KR101765397B1 - The use of il-33 to diagnose the susceptibility of intravaginal viral infection - Google Patents

Abstract

The present invention relates to the use of IL-33 as a vulnerable diagnostic marker for viral infections through the vaginal mucosa. When the diagnostic method of the present invention is used, since the accuracy of diagnosis of vaginal viral infection-related diseases is increased, it is expected to be useful for prediction and diagnosis of effective vaginal viral infection-related diseases.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the use of IL-33 as a diagnostic marker for susceptibility to viral infection through the vaginal mucosa. BACKGROUND OF THE INVENTION 1. Field of the Invention < RTI ID = 0.0 >

The present invention relates to the use of interleukin-33 (IL-33) as a marker for susceptibility to viral infection through vaginal mucosa.

Interleukin is a protein that stimulates the immune system to fight against bacteria or harmful substances that enter the body and is made from white blood cells. IL-1, IL-2, IL-3, IL-4, IL-5 and IL-6 have been identified in the physical and chemical description of interleukins in various types of white blood cells. The interleukins act together to cause the leukocytes to react cascade against disease.

IL-33 is a recently introduced interleukin belonging to the superfamily of IL-1 that signals through ST2, a heterodimer accepting complex composed of ST2L and IL-1R accessory proteins (Liew, FY et al. (2010), Nat. Rev. Immunol. 10: 103-110; Kurowska-Stolarska, M. et al., (2011) J. Intern Med 269: 2935). IL-33 acts as an alarming, similar to high-mobility group box protein 1 (HMGB1) and IL-1α to signal the risk of innate immune system cells in response to trauma or tissue damage during infection (Luthi, AU et al., (2009), Immunity 31: 84-98; Oboki, K. et al., (2010), Proc. Natl. Acad Sci. USA 107: 18581-18586). IL-33 is targeted to cells that are released into the extracellular matrix and induce inflammation after cell injury, mechanical injury, or necrosis. IL-33 may also be used in the treatment of macrophages, dendritic cells, mast cells, basophils, eosinophils, natural helper cells, nuocytes, It is also known to act on multipotent progenitors. IL-33 is also an important cytokine of chronic inflammatory arthritis that integrates activation of fibroblasts and Th1 and Th17 effecting T cells working through mast cell-dependent pathways (Xu, D. et al., (2008 ), Proc Natl Acad Sci USA 105: 10913-10918).

The present invention confirms the use of IL-33 in the vaginal mucosa, which differs from previous studies that IL-33 acts against virus-like pathogens through the activation of innate immune cells and promotes immune responses. In conclusion, IL-33 secreted from the vaginal epithelium inhibits antiviral immunity, and it is expected that the use of IL-33 as a novel diagnostic marker for viral infection-related diseases through vaginal mucosa is expected.

The present invention provides a pharmaceutical composition for the treatment of vaginal mucosal viral diseases, including a diagnosis, a diagnostic kit, a screening method, and an IL-33 inhibitor through the confirmation of expression level of IL-33 gene or protein in the vaginal mucosa .

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

Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, for purposes of complete understanding of the present invention, various specific details are set forth, such as specific forms, compositions and processes, and the like. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well-known processes and techniques of manufacture are not described in any detail, in order not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "embodiment" means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Accordingly, the appearances of the phrase " in one embodiment "or" an embodiment "in various places throughout this specification are not necessarily indicative of the same embodiment of the present invention. In addition, a particular feature, form, composition, or characteristic may be combined in any suitable manner in one or more embodiments.

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

In one embodiment of the present invention, "IL-33" refers to IL-33 secreted from vaginal epithelium after oral antibiotic treatment. IL-33 in the vaginal mucosa inhibits the secretion of IFN-γ, a major antiviral cytokine secreted in vaginal tissue, which is a function of effective T cells, resulting in antiviral immunity against mucosal HSV-2 (herpes simplex virus 2) infection .

In one embodiment of the present invention, "vaginal mucosal viral disease" means a disease caused by herpes simplex virus (HSV). There are two types of herpes simplex virus: HSV-1 and HSV-2. HSV-1 is responsible for herpes zoster around the mouth, HSV-2 is the most common herpes that causes inflammation in the male and female reproductive system, and vaginal viral disease in the present invention is a disease caused by HSV-2 Refers to genital herpes or cervicitis.

In one embodiment of the present invention, the term " diagnosis "means identifying the presence or characteristic of a pathological condition. For the purpose of the present invention, the diagnosis is to check whether a viral infection-related disease is caused through the vaginal mucosa. In the present invention, the presence of at least one IL-33 mRNA or protein in the diagnosis of viral infection-related diseases through the vaginal mucosa indicates that the expression level of mRNA or protein encoding IL-33 is increased And the like.

The term "diagnostic marker, marker for diagnosis or diagnostic marker" in the present invention refers to a substance capable of distinguishing a target cell from normal cells and includes a polypeptide or nucleic acid (Such as mRNA), lipids, glycolipids, glycoproteins, or organic biomolecules such as sugars (monosaccharides, disaccharides, oligosaccharides, etc.). For the purpose of the present invention, the disease diagnostic marker of the present invention is a mRNA of the IL-33 gene which is expressed specifically at a low level in viral infection-related diseases through the vaginal mucosa, It is a protein.

The above-mentioned "measurement of mRNA expression level " is a process of confirming the presence and expression level of mRNA of disease marker genes in a biological sample in order to diagnose disease, and can be determined by measuring the amount of mRNA. (RT-PCR), Competitive RT-PCR, Real-time RT-PCR, RNase protection assay (RPA), Northern blotting Northern blotting, or DNA chip, but are not limited thereto.

The agent for measuring mRNA expression level of the gene refers to a molecule that can be used for detecting a marker by confirming the expression level of the IL-33 gene whose expression is increased in a target cell, Or a probe or a primer that specifically binds to the target. Based on the polynucleotide sequence of IL-33, one skilled in the art can design primers or probes that specifically amplify specific regions of these genes.

The term "primer" in the present invention refers to a nucleotide sequence having a short free 3 'hydroxyl group, capable of forming a base pair with a template complementary to the template, ≪ / RTI > The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i.e., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. In the present invention, PCR amplification using a sense and antisense primer of IL-33 polynucleotide can be used to diagnose disease through the production of a desired product. The PCR conditions, the lengths of the sense and antisense primers can be modified based on what is known in the art.

The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, capping, substitution of one or more natural nucleotides with one or more homologues, and modifications between nucleotides, such as uncharged linkers such as methylphosphonate, phosphotriester, (E.g., dodecanedioic acid, dodecanedioic acid, dodecanedioic acid, tartaric acid, tartaric acid, tartaric acid,

The term " measurement of protein expression level " in the present invention means a process for confirming the presence and the expression level of a protein expressed from a disease marker gene in a biological sample in order to diagnose disease, The amount of protein can be confirmed using an antibody that binds to the protein. Methods for analysis include Western blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, Rocket Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), Protein Chip, and the like, but are not limited thereto. It is not.

The agent for measuring the expression level of the protein refers to a molecule that can be used for detecting a marker by confirming the expression level of the protein expressed from the IL-33 gene, which is a marker for increasing expression in a target cell, And preferably an antibody specific for the protein. Based on the amino acid sequence of the protein expressed from the IL-33 gene, one skilled in the art can design antibodies specific to the protein.

In one embodiment of the invention, "antibody" refers to a specific protein molecule as described above, directed against an antigenic site. For the purpose of the present invention, the antibody refers to an antibody that specifically binds to the IL-33 peptide of the present invention, and the monoclonal antibody to the protein may be produced by a method for producing a monoclonal antibody common in the art Or a commercially available product may be used. In addition, a polyclonal antibody recognizing the protein may be used instead of the monoclonal antibody, or it may be produced and used through an antiserum preparation method customary in the art. The antibody of the present invention also includes partial peptides that can be made from the protein. Partial peptides of the present invention include at least 7 amino acids, preferably 9 amino acids, more preferably 12 amino acids do. The form of the antibody of the present invention is not particularly limited, and any polyclonal antibody, monoclonal antibody or antigen-binding antibody thereof may be included in the antibody of the present invention and include all immunoglobulin antibodies. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies. Antibodies used in the detection of disease diagnostic markers 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 refers to a fragment having at least an antigen-binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

In one embodiment of the present invention, the term "peptide" refers to a polymer of amino acids, wherein the linkage between amino acids is a polymer comprising an amide bond or a peptide bond.

In one embodiment of the present invention, "nucleic acid" is a polymer organic substance in which a nucleotide is polymerized in a long chain shape, and is genetic information encoding a specific peptide in the present specification.

In one embodiment of the present invention, the term "diagnostic kit " refers to a collection of compositions and accessories necessary for a particular purpose. For the purpose of the present invention, the diagnostic kit of the present invention is to confirm the onset of a viral infection-related disease through the vaginal mucosa. The kit of the present invention includes a primer, a probe, an antibody that selectively recognizes a peptide, or a specific peptide that specifically increases expression upon infection with a viral infection-related disease through a vaginal mucosa, As well as one or more other component compositions, solutions or devices suitable for the assay method.

In one embodiment of the present invention, there is provided a method of identifying an IL-33 mRNA, comprising: determining the level of IL-33 mRNA expression from a biological sample isolated from a desired vaginal mucosal-derived subject; Confirming the level of IL-33 mRNA expression from a biological sample isolated from a normal subject; And comparing the expression level of the IL-33 mRNA of the normal subject with the target subject. When the expression level of the IL-33 mRNA of the target subject is higher than that of the normal subject, Provides a way to provide information about the diagnosis that is relevant. In this embodiment, a method of providing diagnostic information, characterized in that said step of IL-33 is treated with an antibiotic prior to the determination of expression level, wherein in said embodiment, specifically binding to IL-33 mRNA Wherein the vaginal viral disease is selected from the group consisting of a herpes zoster virus or a cervicitis virus. The method of claim 1, The method comprising the steps of:

In one embodiment of the present invention, there is provided a method of identifying an IL-33 protein, comprising: determining the level of expression of an IL-33 protein from a biological sample isolated from a desired vaginal mucosal-derived subject; Confirming the expression level of the IL-33 protein from the biological sample separated from the normal subject; And comparing the expression level of IL-33 of the normal subject with that of the subject. If the level of IL-33 expression in the subject of interest is lower than that of the normal subject, And provides a method of providing information about the diagnosis that can be found. In this embodiment, there is provided a method of providing diagnostic information, characterized in that said step of IL-33 is treated with an antibiotic prior to the determination of expression level, wherein in said embodiment, Wherein the vaginal viral disease is one of genital herpes or cervicitis. The method of claim 1, wherein the viral mucosal viral disease is one of vaginal herpesvirus or cervicitis And provides a method for providing diagnostic information.

In one embodiment of the present invention, there is provided a diagnostic kit for vaginal viral disease, comprising a primer or a probe that specifically binds IL-33 mRNA. In this embodiment, the vaginal viral disease is one of genital herpes or cervicitis.

In one embodiment of the present invention, there is provided a diagnostic kit for vaginal viral disease, comprising an antibody that specifically binds to an IL-33 protein, wherein said vaginal mucosal viral disease is a herpes zoster or cervicitis And a diagnostic kit for vaginal viral disease.

In one embodiment of the present invention, the step of contacting a sample to be analyzed with a cell expressing the IL-33 gene; Measuring the expression level of the gene; And a step of determining that the sample is a substance for the prevention or treatment of vaginal viral disease when the expression level of the IL-33 gene is measured to be down-regulated, for the prevention or treatment of vaginal viral diseases A method for screening a substance is provided.

In one embodiment of the invention, contacting the sample to be analyzed with a cell expressing the IL-33 protein; Measuring the amount or activity of the protein; And determining that the sample is a substance for the prevention or treatment of vaginal mucosal viral disease when the amount or activity of the IL-33 protein is measured as being down-regulated. ≪ / RTI >

In one embodiment of the invention, there is provided a pharmaceutical composition for the treatment of vaginal mucosal viral diseases comprising an IL-33 inhibitor. In the above embodiment, the vaginal viral disease is one of genital herpes or cervicitis, and the pharmaceutical composition for treating vaginal viral diseases is provided. In this embodiment, the present invention provides a pharmaceutical composition for treating vaginal viral disease, wherein the IL-33 inhibitor is an anti-IL-33 antibody.

The present invention aims to confirm the expression level of IL-33 in viral infectious diseases through the vaginal mucosa, and the expression pattern of IL-33 gene is used to diagnose a viral infection disease through the vaginal mucosa promptly and more accurately. And it is expected to be useful for the prediction and diagnosis of viral infectious diseases through effective vaginal mucosa.

FIG. 1 shows immunity control of mucosal HSV-2 in a group of symbiotic microorganisms according to an embodiment of the present invention, comparing the survival of mice treated with antibiotic (ABX) and HSV-2.
Figure 2 shows that IFN- [gamma] and IFN-inducible chemokines CXCL9 and CXCL10 are reduced in production after mucosal HSV-2 infection according to one embodiment of the present invention.
FIG. 3 shows that IL-33 secretion according to an embodiment of the present invention is induced in vaginal mucosa after treatment with antibiotics, and shows albumin and IL-33 levels measured in vaginal rinse solution of mice administered with antibiotics.
Figure 4 shows that IL-33 in accordance with one embodiment of the present invention inhibits immunosuppression against mucosal HSV-2 infection, indicating that IFN-y production is inhibited and defective in effect T cell recruitment .

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example  1: Preparation of mouse

Example  1-1. C57BL / 6 and IL-33 ^{- / -}  mouse

6-8 weeks female C57BL / 6 specific pathogen-free mice were treated with DBL Co. Ltd, Korea. IL-33 ^{- / -} mice were received from the RIKEN Center for Developmental Biology. All mice were raised at KAIST's specific pathogen-free facility. All animal-related experiments were in accordance with guidelines and policies for rodent experiments provided by KAIST's Institutional Animal Care and Use Committee (IACUC). The protocol of the present invention was approved by IACUC (IACUC-13-140) of KAIST. Exclusion criteria For example, low cell yields due to inadequate staining or technical problems have been previously determined. The animals were randomly assigned to the test group. Mouse studies were not performed in a blinded fashion.

Example  1-2. Antibiotic treatment

Mice were treated with ampicillin (500 mg / L, AG Scientific), vancomycin (250 mg / L, AG Scientific), neomycin sulfate (500 mg / L, AG Scientific), gentamicin (500 mg / L, AG scientific) and metronidazole 500 mg / L, Sigma) for 4 weeks. Antibiotic treatment started 4 weeks before infection and persisted for the duration of the experiment. In some experiments, individual antibiotics (indicated concentrations) were administered to drinking water.

Example  1-3. Viruses and Vagina  infection

WT and TK-HSV-2 strains were supplied to A. Iwasaki (Yale University, New Haven, CT) and used for all experiments. HSV-2 was propagated and titrated in a Vero Cell using a plaque assay. In vaginal viral infection, mice were inoculated subcutaneously with medroxyprogesterone acetate (Tokyo Chemical Industry Co., Ltd.) at 2 mg / mouse 5 to 7 days before infection, wiped with calcium alginate , 10 ⁴ pfu WT or 10 ⁶ pfu TK-HSV-2 in 10 μl was vaginally inoculated using a blunt-ended micropipette tip. Upon WT HSV-2 inoculation, disease severity was scored as follows: 0, no response; 1, mild genital erythema and edema; 2, moderate genital inflammation; 3, purulent genital tract; 4, hind limb paralysis; 5, Premoribund.

Example  1-4. TLR  Local or terminal stimuli as an acting agent

In vaginal infusion, water-fed and antibiotic-treated mice are wiped with calcium alginate, and Escherichia 10 μl of PBS containing LPS in E. coli (50 μg, Sigma-Aldrich) was injected 24 hours before vaginal viral infection. In intrarectal inoculation, mice were injected with Escherichia 50 [mu] l of PBS containing LPS in E. coli (5 [mu] g, Sigma-Aldrich) was inoculated rectally at the same time or 24 hours before virus infection. Upon oral administration of a TLR agonist, mouse Escherichia 100 μl of PBS containing LPS in E. coli (25 mg / kg, Sigma-Aldrich) was supplied every two weeks before viral infection for 2 weeks.

Example  1-5. Vagina Papain papain injection

Mice were injected subcutaneously 7 days before the infection with 2 mg / mouse of medroxyprogesterone acetate (Tokyo Chemical Industry Co., Ltd.) in 100 占 퐇, wiped with calcium alginate, and inoculated with 200 占 퐂 papain (Calbiochem).

Example  1-6. Recombinant IL-33 treatment

Recombinant mouse IL-33 (rIL-33) was purchased from BioLegend. 2 μg of rIL-33 was intraperitoneally injected into the mice for 8 days every day before vaginal viral infection.

Example  1-7. Recombinant IL- 1β  process

Recombinant mouse IL-1? (RIL-1?) Was purchased from PeproTech. In vaginal infusion, drinking water-treated and antibiotic-treated mice were injected with 100 ng of rIL-1 [beta] 4 days before and 5 days after vaginal infection. During intraperitoneal injection, 500 ng of rIL-1 [beta] was injected into the mice one day before and one day and three days after vaginal infection.

Example  2: Performing inspection and analysis for diagnosis

Example  2-1. Cytokine and albumin measurement

The vaginal solution was collected from 0 to 6 days after infection with PBS (50 μl) by pipetting 20 times in and out of the vagina. IFN- ?, IL-33, IL-25, TSLP (eBiosciences), IL-1 ?, IL-12p40, IL-6 (BD Biosciences), IL-4, IFN- ?, TNF- The levels of IL-1 alpha (BioLegend), CXCL9, CXCL10 (R & D Systems), IL-18 (MBL Co., Ltd) and albumin (Immunology Consultants Laboratory Inc.) were measured using an ELISA kit according to the manufacturer's instructions . The levels of albumin and IL-33 collected in vaginal washes of C57BL / 6 mice receiving antibiotics are shown in FIG.

Example  2-2. Quantitative In PCR  by HSV -2 glycoprotein ( gylcoprotein ) B DNA detection

After infection, vaginal tissue was collected and whole genomic DNA was extracted according to the manufacturer's instructions (QIAamp DNA Mini Kit, QIAGEN). HSV-2 was measured by quantitative PCR (Bio-Rad) using primers for glycoprotein B (gB) (forward direction: 5'-CACCGCTACTCCCAGTTTATG-3 '; reverse direction: 5'-CGGTGGTCTCCATGTTGTT-3'). The purified DNA in WT HSV-2 was used as a basis for calculating pfu equivalents.

Example  2-3. RNA isolation, cDNA production and Reverse transcription - PCR

RNA was isolated from vaginal tissue by mechanical homogenization and RNeasy plus mini kit (QIAGEN), and cDNA was synthesized using M-MLV cDNA synthesis kit (Enzynomics). Quantitative PCR was performed using the SYBR Green-based Quantification (Bio-Rad) CFX96 real-time PCR detection system. The gene of interest was normalized to Hprt and showed differences for uninfected drinking water-treated control mice.

Example  2-4. CD4 and CD8 T-cell responses

HSV-specific T-cell responses were analyzed as described. On post-infection day 6, CD4 and CD8 T cells were transfected with 10 ⁶ pfu of TK-HSV-T cells using anti-CD4- or anti-CD8-conjugated microbeads (Miltenyi Biotech) 2 < / RTI > from a raining lymph node of a vaginally infected mouse. 1x10 ⁵ CD4 and CD8 T cells were re-stimulated with varying amounts of heat-inactivated HSV-2 or HSV gB peptide (SSIEFARL) for 72 hours at 37 ° C. IFN-y produced by CD4 and CD8 isolated from the draining lymph node was then measured by ELISA (Fig. 4). Inflow levels of CD4 and CD8 into vaginal tissue were measured (Figure 4).

Example  2-5. Flow cell  Analyzer

Single cell suspensions were prepared from iliac lymph nodes and vagina. Lymph nodes were cut with collagenase type IV (Worthington) and DNase I (Roche). The vials were treated with Dispase II (Roche) for 15 minutes and cut with collagenase type IV, DNase I and hyaluronidase (MP Biomedicals) for 45 minutes. Single cells were pretreated with anti-CD16 / 32 antibody (2.4G2) and stained with the following antibodies: CD8α (53-6.7), B220 (RA3-6B2), Ly6C (AL-21) CD6b (1A8), CD45.2 (104), CD11c (HL3), CD11b (M1 / 70), CD44 (IM7), Siglec-F (E50-2440) and NK 1.1 (PK136) (BD Biosciences); CD3ε (145-2C11), CD4 (GK1.5), CD11c (N418), CD317 (BST-2, eBio927), CD62L (MEL-14), FcεRI -kit (2B8) and IL-7R (CD127, A7R34) (eBiosciences); MHCII (M5 / 114.15.2) and ST2 (DIH9) (BioLegend). Leukocytes were gated based on forward and side-scatter characteristics and living cells were excised from 4 ', 6-diamidino-2-phenylindole (DAPI) (Invitrogen) or propidium iodide (PI) propidium iodide exclusion). All samples were obtained with an LSR Fortessa cell analyzer (BD Biosciences). Leukocytes from iliac lymph nodes and vagina of infected mice were incubated for 5 hours in the presence of 50 ng / ml of porphyrin myristate acetate (PMA) (Sigma-Aldrich) and 1 ug / ml ionomycin (Sigma-Aldrich) GolgiStop (BD Biosciences) was added for the last 2 hours. Cells were surface stained with CD3ε (145-2C11), CD4 (GK1.5) (eBiosciences), CD8α (53-6.7) and CD44 (IM Biosciences) and cultured in Cytofix / Cytoperm kit Biosciences) and permeabilized. APC-labeled anti-mouse IFN-y antibody (XMG1.2; BD Biosciences) or PE-labeled anti-mouse IL-4 antibody (11B11; BD Biosciences) was used for intracellular cytokine staining. All data were analyzed with Flow Jo (Treestar).

Example  2-6. Immunoblot ( Immunoblot ) analysis

The vaginal tissue was isolated and lysed. Tissue lysates were digested by SDS-PAGE, transferred to PVDF membranes and examined using antibodies against IL-1 (R & D Systems) and ASC (Santa Cruz Biotechnology).

Example  2-7. Bacterial recovery and identification

A stool specimen collected directly from the anus of the mouse was weighed and homogenized in PBS. The washes were collected as described. The specimens are spread on selective agar plates on gram-positive (colistin, nalidix acid), gram-negative (MacConkey) and anaerobic (anaerobic basal agar) bacteria (Oxoid). Bacterial colonies were counted after incubation at 35 ° C for 48 hours (gram-positive and gram-negative conditions) or after incubation under anaerobic conditions (anaerobic bacteria) for 5 days at 37 ° C.

Example  2-8. 16S rDNA  Acquisition and quantification

Stool pellets were collected and extracted with QIAamp DNA Stool Mini Kit (QIAGEN) according to the manufacturer's instructions. In vaginal cleansing specimens, the washing solution was spun down to collect the pellets and whole DNA was extracted using FastDNA ™ SPIN Kit for Soil (MP Biomedicals) according to the manufacturer's instructions. 16S rDNA was quantified by quantitative PCR with a primer (forward direction: 5'-AGAGTTTGATCCTGGCTCAG-3 '; reverse direction: 5'-CTGCTGCCTYCCGTA-3') for detecting 16S rDNA of bacteria.

Example  2-9. DNA extraction and manipulation

Bacterial DNA was extracted from vaginal washes using a protocol published by Fujimoto, et al. (Fujimoto et al., (2004), Memoirs of School of Medicine, Kyushu University 3, 33-37). The extracted nucleic acid was maintained at -196 캜 in liquid nitrogen until use. The 5'-CCTATCCCCTGTGTGCCTTGGCAGTC-TCAG-AC- AGAGTTTGATCMTGGCTCAG- 3 '(the underlined sequence represents the target region primer) and the 541R (5'-CCATCTCATCCCTGCGTGTCTCCGAC-TCAG-X-AC-ATTACCGCGGCTGCTGG-3 ', wherein' X 'represents a specific barcode for each subject). The following PCR reaction conditions were applied: after denaturation at 95 ° C for 5 minutes, denaturation to 30 cycles at 95 ° C for 30 seconds, primer annealing at 55 ° C for 30 seconds, finalization at 72 ° C for 5 minutes And elongated at 72 ° C for 30 seconds. The amplified product was purified with a QIAquick PCR purification kit (QIAGEN). Equal concentrations of purified PCR products were pooled together and short fragments (non-target products) were removed using Ampure bead kit (Agencourt Bioscience). Quality and product size were evaluated using a DNA 7500 chip on a Bioanalyzer 2100 (Agilent). The PCR product was pyrosequenced using a GS Junior Sequencing System (Roche) in Chunlab, Inc. (Seoul, Korea) according to the manufacturer's instructions.

Example  2-10. Bioinformatics analysis

A basic analysis of microbial diversity was carried out. Ambiguous nucleotides, low-quality scores (average score <25), or eliminates readings shorter than 300 bp. Potential chimeric sequences were identified and removed using the bellerophone method to compare BLASTN search results between forward half-sequence and reverse half-sequence. Strain 16S rRNA gene with a representative species-level phylotype of entries in the GeneBank database, which is cultured or not cultured, which is a complete systematic taxonomic classification from valid published names and phylum to specie The EzTaxon-e database containing sequences was used to determine the taxonomic location of each reading.

Example  2-11. In monoacid analysis  Materials and Chemicals

(ABC), dithiothreitol (DTT), formic acid, iodoacetamide (IAA) and L-cysteine were purchased from Sigma-Aldrich (St. Louis, Mo., USA). Sequence grade trypsin was obtained from Promega Corp. (Madison, Wis., USA). Oasis HLB cartridges were purchased from Waters (Milford, MA, USA). With respect to the relative quantitative profiling of the proteome (proteome) in the vaginal washing liquid, to obtain the iTRAQ reagents ^® -4plex in AB Sciex (Framingham, MA, USA ). HPLC-grade acetonitrile and water for binary gradient elution were obtained from Burdick & Jackson (Ulsan, Korea). Molten-silica capillaries (25, 50, 75 and 100 μm-id; 360 μm-od) were used for capillary LC columns and tubing connections were obtained from Polymicro Technology LLC (Phoenix, AZ, USA). Magic C18AQ (3 탆, 100 Å) and Magic C18AQ (3 탆, 200 Å) resin were purchased from Michrom BioResources Inc. (Auburn, CA, USA). Fittings, adapters and PEEK tubing were purchased from Upchurch Scientific ^® (Oak Harbor, WA, USA) from IDEX Health & Science LCC.

Example  2-12. Cleavage by trypsin ( Tryptic  digestion) and iTRAQ  sign

Mice treated with antibiotics and three different antibiotic-treated mice, designated MVABX-0, MVABX-7, MVABX-14 and MVABX-21, respectively, on days 7, 14 and 21 before proteolysis with trypsin The protein concentration of the vaginal wash liquor sample was determined by the Bradford assay (Bio-Rad Protein Assay). For cleavage by trypsin, each vaginal wash specimen was separately dissolved in a 50 mM ABC solution containing 10 mM DTT and incubated at 37 [deg.] C for 2 hours. To allylate the remaining thiol groups, each aliquot was treated with 20 mM IAA in the dark at room temperature (RT) for 30 minutes and then with L-cysteine (40-fold excess of IAA) Excess IAA was removed for 30 minutes. Four vaginal specimens were then cut at 37 째 C for 18 hours using sequence grade trypsin (1/40 of the target protein volume). The result of cleavage by trypsin was demineralized with an Oasis HLB cartridge, lyophilized using SpeedVac, and reduced for the following isobaric tagging treatment at pH 8.5 with 50 mM triethylammonium bicarbonate (TEAB). In the quantitative evaluation, each vaginal excision (digest) was labeled with the iTRAQ reagent according to the manufacturer's instructions. Namely, the cleavage by four trypsins of MVABX-0, MVABX-7, MVABX-14 and MVABX-21 were individually reacted with iTRAQ reagents -114, -115, -116 and -117, respectively, at room temperature for 2 hours . The differentially labeled cuts were then mixed equally, re-lyophilized and then reduced for the next nLC-ESI-MS / MS process with 0.1% formic acid.

Example  2-13. nLC - ESI -MS / MS analysis

Nano-flow fluid chromatography-electrospray-tandem mass spectrometry (nLC-ESI-MS / MS) was used to qualitatively / quantitatively analyze antibiotic-derived mouse proteomics. The nLC-ESI-MS / MS analysis was performed by connecting the binary slope elution and Agilent Technologies Model 1260 capillary LC system to Q-Exactive FT orbitrap-mass spectrometer (FT orbitrap-MS / MS) from Thermo Scientific Respectively. A pre-column (RP) for capturing the peptide by trypsin was used with an analytical capillary RPLC column (150mM x 75μm-id). The pulled tip analytical capillary column used in the present invention was prepared with Magic C18AQ (3 탆, 100 Å) resin. For the purpose of RPLC separation of the peptide mixture, a trap column was prepared in a capillary (100 mu m-id, 360 mu m-od) with an end frit (2 mM length) and the capillary was placed in a Magic C18AQ 3 mu m, 200 ANGSTROM) resin. Trap column and analytical column were connected via PEEK microcross. During the RPLC process, two buffer solutions were used as mobile phase solutions: (A) 2% ACN in water containing 0.1% formic acid and (B) 93% ACN containing 2% DMSO in water. The flow rate for the RPLC process is 200 nL / min and the binary slope process conditions are as follows: A sample is added to the trap column for the first 10 minutes at 0% B, the initially increased 8% B for 2 minutes The RP column was maintained for 2 minutes at 80% for 15 minutes, 80% for 3 minutes, 80% for 32 minutes, and 20 minutes at 15% , And the RP column was injected for 20 minutes prior to maintenance to re-balance. The peptide eluted from the capillary column was directly injected into the FT orbitrap-MS via electrospray ionization in a cation mode at a capillary temperature of 270 ° C and a voltage of 2. kV was applied to the microcross through a lead wire. Data-dependent MS / MS analysis was performed on the ten most important precursor ions at each precursor scan (m / z 300-1800). The dynamic exclusion duration performed for the ten most significant MS / MSs was 30 seconds, and the normalized impact energy was 27 eV in the high-energy impact separation (HCD) experiment. The mass resolution for MS and MS / MS scans was set at 70,000 and 35,000, respectively, with a maximum injection time of 60ms. MS / MS spectra obtained through nLC-ESI-MS / MS analysis were analyzed using the Proteome Discoverer software (Version 1.3.0.339), a 1% false positive selection option based on the mouse proteome database (UniProtKB; date 7/9/2014; 43,539 entries) ). The mass tolerance value for the precursor ion is 1.2 Da and for the product ion is 0.5 Da. Proteomic results were considered according to the following requirements; The cross-correlation (Xcorr) values are greater than 2.7, 3.5, and 4.3, respectively, for 2-fold, 3-fold and 50-fold charged ions. Fixed modifications were set to carbamidomethylation (+ 57.021464 Da) of cysteine as well as lysine and N-terminal iTRAQ markers (+ 144.10206 Da). Oxidation of methionine (+ 15.99492 Da) and iTRAQ labeling of serine, threonine and tyrosine were included as various mutations.

Example  2-14. Statistical analysis

 The experimental results are expressed as mean standard deviation (mean ± sem). Differences between groups at each specific time were analyzed by independent Student's t-test on both sides. The disease scores were analyzed by two-way ANOVA test. The difference in survival was assessed by a log-rank test. Statistical analysis was performed using GraphPad Prism 5.01 software (GraphPad Software, San Diego, Calif.).

Claims (17)

Confirming the expression level of IL-33 mRNA from a biological sample isolated from a desired vaginal mucosal-derived subject;
Confirming the level of IL-33 mRNA expression from a biological sample isolated from a normal subject; And
And comparing the expression level of mRNA of IL-33 of the target subject with the normal subject. When the expression level of IL-33 mRNA of the target subject is higher than that of the normal subject, vaginal viral disease Of the cases,
Wherein the vaginal viral disease is vaginal herp or cervicitis caused by a viral infection. The method according to claim 1,
The step of confirming the level of mRNA expression of IL-33 from the biological sample isolated from the desired vaginal mucosa-derived subject is followed by the step of confirming the presence of bacterial 16sRNA through pyroseqeuncing from the sample Further comprising:
And the possibility that the viral mucosal viral disease is likely to occur when the 16sRNA is not present is higher than that of the normal subject. The method according to claim 1,
A primer or a probe that specifically binds to IL-33 mRNA. delete Confirming the expression level of the IL-33 protein from the biological sample isolated from the desired vaginal mucosal-derived subject;
Confirming the expression level of the IL-33 protein from the biological sample separated from the normal subject; And
And comparing the expression level of IL-33 between the desired subject and the normal subject. When the level of IL-33 expression in the subject is higher than that of the normal subject, the possibility of developing vaginal viral disease However,
Wherein the vaginal viral disease is vaginal herp or cervicitis caused by a viral infection. 6. The method of claim 5,
The step of confirming the level of protein expression of IL-33 from the biological sample isolated from the desired vaginal mucosal-derived subject is followed by the step of confirming the presence of bacterial 16sRNA through pyroseqeuncing from the sample Further comprising:
And the possibility that the viral mucosal viral disease is likely to occur when the 16sRNA is not present is higher than that of the normal subject. 6. The method of claim 5,
An antibody that specifically binds to an IL-33 protein. delete A primer or a probe that specifically binds to IL-33 mRNA,
A diagnostic kit for predicting the incidence of vaginal herpes or cervicitis vaginal viral disease due to viral infection. 10. The method of claim 9,
Wherein the diagnostic kit further comprises a primer or a probe that specifically binds to 16s RNA of bacteria. An antibody that specifically binds to an IL-33 protein,
A diagnostic kit for predicting the incidence of vaginal herpes or cervicitis vaginal viral disease due to viral infection. 12. The method of claim 11,
Wherein the diagnostic kit further comprises a primer or a probe that specifically binds to 16s RNA of bacteria. Contacting the sample to be analyzed with the cell expressing the IL-33 gene, in vitro;
Measuring the expression level of the gene; And
And determining that the sample is a substance for preventing or treating a viral mucosal viral disease when measured that the expression level of the IL-33 gene is down regulation,
Wherein the vaginal mucosal viral disease is a genital herp or cervicitis caused by a viral infection. Contacting the sample to be analyzed with a cell expressing the IL-33 protein, in vitro;
Measuring the amount or activity of the protein; And
And determining that the sample is a substance for preventing or treating vaginal viral disease when the amount or activity of the IL-33 protein is measured as being down-regulated,
Wherein the vaginal mucosal viral disease is a genital herp or cervicitis caused by a viral infection. delete delete delete

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