samples of the normal group and the periodontitis patient group as a heatmap. Heatmap visualizes the comparison of protein quantification between groups. The heatmap was made of 4043 quantified proteins, and the quantitative difference of proteins between groups can be compared.

<Example 2> Analysis of expression markers in gingival crevicular fluid (GCF)

Example 2-1: Method for analyzing gingival fissure fluid

GCFs of normal group (normal1-6) and periodontitis patient group (perio1-7) were prepared.

In order to analyze the level of protein expression in the gingival sulcus fluid, the analysis was performed in the following steps.

1) Lysis was performed by performing high frequency sonication for 20 minutes with dental floss coated with GCF in 8M urea, and a protein mixutre was obtained.

2) Protein was quantified through BCA protein assay for the lysed protein mixture obtained above.

3) 5mM TCEP (tris (2-carboxyethyl) phosphine) was treated as a reducing agent, and incubation was performed at 37 ° C. for 30 minutes.

4) 15mM IAA (Iodoacetamide) was treated and incubation was performed at 25° C. for 30 minutes in a dark environment.

5) 3 times the volume of the sample was treated with 1M Tris, and pH 8-9 was confirmed.

6) Trypsin was treated at a ratio of 1:50 to the amount of protein and incubated at 37 °C for 16 hours.

7) Formic acid was treated to confirm pH 2-3.

8) A desalting process was performed on the sample.

9) Protein was analyzed using LC-MS/MS (Q-Exactive).

10) The data derived through Thermo Scientific™ Proteome Discoverer 2.5.0.400 were analyzed.

Example 2-2: Protein quantification method (BCA protein assay)

The protein amount (concentration) of the sample was confirmed using the Thermo Scientific™ Pierce™ BCA Protein Assay Kit.

Absorbance was measured for each concentration using a BCA protein assay standard solution, and the results are shown in Tables 4 and 5 below.

Table 4 below is the absorbance in periodontitis patient samples, and Table 5 is the absorbance in the BCA protein assay of normal people.

[Table 4]

[Table 5]

Based on the absorbance data, a calibration curve of y = ax + b was plotted as a BCA standard curve. Figure 6a is a BCA standard curve of a GCF sample from a patient with periodontitis, and Figure 6b is a BCA standard curve of a GCF sample from a normal person.

In addition, the amount of protein measured in the BCA Protein Assay in repeated samples for each sample is shown in the table below.

Table 6 below shows protein quantification results in 7 samples from patients with periodontitis, and Table 7 shows protein quantification results in 6 samples from normal people.

[Table 6]

[Table 7]

Example 2-3: Derivation of expressed protein

Label-free quantification using LC-MS/MS (Q-Exactive) was performed to analyze the protein. Chromatogram results of protein analysis for each sample are shown in FIGS. 7a and 7b.

7a and 7b show base peak chromatograms of protein LC-MS/MS analysis results for each tissue sample of a normal group and a periodontitis patient group. The analysis time is 180 minutes, and the peptides separated by the gradient of the solvent and eluted are analyzed by MS/MS and appear as peaks. The conditions are shown in Table 8 below.

[Table 8]

Example 2-4: Proteomics analysis

*Based on the LC-MS/MS analysis results, proteomics analysis was performed using Thermo Scientific™ Proteome Discoverer 2.5.0.400.

First, the LC-MS / MS analysis results of the periodontal disease patient group and the normal group were compared by performing Volcano plots analysis, and the results are shown in FIG. 8 . (P-value = 0.05, Log2 fold change = 1)

8 shows the DEP with P-value < 0.05 and Log2 ratio > 1 or < -1 by t-test statistical analysis of GCF samples of the normal group and periodontitis patient group using Volcano plot. The x-axis represents Log2 ratio and the y-axis represents -Log10 (P-value). Among the overexpressed proteins in the periodontitis patient group, there were 332 proteins with a Log2 ratio > 1 and 166 proteins with a P-value < 0.05. Among the low-expression proteins in the periodontitis patient group, there were 444 proteins with a Log2 ratio < -1 and 148 proteins with a P-value < 0.05. A total of 314 different DEPs in the tissue samples of the normal group and the periodontitis patient group were determined as biomarker candidates.

In addition, principal component analysis (PCA) was performed on the LC-MS/MS analysis results of the periodontal disease patient group and the normal group (X data = PC1, Y data = PC2). The results are shown in FIG. 9 .

Figure 9 shows the difference between each group through PCA results by statistically analyzing the protein quantification of GCF samples from the normal group and the periodontitis patient group with the intensity value of each protein peak. PCA shows an overall understanding of DEP and how the quantification of proteins differs between groups.

In addition, heat map analysis was performed based on the protein analysis results of periodontal patients and normal groups, and the results are shown in FIG. 10 .

10 shows the statistical analysis of the protein quantification of the GCF samples of the normal group and the periodontitis patient group and shows them as a heatmap. Heatmap visualizes the comparison of protein quantification between groups. The heatmap was made with 1491 quantified proteins, and the quantitative difference of proteins between groups can be compared.

<Example 3> Marker discovery through Differentially Expressed Protein (DEP)

Example 3-1: DEP analysis of periodontal tissue and GCF samples

DEP analysis was performed on the expression patterns of proteins identified in tissue and GCF samples of patients with periodontitis and normal subjects, and the protein expression patterns identified in both samples were cross-grouped, and 38 proteins (2.7%) appeared in the intersection (Fig. 11). Among these 38 proteins, 20 biomarker candidates whose expression level was increased or decreased according to the DEP result were identified. The results are shown in Table 9 below.

[Table 9]

Example 3-2: Identification of novel biomarkers

Among the 20 biomarker candidates, proteins previously unknown as biomarkers of periodontal disease were classified, and the results are shown in the table below.

[Table 10]

Claims (1)

A kit for diagnosing the subject's periodontal disease by measuring the expression level of at least one protein selected from the group consisting of A0A087X0N5, E9PH25, A0A0B4J1V0, H0YI36, Q9NZT2, and P07305 from the gingival sulcus fluid (GCF) of the subject or a gene encoding the same. in
The expression level of one or more proteins selected from the group consisting of A0A0B4J1V0, HOYI36, and Q9NZT2 or a gene encoding the same is overexpressed in a sample of a patient with periodontal disease compared to a sample of a normal person,
The expression level of at least one protein selected from the group consisting of A0A087X0N5, E9PH25, and P07305 or a gene encoding the same is lower in samples of patients with periodontal disease than in samples of normal people,
The kit is a reverse transcription polymerase reaction (RT-PCR) kit, a DNA chip kit, or a protein chip kit, which includes essential elements necessary for performing a reverse transcription polymerase reaction, a DNA chip, a protein chip, or an ELISA,
A kit for diagnosing periodontal disease.