WO2023157460A1 - Method for acquisition of data on autoimmune response involved in depression, aging and the like, and use of same - Google Patents

Method for acquisition of data on autoimmune response involved in depression, aging and the like, and use of same Download PDF

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WO2023157460A1
WO2023157460A1 PCT/JP2022/046880 JP2022046880W WO2023157460A1 WO 2023157460 A1 WO2023157460 A1 WO 2023157460A1 JP 2022046880 W JP2022046880 W JP 2022046880W WO 2023157460 A1 WO2023157460 A1 WO 2023157460A1
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sfpq
amino acid
antibody
acid sequence
seq
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PCT/JP2022/046880
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French (fr)
Japanese (ja)
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一博 近藤
直美 岡
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株式会社ウイルス医科学研究所
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Publication of WO2023157460A1 publication Critical patent/WO2023157460A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials

Definitions

  • the present invention relates to a method for obtaining data on autoimmune reactions involved in depression, aging, etc. and its use.
  • Typical symptoms of depression include depressed mood, loss of interest/pleasure, anorexia, overeating, sleep disturbance, hypersomnia, and psychomotor activity, as defined in the DSM-5 diagnostic criteria for major depression.
  • Known symptoms include sexual impatience or inhibition, fatigability, worthlessness and guilt, decreased ability to concentrate and think, and suicidal ideation.
  • depressed patients frequently develop aging-like symptoms such as cognitive function and memory decline, decreased libido, cardiovascular disease, osteoporosis, and diabetes. Are known. The frequent appearance of such aging-like symptoms has a great impact on the healthy life expectancy of depressed patients.
  • herpesviruses including human herpesvirus-6 (HHV-6 (HHV; humanherpesvirus) are known to have three states: primary infection, latent infection, and reactivation. Of these, the virus proliferates in the primary infection and reactivation states, and is known to cause various diseases. On the other hand, viral multiplication does not occur in the state of latent infection. Some viruses, such as Epstein-Barr virus, which is an oncogenic herpes virus, and Kaposi's sarcoma-associated herpes virus, are known to cause tumors in a latent state. Herpes simplex virus, varicella-zoster virus, cytomegalovirus, and HHV-7 are not known to cause latent disease.
  • Epstein-Barr virus which is an oncogenic herpes virus
  • Kaposi's sarcoma-associated herpes virus are known to cause tumors in a latent state.
  • HHV-6 SITH- 1 the Intermediate stage Transcript of HHV-6-1
  • Patent Document 1 discloses that antibodies against SITH-1 can be used to diagnose whether a subject is suffering from a mood disorder.
  • one aspect of the present invention is to identify a factor involved in depression and aging and to provide a method for using the same.
  • the method of obtaining data for diagnosing or testing SFPQ-related dysfunction includes a measuring step of measuring the antibody titer of an anti-SFPQ antibody in a sample isolated from a subject. Methods of obtaining data for diagnosing or testing related impairments.
  • the diagnostic agent for SFPQ-related dysfunction is at least one selected from the following (a1) to (a3), (b1) to (b3), and (c1) to (c3) is a diagnostic for SFPQ-related dysfunctions, including: (a1) a polypeptide represented by SEQ ID NO: 1; (a2) A polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 1; (a3) a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1; (b1) a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 2 or 3; (b2) a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 2 or 3, and having the ability to bind to an anti-SFPQ antibody; (b3) a polypeptide consisting of
  • the carrier for blood purification has, on its surface, at least one selected from the following (a1) to (a3), (b1) to (b3), and (c1) to (c3).
  • a blood purification carrier comprising: (a1) a polypeptide represented by SEQ ID NO: 1; (a2) A polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 1; (a3) a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1; (b1) a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 2 or 3; (b2) a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 2 or 3, and having the ability to bind to an anti-SFPQ antibody; (b3) a polypeptide consisting of an amino acid sequence having
  • An SFPQ-related dysfunction model animal is an SFPQ-related dysfunction model animal obtained by administering an SFPQ protein or an anti-SFPQ antibody to a non-human mammal.
  • a method for producing an SFPQ-related dysfunction model animal is a method for producing an SFPQ-related dysfunction model animal, comprising an administration step of administering an SFPQ protein or an anti-SFPQ antibody to a non-human mammal. .
  • the mechanism of occurrence of SFPQ-related dysfunction found by the present inventors can be used in various ways. Therefore, according to one aspect of the present invention, it is possible to provide a method for acquiring data on SFPQ-related dysfunction, a diagnostic agent, a carrier for blood purification, a model animal, and the like.
  • FIG. 2 shows the results of negative gel staining and the results of autoantibody test by Western Blot in Example 1.
  • FIG. FIG. 2 shows the results of SEAP reporter assay and the results of immunoprecipitation with anti-SFPQ antibody in Example 2.
  • FIG. 10 shows the results of analysis of anxious behavior of mice and the results of histological analysis of mouse brains (bridle) in Example 4.
  • FIG. FIG. 10 shows the results of gene expression analysis in mouse brain in Example 4.
  • FIG. 10 shows the results of gene expression analysis in mouse brain and olfactory bulb, and the results of histological analysis of mouse brain (hippocampus) in Example 5.
  • FIG. FIG. 10 shows the results of analysis of mouse spatial working memory and motor coordination ability in Example 5.
  • FIG. FIG. 10 is a diagram showing the results of gene expression analysis in the small intestine of mice and the measurement results of changes in body weight of mice during the immunization period in Example 6.
  • FIG. FIG. 10 is a diagram showing the results of gene expression analysis in mouse testis in Example 7.
  • FIG. 10 shows the results of gene expression analysis in mouse testis and the results of fluorescence immunostaining of testis sections in Example 7.
  • FIG. 10 shows the results of gene expression analysis in the skin of SFPQ-immunized mice in Example 8.
  • FIG. 10 shows the results of histological analysis of the skin of SFPQ-immunized mice in Example 8.
  • FIG. 10 shows the results of gene expression analysis in the heart of SFPQ-immunized mice in Example 9.
  • FIG. 10 shows the results of gene expression analysis in the lungs of SFPQ-immunized mice in Example 10.
  • FIG. FIG. 11 shows the results of analysis of PCR products, the results of measurement of fluorescence intensity of Neuro2a cells, and the results of measurement of cell body size of Neuro2a cells in Example 11.
  • FIG. FIG. 10 shows the results of analysis of gene expression in nerve cells in Example 12.
  • FIG. FIG. 10 is a diagram showing the results of gene expression analysis of H-1 cells in Example 13.
  • FIG. FIG. 10 is a diagram showing the results of gene expression analysis of H-1 cells in Example 13.
  • FIG. 10 shows the measurement results of body weight change and the measurement results of coordination ability of anti-SFPQ mouse monoclonal antibody tail vein-administered mice in Example 14.
  • FIG. FIG. 14 shows the analysis results of a behavioral test of mice to which an anti-SFPQ rabbit polyclonal antibody was administered via the tail vein and the results of gene expression analysis in the brain of mice to which an anti-SFPQ rabbit polyclonal antibody was administered via the tail vein, in Example 14.
  • SITH-1 is an autoimmune reaction against SFPQ, an RNA-binding protein in the body (e.g., autoantibody (antibody) against SFPQ). It was newly found to induce the production of SFPQ antibody). Furthermore, it was newly found that the antibody titer against SITH-1 and the anti-SFPQ antibody titer show a positive correlation, and that the anti-SFPQ antibody is highly positive in patients with depression. Further, as a result of further intensive studies on autoimmune reactions to SFPQ, the present inventors found that autoimmune reactions to SFPQ induce functional disorders similar to depression and aging in living organisms, and developed the present invention. Completed.
  • a data acquisition method for diagnosing or testing SFPQ-related dysfunction comprises a measuring step of measuring the antibody titer of anti-SFPQ antibody in a sample separated from a subject.
  • this data acquisition method may be referred to as “this data acquisition method”.
  • SFPQ-related dysfunction refers to an autoimmune reaction to SFPQ that inhibits the normal function of SFPQ in situations where SFPQ is required for normal functional expression of cells and organs. Intended for bodily dysfunction. More specifically, such SFPQ-related dysfunctions include psychomotor agitation or retardation, fatigability, poor concentration and thinking ability, or anxiety, which are common in depressed patients.
  • telomere dysfunction resembling aging in the brain, intestine, testis, skin, heart and/or lungs; or stem cell maintenance/differentiation, splicing, axonal survival, Tau protein production and phosphorylation, oxidative stress systemic dysfunction such as regulation, maintenance of telomere stability, PDE3A transcription, CD40 transcription, adipocyte differentiation, DNA damage repair, abnormalities in innate immune response;
  • SFPQ-related dysfunction may accompany the pathology of various diseases.
  • Diseases involving such SFPQ-related dysfunction include depression, dementia, Alzheimer's disease, Pick's disease, frontotemporal dementia (FTD), FTLD spectrum disorders, autism spectrum disorders, colon cancer, Nasopharyngeal cancer, breast cancer, prostate cancer, cystic fibrosis, lipid disorders, atherosclerosis, metabolic myopathy, ALS, infertility, frailty, menopause, etc., but not limited thereto. do not have.
  • SFPQ-related dysfunction is a concept that includes diseases associated with these SFPQ-related dysfunctions.
  • SFPQ-related dysfunction develops by being induced by an autoimmune reaction to SFPQ. More specifically, the induction of an autoimmune reaction against SFPQ leads to the production of anti-SFPQ antibodies, which inhibit cell function, resulting in SFPQ-related dysfunction. Therefore, by measuring the antibody titer of an anti-SFPQ antibody in a sample isolated from a subject, it is possible to obtain data for diagnosing or examining the presence or absence and degree of autoimmune reaction to SFPQ in the subject. Yes, in other words, data can be obtained for diagnosing or testing SFPQ-related dysfunctions.
  • the measurement step is not particularly limited as long as the antibody titer of the anti-SFPQ antibody in the sample separated from the subject can be measured.
  • the term "subject” refers to a target animal, preferably a mammal, most preferably a human. Subjects are not particularly limited in terms of age, sex, and the like.
  • sample separated from the subject is not particularly limited, but can be selected from, for example, blood, serum, plasma, urine and saliva.
  • sample refers to pathological specimens prepared by fixing these samples with a fixative (e.g., formalin), or lysates obtained by solubilizing these samples in a desired solution. is a concept that also includes
  • Anti-SFPQ antibody in this data acquisition method means an antibody that specifically recognizes SFPQ as an antigen and has a complementarity determining region (CDR) for binding to the SFPQ antigen.
  • An “anti-SFPQ antibody” can also be said to be an “antibody that reacts with SFPQ”.
  • SFPQ means (a1) a polypeptide (protein) represented by SEQ ID NO: 1, (a2) deletion of one or several amino acids in the amino acid sequence represented by SEQ ID NO: 1, Either a polypeptide consisting of a substituted or added amino acid sequence, or (a3) a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO:1 is intended.
  • the anti-SFPQ antibody can also be said to be an antibody that specifically binds (antigen-antibody reaction) to the polypeptides (a1) to (a3) above.
  • SEQ ID NO: 1 corresponds to the amino acid sequence of the human SFPQ protein (GenAank accession number NP_005057).
  • the upper limit of the number of substituted, deleted, inserted and/or added amino acid residues is, for example, 7, 6, 5, 4, 3, 2 or 1. sell.
  • amino acid sequence identity is, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.
  • amino acid sequences can be analyzed using the Gapped BLAST program. In this case the default parameters can be used. Additions or deletions (such as gaps) may be allowed in order to optimally align the amino acid sequences being compared.
  • antibody titer means the amount of the antibody that can affect the target (antigen), taking into consideration the amount and binding power of the antibody contained in the sample.
  • an “antibody titer” is the amount of antibody, defined as the functional dilution (or working concentration) of an antibody sample required to achieve a minimal level of specific detection in a given assay. It may be the amount of the antibody sample that takes into account both the binding strength and the binding strength.
  • the antibody titer can also be said to be the titer of the antibody.
  • the measurement step in the present data acquisition method is a step of measuring the "antibody titer", which is the sum of the antibody amount and the binding strength of the anti-SFPQ antibody.
  • the antibody titer can be measured using a known method, for example, an indirect fluorescent antibody method (the indirect fluorescent antibody method is sometimes referred to as "IFA" in this specification), dot blot such as assays, Western blots, enzyme-linked immunosorbent assays (ELISAs (including sandwich ELISAs, direct adsorption ELISAs, competitive ELISAs)), radioimmunoassays (RIAs), and immunodiffusion assays.
  • IFA indirect fluorescent antibody method
  • dot blot such assays
  • Western blots Western blots
  • enzyme-linked immunosorbent assays ELISAs (including sandwich ELISAs, direct adsorption ELISAs, competitive ELISAs)
  • RIAs radioimmunoassays
  • diagnosis refers to the presence or absence of SFPQ-related dysfunction in a subject, the severity of SFPQ-related dysfunction in a subject, and/or SFPQ-related dysfunction in a subject, which does not require identification by a physician. It refers to evaluating the risk (degree of risk) of developing a functional disorder. Also, as used herein, “testing” refers to testing for SFPQ-related dysfunction in a subject that does not require identification by a physician. It should be noted that the term “examination” is a concept that includes not only examination for the presence or absence of onset of SFPQ-related dysfunction, but also examination for the possibility of developing SFPQ-related dysfunction. This data acquisition method does not include operations performed by a doctor, such as identification by a doctor, but the data obtained by this data acquisition method is one of the materials for the evaluation of diagnosis and test results by a doctor. may be
  • a method for diagnosing SFPQ-related dysfunction includes a measuring step of measuring the antibody titer of an anti-SFPQ antibody in a sample separated from a subject. .
  • the specific aspect of the measurement step in this diagnostic method is as described in the above section (Measurement step).
  • the antibody titer of the anti-SFPQ antibody in the sample isolated from the subject is a threshold value or higher, SFPQ-related dysfunction has developed, the symptoms of SFPQ-related dysfunction are severe, or It can be diagnosed that SFPQ-related dysfunction is likely to develop.
  • the "threshold" of the antibody titer of the anti-SFPQ antibody the quantitative value (normal value) in healthy subjects, the quantitative value (disease value) in patients with typical SFPQ-related dysfunction, or mild, moderate or severe
  • a person skilled in the art can appropriately set it with reference to quantitative values (disease values) of patients with SFPQ-related dysfunction.
  • the threshold (so-called cut-off value) in this diagnostic method is appropriately set by those skilled in the art according to the purpose based on many measured values of healthy subjects and patients obtained from clinical trials (e.g. , In the case of a screening test, in which the highest priority is not to miss a group of diseases and a definitive diagnosis is made after the secondary test, sensitivity is prioritized over specificity and the cutoff value is set low.
  • SFPQ-related When diagnosing the degree of symptoms of functional impairment, the threshold is set high with reference to the quantitative value of patients with mild, moderate or severe SFPQ-related functional impairment, etc.), from the description disclosed in the present specification, One skilled in the art can readily determine the threshold titer of anti-SFPQ antibodies for diagnosis of SFPQ-related dysfunction.
  • the numerical value measured from a sample isolated from an individual patient with healthy subjects or SFPQ-related dysfunction may be directly used.
  • An average value obtained when the values measured from the samples separated from the disabled patients are used as a population may be used.
  • a diagnostic for SFPQ-related dysfunction includes the following (a1) to (a3), (b1) to (b3), and (c1 ) to (c3): (a1) a polypeptide represented by SEQ ID NO: 1; (a2) in the amino acid sequence represented by SEQ ID NO: 1, one or several amino acids are (a3) a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1; (b1) SEQ ID NO: 2 or 3 (b2) consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 or 3, and an anti-SFPQ antibody (b3) a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO:
  • amino acid sequences shown in SEQ ID NOS: 2 and 3 are epitopes of SFPQ, which the present inventors found as a result of attempting epitope analysis using the amino acid sequence of SFPQ (SFPQ protein).
  • amino acid sequence of SEQ ID NO: 2 corresponds to the sequence of 15 amino acid residues from 36th to 50th of SFPQ (SFPQ36-50)
  • amino acid sequence of SEQ ID NO: 3 corresponds to the sequence of 15 amino acid residues from 452nd to 466th (SFPQ452- 466).
  • this diagnostic agent contains the SFPQ protein itself shown by SEQ ID NO: 1, the SFPQ epitope shown by SEQ ID NO: 2 or 3, and a polypeptide containing the epitope or a variant thereof, the anti-SFPQ antibody can be specifically detected and used to measure the antibody titer of the antibody. Therefore, it can be suitably applied as a diagnostic agent for SFPQ-related dysfunction.
  • the upper limit of the number of substituted, deleted, inserted and/or added amino acid residues can be, for example, two or one.
  • amino acid sequence identity is, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.
  • polypeptide comprising an amino acid sequence refers to a poly(polypeptide) in which any number of additional amino acids are added to the N-terminal amino acid and/or C-terminal amino acid of the amino acid sequence. means peptide.
  • the number of amino acids to be added is not particularly limited, and may be, for example, 500 or less, 300 or less, 200 or less, 100 or less, 50 or less, or 30 or less.
  • whether or not the polypeptide has the ability to bind to an anti-SFPQ antibody can be determined, for example, by binding an anti-SFPQ antibody as described in the Examples below. It can be determined by the immunoprecipitation method used.
  • the ability of the polypeptides (b2) to (b3) and (c1) to (c3) to bind to an anti-SFPQ antibody is 40% or more, 45% or more of that of human SFPQ (SEQ ID NO: 1), 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more.
  • Labeling substances can also be added to the polypeptides shown in (a1) to (a3), (b1) to (b3), and (c1) to (c3) as the present diagnostic agent.
  • the labeling substance include, but are not limited to, fluorescent dyes, enzymes, coenzymes, chemiluminescent substances, radioactive substances, and the like.
  • radioisotopes 32 P, 14 C, 125 I, 3 H, 131 I, etc.
  • fluorescein 32 P, 14 C, 125 I, 3 H, 131 I, etc.
  • fluorescein 32 P, 14 C, 125 I, 3 H, 131 I, etc.
  • rhodamine rhodamine
  • dansyl chloride umbelliferone
  • luciferase peroxidase
  • alkaline phosphatase ⁇ -galactosidase
  • ⁇ -glucosidase horseradish peroxidase
  • glucoamylase lysozyme
  • saccharide oxidase microperoxidase, biotin and the like
  • the diagnostic agent comprises, in addition to the polypeptides shown in (a1) to (a3), (b1) to (b3), and (c1) to (c3), a pharmaceutically acceptable carrier, a lubricant , preservatives, stabilizers, wetting agents, emulsifiers, salts for adjusting osmotic pressure, buffers, coloring agents, antioxidants, viscosity modifiers, activators, nanoparticles, and the like.
  • pharmaceutically acceptable carriers include, but are not limited to, water, various salt solutions, alcohols, vegetable oils, mineral oils, and the like.
  • the blood is passed through a column equipped with a carrier capable of adsorbing the anti-SFPQ antibody.
  • Blood purification therapy which is a method of reducing the concentration of anti-SFPQ antibodies in blood by adsorbing anti-SFPQ antibodies to the column (carrier), is considered to be particularly effective.
  • the method for reducing the anti-SFPQ antibody concentration in blood is not limited to this.
  • the surface contains at least one selected from the following (a1) to (a3), (b1) to (b3), and (c1) to (c3) ( (a1) a polypeptide represented by SEQ ID NO: 1; (a2) a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1 (a3) a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1; (b1) a polypeptide consisting of an amino acid sequence shown in SEQ ID NO: 2 or 3; (b2) a sequence A polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by number 2 or 3, and having the ability to bind to an anti-SFPQ antibody; (b3) SEQ ID NO: (c1) a polypeptide consisting of an amino acid sequence consisting of an amino acid sequence represented by SEQ ID NO: 1; (a2)
  • the carrier for blood purification according to one embodiment of the present invention (hereinafter sometimes referred to as the carrier) has, on its surface, the SFPQ protein itself represented by SEQ ID NO: 1, or an epitope of SFPQ represented by SEQ ID NO: 2 or 3. , and a polypeptide containing the epitope or a variant thereof, so that the anti-SFPQ antibody can be specifically adsorbed. Therefore, by passing a liquid (especially blood) through a blood purification column according to an embodiment of the present invention (hereinafter sometimes referred to as the present column) comprising the present carrier, the anti-SFPQ antibody in the liquid is removed. can do.
  • polypeptides represented by (a1) to (a3), (b1) to (b3) and (c1) to (c3) are described in [2. Acquisition method of data for diagnosing or examining SFPQ-related dysfunction] and [3. Diagnostic agent for SFPQ-related dysfunction] section is incorporated as appropriate.
  • the shape of the present carrier is not particularly limited, and may be any shape such as particulate, fibrous, or membrane (including hollow fiber membrane). From the viewpoint of easiness, it is preferably particulate or fibrous.
  • the carrier has the ability to adsorb anti-SFPQ antibodies in blood components, there are no particular restrictions on whether or not it has the ability to adsorb other substances, but it is preferable that it can specifically adsorb only anti-SFPQ antibodies.
  • the present column is not particularly limited as long as it comprises the present carrier.
  • an inlet for introducing a liquid containing an anti-SFPQ antibody (especially blood) into a container (inside a case described later) and an outlet for discharging the liquid may be a container having a case portion filled with the present carrier.
  • the material of the container is not particularly limited, and may be glass, stainless steel, polyethylene, polypropylene, polycarbonate, polystyrene, polymethyl methacrylate, or the like. Among them, polypropylene or polycarbonate is preferable in consideration of handling properties such as sterilization. Also, the shape of the container is not particularly limited, but it is preferably cylindrical.
  • the column may be filled with the carrier alone, or may be filled with other carriers for blood purification or various spacers in combination with the carrier.
  • spacers include sheet-shaped fibers such as knitted fabrics, woven fabrics and non-woven fabrics, membranes, beads, hydrogels and the like.
  • a method of treating or preventing SFPQ-related dysfunction comprising a purification step of passing a subject's blood through the column to reduce the amount of anti-SFPQ antibodies in the blood.
  • the method for treating or preventing SFPQ-related dysfunction (hereinafter sometimes referred to as "this treatment or prevention method") is a mechanism for treating or preventing the pathology of SFPQ-related dysfunction (mechanism of action). Introduction) is as described above.
  • treatment refers to the complete cure or alleviation of symptoms of SFPQ-related dysfunction in subjects (especially patients with SFPQ-related dysfunction), or suppression of exacerbation of symptoms of SFPQ-related dysfunction.
  • prevention refers to the prevention of SFPQ-related dysfunction in a subject (particularly, a subject who has not developed symptoms of SFPQ-related dysfunction or a subject who is likely to develop SFPQ-related dysfunction). intended to suppress or delay the onset of symptoms of
  • treatment means “prevention” if the subject does not develop symptoms of SFPQ-related dysfunction.
  • an animal model of SFPQ-related dysfunction is provided by administering SFPQ protein or anti-SFPQ antibody to a non-human mammal.
  • An SFPQ-related dysfunction model animal (hereinafter sometimes referred to as "this model animal") is used to study the cause of SFPQ-related dysfunction, a method for treating or preventing SFPQ-related dysfunction, and or to develop or screen a therapeutic or prophylactic agent, it can be suitably used as an animal experiment.
  • this model animal is not particularly limited as long as it is a non-human mammal (mammal other than human) that can be used as an experimental animal, and can be appropriately selected depending on the intended use of the model animal to be produced.
  • non-human mammals that can be used as this model animal include mice, rats, guinea pigs, dogs, rabbits, monkeys, and chimpanzees.
  • This model animal can be produced by administering SFPQ protein or anti-SFPQ antibody to non-human mammals. That is, in one embodiment of the present invention, there is provided a method for producing an SFPQ-related dysfunction model animal, comprising an administration step of administering SFPQ protein or anti-SFPQ antibody to a non-human mammal.
  • the administration step includes adding SFPQ protein or anti-SFPQ to a non-human mammal. This is the step of administering the antibody.
  • the administration step can also be said to be a step of immunizing a non-human mammal with the SFPQ protein or anti-SFPQ antibody.
  • the SFPQ protein administered in the administration step may be a human SFPQ protein, a model animal-derived SFPQ protein to be produced, or an artificially synthesized SFPQ protein.
  • a protein (polypeptide) in which an epitope of the SFPQ protein eg, a polypeptide represented by the amino acid sequence shown in SEQ ID NO: 1 or 2) is conjugated to a carrier protein such as KLH can also be used.
  • the anti-SFPQ antibody in the administration step it is desirable to use an antibody from an animal of the same species as the model animal, but it is not limited to this.
  • the antibody may be a monoclonal antibody or a polyclonal antibody.
  • the anti-SFPQ antibody in addition to natural antibodies, synthetic antibodies, recombinant antibodies, mutagenized antibodies, or artificial antibodies such as graft-bonded antibodies can also be used depending on the purpose of use.
  • the definition of "anti-SFPQ antibody” is as described above.
  • antibody as used herein includes immunoglobulins (IgA, IgD, IgE, IgG, IgM), functional fragments of antibodies, and functional fragments of antibodies mutated to the extent that they maintain reactivity. It is a concept.
  • SFPQ protein and anti-SFPQ antibody in addition to the SFPQ protein and anti-SFPQ antibody, other substances such as solvents (e.g., pure water, saline), adjuvants, pH adjusters, buffers, stabilizers, and/or preservatives are added.
  • Ingredients may be administered.
  • SFPQ proteins and anti-SFPQ antibodies may be administered to non-human mammals in admixture with these other ingredients.
  • the dosage of the SFPQ protein and anti-SFPQ antibody in the administration step may be administered in an appropriate amount at an appropriate frequency, taking into account the type, body weight, etc. of the non-human mammal to be administered.
  • SFPQ proteins and anti-SFPQ antibodies may be administered to a subject non-human mammal at 0.01 ⁇ g to 1000.0 ⁇ g/kg body weight, and 0.1 ⁇ g to 500.0 ⁇ g/kg body weight. 1.0 ⁇ g to 500.0 ⁇ g/kg body weight, or 1.0 ⁇ g to 300.0 ⁇ g/kg body weight.
  • the administration interval of the SFPQ protein and anti-SFPQ antibody is, for example, once an hour, once every 1 to 6 hours, once every 6 to 12 hours, once every 12 hours to once a day, once every 1 to 3 days, 1 day to It can be once every 5 days, once every 1-7 days, once every 7-14 days, once every 14-21 days, once per month, and the like.
  • One embodiment of the present invention includes the following configurations.
  • a method of obtaining data for diagnosing or testing SFPQ-related dysfunction comprising a measuring step of measuring the antibody titer of an anti-SFPQ antibody in a sample isolated from a subject.
  • the sample is selected from blood, serum, plasma, urine and saliva.
  • a diagnostic agent for SFPQ-related dysfunction including at least one selected from the following (a1) to (a3), (b1) to (b3), and (c1) to (c3): (a1) a polypeptide represented by SEQ ID NO: 1; (a2) A polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 1; (a3) a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1; (b1) a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 2 or 3; (b2) a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 2 or 3, and having the ability to bind to an anti-SFPQ antibody; (b3) a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO:
  • a carrier for blood purification the surface of which contains at least one selected from the following (a1) to (a3), (b1) to (b3), and (c1) to (c3): (a1) a polypeptide represented by SEQ ID NO: 1; (a2) A polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 1; (a3) a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1; (b1) a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 2 or 3; (b2) a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 2 or 3, and having the ability to bind to an anti-SFPQ antibody; (b3) a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO:
  • a blood purification column comprising the blood purification carrier according to ⁇ 5>.
  • An SFPQ-related dysfunction model animal obtained by administering an SFPQ protein or an anti-SFPQ antibody to a non-human mammal.
  • a method for producing an SFPQ-related dysfunction model animal comprising the step of administering an SFPQ protein or an anti-SFPQ antibody to a non-human mammal.
  • mice 6-week-old mice 2 weeks after the first administration of the DNA vaccine and 8-week-old mice 4 weeks after the administration of the first DNA vaccine were similarly administered the DNA vaccine. Blood was collected from the mice that had been administered the DNA vaccine a total of three times, and serum was collected one week after the third administration of the DNA vaccine (9 weeks old). Mice that received a total of three DNA vaccinations are referred to as SITH-1 DNA-vaccinated mice.
  • 0.1 M Citrate pH 2-3 was added for elution in order to detect self-antigens presenting. After that, a sample buffer was added, elution was performed by heating under reduction, and SDS-PAGE (electrophoresis) was performed.
  • SITH-1-expressing mice An adenovirus vector (SITH-1/Adv) incorporating the SITH-1 gene downstream of the GFAP promoter was produced, and purified using Adeno-XTM Maxi Purification Kit (TaKaRa). did. 25 ⁇ L of 1 ⁇ 10 9 ifu/mL SITH-1/Adv solution was nasally administered to 8-week-old mice (C57BL/6NCrSlc) and maintained for 4 weeks. SITH-1/Adv was intranasally administered in the same manner to 10-week-old and 12-week-old mice during breeding, and SITH-1/Adv was administered 3 times in total to induce expression of SITH-1. A group (SITH-1 group) was created.
  • mice As a control, 8-week-old mice (C57BL/6NCrSlc) were intranasally administered three times with an adenoviral vector (Vector/Adv) that did not express anything (gene was not integrated) (Vector group).
  • Vector/Adv adenoviral vector
  • the left figure in Fig. 1 shows photographs of negative stains obtained by negative gel staining in the operations (1) to (3).
  • two bands of 60 kDa and 68 kDa were obtained by the negative gel staining, and as a result of protein identification by PMF analysis, both were Splicing factor, proline- and glutamine-rich (SFPQ). ) was a protein.
  • mice in which SITH-1 expression was induced are shown in the right figure of FIG.
  • 68.8% of the mice in the SITH-1 group were autoantibody-positive.
  • the control Vector group mice only 33.3% of the mice were autoantibody-positive.
  • SITH-1 protein induces the production of anti-SFPQ antibodies as autoantibodies with SFPQ as the autoantigen.
  • Example 2 Induction of autoantibody (anti-SFPQ antibody) by SITH-1 ⁇ Method> It is known that SITH-1 binds to CAML and increases the intracellular amount of CAML, and CAML binds to the immunosuppressive receptor TACI of B cells. Therefore, it was confirmed that CAML suppresses the TACI signal and induces an autoimmune reaction, and that CAML binds to SFPQ and attracts SFPQ as an antigen to the site of the autoimmune reaction.
  • HEK293T cells were transformed by mixing with Lipofectamine LTX (ThermoFisher).
  • Lipofectamine LTX Lipofectamine LTX
  • hCAML-expressing cells in addition to the pNF ⁇ B-SEAP reporter, hCAML-expressing cells, hTACI-expressing cells, and both hCAML and hTACI-expressing cells were prepared, and each ligand (100 ng/ml BLyS or 200 ng/ml APRIL ) and cultured at 37° C. for 18 hours.
  • SEAP reporter assay was performed using Phospha-Light SEAP Reporter Gene Assay System (ThermoFisher).
  • CAML-expressing HEK293T cells were solubilized and immunoprecipitation with an anti-SFPQ antibody was attempted.
  • hCAML was expressed in HEK293T cells using Lipofectamine LTX as in Example 2, and solubilized with an SDS-free RIPA buffer to prepare a soluble fraction.
  • SDS-free RIPA buffer As a control, a soluble fraction of HEK293T cells expressing SITH-1 was prepared in a similar manner.
  • Anti-SFPQ antibody (abcam)-immobilized magnetic beads prepared in the same manner as in Example 1 were added to each soluble fraction, reacted at room temperature for 1 hour, and proteins bound to the magnetic beads were fractionated by SDS-PAGE. Then, the presence or absence of binding between SFPQ and CAML was detected by Western Blot using an anti-CAML antibody (abcam).
  • Example 3 SFPQ antibody measurement in healthy subjects, SITH-1 positive subjects, and depressed patients ⁇ Method> (1) Epitope analysis of SFPQ protein As a result of attempting epitope analysis using the amino acid sequence of human SFPQ protein, 15 amino acid residues from 36th to 50th (SFPQ36-50, SEQ ID NO: 2) and from 452nd to 466th 15 amino acid residues (SFPQ452-466, SEQ ID NO:3) were nominated. Therefore, it was decided to synthesize peptides of 16 amino acid residues by adding cysteine to the N-terminus of these amino acid sequences and use them as antigens.
  • Non-Patent Document 1 Detection of anti-SITH-CAML antibody by indirect fluorescent antibody method
  • serum collected from healthy subjects or depressed patients was diluted 40-fold with PBS containing 2% BSA and 0.05% Tween 20, and stained with an indirect fluorescent antibody method (IFA).
  • IFA indirect fluorescent antibody method
  • Alexa Fluor 488 goat anti-human secondary antibodies Molecular Probes
  • PBS containing 2% BSA and 0.05% Tween 20 As an antigen expression plasmid, a plasmid in which SITH-CAML was inserted between the CMV promoter and the FLAG tag of pCMV-FLAG-5a and LITMUS28i as a control were used.
  • Each plasmid was transfected with Lipofectamine LTX (Invitrogen) into HEK293T cells cultured on Lab-Tek chamber slides (Nunc), and then cultured for 48 hours. Fixed for 5 minutes and used as IFA antigen. These cells were observed with an indirect fluorescent antibody method (IFA) using a fluorescence microscope, and the fluorescence intensity of the IFA-stained cells was quantified using image analysis software ImageJ.
  • IFA indirect fluorescent antibody method
  • Table 1 shows the sequences of synthetic peptides of SFPQ36-50 or SFPQ452-466, which are epitopes of SFPQ. Cysteine is added to the N-terminus of the synthetic peptide.
  • Figure 3 shows the anti-SFPQ antibody titer by ELISA in Example 3, and Westen The relationship between the anti-SFPQ antibody titer by Blot and the detection result of the anti-SITH-CAML antibody titer by the indirect fluorescent antibody method (IFA) (the correlation between the anti-SFPQ antibody titer and the SITH-CAML antibody titer) is shown.
  • IFA indirect fluorescent antibody method
  • FIG. 3 shows the results of anti-SFPQ antibody titer detection by ELISA using synthetic peptides.
  • the amount of anti-SFPQ antibody in depressed patients is significantly higher than in healthy subjects, regardless of whether the antigen is either SFPQ36-50 or SFPQ452-466. It has been shown. ***P ⁇ 0.0001.
  • the lower left figure in Figure 3 shows the correlation plot between the anti-SFPQ antibody titer and the SITH-CAML antibody titer by Western Blot.
  • a correlation was observed between the anti-SFPQ antibody titer and the SITH-CAML antibody titer, particularly in humans under the age of 60. This result indicates that the higher the anti-SITH-CAML antibody titer, the higher the anti-SFPQ antibody titer in a person who repeatedly expresses the SITH-1 protein.
  • the lower right figure of Figure 3 shows the anti-SFPQ antibody positive rate by age group by Western Blot. As is clear from the lower right figure of FIG. 3, there is a tendency that the higher the age, the higher the ratio of holding anti-SFPQ antibodies. This indicated that the retention rate of the anti-SFPQ antibody increased with aging.
  • mice Occurrence of depression-related symptoms by immunization with SFPQ ⁇ Method>
  • mice were immunized as described below, and behavioral tests, histological analyses, and gene expression analyses were attempted.
  • mice 1 mg/mL KLH conjugated SFPQ synthetic peptide solution (SFPQ36-50 or SFPQ452-466) and an equal amount of Freund T s Complete Adjuvant (Fujifilm Wako Pure Chemical) were mixed to form an emulsion. A solution was prepared. As a control, an emulsion solution was made using a 1 mg/mL KLH protein solution. This emulsion solution was subcutaneously administered to 4-week-old C57BL/6 male mice at 400 ⁇ L each (antigen dose was 0.2 mg/mouse) (first immunization), returned to their home cages and reared normally.
  • SFPQ36-50 or SFPQ452-466 1 mg/mLH conjugated SFPQ synthetic peptide solution
  • Freund T s Complete Adjuvant Freund T s Complete Adjuvant
  • a 1 mg/mL KLH conjugated SFPQ synthetic peptide solution (SFPQ36-50 or SFPQ452-466) and an equal amount of Freund T 's Incomplete Adjuvant (Fujifilm Wako Pure Chemical Industries) are mixed to prepare an emulsion solution. 200 ⁇ L each subcutaneously of the 6-week-old (2 weeks after the first immunization) mouse (C57BL/6 male mouse that was immunized for the first time) (antigen dose was 0.1 mg/animal) ) (second immunization), returned to their home cages and reared normally.
  • mice Two weeks later, the 8-week-old mice were administered the emulsion solution in the same manner as the second immunization (third immunization), and were then fed normally for another week (until they reached the age of 9 weeks). In this way, SFPQ-immunized mice, which were immunized a total of three times, were produced.
  • mice (3) Analysis of anxiety behavior in mice (1) An elevated plus maze with a height of 500 mm was used to analyze the anxiety behavior of generated SFPQ-immunized mice. SFPQ-immunized mice were placed in the center of the elevated plus maze, allowed to move freely for 10 minutes, and the number of open arm entries counted. In an experiment using an elevated plus maze, it is believed that the smaller the number of intrusions, the more anxious behavior is exhibited.
  • mice Two weeks after the third SFPQ immunization, the brains of 10-week-old SFPQ-immunized mice were excised and fixed with a 10% neutral formalin solution. The fixed brain was embedded in paraffin, and a coronal section (brain section) was prepared at a position where the reins (Bregma-1.70 mm) could be observed. The prepared brain sections were subjected to antigen retrieval treatment after deparaffinization, and fluorescent immunostaining was performed with an anti-choline acetyltransferase (ChAT) antibody (manufactured by abcam).
  • ChAT anti-choline acetyltransferase
  • FIG. 4 shows the results of analysis of anxious behavior of mice, the results of histological analysis of mouse brains (bridle), and FIG. 5 shows the results of gene expression analysis in mouse brains in Example 4.
  • FIG. 4 shows a photograph of observing choline acetyltransferase-producing cells in the habenula and the number of cholinergic neurons as the results of histological analysis of the mouse brain.
  • SFPQ-immunized mice have medial habenula (MHb), which is involved in anhedonia, and lateral habenula (LHb), which is involved in sleep, circadian rhythm, reward system, and cognitive function. It was shown that there was a decrease in choline acetyltransferase-producing cells in
  • a 1 mg/mL KLH conjugated SFPQ synthetic peptide solution (SFPQ36-50 or SFPQ452-466) and an equal amount of Freund T 's Incomplete Adjuvant (Fujifilm Wako Pure Chemical Industries) are mixed to prepare an emulsion solution. 200 ⁇ L each subcutaneously of the 6-week-old (2 weeks after the first immunization) mouse (C57BL/6 male mouse that was immunized for the first time) (antigen dose was 0.1 mg/animal) ) (second immunization), returned to their home cages and reared normally.
  • mice Two weeks later, the 8-week-old mice were administered the emulsion solution in the same manner as the second immunization (third immunization), and were then fed normally for another week (until they reached the age of 9 weeks). In this way, SFPQ-immunized mice, which were immunized a total of three times, were produced.
  • Rotarod test was used to analyze the coordinated motor ability of the prepared SFPQ-immunized mice. Specifically, the SFPQ-immunized mouse was placed on a rod rotating at 4 rpm for a certain period of time, and after habituation, the rotation speed was increased from 4 rpm to 60 rpm in 1 minute, and the time until the mouse fell was measured. did.
  • FIG. 6 shows the expression level of the Edn-1 gene, a marker of aging, in the brain and olfactory bulb of SFPQ-immunized mice.
  • increased expression of the Edn-1 gene was confirmed in all SFPQ-immunized mice compared with control mice.
  • FIG. 6 shows a photograph of observation of DCX-producing cells, which is a neurogenesis marker, in the hippocampus of SFPQ-immunized mice, and a graph of measurement of DCX-positive cells.
  • all SFPQ-immunized mice showed decreased neurogenesis in the hippocampus.
  • FIG. 7 shows the behavioral analysis results of SFPQ-immunized mice using the Y-maze and Rotarod test. As is clear from FIG. 7, all SFPQ-immunized mice had impaired spatial working memory, and decreased coordination was also observed. Therefore, it was shown that all SFPQ antibody-producing mice exhibit senescence-like behavior.
  • This emulsion solution was subcutaneously administered to 4-week-old C57BL/6 male mice (first immunization) so that the dose of antigen was 125 ⁇ g/mouse, and the mice were returned to their home cages and reared normally.
  • the antigen solution alone was subcutaneously administered to the mice so that the dose of the antigen was 125 ⁇ g/mouse.
  • the antigen solution was subcutaneously administered to the mice so that the antigen dose was 125 ⁇ g/mouse.
  • mice 4 weeks after the first immunization were used as single-immune mice
  • mice 4 weeks after the second immunization were used as mice with two immunizations
  • three-time immunization mice were used.
  • mice 4 weeks after the third immunization were used.
  • the body weight of the mice was measured every two weeks.
  • mice immunized once and mice immunized three times were collected, and mouse small intestine cDNA was synthesized in the same manner as in Example 4 (6).
  • stem cell markers Lgr5 and Sox2 were used as a template, and senescence marker cyclin-dependent kinase Gene expression of inhibitor 2A (Cdkn2a) and Edn-1 was analyzed by RT-qPCR.
  • FIG. 8 shows the results of gene expression analysis in the small intestine of mice and the measurement results of changes in body weight of mice during the immunization period in Example 6.
  • the upper diagram of FIG. 8 shows the gene expression analysis results of Cdkn2a and Edn-1, which are senescence markers, and Sox2, a stem cell marker, whose changes were observed after a single immunization.
  • Cdkn2a and Edn-1 which are senescence markers, and Sox2, a stem cell marker, whose changes were observed after a single immunization.
  • Sox2 a stem cell marker
  • the lower left figure in Figure 8 shows the gene expression analysis results of Lgr5, an intestinal stem cell marker, in which changes were observed after three immunizations. As is clear from the lower left diagram of FIG. 8, all SFPQ-immunized mice had reduced Lgr5 expression compared to control mice.
  • weight gain was suppressed in all SFPQ-immunized mice compared to control mice.
  • Example 7 Occurrence of testicular aging phenomenon by immunization with SFPQ ⁇ Method> Mice were immunized with SFPQ in the same manner as in Example 6 to obtain SFPQ-immunized mice that produced anti-SFPQ antibodies. After that, the testis of the mouse immunized once and the mouse immunized three times was collected, and cDNA of the mouse testis was synthesized in the same manner as in the operation of Example 4 (6). Using the synthesized cDNA as a template, gene expressions of stem cell markers Lgr4, Lgr5 and Sox2 and testosterone production marker steroidogenic acute regulatory protein (StAR) gene expression were analyzed by RT-qPCR.
  • StAR steroidogenic acute regulatory protein
  • testis section was prepared in the same manner as in Example 4 (5).
  • the prepared testis sections were subjected to antigen retrieval treatment after deparaffinization, and fluorescent immunostaining was performed with an anti-Leydig cell marker HSD3B1 antibody (abcam).
  • Fig. 9 shows the analysis results of gene expression in the testis of SFPQ-immunized mice 4 weeks after the first SFPQ immunization.
  • the testicular stem cell marker Lgr5 and the testosterone production marker StAR were decreased in the SFPQ-immunized mice due to the anti-SFPQ antibody produced by the single immunization.
  • the upper diagram of FIG. 10 shows the analysis results of gene expression in the testis of SFPQ-immunized mice 4 weeks after the third SFPQ immunization.
  • the effects of the anti-SFPQ antibody produced in the three immunizations reduced each marker confirmed in the first immunization. and Sox2 decreased.
  • Example 8 Occurrence of skin aging phenomenon by immunization with SFPQ ⁇ Method> Mice were immunized with SFPQ in the same manner as in Example 6 to obtain SFPQ-immunized mice that produced anti-SFPQ antibodies. After that, the skin of the mice immunized three times was collected, and the cDNA of the mouse skin was synthesized in the same manner as in the operation of Example 4 (6).
  • Synthesized cDNA was used as a template for stem cell marker Lgr4, senescence marker Cdkn2a, inflammatory cytokines IL-1 ⁇ and IL-6, inflammation-causing inflammasome Nlrp3, and apoptosis-related factors Bax and Bcl-2 genes. Expression was analyzed by RT-qPCR.
  • FIG. 11 shows the results of gene expression analysis in the skin of SFPQ-immunized mice in Example 8
  • FIG. 12 shows the results of histological analysis of the skin of SFPQ-immunized mice.
  • FIG. 11 shows the results of gene expression analysis in the skin of SFPQ-immunized mice 4 weeks after the third SFPQ immunization.
  • the cutaneous stem cell marker Lgr4 decreased, the senescence marker Cdkn2a increased, the inflammatory cytokines IL-1 ⁇ and IL-6 increased, An increase in inflammasome Nlrp3, which causes inflammation, was observed.
  • the Bax/Bcl-2 ratio a marker of apoptosis, was increased.
  • Fig. 12 shows a photograph of hair loss in SFPQ-immunized mice that produced anti-SFPQ antibodies that recognize SFPQ452-466, which was observed during the immunization procedure.
  • the lower diagram of FIG. 12 shows the results of histological analysis by fluorescent immunostaining of mouse skin sections (hair follicles).
  • a decrease in Lgr5-positive cells which are stem cells of hair follicles, and an increase in Cdkn2a-positive cells were observed.
  • Example 9 Occurrence of Cardiac Aging by Immunization with SFPQ ⁇ Method> Mice were immunized with SFPQ in the same manner as in Example 6 to obtain SFPQ-immunized mice that produced anti-SFPQ antibodies. Thereafter, the hearts of the three-immunized mice were harvested, and the mouse heart cDNA was synthesized in the same manner as in Example 4 (6). Gene expressions of the senescence marker Cdkn2a, the inflammatory cytokine IL-1 ⁇ , and the inflammasome Nlrp3 were analyzed by RT-qPCR using the synthesized cDNA as a template.
  • Fig. 13 shows the results of gene expression analysis in mouse hearts 4 weeks after the third SFPQ immunization. As is clear from z13, increased expression of cardiac senescence marker Cdkn2a, inflammatory cytokine IL-1 ⁇ , and inflammasome Nlrp3 was observed in SFPQ-immunized mice.
  • Example 10 Occurrence of pulmonary aging phenomenon by immunization with SFPQ ⁇ Method> Mice were immunized with SFPQ in the same manner as in Example 6 to obtain SFPQ-immunized mice that produced anti-SFPQ antibodies. After that, the lungs of the mouse immunized once and the mouse immunized three times were collected, and mouse lung cDNA was synthesized in the same manner as in Example 4 (6). Gene expressions of the senescence marker Edn-1 and the apoptosis-related factors Bax and Bcl-2 were analyzed by RT-qPCR using the synthesized cDNA as a template.
  • Fig. 14 shows the results of gene expression analysis of the lungs of mice 4 weeks after the first SFPQ immunization and 4 weeks after the third SFPQ immunization.
  • the higher the number of immunizations that is, the higher the amount of anti-SFPQ antibody
  • the higher the expression level of endothelin Edn1 a lung aging marker
  • the marker of apoptosis induction An increase in the Bax/Bcl-2 ratio was observed.
  • Example 11 Effect of anti-SFPQ antibody on Tau protein ⁇ Method> Increased phosphorylation of Tau protein has been reported to be associated with aging and dementia. Therefore, an anti-SFPQ antibody was added to the neuronal cell line Neuro2a to examine whether the presence of the anti-SFPQ antibody affects Tau gene splicing and increases phosphorylation. In addition, we investigated whether the addition of anti-SFPQ antibody to the neuronal cell line Neuro2a promotes the phosphorylation of Tau protein.
  • Antiserum immunized with purified GST-SFPQ-His protein was used as anti-SFPQfull antiserum
  • antiserum immunized with KLH-conjugated synthetic peptide SFPQ36-50 was used as anti-SFPQ36-50 antiserum
  • KLH-conjugated synthetic peptide SFPQ452-466 was used as antiserum.
  • the immunizing antiserum was designated as anti-SFPQ452-466 antiserum.
  • PCR reaction was performed using primers (Forward: TCCCCCTAAGTCACCATCAG (SEQ ID NO: 4), Reverse: GCCAATCTTCGACTGGACTC (SEQ ID NO: 5)) that amplify the exon 10 region of the Tau gene.
  • primers Forward: TCCCCCTAAGTCACCATCAG (SEQ ID NO: 4), Reverse: GCCAATCTTCGACTGGACTC (SEQ ID NO: 5)
  • the solution after the PCR reaction was applied to a 15% polyacrylamide gel e-pagel (ATTO) and subjected to electrophoresis. After electrophoresis, the polyacrylamide gel was stained with ethidium bromide, and the band of the PCR product detected by UV light irradiation was quantified using software Image Lab (manufactured by Bio-Rad). A value was calculated by dividing the numerical value of the band containing exon10 amplified by Tau phosphorylation (exon10+) by the numerical value of the band not
  • Neuro2a cultured in the presence of anti-SFPQ was immunofluorescently stained with an anti-phosphorylated Tau antibody (AT8). Then, the fluorescence intensity of each cell was measured with an image analyzer ArrayScan XT (ThermoFisher). Similarly, the cells were fluorescently immunostained with a neuron marker Microtubule-associated protein 2 (MAP2) antibody, and the cell body size of each cell was measured with an image analyzer ArrayScan XT.
  • MAP2 Microtubule-associated protein 2
  • FIG. 15 shows the results of PCR product analysis, the measurement results of the fluorescence intensity of Neuro2a cells, and the measurement results of the cell body size of Neuro2a cells in Example 11, respectively.
  • the lower left diagram of FIG. 15 shows the results of staining Neuro2a cells in the presence of 20 ⁇ M RA and anti-SFPQ antibody.
  • Neuro2a cells in the presence of 20 ⁇ M RA and anti-SFPQ antibody were shown to be significantly strongly stained with anti-phosphorylated Tau antibody. Therefore, the presence of anti-SFPQ antibody was shown to increase phosphorylated Tau protein in neuronal cells.
  • the lower right diagram of FIG. 15 shows changes in the size of Neuro2a cell bodies in the presence of 20 ⁇ M RA and anti-SFPQ antibody.
  • Neuro2a cells in the presence of 20 ⁇ M RA and anti-SFPQ antibody, which promote phosphorylation of Tau protein, showed swelling of cells and cell nuclei due to toxicity.
  • Example 12 Relationship between the presence or absence of anti-SFPQ antibody and survival of neural stem cells ⁇ Method>
  • mouse ES cell H-1 cultured in a serum-free feeder-free medium Cellartis 2i mES/iPSC Culture Medium (TaKaRa) was added at a concentration of 2% in the culture medium.
  • Any one of the three types of anti-SFPQ antisera (SFPQfull, SFPQ36-50, SFPQ452-466) was added to the cells so that the cells were cultured for 24 hours or 48 hours. Rabbit serum before immunization with SFPQ was used as a control.
  • synthesized cDNA was synthesized using gene expressions of radial glial cell marker Nestin, neuroblast marker Doublecortin (DCX), immature neuron marker Calretinin, and mature neuron marker Calbindin were analyzed by RT-qPCR.
  • FIG. 16 shows the results of analysis of gene expression in neurons in Example 12.
  • the expression of the radial glial cell marker Nestin and the neuroblast marker DCX decreased after 24 hours in the presence of the anti-SFPQ antiserum.
  • the expression of Calretinin, an immature neuron marker, and Calbindin, a differentiated mature neuron marker decreased after 48 hours in the presence of anti-SFPQ antiserum. This result suggests that the anti-SFPQ antibody inhibits neuronal differentiation.
  • Example 13 Relationship between presence or absence of anti-SFPQ antibody and survival of undifferentiated stem cells ⁇ Method>
  • three types of anti-SFPQ were added to mouse ES cells H-1 cultured in a serum-free feeder-free medium Cellartis 2i mES/iPSC Culture Medium (TaKaRa) at 2%. Any one of the antisera (SFPQfull, SFPQ36-50, SFPQ452-466) was added and cultured for 24 hours or 48 hours. Rabbit serum before immunization with SFPQ was used as a control. Thereafter, cDNA was prepared from each cell in the same manner as in Example 5, and gene expression of stem cell markers Lgr4, Lgr5, Lgr6 and Sox2 was analyzed by RT-qPCR.
  • FIG. 17 and the upper figure in FIG. 18 show the gene expression of H-1 cells 24 hours after the addition of anti-SFPQ antiserum, and the lower figure in FIG. 17 and the lower figure in FIG. -1 cell gene expression.
  • FIG. 17 shows the gene expression of stem cell markers Lgr4 and Lgr5 from the left
  • FIG. 18 shows the gene expression of stem cell markers Lgr6 and Sox2 from the left.
  • Anti-SFPQ full antiserum, anti-SFPQ36-50 antiserum, or anti-SFPQ452-466 antiserum obtained by the method described in Example 11 was administered to 6-week-old C57BL/ 200 ⁇ L/mouse was administered to 6NCrSlc male mice through the tail vein.
  • Behavioral test of anti-SFPQ rabbit polyclonal antibody tail vein administration mice One week after tail vein administration, to examine the phenotype of anti-SFPQ rabbit polyclonal antibody tail vein administration mice, it is used to evaluate depression-like behavior. A forced swim test (FST) and a tail suspension test (TST) were performed.
  • FST forced swim test
  • TST tail suspension test
  • tail suspension test the tail of the mouse was fixed, suspended for 10 minutes, recorded, analyzed with image analysis software TailSuspScan (CleverSys Inc), and immobility time was measured.
  • Anti-SFPQ rabbit polyclonal antibody was administered three times to the tail vein of mice. Brain cDNA was synthesized. Gene expressions of brain-derived neurotrophic factor BDNF, apoptosis-related factors Bax and Bcl-2, and inflammation suppressor Zfp36 were analyzed by RT-qPCR using the synthesized cDNA as a template.
  • Figure 19 shows the measurement results of body weight change and the measurement results of coordinated motor ability of mice administered with anti-SFPQ mouse monoclonal antibody tail vein in Example 14.
  • FIG. 20 shows the results of gene expression analysis in the brains of mice to which the anti-SFPQ rabbit polyclonal antibody was administered via the tail vein.
  • the upper diagram of FIG. 20 shows the results of behavioral test analysis of mice to which anti-SFPQ rabbit polyclonal antibody was administered through the tail vein.
  • mice administered with anti-SFPQ rabbit polyclonal antibody increased Float time and shortened Struggle time in the forced swimming test, which are indicators of depression-like behavior, and immobility time in the tail suspension test. was observed. Therefore, it was revealed that mice administered with anti-SFPQ rabbit polyclonal antibody exhibited depression-like behavior.
  • the lower figure in FIG. 20 shows the results of gene expression analysis in the brain of mice to which anti-SFPQ rabbit polyclonal antibody was administered via the tail vein.
  • the brain-derived neurotrophic factor BDNF observed in depression decreased, and the Bax/Bcl-2 ratio, which is an index of apoptosis induction, increased. was observed, and a decrease in the expression of the inflammation suppressor Zfp36 in the brain was also observed.

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Abstract

The present invention addresses the problem of specifying a factor involved in depression and aging and providing a method for the use thereof. This problem is solved by a method for acquisition of data for diagnosis or testing of SFPQ-related dysfunction, said method comprising a measuring step for measuring the antibody titer of anti-SFPQ antibodies in a specimen removed from a subject.

Description

うつ病と老化等に関与する自己免疫反応のデータの取得方法およびその利用Acquisition method and use of data on autoimmune reactions involved in depression, aging, etc.
　本発明は、うつ病と老化等に関与する自己免疫反応のデータの取得方法およびその利用に関する。 The present invention relates to a method for obtaining data on autoimmune reactions involved in depression, aging, etc. and its use.
　うつ病の代表的な症状としては、ＤＳＭ－５による大うつ病の診断基準にあるように、抑うつ気分、興味関心・喜びの感情の喪失、食欲不振、過食、睡眠障害、過眠、精神運動性の焦燥または制止、易疲労性、無価値感や罪責感、集中力の低下や思考力の低下、あるいは、自殺念慮、等の症状が知られている。しかし、これらの代表的な症状以外にも、うつ病患者は、認知機能や記憶力の低下、性欲減退、心血管疾患、骨粗鬆症、糖尿病等の、老化様の症状を、高頻度で発現することが知られている。このような老化様の症状の高頻度の発現は、うつ病患者の健康寿命に大きな影響を与えている。 Typical symptoms of depression include depressed mood, loss of interest/pleasure, anorexia, overeating, sleep disturbance, hypersomnia, and psychomotor activity, as defined in the DSM-5 diagnostic criteria for major depression. Known symptoms include sexual impatience or inhibition, fatigability, worthlessness and guilt, decreased ability to concentrate and think, and suicidal ideation. However, in addition to these typical symptoms, depressed patients frequently develop aging-like symptoms such as cognitive function and memory decline, decreased libido, cardiovascular disease, osteoporosis, and diabetes. Are known. The frequent appearance of such aging-like symptoms has a great impact on the healthy life expectancy of depressed patients.
　老化（および老化と関連する疾患）の発生メカニズムには、多くのメカニズムが作用していると考えられており、遺伝子変異の蓄積やエピジェネティックな要因等、様々な因子の作用が提唱されている。 Many mechanisms are thought to act in the mechanism of aging (and diseases related to aging), and various factors such as the accumulation of gene mutations and epigenetic factors have been proposed. .
　ところで、ヒトヘルペスウイルス－６（ＨＨＶ－６（ＨＨＶ；ｈｕｍａｎｈｅｒｐｅｓｖｉｒｕｓ））を含むヘルペスウイルスには、初感染、潜伏感染、再活性化の３種類の状態が知られている。このうち、初感染と再活性化の状態ではウイルスが増殖しており、様々な疾患の原因となることが知られている。一方で、潜伏感染の状態ではウイルスの増殖は生じない。発がん性のヘルペスウイルスであるエプシュタインバーウイルスやカポジ肉腫関連ヘルペスウイルス等の一部のウイルスでは、潜伏感染状態で腫瘍が発症することがわかっているが、このような発がん性ヘルペスウイルスではない、単純ヘルペスウイルス、水痘帯状疱疹ウイルス、サイトメガロウイルス、ＨＨＶ－７では潜伏感染状態で生じる疾患は知られていない。 By the way, herpesviruses including human herpesvirus-6 (HHV-6 (HHV; humanherpesvirus)) are known to have three states: primary infection, latent infection, and reactivation. Of these, the virus proliferates in the primary infection and reactivation states, and is known to cause various diseases. On the other hand, viral multiplication does not occur in the state of latent infection. Some viruses, such as Epstein-Barr virus, which is an oncogenic herpes virus, and Kaposi's sarcoma-associated herpes virus, are known to cause tumors in a latent state. Herpes simplex virus, varicella-zoster virus, cytomegalovirus, and HHV-7 are not known to cause latent disease.
　ＨＨＶ－６の潜伏感染状態では、ウイルス増殖は行われないが、ＨＨＶ－６には、潜伏感染状態で発現するタンパク質（潜伏感染タンパク質）としてＳｍａｌｌｐｒｔｅｉｎｅｎｃｏｄｅｄｂｙｔｈｅＩｎｔｅｒｍｅｄｉａｔｅｓｔａｇｅＴｒａｎｓｃｒｉｐｔｏｆＨＨＶ－６－１（以下、「ＨＨＶ－６　ＳＩＴＨ－１」あるいは、「ＳＩＴＨ－１」と称する場合がある）が同定されている。 In the latent infection state of HHV-6, viral propagation does not occur, but HHV-6 has Small protein encoded by the Intermediate stage Transcript of HHV-6-1 (hereinafter referred to as "HHV-6 SITH- 1” or sometimes referred to as “SITH-1”) has been identified.
　本発明者らは、以前、このＳＩＴＨ－１に対する抗体が、うつ病、疲労などの様々な疾患の患者から特異的に検出されること、またＳＩＴＨ－１の発現がこれらの疾患に関与していることを報告している。例えば、特許文献１には、ＳＩＴＨ－１に対する抗体を用いて、被験者が気分障害に罹患しているかどうかを診断できることが開示されている。 The present inventors have previously found that antibodies against this SITH-1 are specifically detected in patients with various diseases such as depression and fatigue, and that the expression of SITH-1 is involved in these diseases. are reporting that there are For example, Patent Document 1 discloses that antibodies against SITH-1 can be used to diagnose whether a subject is suffering from a mood disorder.
ＷＯ２０１７／１１５７８７８号公報WO2017/1157878
　しかしながら、老化の発生メカニズムには、未だ不明な点が多く、科学的な根拠を伴った積極的な老化の予防方法や治療方法を確立するために、該老化の発生のメカニズムを解明することが期待されている。 However, there are still many unclear points about the mechanism of aging, and it is necessary to clarify the mechanism of aging in order to establish positive methods for preventing and treating aging with scientific grounds. Expected.
　このような状況にあって、本発明者らは、うつ病と老化と関連する疾患との関係性に着目し、うつ病と老化の関係性を明らかにできれば、老化の発生メカニズムの解明に寄与し得る知見を得られるのではないかと考えた。したがって、本発明の一態様は、うつ病と老化に関与する因子を特定し、その利用法を提供することにある。 Under these circumstances, the present inventors focused on the relationship between depression and aging-related diseases, and if the relationship between depression and aging could be clarified, it would contribute to the elucidation of the mechanism of aging. I thought that I might be able to obtain knowledge that could be done. Accordingly, one aspect of the present invention is to identify a factor involved in depression and aging and to provide a method for using the same.
　本発明者らは、上記課題について鋭意検討を進めるうちに、（１）うつ病患者において、生体内のＲＮＡ結合タンパク質であるSplicing factor, proline- and glutamine-rich（ＳＦＰＱ）に対する自己抗体（抗ＳＦＰＱ抗体）が高い確率で陽性であること、ならびに、（２）抗ＳＦＰＱ抗体よって、うつ病様症状および／または老化様症状が誘導されることを新たに見出した。換言すると、ＳＦＰＱに対する自己免疫反応が、うつ病と老化等に関与していることを新たに見出し、本発明を完成させるに至った。なお、本明細書において、ＳＦＰＱに対する自己免疫反応により誘導される、うつ病、老化等の生体機能の障害を、ＳＦＰＱ関連機能障害と称する場合がある。 While conducting intensive studies on the above problems, the present inventors found that (1) autoantibodies (anti-SFPQ and (2) anti-SFPQ antibody induces depression-like symptoms and/or aging-like symptoms. In other words, the present inventors have newly discovered that autoimmune reactions to SFPQ are involved in depression, aging, and the like, and have completed the present invention. In the present specification, disorders of biological functions such as depression and aging induced by autoimmune reactions to SFPQ are sometimes referred to as SFPQ-related dysfunctions.
　すなわち、本発明の一実施形態に係るＳＦＰＱ関連機能障害の診断または検査するためのデータの取得方は、被験体から分離した試料中の抗ＳＦＰＱ抗体の抗体価を測定する測定工程を含む、ＳＦＰＱ関連機能障害の診断または検査するためのデータの取得方法である。 That is, the method of obtaining data for diagnosing or testing SFPQ-related dysfunction according to one embodiment of the present invention includes a measuring step of measuring the antibody titer of an anti-SFPQ antibody in a sample isolated from a subject. Methods of obtaining data for diagnosing or testing related impairments.
　本発明の一実施形態に係るＳＦＰＱ関連機能障害の診断薬は、以下の（ａ１）～（ａ３）、（ｂ１）～（ｂ３）、および、（ｃ１）～（ｃ３）から選択される少なくとも１つを含む、ＳＦＰＱ関連機能障害の診断薬である：
　（ａ１）配列番号１で表されるポリペプチド；
　（ａ２）配列番号１で示されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換または付加されたアミノ酸配列からなるポリペプチド；
　（ａ３）配列番号１で示されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列からなるポリペプチド；
　（ｂ１）配列番号２または３で示されるアミノ酸配列からなるポリペプチド；
　（ｂ２）配列番号２または３で示されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換または付加されたアミノ酸配列からなり、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；
　（ｂ３）配列番号２または３で示されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列からなり、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；
　（ｃ１）配列番号２または３で示されるアミノ酸配列を含み、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；
　（ｃ２）配列番号２または３で示されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換または付加されたアミノ酸配列を含み、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；
　（ｃ３）配列番号２または３で示されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含み、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド。 The diagnostic agent for SFPQ-related dysfunction according to one embodiment of the present invention is at least one selected from the following (a1) to (a3), (b1) to (b3), and (c1) to (c3) is a diagnostic for SFPQ-related dysfunctions, including:
(a1) a polypeptide represented by SEQ ID NO: 1;
(a2) A polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 1;
(a3) a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1;
(b1) a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 2 or 3;
(b2) a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 2 or 3, and having the ability to bind to an anti-SFPQ antibody;
(b3) a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or 3 and having the ability to bind to an anti-SFPQ antibody;
(c1) a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 2 or 3 and capable of binding to an anti-SFPQ antibody;
(c2) A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 2 or 3, and having the ability to bind to an anti-SFPQ antibody;
(c3) A polypeptide comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2 or 3 and having the ability to bind to an anti-SFPQ antibody.
　本発明の一実施形態に係る血液浄化用担体は、表面に、以下の（ａ１）～（ａ３）、（ｂ１）～（ｂ３）、および、（ｃ１）～（ｃ３）から選択される少なくとも１つを含む、血液浄化用担体である：
　（ａ１）配列番号１で表されるポリペプチド；
　（ａ２）配列番号１で示されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換または付加されたアミノ酸配列からなるポリペプチド；
　（ａ３）配列番号１で示されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列からなるポリペプチド；
　（ｂ１）配列番号２または３で示されるアミノ酸配列からなるポリペプチド；
　（ｂ２）配列番号２または３で示されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換または付加されたアミノ酸配列からなり、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；
　（ｂ３）配列番号２または３で示されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列からなり、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；
　（ｃ１）配列番号２または３で示されるアミノ酸配列を含み、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；
　（ｃ２）配列番号２または３で示されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換または付加されたアミノ酸配列を含み、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；
　（ｃ３）配列番号２または３で示されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含み、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド。 The carrier for blood purification according to one embodiment of the present invention has, on its surface, at least one selected from the following (a1) to (a3), (b1) to (b3), and (c1) to (c3). A blood purification carrier comprising:
(a1) a polypeptide represented by SEQ ID NO: 1;
(a2) A polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 1;
(a3) a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1;
(b1) a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 2 or 3;
(b2) a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 2 or 3, and having the ability to bind to an anti-SFPQ antibody;
(b3) a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or 3 and having the ability to bind to an anti-SFPQ antibody;
(c1) a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 2 or 3 and capable of binding to an anti-SFPQ antibody;
(c2) A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 2 or 3, and having the ability to bind to an anti-SFPQ antibody;
(c3) A polypeptide comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2 or 3 and having the ability to bind to an anti-SFPQ antibody.
　本発明の一実施形態に係るＳＦＰＱ関連機能障害モデル動物は、非ヒト哺乳動物にＳＦＰＱタンパク質または抗ＳＦＰＱ抗体を投与してなる、ＳＦＰＱ関連機能障害モデル動物である。 An SFPQ-related dysfunction model animal according to one embodiment of the present invention is an SFPQ-related dysfunction model animal obtained by administering an SFPQ protein or an anti-SFPQ antibody to a non-human mammal.
　本発明の一実施形態に係るＳＦＰＱ関連機能障害モデル動物の製造方法は、非ヒト哺乳動物に、ＳＦＰＱタンパク質または抗ＳＦＰＱ抗体を投与する投与工程を含む、ＳＦＰＱ関連機能障害モデル動物の製造方法である。 A method for producing an SFPQ-related dysfunction model animal according to one embodiment of the present invention is a method for producing an SFPQ-related dysfunction model animal, comprising an administration step of administering an SFPQ protein or an anti-SFPQ antibody to a non-human mammal. .
　本発明者らの見出した、ＳＦＰＱ関連機能障害の発生メカニズムは、様々な方法で利用することができる。したがって、本発明の一態様によれば、ＳＦＰＱ関連機能障害のデータの取得方法、診断薬、血液浄化用担体、および、モデル動物等を提供することができる。 The mechanism of occurrence of SFPQ-related dysfunction found by the present inventors can be used in various ways. Therefore, according to one aspect of the present invention, it is possible to provide a method for acquiring data on SFPQ-related dysfunction, a diagnostic agent, a carrier for blood purification, a model animal, and the like.
実施例１における、ネガティブゲル染色の結果およびＷｅｓｔｅｒｎ　Ｂｌｏｔによる自己抗体の検査の結果を示す図である。FIG. 2 shows the results of negative gel staining and the results of autoantibody test by Western Blot in Example 1. FIG. 実施例２におけるＳＥＡＰレポーターアッセイの結果および抗ＳＦＰＱ抗体による免疫沈降の結果を示す図である。FIG. 2 shows the results of SEAP reporter assay and the results of immunoprecipitation with anti-SFPQ antibody in Example 2. FIG. 実施例３におけるＥＬＩＳＡによる抗ＳＦＰＱ抗体価、および、Ｗｅｓｔｅｎ　Ｂｌｏｔによる、抗ＳＦＰＱ抗体価と、間接蛍光抗体法（ＩＦＡ）による抗ＳＩＴＨ－ＣＡＭＬ抗体価の検出結果との関係を示す図、および、各年代のヒト血清を用いたＷｅｓｔｅｎ　Ｂｌｏｔによる抗ＳＦＰＱ抗体価の変化を示す図である。A diagram showing the relationship between the anti-SFPQ antibody titer by ELISA, the anti-SFPQ antibody titer by Westen Blot, and the anti-SITH-CAML antibody titer by indirect immunofluorescence (IFA) in Example 3, and each FIG. 10 is a diagram showing changes in anti-SFPQ antibody titers by Westen Blot using human sera of different ages. 実施例４における、マウスの不安行動の解析結果、マウスの脳（手綱）の組織学的解析の結果を示す図である。FIG. 10 shows the results of analysis of anxious behavior of mice and the results of histological analysis of mouse brains (bridle) in Example 4. FIG. 実施例４における、マウスの脳における遺伝子発現解析の結果を示す図である。FIG. 10 shows the results of gene expression analysis in mouse brain in Example 4. FIG. 実施例５における、マウスの脳および嗅球における遺伝子発現解析の結果、および、マウスの脳（海馬）の組織学的解析の結果を示す図である。FIG. 10 shows the results of gene expression analysis in mouse brain and olfactory bulb, and the results of histological analysis of mouse brain (hippocampus) in Example 5. FIG. 実施例５における、マウスの空間作業記憶および協調運動能力の解析の結果を示す図である。FIG. 10 shows the results of analysis of mouse spatial working memory and motor coordination ability in Example 5. FIG. 実施例６における、マウスの小腸における遺伝子発現解析の結果および免疫期間中のマウスの体重変化の測定結果を示す図である。FIG. 10 is a diagram showing the results of gene expression analysis in the small intestine of mice and the measurement results of changes in body weight of mice during the immunization period in Example 6. FIG. 実施例７における、マウスの精巣における遺伝子発現解析の結果を示す一図である。FIG. 10 is a diagram showing the results of gene expression analysis in mouse testis in Example 7. FIG. 実施例７における、マウスの精巣における遺伝子発現解析の結果および精巣切片の蛍光免疫染色の結果を示す図である。FIG. 10 shows the results of gene expression analysis in mouse testis and the results of fluorescence immunostaining of testis sections in Example 7. FIG. 実施例８における、ＳＦＰＱ免疫マウスの皮膚における遺伝子発現解析の結果を示す図である。FIG. 10 shows the results of gene expression analysis in the skin of SFPQ-immunized mice in Example 8. FIG. 実施例８における、ＳＦＰＱ免疫マウスの皮膚の組織学的解析の結果を示す図である。FIG. 10 shows the results of histological analysis of the skin of SFPQ-immunized mice in Example 8. FIG. 実施例９における、ＳＦＰＱ免疫マウスの心臓における遺伝子発現解析の結果を示す図である。FIG. 10 shows the results of gene expression analysis in the heart of SFPQ-immunized mice in Example 9. FIG. 実施例１０における、ＳＦＰＱ免疫マウスの肺における遺伝子発現解析の結果を示す図である。FIG. 10 shows the results of gene expression analysis in the lungs of SFPQ-immunized mice in Example 10. FIG. 実施例１１におけるＰＣＲ産物の解析結果、Ｎｅｕｒｏ２ａ細胞の蛍光強度の測定結果、および、Ｎｅｕｒｏ２ａ細胞の細胞体サイズの測定結果を示す図である。FIG. 11 shows the results of analysis of PCR products, the results of measurement of fluorescence intensity of Neuro2a cells, and the results of measurement of cell body size of Neuro2a cells in Example 11. FIG. 実施例実施例１２における、神経細胞の遺伝子発現の解析の結果を示す図である。FIG. 10 shows the results of analysis of gene expression in nerve cells in Example 12. FIG. 実施例１３における、Ｈ－１細胞の遺伝子発現の解析の結果を示す一図である。FIG. 10 is a diagram showing the results of gene expression analysis of H-1 cells in Example 13. FIG. 実施例１３における、Ｈ－１細胞の遺伝子発現の解析の結果を示す一図である。FIG. 10 is a diagram showing the results of gene expression analysis of H-1 cells in Example 13. FIG. 実施例１４における、抗ＳＦＰＱマウスモノクローナル抗体尾静脈投与マウスの体重変化の測定結果および協調運動能力の測定結果を示す図である。FIG. 10 shows the measurement results of body weight change and the measurement results of coordination ability of anti-SFPQ mouse monoclonal antibody tail vein-administered mice in Example 14. FIG. 実施例１４における、抗ＳＦＰＱウサギポリクローナル抗体を尾静脈投与したマウスの行動試験の解析結果および抗ＳＦＰＱウサギポリクローナル抗体を尾静脈投与したマウスの脳における遺伝子発現解析の結果を示す図である。FIG. 14 shows the analysis results of a behavioral test of mice to which an anti-SFPQ rabbit polyclonal antibody was administered via the tail vein and the results of gene expression analysis in the brain of mice to which an anti-SFPQ rabbit polyclonal antibody was administered via the tail vein, in Example 14.
　本発明の一実施形態について説明すると以下の通りであるが、本発明はこれに限定されない。本発明は、以下に説明する各構成に限定されるものではなく、特許請求の範囲に示した範囲で種々の変更が可能であり、異なる実施形態および実施例にそれぞれ開示された技術的手段を適宜組み合わせて得られる実施形態および実施例についても本発明の技術的範囲に含まれる。また、本明細書中に記載された文献の全てが、本明細書中において参考文献として援用される。本明細書中、数値範囲に関して「Ａ～Ｂ」と記載した場合、該記載は「Ａ以上Ｂ以下」を意図する。 An embodiment of the present invention is described below, but the present invention is not limited to this. The present invention is not limited to the configurations described below, and can be modified in various ways within the scope of the claims, and the technical means disclosed in different embodiments and examples. Embodiments and examples obtained by appropriate combinations are also included in the technical scope of the present invention. Also, all of the documents mentioned in this specification are incorporated herein by reference. In this specification, when "A to B" is described with respect to a numerical range, the description means "A or more and B or less".
　〔１．本発明の背景〕
　本発明者らは、うつ病と老化との関係性を検討するにあたり、(ｉ）うつ病患者の７９．８％、オッズ比１２．２において、ＳＩＴＨ－１に対する抗体が陽性であること、(ｉｉ）ＳＩＴＨ‐１が生体のカルシウム関連タンパク質ｃａｌｃｉｕｍ　ｍｏｄｕｌａｔｉｎｇ　ｌｉｇａｎｄ（ＣＡＭＬ）と結合して病原性を発揮すること、および、(ｉｉｉ）ＳＩＴＨ－１を人為的に発現させたマウスにおいて、うつ症状が確認されることを見出した。その結果、ＳＩＴＨ－１の発現が、うつ病の発病に関与しているという新規の知見を得た。 [1. Background of the present invention]
In examining the relationship between depression and aging, the present inventors found that (i) 79.8% of depressed patients with an odds ratio of 12.2 were positive for antibodies to SITH-1, ( ii) SITH-1 exerts pathogenicity by binding to the calcium-related protein calcium modulating ligand (CAML) in vivo, and (iii) depressive symptoms were confirmed in mice in which SITH-1 was artificially expressed. found to be As a result, we obtained a novel finding that the expression of SITH-1 is involved in the onset of depression.
　本発明者らは、上記の新規の知見に基づきＳＩＴＨ‐１に関する研究を鋭意進めるうちに、ＳＩＴＨ－１が、生体のＲＮＡ結合タンパク質であるＳＦＰＱに対する自己免疫反応（例えば、ＳＦＰＱに対する自己抗体（抗ＳＦＰＱ抗体）の産生）を誘導することを新たに見出した。さらに、ＳＩＴＨ－１に対する抗体価と抗ＳＦＰＱ抗体価が正の相関を示すこと、および、うつ病患者において、抗ＳＦＰＱ抗体が高率で陽性であることも新たに見出した。そして、ＳＦＰＱに対する自己免疫反応について、さらに鋭意検討した結果、本発明者らは、ＳＦＰＱに対する自己免疫反応が、生体において、うつ病および老化と類似の機能障害を誘導することを見出し、本発明を完成するに至った。 Based on the above-mentioned new findings, the inventors of the present invention have been earnestly conducting research on SITH-1, and found that SITH-1 is an autoimmune reaction against SFPQ, an RNA-binding protein in the body (e.g., autoantibody (antibody) against SFPQ). It was newly found to induce the production of SFPQ antibody). Furthermore, it was newly found that the antibody titer against SITH-1 and the anti-SFPQ antibody titer show a positive correlation, and that the anti-SFPQ antibody is highly positive in patients with depression. Further, as a result of further intensive studies on autoimmune reactions to SFPQ, the present inventors found that autoimmune reactions to SFPQ induce functional disorders similar to depression and aging in living organisms, and developed the present invention. Completed.
　ＳＦＰＱに対する自己抗体（抗ＳＦＰＱ抗体）は、これまでに全身性エリテマトーデス患者や皮膚筋炎患者での検出が報告されていたが、特定の疾患の重症度、あるいは、発症機構との関係性は一切知られていなかった。そのため、本発明者らの見出した「ＳＦＰＱに対する自己免疫反応が、生体において、老化と類似の機能障害を誘導する」との知見は驚くべき発見であると言える。 Autoantibodies against SFPQ (anti-SFPQ antibodies) have been reported to be detected in patients with systemic lupus erythematosus and dermatomyositis, but their relationship with the severity of specific diseases or the onset mechanism is unknown. It wasn't done. Therefore, it can be said that the finding by the present inventors that "an autoimmune reaction against SFPQ induces a functional disorder similar to aging in a living body" is a surprising finding.
　〔２．ＳＦＰＱ関連機能障害の診断または検査するためのデータの取得方法〕
　本発明の一実施形態に係るＳＦＰＱ関連機能障害の診断または検査するためのデータの取得方法は、被験体から分離した試料中の抗ＳＦＰＱ抗体の抗体価を測定する測定工程を含む。以下、「本発明の一実施形態に係るＳＦＰＱ関連機能障害の診断または検査するためのデータの取得方法」を「本データの取得方法」と称する場合がある。 [2. Method of acquiring data for diagnosing or examining SFPQ-related dysfunction]
A data acquisition method for diagnosing or testing SFPQ-related dysfunction according to one embodiment of the present invention comprises a measuring step of measuring the antibody titer of anti-SFPQ antibody in a sample separated from a subject. Hereinafter, the "data acquisition method for diagnosing or examining SFPQ-related dysfunction according to one embodiment of the present invention" may be referred to as "this data acquisition method".
　本明細書において、「ＳＦＰＱ関連機能障害」とは、細胞や臓器の正常な機能発現にＳＦＰＱを必要とする場面において、ＳＦＰＱに対する自己免疫反応により、ＳＦＰＱの正常な機能が阻害されるために生じる生体の機能障害を意図する。より具体的には、このようなＳＦＰＱ関連機能障害としては、精神運動性の焦燥または制止、易疲労性、集中力の低下や思考力の低下、あるいは、不安、等のうつ病患者でよくみられる機能障害；脳、腸、精巣、皮膚、心臓および／または肺の老化と類似の機能障害；あるいは、幹細胞の維持・分化、スプライシング、軸索の生存、Ｔａｕタンパク質の産生とリン酸化、酸化ストレス調節、テロメア安定性の維持、ＰＤＥ３Ａ転写、ＣＤ４０転写、脂肪細胞分化、ＤＮＡ損傷修復、自然免疫応答の異常、等の全身性の機能障害；等が挙げられる。 As used herein, the term "SFPQ-related dysfunction" refers to an autoimmune reaction to SFPQ that inhibits the normal function of SFPQ in situations where SFPQ is required for normal functional expression of cells and organs. Intended for bodily dysfunction. More specifically, such SFPQ-related dysfunctions include psychomotor agitation or retardation, fatigability, poor concentration and thinking ability, or anxiety, which are common in depressed patients. dysfunction resembling aging in the brain, intestine, testis, skin, heart and/or lungs; or stem cell maintenance/differentiation, splicing, axonal survival, Tau protein production and phosphorylation, oxidative stress systemic dysfunction such as regulation, maintenance of telomere stability, PDE3A transcription, CD40 transcription, adipocyte differentiation, DNA damage repair, abnormalities in innate immune response;
　ＳＦＰＱ関連機能障害の症状は、様々な疾患の病態を伴う場合もある。このようなＳＦＰＱ関連機能障害の関与する疾患としては、うつ病、認知症、アルツハイマー病、ピック病、前頭側頭型認知症（ＦＴＤ）、ＦＴＬＤスペクトル疾患、自閉症スペクトラム障害、大腸がん、鼻咽頭がん、乳がん、前立腺がん、嚢胞繊維症、脂質障害、アテローム性動脈硬化症、代謝性ミオパチー、ＡＬＳ、不妊症、フレイル、更年期障害、等が挙げられるがこれに限定されるものではない。本明細書における、ＳＦＰＱ関連機能障害とは、これらのＳＦＰＱ関連機能障害の関与する疾患を包含する概念である。 　Symptoms of SFPQ-related dysfunction may accompany the pathology of various diseases. Diseases involving such SFPQ-related dysfunction include depression, dementia, Alzheimer's disease, Pick's disease, frontotemporal dementia (FTD), FTLD spectrum disorders, autism spectrum disorders, colon cancer, Nasopharyngeal cancer, breast cancer, prostate cancer, cystic fibrosis, lipid disorders, atherosclerosis, metabolic myopathy, ALS, infertility, frailty, menopause, etc., but not limited thereto. do not have. As used herein, SFPQ-related dysfunction is a concept that includes diseases associated with these SFPQ-related dysfunctions.
　上記のように、ＳＦＰＱ関連機能障害は、ＳＦＰＱに対する自己免疫反応により誘導されることで発症する。より具体的には、ＳＦＰＱに対する自己免疫反応が誘導されることで、抗ＳＦＰＱ抗体が産生され、該抗ＳＦＰＱ抗体が細胞機能を阻害することで、ＳＦＰＱ関連機能障害が生じる。したがって、被験体から分離した試料中における、抗ＳＦＰＱ抗体の抗体価を測定することで、該被験体におけるＳＦＰＱに対する自己免疫反応の有無およびその程度を診断または検査するためのデータを取得することができる、換言すると、ＳＦＰＱ関連機能障害の診断または検査するためのデータを取得することができる。 As described above, SFPQ-related dysfunction develops by being induced by an autoimmune reaction to SFPQ. More specifically, the induction of an autoimmune reaction against SFPQ leads to the production of anti-SFPQ antibodies, which inhibit cell function, resulting in SFPQ-related dysfunction. Therefore, by measuring the antibody titer of an anti-SFPQ antibody in a sample isolated from a subject, it is possible to obtain data for diagnosing or examining the presence or absence and degree of autoimmune reaction to SFPQ in the subject. Yes, in other words, data can be obtained for diagnosing or testing SFPQ-related dysfunctions.
　（測定工程）
　測定工程は、被験体から分離した試料中の抗ＳＦＰＱ抗体の抗体価を測定できる限り、特に限定されない。 (Measurement process)
The measurement step is not particularly limited as long as the antibody titer of the anti-SFPQ antibody in the sample separated from the subject can be measured.
　本明細書において「被験体」とは、対象の動物、好ましくは哺乳類、最も好ましくはヒトを指す。被験体としては、年齢、性別等は特に限定されない。 As used herein, the term "subject" refers to a target animal, preferably a mammal, most preferably a human. Subjects are not particularly limited in terms of age, sex, and the like.
　本明細書において、被験体から分離される「試料」は、特に限定されないが、例えば、血液、血清、血漿、尿および唾液から選択することができる。本明細書における「試料」とは、これらの試料を固定化液（例えば、ホルマリン）によって固定することによって作製された病理標本、あるいは、これらの試料を所望の溶液に可溶化させた可溶化物、も包含する概念である。 As used herein, the "sample" separated from the subject is not particularly limited, but can be selected from, for example, blood, serum, plasma, urine and saliva. As used herein, the term "sample" refers to pathological specimens prepared by fixing these samples with a fixative (e.g., formalin), or lysates obtained by solubilizing these samples in a desired solution. is a concept that also includes
　本データの取得方法における、「抗ＳＦＰＱ抗体」とは、ＳＦＰＱを抗原として特異的に認識し、ＳＦＰＱ抗原に結合するための相補性決定領域（ＣＤＲ）を有する抗体を意図する。「抗ＳＦＰＱ抗体」は、「ＳＦＰＱと反応する抗体」であるともいえる。 "Anti-SFPQ antibody" in this data acquisition method means an antibody that specifically recognizes SFPQ as an antigen and has a complementarity determining region (CDR) for binding to the SFPQ antigen. An "anti-SFPQ antibody" can also be said to be an "antibody that reacts with SFPQ".
　なお本明細書における「ＳＦＰＱ」とは、（ａ１）配列番号１で表されるポリペプチド（タンパク質）、（ａ２）配列番号１で示されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換または付加されたアミノ酸配列からなるポリペプチド、および、（ａ３）配列番号１で示されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列からなるポリペプチドの何れかを意図する。抗ＳＦＰＱ抗体は、上記（ａ１）～（ａ３）のポリペプチドと、特異的に（抗原抗体反応的に）結合する抗体であるともいえる。 In the present specification, "SFPQ" means (a1) a polypeptide (protein) represented by SEQ ID NO: 1, (a2) deletion of one or several amino acids in the amino acid sequence represented by SEQ ID NO: 1, Either a polypeptide consisting of a substituted or added amino acid sequence, or (a3) a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO:1 is intended. The anti-SFPQ antibody can also be said to be an antibody that specifically binds (antigen-antibody reaction) to the polypeptides (a1) to (a3) above.
　（ａ１）に関して、配列番号１は、ヒトＳＦＰＱタンパク質（ＧｅｎＡａｎｋのアクセッション番号ＮＰ＿００５０５７）のアミノ酸配列に対応している。 Regarding (a1), SEQ ID NO: 1 corresponds to the amino acid sequence of the human SFPQ protein (GenAank accession number NP_005057).
　（ａ２）に関して、アミノ酸残基が置換、欠失、挿入および／または付加される数の上限は、例えば、７個、６個、５個、４個、３個、２個または１個でありうる。 Regarding (a2), the upper limit of the number of substituted, deleted, inserted and/or added amino acid residues is, for example, 7, 6, 5, 4, 3, 2 or 1. sell.
　（ａ３）に関して、アミノ酸配列の同一性は、例えば、９０％以上、９１％以上、９２％以上、９３％以上、９４％以上、９５％以上、９６％以上、９７％以上、９８％以上、９９％以上、または１００％である。 Regarding (a3), the amino acid sequence identity is, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.
　本明細書において、アミノ酸配列の同一性は、例えば、BLASTXを利用して決定することができる。この場合のパラメータは、例えば、score=50, wordlength=3を採用できる。あるいは、Gapped BLASTプログラムを用いて、アミノ酸配列を解析することもできる。この場合は、デフォルトのパラメータを使用することができる。比較対象のアミノ酸配列を最適な状態にアラインメントするために、付加または欠失（ギャップなど）を許容してもよい。 In this specification, the identity of amino acid sequences can be determined using, for example, BLASTX. Parameters in this case can be score=50, wordlength=3, for example. Alternatively, amino acid sequences can be analyzed using the Gapped BLAST program. In this case the default parameters can be used. Additions or deletions (such as gaps) may be allowed in order to optimally align the amino acid sequences being compared.
　本明細書における「抗体価」とは、試料中に含まれる該抗体の量および結合力を加味した、該抗体が対象（抗原）に影響を与え得る量を意図する。本発明の一実施形態において、「抗体価」は、所定のアッセイ法において最低限レベルの特異的検出達成に必要となる、抗体サンプルの機能希釈（または作業濃度）として定義される、抗体の量と結合力の両者を加味した、抗体サンプルの量であってもよい。抗体価は、抗体の力価とも言える。すなわち、本データの取得方法における測定工程は、抗ＳＦＰＱ抗体の抗体量および結合力を総合した「抗体価」を測定する工程であるともいえる。抗体の抗体価の測定は、公知の方法を用いて行うことが可能であり、例えば、間接蛍光抗体法（本明細書において間接蛍光抗体法を、「ＩＦＡ」という場合がある。）、ドットブロットアッセイ法、ウエスタンブロット法、酵素結合免疫吸着アッセイ法（ＥＬＩＳＡ（サンドイッチＥＬＩＳＡ法、直接吸着によるＥＬＩＳＡ法、競合によるＥＬＩＳＡ法を含む））、放射性イムノアッセイ法（ＲＩＡ）、および免疫拡散アッセイ法のような、ｉｎ　ｖｉｔｒｏでの免疫組織学的方法を利用したアッセイ法で測定することができる。あるいは、ｉｎ　ｖｉｖｏでの画像解析等によって検出することもできる。 The term "antibody titer" as used herein means the amount of the antibody that can affect the target (antigen), taking into consideration the amount and binding power of the antibody contained in the sample. In one embodiment of the invention, an "antibody titer" is the amount of antibody, defined as the functional dilution (or working concentration) of an antibody sample required to achieve a minimal level of specific detection in a given assay. It may be the amount of the antibody sample that takes into account both the binding strength and the binding strength. The antibody titer can also be said to be the titer of the antibody. That is, it can be said that the measurement step in the present data acquisition method is a step of measuring the "antibody titer", which is the sum of the antibody amount and the binding strength of the anti-SFPQ antibody. The antibody titer can be measured using a known method, for example, an indirect fluorescent antibody method (the indirect fluorescent antibody method is sometimes referred to as "IFA" in this specification), dot blot such as assays, Western blots, enzyme-linked immunosorbent assays (ELISAs (including sandwich ELISAs, direct adsorption ELISAs, competitive ELISAs)), radioimmunoassays (RIAs), and immunodiffusion assays. , can be measured by an assay method using an in vitro immunohistological method. Alternatively, it can be detected by in vivo image analysis or the like.
　本明細書において「診断」とは、医師による同定を必須としない、被験体におけるＳＦＰＱ関連機能障害の発症の有無、被験体におけるＳＦＰＱ関連機能障害の重症度、および／または、被験体におけるＳＦＰＱ関連機能障害発症するリスク（危険度）を評価することをいう。また、本明細書において「検査」とは、医師による同定を必須としない、被験体における、ＳＦＰＱ関連機能障害の検査を指す。なお、「検査」とは、ＳＦＰＱ関連機能障害の発症の有無の検査のみならず、ＳＦＰＱ関連機能障害の発症する可能性を検査することも含む概念である。本データの取得方法は、医師による同定等の、医師によってなされる操作を含む物ではないが、本データの取得方法によって得られたデータは、医師によってなされる診断・検査結果の評価の一材料となってもよい。 As used herein, "diagnosis" refers to the presence or absence of SFPQ-related dysfunction in a subject, the severity of SFPQ-related dysfunction in a subject, and/or SFPQ-related dysfunction in a subject, which does not require identification by a physician. It refers to evaluating the risk (degree of risk) of developing a functional disorder. Also, as used herein, "testing" refers to testing for SFPQ-related dysfunction in a subject that does not require identification by a physician. It should be noted that the term “examination” is a concept that includes not only examination for the presence or absence of onset of SFPQ-related dysfunction, but also examination for the possibility of developing SFPQ-related dysfunction. This data acquisition method does not include operations performed by a doctor, such as identification by a doctor, but the data obtained by this data acquisition method is one of the materials for the evaluation of diagnosis and test results by a doctor. may be
　（ＳＦＰＱ関連機能障害の診断方法）
　本発明の一実施形態において、ＳＦＰＱ関連機能障害の診断方法を提供する。本発明の一実施形態に係るＳＦＰＱ関連機能障害の診断方法（以下、本診断方法と称する場合がある）は、被験体から分離した試料中の抗ＳＦＰＱ抗体の抗体価を測定する測定工程を含む。なお、本診断方法における、測定工程の具体的な態様は、上記（測定工程）項に記載の通りである。 (Method for diagnosing SFPQ-related dysfunction)
In one embodiment of the present invention, a method for diagnosing SFPQ-related dysfunction is provided. A method for diagnosing SFPQ-related dysfunction according to one embodiment of the present invention (hereinafter sometimes referred to as this diagnostic method) includes a measuring step of measuring the antibody titer of an anti-SFPQ antibody in a sample separated from a subject. . In addition, the specific aspect of the measurement step in this diagnostic method is as described in the above section (Measurement step).
　本診断方法においては、測定工程における、被験体から分離した試料中の抗ＳＦＰＱ抗体の抗体価の測定結果に基づき、ＳＦＰＱ関連機能障害の発症の有無、ＳＦＰＱ関連機能障害の症状の程度、および／またはＳＦＰＱ関連機能障害の発症の可能性を診断する。 In this diagnostic method, the presence or absence of onset of SFPQ-related dysfunction, the degree of symptoms of SFPQ-related dysfunction, and/or or to diagnose the possible development of SFPQ-related dysfunction.
　本診断方法において、被験体から分離した試料中の抗ＳＦＰＱ抗体の抗体価が閾値以上である場合に、ＳＦＰＱ関連機能障害が発症している、ＳＦＰＱ関連機能障害の症状が重度である、あるいは、ＳＦＰＱ関連機能障害が発症する可能性が高い、と診断することができる。ここで、抗ＳＦＰＱ抗体の抗体価の「閾値」については、健常者における定量値（正常値）、典型的なＳＦＰＱ関連機能障害患者の定量値（疾患値）、又は軽度、中度もしくは重度のＳＦＰＱ関連機能障害患者の定量値（疾患値）を参考に、当業者が適宜設定することができる。すなわち、本診断方法における閾値（いわゆるカットオフ値）は、臨床試験から得られた健常者や患者の多くの測定値を基に、その目的に合わせて当業者が適宜設定するものであり（例えば、スクリーニング検査のように、疾患群を見逃さないことを最優先として二次検査以降で確定診断するような場合には、特異度よりも感度を優先し、カットオフ値を低く設定する、ＳＦＰＱ関連機能障害の症状の程度を診断する場合には、軽度、中度または重度のＳＦＰＱ関連機能障害患者の定量値を参考に閾値を高く設定する等）、本明細書に開示されている記載から、当業者は容易にＳＦＰＱ関連機能障害の診断のための、抗ＳＦＰＱ抗体の抗体価の閾値を決定することができる。なお、本診断方法に用いられる閾値の数値については、健常者またはＳＦＰＱ関連機能障害患者個人から分離した試料から測定された数値を直接利用してもよく、一定の人数の健常者又はＳＦＰＱ関連機能障害患者分離した試料から測定された数値を母集団としたときに得られる平均値を利用してもよい。 In this diagnostic method, when the antibody titer of the anti-SFPQ antibody in the sample isolated from the subject is a threshold value or higher, SFPQ-related dysfunction has developed, the symptoms of SFPQ-related dysfunction are severe, or It can be diagnosed that SFPQ-related dysfunction is likely to develop. Here, for the "threshold" of the antibody titer of the anti-SFPQ antibody, the quantitative value (normal value) in healthy subjects, the quantitative value (disease value) in patients with typical SFPQ-related dysfunction, or mild, moderate or severe A person skilled in the art can appropriately set it with reference to quantitative values (disease values) of patients with SFPQ-related dysfunction. That is, the threshold (so-called cut-off value) in this diagnostic method is appropriately set by those skilled in the art according to the purpose based on many measured values of healthy subjects and patients obtained from clinical trials (e.g. , In the case of a screening test, in which the highest priority is not to miss a group of diseases and a definitive diagnosis is made after the secondary test, sensitivity is prioritized over specificity and the cutoff value is set low. SFPQ-related When diagnosing the degree of symptoms of functional impairment, the threshold is set high with reference to the quantitative value of patients with mild, moderate or severe SFPQ-related functional impairment, etc.), from the description disclosed in the present specification, One skilled in the art can readily determine the threshold titer of anti-SFPQ antibodies for diagnosis of SFPQ-related dysfunction. As for the numerical value of the threshold value used in this diagnostic method, the numerical value measured from a sample isolated from an individual patient with healthy subjects or SFPQ-related dysfunction may be directly used. An average value obtained when the values measured from the samples separated from the disabled patients are used as a population may be used.
　〔３．ＳＦＰＱ関連機能障害の診断薬〕
　本発明の一実施形態において、ＳＦＰＱ関連機能障害の診断薬を提供する。本発明の一実施形態に係るＳＦＰＱ関連機能障害の診断薬（以下、「本診断薬」と称する）は、以下の（ａ１）～（ａ３）、（ｂ１）～（ｂ３）、および、（ｃ１）～（ｃ３）から選択される少なくとも１つの何れかを含む：（ａ１）配列番号１で表されるポリペプチド；（ａ２）配列番号１で示されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換または付加されたアミノ酸配列からなるポリペプチド；（ａ３）配列番号１で示されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列からなるポリペプチド；（ｂ１）配列番号２または３で示されるアミノ酸配列からなるポリペプチド；（ｂ２）配列番号２または３で示されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換または付加されたアミノ酸配列からなり、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；（ｂ３）配列番号２または３で示されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列からなり、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；（ｃ１）配列番号２または３で示されるアミノ酸配列を含み、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；（ｃ２）配列番号２または３で示されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換または付加されたアミノ酸配列を含み、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；（ｃ３）配列番号２または３で示されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含み、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド。 [3. Diagnostic agent for SFPQ-related dysfunction]
In one embodiment of the invention, a diagnostic for SFPQ-related dysfunction is provided. The diagnostic agent for SFPQ-related dysfunction according to one embodiment of the present invention (hereinafter referred to as "this diagnostic agent") includes the following (a1) to (a3), (b1) to (b3), and (c1 ) to (c3): (a1) a polypeptide represented by SEQ ID NO: 1; (a2) in the amino acid sequence represented by SEQ ID NO: 1, one or several amino acids are (a3) a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1; (b1) SEQ ID NO: 2 or 3 (b2) consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 or 3, and an anti-SFPQ antibody (b3) a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2 or 3, and having the ability to bind to an anti-SFPQ antibody; (c1) a polypeptide comprising the amino acid sequence shown by SEQ ID NO: 2 or 3 and having the ability to bind to an anti-SFPQ antibody; (c2) in the amino acid sequence shown by SEQ ID NO: 2 or 3, one or several A polypeptide containing an amino acid sequence in which amino acids have been deleted, substituted or added and having the ability to bind to an anti-SFPQ antibody; and having the ability to bind to an anti-SFPQ antibody.
　上記（ａ１）～（ａ３）に関しては、上記〔２．ＳＦＰＱ関連機能障害の診断または検査するためのデータの取得方法〕項の説明を適宜援用する。 Regarding the above (a1) to (a3), the above [2. Acquisition Method of Data for Diagnosing or Examining SFPQ-Related Dysfunction].
　（ｂ１）および（ｃ１）に関して、配列番号２および３で示されるアミノ酸配列は、本発明者らがＳＦＰＱ（ＳＦＰＱタンパク質）のアミノ酸配列を用いて、エピトープ解析を試みた結果見出した、ＳＦＰＱのエピトープのアミノ酸配列である。配列番号２は、ＳＦＰＱの３６番目から５０番目の１５アミノ酸残基（ＳＦＰＱ３６－５０、）の配列に対応し、配列番号３のアミノ酸配列は、４５２番目から４６６番目の１５アミノ酸残基（ＳＦＰＱ４５２－４６６）の配列に対応する。 Regarding (b1) and (c1), the amino acid sequences shown in SEQ ID NOS: 2 and 3 are epitopes of SFPQ, which the present inventors found as a result of attempting epitope analysis using the amino acid sequence of SFPQ (SFPQ protein). is the amino acid sequence of SEQ ID NO: 2 corresponds to the sequence of 15 amino acid residues from 36th to 50th of SFPQ (SFPQ36-50), and the amino acid sequence of SEQ ID NO: 3 corresponds to the sequence of 15 amino acid residues from 452nd to 466th (SFPQ452- 466).
　本診断薬は、配列番号１で示されるＳＦＰＱタンパク質そのもの、配列番号２または３で示されるＳＦＰＱのエピトープ、および、該エピトープを含むポリペプチドあるいはそれらのバリアントであるポリペプチドを含むため、抗ＳＦＰＱ抗体を特異的に検出することができ、該抗体の抗体価の測定に利用することができる。そのため、ＳＦＰＱ関連機能障害の診断薬として好適に適用できる。 Since this diagnostic agent contains the SFPQ protein itself shown by SEQ ID NO: 1, the SFPQ epitope shown by SEQ ID NO: 2 or 3, and a polypeptide containing the epitope or a variant thereof, the anti-SFPQ antibody can be specifically detected and used to measure the antibody titer of the antibody. Therefore, it can be suitably applied as a diagnostic agent for SFPQ-related dysfunction.
　本診断薬を適用し得るＳＦＰＱ関連機能障害については、〔２．ＳＦＰＱ関連機能障害の診断または検査するためのデータの取得方法〕項の記載を適宜援用する。 For SFPQ-related dysfunctions to which this diagnostic agent can be applied, see [2. Acquisition method of data for diagnosing or examining SFPQ-related dysfunction].
　（ｂ２）および（ｃ２）に関して、アミノ酸残基が置換、欠失、挿入および／または付加される数の上限は、例えば、２個または１個でありうる。 Regarding (b2) and (c2), the upper limit of the number of substituted, deleted, inserted and/or added amino acid residues can be, for example, two or one.
　（ｂ３）および（ｃ３）に関して、アミノ酸配列の同一性は、例えば、９０％以上、９１％以上、９２％以上、９３％以上、９４％以上、９５％以上、９６％以上、９７％以上、９８％以上、９９％以上、または１００％である。 Regarding (b3) and (c3), the amino acid sequence identity is, for example, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%.
　上記（ｃ１）～（ｃ３）に関し、「アミノ酸配列を含むポリペプチド」とは、当該アミノ酸配列のＮ末端のアミノ酸および／またはＣ末端のアミノ酸に、更なる任意の数のアミノ酸が付加されたポリペプチドを意味する。ここで、付加されるアミノ酸の個数は特に限定されず、例えば、５００個以下、３００個以下、２００個以下、１００個以下、５０個以下、あるいは、３０個以下、等であり得る。 Regarding (c1) to (c3) above, the term "polypeptide comprising an amino acid sequence" refers to a poly(polypeptide) in which any number of additional amino acids are added to the N-terminal amino acid and/or C-terminal amino acid of the amino acid sequence. means peptide. Here, the number of amino acids to be added is not particularly limited, and may be, for example, 500 or less, 300 or less, 200 or less, 100 or less, 50 or less, or 30 or less.
　上記（ｂ２）～（ｂ３）および（ｃ１）～（ｃ３）に関して、ポリペプチドが、抗ＳＦＰＱ抗体との結合能を有するか否かは、例えば、後述する実施例に記載の、抗ＳＦＰＱ抗体を用いた免疫沈降法によって判定できる。一実施形態において、（ｂ２）～（ｂ３）および（ｃ１）～（ｃ３）のポリペプチドの抗ＳＦＰＱ抗体との結合能は、ヒトＳＦＰＱ（配列番号１）の、４０％以上、４５％以上、５０％以上、５５％以上、６０％以上、６５％以上、７０％以上、７５％以上、８０％以上、８５％以上、９０％以上または９５％以上である。 Regarding (b2) to (b3) and (c1) to (c3) above, whether or not the polypeptide has the ability to bind to an anti-SFPQ antibody can be determined, for example, by binding an anti-SFPQ antibody as described in the Examples below. It can be determined by the immunoprecipitation method used. In one embodiment, the ability of the polypeptides (b2) to (b3) and (c1) to (c3) to bind to an anti-SFPQ antibody is 40% or more, 45% or more of that of human SFPQ (SEQ ID NO: 1), 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more.
　本診断薬としては、（ａ１）～（ａ３）、（ｂ１）～（ｂ３）、および、（ｃ１）～（ｃ３）で示されるポリペプチドに、標識物質を付加することも可能である。標識物質としては、特に限定されないが、例えば、蛍光色素、酵素、補酵素、化学発光物質、放射性物質等が使用される。具体的には、ラジオアイソトープ（３２Ｐ、１４Ｃ、１２５Ｉ、３Ｈ、１３１Ｉ等）、フルオレセイン、ローダミン、ダンシルクロリド、ウンベリフェロン、ルシフェラーゼ、ペルオキシダーゼ、アルカリホスファターゼ、β-ガラクトシダーゼ、β-グルコシダーゼ、ホースラディッシュパーオキシダーゼ、グルコアミラーゼ、リゾチーム、サッカリドオキシダーゼ、マイクロペルオキシダーゼ、ビオチン等が使用され得る。 Labeling substances can also be added to the polypeptides shown in (a1) to (a3), (b1) to (b3), and (c1) to (c3) as the present diagnostic agent. Examples of the labeling substance include, but are not limited to, fluorescent dyes, enzymes, coenzymes, chemiluminescent substances, radioactive substances, and the like. Specifically, radioisotopes ( 32 P, 14 C, 125 I, 3 H, 131 I, etc.), fluorescein, rhodamine, dansyl chloride, umbelliferone, luciferase, peroxidase, alkaline phosphatase, β-galactosidase, β-glucosidase , horseradish peroxidase, glucoamylase, lysozyme, saccharide oxidase, microperoxidase, biotin and the like can be used.
　本診断薬は、前記（ａ１）～（ａ３）、（ｂ１）～（ｂ３）、および、（ｃ１）～（ｃ３）で示されるポリペプチドの他に、薬学的に許容される担体、潤滑剤、保存剤、安定剤、湿潤剤、乳化剤、浸透圧調整用の塩類、緩衝剤、着色剤、抗酸化剤、粘度調整剤、賦活剤、またはナノ粒子等を含んでいてもよい。薬学的に許容される担体としては、例えば、水、各種塩溶液、アルコール、植物油、およびミネラルオイル等が挙げられるが、これらに限定されない。 The diagnostic agent comprises, in addition to the polypeptides shown in (a1) to (a3), (b1) to (b3), and (c1) to (c3), a pharmaceutically acceptable carrier, a lubricant , preservatives, stabilizers, wetting agents, emulsifiers, salts for adjusting osmotic pressure, buffers, coloring agents, antioxidants, viscosity modifiers, activators, nanoparticles, and the like. Examples of pharmaceutically acceptable carriers include, but are not limited to, water, various salt solutions, alcohols, vegetable oils, mineral oils, and the like.
　〔４．血液浄化用担体および血液浄化カラム〕
　本発明者らは、ＳＦＰＱ関連機能障害について研究を進めるうちに、ＳＦＰＱ関連機能障害を発症している患者において、血液中の抗ＳＦＰＱ抗体の濃度を低下させることにより、該機能障害の病態を抑制し得ることを見出した。このことは、実施例（実施例～）において、認知症の原因となるＴａｕタンパク質のリン酸化、脳の老化の原因となる神経幹細胞の消耗、全身的な老化の原因となる未分化幹細胞の消耗、老化の指標となる体重減少、うつ病の悪化を示す指標、といった、ＳＦＰＱ関連機能障害の重要な病態が抗ＳＦＰＱ抗体の存在によってもたらされていることによって裏付けられている。また、ＳＦＰＱ関連機能障害を発症していない被験体においても、血液中の抗ＳＦＰＱ抗体の濃度を低下させることで、ＳＦＰＱ関連機能障害の発症を予防できると考えられる。 [4. Carrier for Blood Purification and Blood Purification Column]
While conducting research on SFPQ-related dysfunction, the present inventors found that by reducing the concentration of anti-SFPQ antibodies in the blood of patients with SFPQ-related dysfunction, the pathology of the dysfunction was suppressed. I found what I could do. In Examples (Examples to ), phosphorylation of Tau protein that causes dementia, exhaustion of neural stem cells that causes brain aging, and exhaustion of undifferentiated stem cells that causes systemic aging , weight loss as an indicator of aging, and an indicator of exacerbation of depression, important pathologies of SFPQ-related dysfunction are supported by the presence of anti-SFPQ antibodies. In addition, even in subjects who have not developed SFPQ-related dysfunction, it is believed that the development of SFPQ-related dysfunction can be prevented by reducing the concentration of anti-SFPQ antibodies in the blood.
　血液中の抗ＳＦＰＱ抗体濃度を低下させるためには、血漿交換、大量の抗体を静脈内投与する大量ガンマグロブリン療法、血液浄化療法が適応可能と考えられる。中でも、身体的な負担が比較的小さく、かつ、確実に抗ＳＦＰＱ抗体を低下させることができるという理由から、抗ＳＦＰＱ抗体を吸着し得る担体を備えるカラムに血液を通過させることで、血中の抗ＳＦＰＱ抗体を該カラム（担体）に吸着させ、血液中の抗ＳＦＰＱ抗体濃度を低下させる方法である、血液浄化療法が特に有効であると考えられる。ただし、血液中の抗ＳＦＰＱ抗体濃度を低下させる方法はこれに限定されるものではない。 In order to reduce the anti-SFPQ antibody concentration in the blood, plasmapheresis, high-dose gamma globulin therapy that intravenously administers a large amount of antibody, and blood purification therapy are considered applicable. Among them, since the physical burden is relatively small and the anti-SFPQ antibody can be reliably reduced, the blood is passed through a column equipped with a carrier capable of adsorbing the anti-SFPQ antibody. Blood purification therapy, which is a method of reducing the concentration of anti-SFPQ antibodies in blood by adsorbing anti-SFPQ antibodies to the column (carrier), is considered to be particularly effective. However, the method for reducing the anti-SFPQ antibody concentration in blood is not limited to this.
　（血液浄化用担体および血液浄化カラム）
　本発明の一実施形態において、表面に、以下の（ａ１）～（ａ３）、（ｂ１）～（ｂ３）、および、（ｃ１）～（ｃ３）から選択される少なくとも１つの何れかを含む（備える）：（ａ１）配列番号１で表されるポリペプチド；（ａ２）配列番号１で示されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換または付加されたアミノ酸配列からなるポリペプチド；（ａ３）配列番号１で示されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列からなるポリペプチド；（ｂ１）配列番号２または３で示されるアミノ酸配列からなるポリペプチド；（ｂ２）配列番号２または３で示されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換または付加されたアミノ酸配列からなり、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；（ｂ３）配列番号２または３で示されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列からなり、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；（ｃ１）配列番号２または３で示されるアミノ酸配列を含み、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；（ｃ２）配列番号２または３で示されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換または付加されたアミノ酸配列を含み、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；（ｃ３）配列番号２または３で示されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含み、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド。 (Blood purification carrier and blood purification column)
In one embodiment of the present invention, the surface contains at least one selected from the following (a1) to (a3), (b1) to (b3), and (c1) to (c3) ( (a1) a polypeptide represented by SEQ ID NO: 1; (a2) a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1 (a3) a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1; (b1) a polypeptide consisting of an amino acid sequence shown in SEQ ID NO: 2 or 3; (b2) a sequence A polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by number 2 or 3, and having the ability to bind to an anti-SFPQ antibody; (b3) SEQ ID NO: (c1) a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by 2 or 3 and having the ability to bind to an anti-SFPQ antibody; (c1) the amino acid sequence represented by SEQ ID NO: 2 or 3 (c2) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 or 3 and a polypeptide having the ability to bind to an anti-SFPQ antibody; (c3) comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2 or 3, and binding to an anti-SFPQ antibody A polypeptide having an ability.
　本発明の一実施形態に係る血液浄化用担体（以下、本担体と称する場合がある）は、その表面に、配列番号１で示されるＳＦＰＱタンパク質そのもの、配列番号２または３で示されるＳＦＰＱのエピトープ、および、該エピトープを含むポリペプチドあるいはそれらのバリアントであるポリペプチドを含むため、抗ＳＦＰＱ抗体を特異的に吸着することができる。したがって、本担体を備える、本発明の一実施形態に係る血液浄化カラム（以下、本カラムと称する場合がある）に液体（特に血液）を通過させることにより、該液体中の抗ＳＦＰＱ抗体を除去することができる。なお、本担体および本カラムに関し、（ａ１）～（ａ３）、（ｂ１）～（ｂ３）および（ｃ１）～（ｃ３）で表されるポリペプチドについては、上記〔２．ＳＦＰＱ関連機能障害の診断または検査するためのデータの取得方法〕項および〔３．ＳＦＰＱ関連機能障害の診断薬〕項の記載を適宜援用する。 The carrier for blood purification according to one embodiment of the present invention (hereinafter sometimes referred to as the carrier) has, on its surface, the SFPQ protein itself represented by SEQ ID NO: 1, or an epitope of SFPQ represented by SEQ ID NO: 2 or 3. , and a polypeptide containing the epitope or a variant thereof, so that the anti-SFPQ antibody can be specifically adsorbed. Therefore, by passing a liquid (especially blood) through a blood purification column according to an embodiment of the present invention (hereinafter sometimes referred to as the present column) comprising the present carrier, the anti-SFPQ antibody in the liquid is removed. can do. Regarding the present carrier and the present column, the polypeptides represented by (a1) to (a3), (b1) to (b3) and (c1) to (c3) are described in [2. Acquisition method of data for diagnosing or examining SFPQ-related dysfunction] and [3. Diagnostic agent for SFPQ-related dysfunction] section is incorporated as appropriate.
　本担体の形状は特に限定されず、粒子状、繊維状、膜状（中空糸膜も含む）などいずれの形状であっても良いが、通液性、表面積確保、吸着材調製時の取扱いの容易さなどの観点から、粒子状または繊維状であることが好ましい。 The shape of the present carrier is not particularly limited, and may be any shape such as particulate, fibrous, or membrane (including hollow fiber membrane). From the viewpoint of easiness, it is preferably particulate or fibrous.
　本担体としては、血液成分中の抗ＳＦＰＱ抗体の吸着性能を有していれば、その他の物質に対する吸着性能の有無については特に制限されないが、抗ＳＦＰＱ抗体のみを特異的に吸着できることが好ましい。 As long as the carrier has the ability to adsorb anti-SFPQ antibodies in blood components, there are no particular restrictions on whether or not it has the ability to adsorb other substances, but it is preferable that it can specifically adsorb only anti-SFPQ antibodies.
　本カラムとしては、本担体を備える限り特に限定されないが、例えば、抗ＳＦＰＱ抗体を含む液体（特に血液）を容器内（後述するケース部内）に導入する入口部および該液体を排出する出口部、および、本担体を充填するケース部を有する容器であり得る。 The present column is not particularly limited as long as it comprises the present carrier. For example, an inlet for introducing a liquid containing an anti-SFPQ antibody (especially blood) into a container (inside a case described later) and an outlet for discharging the liquid, And, it may be a container having a case portion filled with the present carrier.
　容器の材質は特に限定されず、ガラス、ステンレス鋼、ポリエチレン、ポリプロピレン、ポリカーボネート、ポリスチレン、ポリメチルメタクリレート等でありえる。中でも、滅菌などの取扱い性を考慮するとポリプロピレンまたはポリカーボネートが好ましい。また、容器の形状についても特に限定されないが、円筒状であることが好ましい。 The material of the container is not particularly limited, and may be glass, stainless steel, polyethylene, polypropylene, polycarbonate, polystyrene, polymethyl methacrylate, or the like. Among them, polypropylene or polycarbonate is preferable in consideration of handling properties such as sterilization. Also, the shape of the container is not particularly limited, but it is preferably cylindrical.
　また、本カラム内には、本担体を単独で充填してもよく、他の血液浄化用担体、または、各種スペーサー等を本担体と組み合わせて充填してもよい。このようなスペーサーとしては、例えば、編地、織物、不織布等シート形状にした繊維や、膜、ビーズ、ハイドロゲル等が挙げられる。 In addition, the column may be filled with the carrier alone, or may be filled with other carriers for blood purification or various spacers in combination with the carrier. Examples of such spacers include sheet-shaped fibers such as knitted fabrics, woven fabrics and non-woven fabrics, membranes, beads, hydrogels and the like.
　（ＳＦＰＱ関連機能障害の治療または予防方法）
　本発明の一実施形態において、本カラムに被験体の血液を通過させ、該血液中の抗ＳＦＰＱ抗体の量を減少させる、浄化工程を含む、ＳＦＰＱ関連機能障害の治療または予防方法を提供する。本発明の一実施形態にＳＦＰＱ関連機能障害の治療または予防方法（以下、「本治療または予防方法」と称する場合がある）により、ＳＦＰＱ関連機能障害の病態を治療または予防する機序（作用機序）については、上述の通りである。 (Methods for treating or preventing SFPQ-associated dysfunction)
In one embodiment of the invention, a method of treating or preventing SFPQ-related dysfunction is provided, comprising a purification step of passing a subject's blood through the column to reduce the amount of anti-SFPQ antibodies in the blood. In one embodiment of the present invention, the method for treating or preventing SFPQ-related dysfunction (hereinafter sometimes referred to as "this treatment or prevention method") is a mechanism for treating or preventing the pathology of SFPQ-related dysfunction (mechanism of action). Introduction) is as described above.
　本明細書において、「治療」とは、被験体（特にＳＦＰＱ関連機能障害の患者）において、ＳＦＰＱ関連機能障害の症状を完治または軽減させること、あるいは、ＳＦＰＱ関連機能障害の症状の悪化を抑制することを意図する。また、本明細書において、「予防」とは、被験体（特に、ＳＦＰＱ関連機能障害の症状を生じていない被験体またはＳＦＰＱ関連機能障害の発症の可能性がある被験体）においてＳＦＰＱ関連機能障害の症状の発症を抑制すること、または遅延させることを意図する。 As used herein, the term “treatment” refers to the complete cure or alleviation of symptoms of SFPQ-related dysfunction in subjects (especially patients with SFPQ-related dysfunction), or suppression of exacerbation of symptoms of SFPQ-related dysfunction. intended to be Also, as used herein, "prevention" refers to the prevention of SFPQ-related dysfunction in a subject (particularly, a subject who has not developed symptoms of SFPQ-related dysfunction or a subject who is likely to develop SFPQ-related dysfunction). intended to suppress or delay the onset of symptoms of
　本明細書において、「治療」および「予防」の定義は前述のとおりであるが、いずれの場合も本治療または予防方法の作用機序は同じである。従って、「治療方法」は、「予防方法」に置換可能である。すなわち、対象者がＳＦＰＱ関連機能障害の症状を生じていない場合、「治療」は「予防」を意味する。 In the present specification, the definitions of "treatment" and "prevention" are as described above, but in both cases the mechanism of action of this treatment or prevention method is the same. Therefore, "therapeutic method" can be replaced with "preventive method". That is, "treatment" means "prevention" if the subject does not develop symptoms of SFPQ-related dysfunction.
　〔５．ＳＦＰＱ関連機能障害モデル動物およびその製造方法〕
　（ＳＦＰＱ関連機能障害モデル動物）
　本発明の一実施形態において、非ヒト哺乳動物にＳＦＰＱタンパク質または抗ＳＦＰＱ抗体を投与してなる、ＳＦＰＱ関連機能障害モデル動物を提供する。 [5. SFPQ-related dysfunction model animal and its production method]
(SFPQ-related dysfunction model animals)
In one embodiment of the present invention, an animal model of SFPQ-related dysfunction is provided by administering SFPQ protein or anti-SFPQ antibody to a non-human mammal.
　本発明の一実施形態に係るＳＦＰＱ関連機能障害モデル動物（以下、「本モデル動物」と称する場合がある）は、ＳＦＰＱ関連機能障害の発症原因を研究する、ＳＦＰＱ関連機能障害の治療または予防方法を開発する、あるいは、治療薬または予防薬を開発またはスクリーニングする上で、動物実験として好適に用いることができる。 An SFPQ-related dysfunction model animal according to one embodiment of the present invention (hereinafter sometimes referred to as "this model animal") is used to study the cause of SFPQ-related dysfunction, a method for treating or preventing SFPQ-related dysfunction, and or to develop or screen a therapeutic or prophylactic agent, it can be suitably used as an animal experiment.
　本モデル動物としては、実験動物として用いることができる非ヒト哺乳動物（ヒト以外の哺乳動物）であれば種類は特に限定されず、製造するモデル動物の使用目的によって適宜選択することができる。本モデル動物として使用できる非ヒト哺乳動物の例としては、マウス、ラット、モルモット、イヌ、ウサギ、サル、チンパンジーなどを挙げることができる。 The type of this model animal is not particularly limited as long as it is a non-human mammal (mammal other than human) that can be used as an experimental animal, and can be appropriately selected depending on the intended use of the model animal to be produced. Examples of non-human mammals that can be used as this model animal include mice, rats, guinea pigs, dogs, rabbits, monkeys, and chimpanzees.
　（ＳＦＰＱ関連機能障害モデル動物の製造方法）
　本モデル動物は、非ヒト哺乳動物にＳＦＰＱタンパク質または抗ＳＦＰＱ抗体を投与することで製造することができる。すなわち、本発明の一実施形態において、非ヒト哺乳動物に、ＳＦＰＱタンパク質または抗ＳＦＰＱ抗体を投与する投与工程を含む、ＳＦＰＱ関連機能障害モデル動物の製造方法を提供する。 (Method for producing SFPQ-related dysfunction model animal)
This model animal can be produced by administering SFPQ protein or anti-SFPQ antibody to non-human mammals. That is, in one embodiment of the present invention, there is provided a method for producing an SFPQ-related dysfunction model animal, comprising an administration step of administering SFPQ protein or anti-SFPQ antibody to a non-human mammal.
　（投与工程）
　本発明の一実施形態に係るＳＦＰＱ関連機能障害モデル動物の製造方法（以下、「本モデル動物の製造方法」と称する場合がある）における投与工程は、非ヒト哺乳動物に、ＳＦＰＱタンパク質または抗ＳＦＰＱ抗体を投与する工程である。投与工程は、非ヒト哺乳動物に、ＳＦＰＱタンパク質または抗ＳＦＰＱ抗体を免疫する工程であるともいえる。 (Administration step)
In the method for producing an SFPQ-related dysfunction model animal according to one embodiment of the present invention (hereinafter sometimes referred to as "the present model animal production method"), the administration step includes adding SFPQ protein or anti-SFPQ to a non-human mammal. This is the step of administering the antibody. The administration step can also be said to be a step of immunizing a non-human mammal with the SFPQ protein or anti-SFPQ antibody.
　投与工程において投与されるＳＦＰＱタンパク質としては、ヒトＳＦＰＱタンパク質であってもよく、製造するモデル動物由来のＳＦＰＱタンパク質であってもよく、人工的に合成したものであってもよい。また、ＳＦＰＱタンパク質のエピトープ（例えば、配列番号１または２で示されるアミノ酸配列で表されるポリペプチド）を、ＫＬＨなどのキャリアタンパク質にコンジュゲートしたタンパク質（ポリペプチド）を使用することもできる。 The SFPQ protein administered in the administration step may be a human SFPQ protein, a model animal-derived SFPQ protein to be produced, or an artificially synthesized SFPQ protein. Alternatively, a protein (polypeptide) in which an epitope of the SFPQ protein (eg, a polypeptide represented by the amino acid sequence shown in SEQ ID NO: 1 or 2) is conjugated to a carrier protein such as KLH can also be used.
　投与工程において抗ＳＦＰＱ抗体としては、モデル動物と同種の動物の抗体を用いることが望ましいが、これに限定されない。また、抗体はモノクローナル抗体でもポリクローナル抗体でもよい。また、抗ＳＦＰＱ抗体としては、天然型の抗体に加えて、使用目的に応じて、合成抗体、組換え抗体、変異導入抗体、または、グラフト結合抗体等の人工の抗体を使用することもできる。なお、「抗ＳＦＰＱ抗体」の定義については、上述の通りである。また、本明細書における「抗体」とは、免疫グロブリン（ＩｇＡ、ＩｇＤ、ＩｇＥ、ＩｇＧ、ＩｇＭ）ならびに抗体の機能的断片および反応性を維持する範囲において変異導入された抗体の機能的断片を含む概念である。 As the anti-SFPQ antibody in the administration step, it is desirable to use an antibody from an animal of the same species as the model animal, but it is not limited to this. Also, the antibody may be a monoclonal antibody or a polyclonal antibody. As the anti-SFPQ antibody, in addition to natural antibodies, synthetic antibodies, recombinant antibodies, mutagenized antibodies, or artificial antibodies such as graft-bonded antibodies can also be used depending on the purpose of use. The definition of "anti-SFPQ antibody" is as described above. In addition, the term "antibody" as used herein includes immunoglobulins (IgA, IgD, IgE, IgG, IgM), functional fragments of antibodies, and functional fragments of antibodies mutated to the extent that they maintain reactivity. It is a concept.
　投与工程においては、ＳＦＰＱタンパク質および抗ＳＦＰＱ抗体に加えて、溶媒（たとえば、純水、生理食塩水）、アジュバンド、ｐＨ調製剤、緩衝剤、安定化剤、および／または保存剤等のその他の成分を投与してもよい。本発明の一実施形態において、ＳＦＰＱタンパク質および抗ＳＦＰＱ抗体は、これらのその他の成分と混合した状態で非ヒト哺乳動物に投与されてもよい。 In the administration step, in addition to the SFPQ protein and anti-SFPQ antibody, other substances such as solvents (e.g., pure water, saline), adjuvants, pH adjusters, buffers, stabilizers, and/or preservatives are added. Ingredients may be administered. In one embodiment of the invention, SFPQ proteins and anti-SFPQ antibodies may be administered to non-human mammals in admixture with these other ingredients.
　投与工程におけるＳＦＰＱタンパク質および抗ＳＦＰＱ抗体の投与量は、投与対象の非ヒト哺乳動物の種別、体重等を考慮し、適切な量を適切な頻度で投与すればよい。例えば、ＳＦＰＱタンパク質および抗ＳＦＰＱ抗体は、対象の非ヒト哺乳動物に対して、０．０１μｇ～１０００．０μｇ／ｋｇ体重となるように投与されてもよく、０．１μｇ～５００．０μｇ／ｋｇ体重となるように投与されてもよく、１．０μｇ～５００．０μｇ／ｋｇ体重となるように投与されてもよく、１．０μｇ～３００．０μｇ／ｋｇ体重となるように投与されてもよい。 The dosage of the SFPQ protein and anti-SFPQ antibody in the administration step may be administered in an appropriate amount at an appropriate frequency, taking into account the type, body weight, etc. of the non-human mammal to be administered. For example, SFPQ proteins and anti-SFPQ antibodies may be administered to a subject non-human mammal at 0.01 μg to 1000.0 μg/kg body weight, and 0.1 μg to 500.0 μg/kg body weight. 1.0 μg to 500.0 μg/kg body weight, or 1.0 μg to 300.0 μg/kg body weight.
　また、ＳＦＰＱタンパク質および抗ＳＦＰＱ抗体の、対象の非ヒト哺乳動物への投与間隔に制限はない。前記投与間隔は、例えば、１時間に１回、１～６時間に１回、６～１２時間に１回、１２時間～１日あたり１回、１日～３日あたり一回、１日～５日あたり１回、１日～７日あたり１回、７日～１４日あたり１回、１４日～２１日あたり１回、１カ月あたり１回、等であり得る。 In addition, there is no restriction on the administration interval of the SFPQ protein and anti-SFPQ antibody to the target non-human mammal. The administration interval is, for example, once an hour, once every 1 to 6 hours, once every 6 to 12 hours, once every 12 hours to once a day, once every 1 to 3 days, 1 day to It can be once every 5 days, once every 1-7 days, once every 7-14 days, once every 14-21 days, once per month, and the like.
　本発明の一実施形態は、以下の構成を含む。
＜１＞被験体から分離した試料中の抗ＳＦＰＱ抗体の抗体価を測定する測定工程を含む、ＳＦＰＱ関連機能障害の診断または検査するためのデータの取得方法。
＜２＞前記ＳＦＰＱ関連機能障害は、脳、腸、精巣、皮膚、心臓および／または肺の老化と類似の機能障害である、＜１＞に記載のデータの取得方法。
＜３＞前記試料は、血液、血清、血漿、尿および唾液から選択される、＜１＞または＜２＞に記載のデータの取得方法。
＜４＞以下の（ａ１）～（ａ３）、（ｂ１）～（ｂ３）、および、（ｃ１）～（ｃ３）から選択される少なくとも１つを含む、ＳＦＰＱ関連機能障害の診断薬：
　（ａ１）配列番号１で表されるポリペプチド；
　（ａ２）配列番号１で示されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換または付加されたアミノ酸配列からなるポリペプチド；
　（ａ３）配列番号１で示されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列からなるポリペプチド；
　（ｂ１）配列番号２または３で示されるアミノ酸配列からなるポリペプチド；
　（ｂ２）配列番号２または３で示されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換または付加されたアミノ酸配列からなり、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；
　（ｂ３）配列番号２または３で示されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列からなり、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；
　（ｃ１）配列番号２または３で示されるアミノ酸配列を含み、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；
　（ｃ２）配列番号２または３で示されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換または付加されたアミノ酸配列を含み、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；
　（ｃ３）配列番号２または３で示されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含み、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド。
＜５＞表面に、以下の（ａ１）～（ａ３）、（ｂ１）～（ｂ３）、および、（ｃ１）～（ｃ３）から選択される少なくとも１つを含む、血液浄化用担体：
　（ａ１）配列番号１で表されるポリペプチド；
　（ａ２）配列番号１で示されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換または付加されたアミノ酸配列からなるポリペプチド；
　（ａ３）配列番号１で示されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列からなるポリペプチド；
　（ｂ１）配列番号２または３で示されるアミノ酸配列からなるポリペプチド；
　（ｂ２）配列番号２または３で示されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換または付加されたアミノ酸配列からなり、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；
　（ｂ３）配列番号２または３で示されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列からなり、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；
　（ｃ１）配列番号２または３で示されるアミノ酸配列を含み、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；
　（ｃ２）配列番号２または３で示されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換または付加されたアミノ酸配列を含み、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；
　（ｃ３）配列番号２または３で示されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含み、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド。
＜６＞＜５＞に記載の血液浄化用担体を備える、血液浄化カラム。
＜７＞非ヒト哺乳動物にＳＦＰＱタンパク質または抗ＳＦＰＱ抗体を投与してなる、ＳＦＰＱ関連機能障害モデル動物。
＜８＞非ヒト哺乳動物に、ＳＦＰＱタンパク質または抗ＳＦＰＱ抗体を投与する投与工程を含む、ＳＦＰＱ関連機能障害モデル動物の製造方法。 One embodiment of the present invention includes the following configurations.
<1> A method of obtaining data for diagnosing or testing SFPQ-related dysfunction, comprising a measuring step of measuring the antibody titer of an anti-SFPQ antibody in a sample isolated from a subject.
<2> The method for acquiring data according to <1>, wherein the SFPQ-related dysfunction is a dysfunction similar to aging of the brain, intestine, testis, skin, heart and/or lungs.
<3> The data acquisition method according to <1> or <2>, wherein the sample is selected from blood, serum, plasma, urine and saliva.
<4> A diagnostic agent for SFPQ-related dysfunction, including at least one selected from the following (a1) to (a3), (b1) to (b3), and (c1) to (c3):
(a1) a polypeptide represented by SEQ ID NO: 1;
(a2) A polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 1;
(a3) a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1;
(b1) a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 2 or 3;
(b2) a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 2 or 3, and having the ability to bind to an anti-SFPQ antibody;
(b3) a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or 3 and having the ability to bind to an anti-SFPQ antibody;
(c1) a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 2 or 3 and capable of binding to an anti-SFPQ antibody;
(c2) A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 2 or 3, and having the ability to bind to an anti-SFPQ antibody;
(c3) A polypeptide comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2 or 3 and having the ability to bind to an anti-SFPQ antibody.
<5> A carrier for blood purification, the surface of which contains at least one selected from the following (a1) to (a3), (b1) to (b3), and (c1) to (c3):
(a1) a polypeptide represented by SEQ ID NO: 1;
(a2) A polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 1;
(a3) a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1;
(b1) a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 2 or 3;
(b2) a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 2 or 3, and having the ability to bind to an anti-SFPQ antibody;
(b3) a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or 3 and having the ability to bind to an anti-SFPQ antibody;
(c1) a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 2 or 3 and capable of binding to an anti-SFPQ antibody;
(c2) A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 2 or 3, and having the ability to bind to an anti-SFPQ antibody;
(c3) A polypeptide comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2 or 3 and having the ability to bind to an anti-SFPQ antibody.
<6> A blood purification column comprising the blood purification carrier according to <5>.
<7> An SFPQ-related dysfunction model animal obtained by administering an SFPQ protein or an anti-SFPQ antibody to a non-human mammal.
<8> A method for producing an SFPQ-related dysfunction model animal, comprising the step of administering an SFPQ protein or an anti-SFPQ antibody to a non-human mammal.
　本発明は上述した各実施形態に限定されるものではなく、請求項に示した範囲で種々の変更が可能であり、異なる実施形態にそれぞれ開示された技術的手段を適宜組み合わせて得られる実施形態についても本発明の技術的範囲に含まれる。 The present invention is not limited to the above-described embodiments, but can be modified in various ways within the scope of the claims, and can be obtained by appropriately combining technical means disclosed in different embodiments. is also included in the technical scope of the present invention.
　以下に実施例を挙げて本発明の一実施形態を詳説するが、本発明はこれによって限定されるものではない。 An embodiment of the present invention will be described in detail below with reference to examples, but the present invention is not limited thereto.
　〔実施例１〕
　ＳＩＴＨ‐１によって誘導される自己抗体の同定
　＜方法＞
　（１）ＳＩＴＨ－１　ＤＮＡワクチンマウスの作製
　４週齢のＣ５７ＢＬ／６ＮＣｒＳｌｃ雄マウスの後肢を除毛し、前脛骨筋に１０μＭカルディオトキシン溶液１００μＬを筋肉内注射した。該マウスを４日間通常飼育した後に、５０μｇ相当の哺乳類細胞ＳＩＴＨ－１発現プラスミド（ＳＩＴＨ－１／ｐＦｌａｇ－ＣＭＶ－５ｂ）溶液を、前記カルディオトキシン溶液を筋肉内注射した部位に注入した。この一連の作業を「ＤＮＡワクチン投与」と称する。最初のＤＮＡワクチン投与から２週間後の６週齢のマウス、および、４週間後の８週齢のマウスについても同様にＤＮＡワクチン投与を実施した。計３回のＤＮＡワクチン投与を実施したマウスについて、３回目のＤＮＡワクチン投与から１週間後（９週齢）に採血を行い、血清を採取した。この計３回のＤＮＡワクチン投与を実施したマウスを、ＳＩＴＨ－１　ＤＮＡワクチンマウスと称する。 [Example 1]
Identification of autoantibodies induced by SITH-1 <Method>
(1) Production of SITH-1 DNA Vaccinated Mice The hind limbs of 4-week-old C57BL/6NCrSlc male mice were shaved, and 100 μL of 10 μM cardiotoxin solution was injected intramuscularly into the tibialis anterior muscle. After the mice were normally fed for 4 days, a mammalian cell SITH-1 expression plasmid (SITH-1/pFlag-CMV-5b) solution equivalent to 50 μg was injected into the intramuscular injection site of the cardiotoxin solution. This series of operations is called "DNA vaccination". 6-week-old mice 2 weeks after the first administration of the DNA vaccine and 8-week-old mice 4 weeks after the administration of the first DNA vaccine were similarly administered the DNA vaccine. Blood was collected from the mice that had been administered the DNA vaccine a total of three times, and serum was collected one week after the third administration of the DNA vaccine (9 weeks old). Mice that received a total of three DNA vaccinations are referred to as SITH-1 DNA-vaccinated mice.
　（２）血中抗体を用いた免疫沈降
　ＳＩＴＨ－１　ＤＮＡワクチンマウスから採取した血清を、磁性ビーズＤｙｎａｂｅａｄｓ　ｐｒｏｔｅｉｎ　Ｇ（ＴｈｅｒｍｏＦｉｓｈｅｒ社）と混合し、血清中に含まれる抗体を磁性ビーズに固定化（抗体固定化ビーズ）した。一方、マウス全脳を前タンパク質溶解試薬Ｔ－ＰＥＲ（ＴｈｅｒｍｏＦｉｓｈｅｒ社）で可溶化し、可溶性画分（Ｂｒａｉｎ　Ｌｙｓａｔｅ）を調製した。得られた抗体固定化ビーズとＢｒａｉｎ　Ｌｙｓａｔｅを室温で１時間混合した後、該抗体固定化ビーズを、洗浄バッファー（０．２％　Ｔｗｅｅｎ／ＴＢＳ）を用いて洗浄し、該抗体固定化ビーズに結合している自己抗原を検出するために０．１Ｍ　Ｃｉｔｒａｔｅ（ｐＨ　２～３）を添加して溶出した。その後、サンプルバッファーを加えて還元加熱溶出し、ＳＤＳ－ＰＡＧＥ（電気泳動）に供した。 (2) Immunoprecipitation using blood antibodies Serum collected from SITH-1 DNA vaccine mice is mixed with magnetic beads Dynabeads protein G (ThermoFisher), and antibodies contained in the serum are immobilized on magnetic beads (antibody immobilization beads). On the other hand, the whole mouse brain was solubilized with a proprotein lysing reagent T-PER (ThermoFisher) to prepare a soluble fraction (Brain Lysate). After mixing the obtained antibody-immobilized beads and Brain Lysate at room temperature for 1 hour, the antibody-immobilized beads were washed with a washing buffer (0.2% Tween/TBS) and bound to the antibody-immobilized beads. 0.1 M Citrate (pH 2-3) was added for elution in order to detect self-antigens presenting. After that, a sample buffer was added, elution was performed by heating under reduction, and SDS-PAGE (electrophoresis) was performed.
　（３）自己抗体に結合する自己抗原の同定
　電気泳動後のゲルをネガティブゲル染色ＭＳキット（富士フイルム和光純薬）で染色し、検出された６０ｋＤａおよび６８ｋＤａのバンドを切り出した。切り出したゲル中に含まれる自己抗原と考えられるタンパク質を同定するために、ＭＡＬＤＩ－ＴＯＦ　ＭＡＳＳによるＰＭＦ（Ｐｅｐｔｉｄｅ　Ｍａｓｓ　Ｆｉｎｇｅｒｐｒｉｎｔ）解析を行った。 (3) Identification of autoantigen that binds to autoantibody After electrophoresis, the gel was stained with a negative gel staining MS kit (Fujifilm Wako Pure Chemical Industries, Ltd.), and the detected 60 kDa and 68 kDa bands were excised. PMF (Peptide Mass Fingerprint) analysis by MALDI-TOF MASS was performed in order to identify a protein considered to be an autoantigen contained in the excised gel.
　（４）反復ＳＩＴＨ－１発現マウスの作製
　ＧＦＡＰプロモーターの下流にＳＩＴＨ－１遺伝子を組み込んだアデノウイルスベクター（ＳＩＴＨ－１／Ａｄｖ）を作製し、Ａｄｅｎｏ－ＸＴＭ　Ｍａｘｉ　Ｐｕｒｉｆｉｃａｔｉｏｎ　Ｋｉｔ（ＴａＫａＲａ社製）で精製した。１×１０９　ｉｆｕ／ｍＬのＳＩＴＨ－１／Ａｄｖ溶液２５μＬを、８週齢のマウス（Ｃ５７ＢＬ／６ＮＣｒＳｌｃ）に鼻腔投与し、４週間飼育した。飼育中、１０週齢および１２週齢達したマウスについて、同様の方法でＳＩＴＨ－１／Ａｄｖを鼻腔投与し、計３回ＳＩＴＨ－１／Ａｄｖを投与し、ＳＩＴＨ－１の発現を誘導したマウス群（ＳＩＴＨ－１群）を作製した。対照として、８週齢のマウス（Ｃ５７ＢＬ／６ＮＣｒＳｌｃ）に何も発現しない（遺伝子を組み込んでいない）アデノウイルスベクター（Ｖｅｃｔｏｒ／Ａｄｖ）を３回鼻腔投与したマウス群（Ｖｅｃｔｏｒ群）を用いた。 (4) Production of recurrent SITH-1-expressing mice An adenovirus vector (SITH-1/Adv) incorporating the SITH-1 gene downstream of the GFAP promoter was produced, and purified using Adeno-XTM Maxi Purification Kit (TaKaRa). did. 25 μL of 1×10 9 ifu/mL SITH-1/Adv solution was nasally administered to 8-week-old mice (C57BL/6NCrSlc) and maintained for 4 weeks. SITH-1/Adv was intranasally administered in the same manner to 10-week-old and 12-week-old mice during breeding, and SITH-1/Adv was administered 3 times in total to induce expression of SITH-1. A group (SITH-1 group) was created. As a control, 8-week-old mice (C57BL/6NCrSlc) were intranasally administered three times with an adenoviral vector (Vector/Adv) that did not express anything (gene was not integrated) (Vector group).
　（５）Ｗｅｓｔｅｒｎ　Ｂｌｏｔによる自己抗体の検出
　マウスのＢｒａｉｎ　ＬｙｓａｔｅをＳＤＳ－ＰＡＧＥし、ゲル中に分画されたタンパク質をＰＶＤＦメンブレンに転写した。このメンブレンに反復ＳＩＴＨ－１発現マウスおよび対照マウスの血清を反応させた後に、二次抗体としてＨｏｒｓｅｒａｄｉｓｈ　ｐｅｒｏｘｉｄａｓｅ（ＨＲＰ）標識した抗マウスＩｇＧ抗体（Ｊａｃｋｓｏｎ社製）を反応させ、化学発光により自己抗体が検出されるかどうかを検討した。なお、ヒトとマウスのＳＦＰＱのホモロジーはＩｄｅｎｔｉｔｙ：６６５／７０７（９４％）である。 (5) Detection of Autoantibody by Western Blot Mouse Brain Lysate was subjected to SDS-PAGE, and proteins fractionated in the gel were transferred to a PVDF membrane. This membrane was reacted with sera of repetitive SITH-1-expressing mice and control mice, and then reacted with a horseradish peroxidase (HRP)-labeled anti-mouse IgG antibody (manufactured by Jackson) as a secondary antibody, and autoantibodies were detected by chemiluminescence. See if it can be detected. The homology between human and mouse SFPQ is Identity: 665/707 (94%).
　＜結果＞
　実施例１における、ネガティブゲル染色の結果およびＷｅｓｔｅｒｎ　Ｂｌｏｔによる自己抗体の検査の結果を図１に示す。 <Results>
The results of negative gel staining and Western Blot autoantibody test in Example 1 are shown in FIG.
　図１左図に（１）～（３）の操作におけるネガティブゲル染色で得られたＮｅｇａｔｉｖｅ　ｓｔａｉｎの写真を示す。図１左図より明らかなように、該ネガティブゲル染色では、６０ｋＤａおよび６８ｋＤａの２本のバンドを得たが、ＰＭＦ解析によるタンパク質同定の結果、いずれもＳｐｌｉｃｉｎｇ　ｆａｃｔｏｒ，　ｐｒｏｌｉｎｅ－　ａｎｄ　ｇｌｕｔａｍｉｎｅ－ｒｉｃｈ（ＳＦＰＱ）タンパク質であった。 The left figure in Fig. 1 shows photographs of negative stains obtained by negative gel staining in the operations (1) to (3). As is clear from the left figure of FIG. 1, two bands of 60 kDa and 68 kDa were obtained by the negative gel staining, and as a result of protein identification by PMF analysis, both were Splicing factor, proline- and glutamine-rich (SFPQ). ) was a protein.
　ＳＩＴＨ‐１発現の発現を誘導したマウスにおける、Ｗｅｓｔｅｒｎ　Ｂｌｏｔによる自己抗体の検査（（４）～（５）の操作）の結果を図１右図に示す。図１右図より明らかなように、ＳＩＴＨ－１群のマウスの６８．８％が自己抗体陽性となった。一方で、対照のＶｅｃｔｏｒ群マウスでは、３３．３％のマウスのみが自己抗体陽性であった。 The results of autoantibody tests by Western Blot (operations (4) to (5)) in mice in which SITH-1 expression was induced are shown in the right figure of FIG. As is clear from the right panel of FIG. 1, 68.8% of the mice in the SITH-1 group were autoantibody-positive. On the other hand, in the control Vector group mice, only 33.3% of the mice were autoantibody-positive.
　以上の結果から、ＳＩＴＨ－１タンパク質の発現により、ＳＦＰＱを自己抗原とする自己抗体として、抗ＳＦＰＱ抗体の産生が誘導されることが示された。 From the above results, it was shown that the expression of SITH-1 protein induces the production of anti-SFPQ antibodies as autoantibodies with SFPQ as the autoantigen.
　〔実施例２〕
　ＳＩＴＨ‐１による自己抗体（抗ＳＦＰＱ抗体）の誘導
　＜方法＞
　ＳＩＴＨ‐１はＣＡＭＬに結合し細胞内でのＣＡＭＬ量を増加させること、ＣＡＭＬはＢ細胞の免疫抑制性受容体ＴＡＣＩに結合することが分かっている。このため、ＣＡＭＬがＴＡＣＩシグナルを抑制し自己免疫反応を生じさせることと、ＣＡＭＬがＳＦＰＱに結合して自己免疫反応の場にＳＦＰＱを抗原として引き寄せてくることを確認した。 [Example 2]
Induction of autoantibody (anti-SFPQ antibody) by SITH-1 <Method>
It is known that SITH-1 binds to CAML and increases the intracellular amount of CAML, and CAML binds to the immunosuppressive receptor TACI of B cells. Therefore, it was confirmed that CAML suppresses the TACI signal and induces an autoimmune reaction, and that CAML binds to SFPQ and attracts SFPQ as an antigen to the site of the autoimmune reaction.
　（１）ＣＡＭＬによるＴＡＣＩシグナルの抑制
　ＨＥＫ２９３Ｔ細胞にヒトＴＡＣＩ（ｈＴＡＣＩ）を発現させ、ヒトＣＡＭＬ（ｈＣＡＭＬ）存在下でｈＴＡＣＩのリガンドであるＢＬｙＳもしくはＡＰＲＩＬを添加した際に、シグナル伝達経路が活性化するかどうかを検討するために、以下の実験を実施した。まず、哺乳類細胞ｈＴＡＣＩ発現プラスミド（ｈＴＡＣＩ／ｐＦｌａｇ－ＣＭＶ－５ａ）、哺乳類細胞ｈＣＡＭＬ発現プラスミド（ｈＣＡＭＬ／ｐＣＭＶ－ＨＡ）およびｐＮＦκＢ－ＳＥＡＰレポーターベクター（ＴａＫａＲａ社製）をＰｌｕｓ　Ｒｅａｇｅｎｔ（ＴｈｅｒｍｏＦｉｓｈｅｒ社）存在下でＬｉｐｏｆｅｃｔａｍｉｎｅ　ＬＴＸ（ＴｈｅｒｍｏＦｉｓｈｅｒ社）と混合し、ＨＥＫ２９３Ｔ細胞の形質転換を行った。試験区としては、ｐＮＦκＢ－ＳＥＡＰレポーターに加えて、ｈＣＡＭＬ発現細胞、ｈＴＡＣＩ発現細胞、ｈＣＡＭＬとｈＴＡＣＩの両発現細胞を作製し、これらの細胞に各リガンド（１００ｎｇ／ｍｌのＢＬｙＳまたは２００ｎｇ／ｍｌのＡＰＲＩＬ）を添加した後、３７℃で１８時間培養した。これらの培養後の細胞を用いてＰｈｏｓｐｈａ－Ｌｉｇｈｔ　ＳＥＡＰ　Ｒｅｐｏｒｔｅｒ　Ｇｅｎｅ　Ａｓｓａｙ　Ｓｙｓｔｅｍ（ＴｈｅｒｍｏＦｉｓｈｅｒ社）を用いたＳＥＡＰレポーターアッセイを行った。 (1) Suppression of TACI Signal by CAML Human TACI (hTACI) is expressed in HEK293T cells, and when the hTACI ligand BLyS or APRIL is added in the presence of human CAML (hCAML), the signal transduction pathway is activated. In order to examine whether or not, the following experiment was carried out. First, mammalian cell hTACI expression plasmid (hTACI/pFlag-CMV-5a), mammalian cell hCAML expression plasmid (hCAML/pCMV-HA) and pNFκB-SEAP reporter vector (manufactured by TaKaRa) were added in the presence of Plus Reagent (ThermoFisher). HEK293T cells were transformed by mixing with Lipofectamine LTX (ThermoFisher). As a test group, in addition to the pNFκB-SEAP reporter, hCAML-expressing cells, hTACI-expressing cells, and both hCAML and hTACI-expressing cells were prepared, and each ligand (100 ng/ml BLyS or 200 ng/ml APRIL ) and cultured at 37° C. for 18 hours. Using these cultured cells, SEAP reporter assay was performed using Phospha-Light SEAP Reporter Gene Assay System (ThermoFisher).
　（２）ＣＡＭＬとＳＦＰＱの結合
　ＣＡＭＬとＳＦＰＱの結合を検討するために、ＣＡＭＬ発現ＨＥＫ２９３Ｔ細胞を可溶化し、抗ＳＦＰＱ抗体による免疫沈降を試みた。具体的には、実施例２と同様のＬｉｐｏｆｅｃｔａｍｉｎｅ　ＬＴＸを用いてＨＥＫ２９３Ｔ細胞にｈＣＡＭＬを発現させ、ＳＤＳフリーのＲＩＰＡバッファーで可溶化し、可溶性画分を調製した。対照として、同様の方法でＳＩＴＨ－１を発現させたＨＥＫ２９３Ｔ細胞の可溶性画分を調製した。各可溶性画分に実施例１と同様の方法で調整した抗ＳＦＰＱ抗体（ａｂｃａｍ）固定化磁性ビーズを添加し、室温で１時間反応させた後に磁性ビーズに結合したタンパク質をＳＤＳ－ＰＡＧＥで分画し、抗ＣＡＭＬ抗体（ａｂｃａｍ）を用いたＷｅｓｔｅｒｎ　ＢｌｏｔによってＳＦＰＱとＣＡＭＬの結合の有無を検出した。 (2) Binding of CAML and SFPQ To examine the binding of CAML and SFPQ, CAML-expressing HEK293T cells were solubilized and immunoprecipitation with an anti-SFPQ antibody was attempted. Specifically, hCAML was expressed in HEK293T cells using Lipofectamine LTX as in Example 2, and solubilized with an SDS-free RIPA buffer to prepare a soluble fraction. As a control, a soluble fraction of HEK293T cells expressing SITH-1 was prepared in a similar manner. Anti-SFPQ antibody (abcam)-immobilized magnetic beads prepared in the same manner as in Example 1 were added to each soluble fraction, reacted at room temperature for 1 hour, and proteins bound to the magnetic beads were fractionated by SDS-PAGE. Then, the presence or absence of binding between SFPQ and CAML was detected by Western Blot using an anti-CAML antibody (abcam).
　＜結果＞
　実施例２におけるＳＥＡＰレポーターアッセイの結果および抗ＳＦＰＱ抗体による免疫沈降の結果を図２に示す。 <Results>
The results of the SEAP reporter assay in Example 2 and the results of immunoprecipitation with an anti-SFPQ antibody are shown in FIG.
　（１）の操作における、ＳＥＡＰアッセイの結果を図２左図および図２中央図に示す。 The results of the SEAP assay in the operation of (1) are shown in the left figure of FIG. 2 and the middle figure of FIG.
　図２左図および図２中央図より明らかなように、ＴＡＣＩの２種類のリガンドであるＢＬｙＳとＡＰＲＩＬにより活性化されたＮＦκＢによる転写活性はいずれもＣＡＭＬの共発現によって抑制された。この結果はＣＡＭＬがＴＡＣＩシグナルを抑制し自己免疫反応を生じさせる可能性を示唆するものである。 As is clear from the left panel of FIG. 2 and the central panel of FIG. 2, the transcriptional activities of NFκB activated by BLyS and APRIL, two types of TACI ligands, were both suppressed by co-expression of CAML. This result suggests the possibility that CAML suppresses TACI signals and induces autoimmune reactions.
　（２）の操作における、抗ＳＦＰＱ抗体による免疫沈降の結果を図２右図に示す。図２右図より明らかなように、抗ＳＦＰＱ抗体を用いてＳＦＰＱタンパク質を免疫沈降すると、ＣＡＭＬが共沈されること、すなわち、ＣＡＭＬとＳＦＰＱが結合していることが示された。 The results of immunoprecipitation with anti-SFPQ antibody in the operation of (2) are shown in the right figure of FIG. As is clear from the right figure of FIG. 2, immunoprecipitation of SFPQ protein using an anti-SFPQ antibody showed coprecipitation of CAML, that is, binding of CAML and SFPQ.
　以上の結果から、ＣＡＭＬがＳＦＰＱに結合して自己免疫反応の場にＳＦＰＱを抗原として引き寄せてくる可能性が示唆された。 The above results suggest the possibility that CAML binds to SFPQ and attracts SFPQ as an antigen to the site of autoimmune reactions.
　〔実施例３〕
　健常人、ＳＩＴＨ－１陽性者、および、うつ病患者におけるＳＦＰＱ抗体測定
　＜方法＞
　（１）ＳＦＰＱタンパク質のエピトープ解析
　ヒトＳＦＰＱタンパク質のアミノ酸配列を用いてエピトープ解析を試みた結果、３６番目から５０番目の１５アミノ酸残基（ＳＦＰＱ３６－５０、配列番号２）および４５２番目から４６６番目の１５アミノ酸残基（ＳＦＰＱ４５２－４６６、配列番号３）が候補に挙がった。そこで、これらのアミノ酸配列のＮ末端にシステインを付加した１６アミノ酸残基のペプチドを合成し、抗原として用いることにした。 [Example 3]
SFPQ antibody measurement in healthy subjects, SITH-1 positive subjects, and depressed patients <Method>
(1) Epitope analysis of SFPQ protein As a result of attempting epitope analysis using the amino acid sequence of human SFPQ protein, 15 amino acid residues from 36th to 50th (SFPQ36-50, SEQ ID NO: 2) and from 452nd to 466th 15 amino acid residues (SFPQ452-466, SEQ ID NO:3) were nominated. Therefore, it was decided to synthesize peptides of 16 amino acid residues by adding cysteine to the N-terminus of these amino acid sequences and use them as antigens.
　（２）ＥＬＩＳＡによる抗ＳＦＰＱ抗体の検出
ＳＦＰＱ３６－５０もしくはＳＦＰＱ４５２－４６６の合成ペプチドを１μＭとなるように炭酸バッファー（１５ｍＭ　Ｎａ２ＣＯ３、３５ｍＭ　ＮａＨＣＯ３、０．０２％　ＮａＮ３）で希釈した。得られた１μＭペプチド溶液を９６ウェルマイクロタイタープレートに加え、３７℃で２時間静置することでペプチドを固定化した。ペプチド固定化プレートを、洗浄バッファー（０．０５％　Ｔｗｅｅｎ２０／ＰＢＳ）を用いて洗浄後、抗体希釈バッファー（３％　ＢＳＡおよび０．０５％　Ｔｗｅｅｎ２０を含むＰＢＳ）で６０００倍希釈した血清と反応させ、二次抗体として５０００倍希釈したＨＲＰ標識抗ヒトＩｇＧ抗体（Ｊａｃｋｓｏｎ社）を反応させた。抗ＳＦＰＱ抗体の結合量を、ＳｕｐｅｒＳｉｇｎａｌ　ＥＬＩＳＡ　Ｐｉｃｏ　Ｓｕｂｓｔｒａｔｅ（ＴｈｅｒｍｏＦｉｓｈｅｒ社）を用いて発光量として測定した。 (2) Detection of Anti-SFPQ Antibody by ELISA A synthetic peptide of SFPQ36-50 or SFPQ452-466 was diluted to 1 μM with a carbonate buffer (15 mM Na 2 CO 3 , 35 mM NaHCO 3 , 0.02% NaN 3 ). The resulting 1 μM peptide solution was added to a 96-well microtiter plate and allowed to stand at 37° C. for 2 hours to immobilize the peptide. After washing the peptide-immobilized plate with a washing buffer (0.05% Tween20/PBS), it was reacted with serum diluted 6000-fold with an antibody dilution buffer (PBS containing 3% BSA and 0.05% Tween20), A 5000-fold diluted HRP-labeled anti-human IgG antibody (Jackson) was reacted as a secondary antibody. The amount of binding of the anti-SFPQ antibody was measured as the amount of luminescence using SuperSignal ELISA Pico Substrate (ThermoFisher).
　（３）間接蛍光抗体法（ＩＦＡ）による抗ＳＩＴＨ－ＣＡＭＬ抗体の検出
　以前、本発明者はＳＩＴＨ－１タンパク質が細胞内に存在するＣＡＭＬタンパク質と結合し、その結合タンパク質（ＳＩＴＨ－ＣＡＭＬ）に対する抗体をうつ病患者が保持していることを報告している（非特許文献１）。そこで、健常者およびうつ病患者における抗ＳＩＴＨ－ＣＡＭＬ抗体価を非特許文献１に記載した方法を用いて算出した。 (3) Detection of anti-SITH-CAML antibody by indirect fluorescent antibody method (IFA) Previously, the present inventors found that SITH-1 protein binds to CAML protein present in cells, and an antibody against the binding protein (SITH-CAML) have been reported to be retained by depressed patients (Non-Patent Document 1). Therefore, the anti-SITH-CAML antibody titers in healthy subjects and depressed patients were calculated using the method described in Non-Patent Document 1.
　具体的には、健常者またはうつ病患者から採取した血清を２％　ＢＳＡおよび０．０５％　Ｔｗｅｅｎ２０を含むＰＢＳで４０倍に希釈し、間接蛍光抗体法（ＩＦＡ）にて染色を行った。二次抗体には、Ａｌｅｘａ　Ｆｌｕｏｒ　４８８　ｇｏａｔ　ａｎｔｉ－ｈｕｍａｎ　ｓｅｃｏｎｄａｒｙ　ａｎｔｉｂｏｄｉｅｓ（Ｍｏｌｅｃｕｌａｒ　Ｐｒｏｂｅｓ）を２％　ＢＳＡおよび０．０５％　Ｔｗｅｅｎ２０を含むＰＢＳで２００倍に希釈して用いた。抗原用の発現プラスミドとして、ＳＩＴＨ－ＣＡＭＬをｐＣＭＶ－ＦＬＡＧ－５ａのＣＭＶプロモーターとＦＬＡＧタグの間に挿入したプラスミドと、コントロールとしてＬＩＴＭＵＳ２８ｉを用いた。それぞれのプラスミドを、Ｌａｂ－Ｔｅｋ　ｃｈａｍｂｅｒ　ｓｌｉｄｅｓ　（Ｎｕｎｃ）で培養したＨＥＫ２９３Ｔ細胞にＬｉｐｏｆｅｃｔａｍｉｎｅ　ＬＴＸ　（Ｉｎｖｉｔｒｏｇｅｎ）でトランスフェクションし、その後４８時間培養したものを、乾燥固定後に－２０℃の５％メタノール添加アセトンで５分間固定し、ＩＦＡ抗原として用いた。これらを間接蛍光抗体法（ＩＦＡ）により、蛍光顕微鏡を用いて観察し、画像解析ソフトＩｍａｇｅＪを使用して、ＩＦＡ染色細胞の蛍光光度を数値化した。 Specifically, serum collected from healthy subjects or depressed patients was diluted 40-fold with PBS containing 2% BSA and 0.05% Tween 20, and stained with an indirect fluorescent antibody method (IFA). As the secondary antibody, Alexa Fluor 488 goat anti-human secondary antibodies (Molecular Probes) were diluted 200-fold with PBS containing 2% BSA and 0.05% Tween 20 and used. As an antigen expression plasmid, a plasmid in which SITH-CAML was inserted between the CMV promoter and the FLAG tag of pCMV-FLAG-5a and LITMUS28i as a control were used. Each plasmid was transfected with Lipofectamine LTX (Invitrogen) into HEK293T cells cultured on Lab-Tek chamber slides (Nunc), and then cultured for 48 hours. Fixed for 5 minutes and used as IFA antigen. These cells were observed with an indirect fluorescent antibody method (IFA) using a fluorescence microscope, and the fluorescence intensity of the IFA-stained cells was quantified using image analysis software ImageJ.
　（４）ＳＦＰＱ抗原の精製
　ヒトＳＦＰＱをコードするＤＮＡ断片をＧＳＴ融合大腸菌発現ベクターｐＥＴ４１ｂ（＋）に挿入し、Ｎ末端にＧＳＴタンパク質、Ｃ末端にＨｉｓタグを融合させたヒトＳＦＰＱ（ＧＳＴ－ＳＦＰＱ－Ｈｉｓ）発現プラスミドベクターを構築した。この発現プラスミドベクターをＲｏｓｅｔｔａ－ｇａｍｉ２（ＤＥ３）等の発現用大腸菌株にトランスフェクションし、１ｍＭのＩＰＴＧを添加してＧＳＴ－ＳＦＰＱタンパク質の発現を誘導した。　その後大腸菌を可溶化し、Ｇｌｕｔａｔｈｉｏｎｅ　Ｓｅｐｈａｒｏｓｅ　４　Ｆａｓｔ　Ｆｌｏｗ（ＭＥＲＣＫ社製）を用いてＧＳＴ－ＳＦＰＱ－Ｈｉｓタンパク質を精製した。対照として、大腸菌発現ベクターｐＥＴ４１ｂ（＋）を大腸菌にトランスフェクションし、ＧＳＴタンパク質単体を発現させた後に精製した画分も調製した。 (4) Purification of SFPQ antigen A DNA fragment encoding human SFPQ was inserted into the GST-fused E. coli expression vector pET41b(+), and human SFPQ (GST-SFPQ- His) expression plasmid vector was constructed. This expression plasmid vector was transfected into an E. coli strain for expression such as Rosetta-gami2(DE3), and 1 mM IPTG was added to induce expression of the GST-SFPQ protein. After that, E. coli was solubilized and the GST-SFPQ-His protein was purified using Glutathione Sepharose 4 Fast Flow (manufactured by MERCK). As a control, the E. coli expression vector pET41b(+) was transfected into E. coli to express the GST protein alone, and then a purified fraction was also prepared.
　（５）Ｗｅｓｔｅｒｎ　Ｂｌｏｔによる抗ＳＦＰＱ抗体の検出
　精製したＧＳＴ－ＳＦＰＱ－Ｈｉｓタンパク質およびＧＳＴタンパク質単体（対照）をＳＤＳ－ＰＡＧＥし、ゲル中に分画されたタンパク質をＰＶＤＦメンブレンに転写した。このメンブレンに検体血清を反応させた後に、二次抗体としてＨｏｒｓｅｒａｄｉｓｈ　ｐｅｒｏｘｉｄａｓｅ（ＨＲＰ）標識した抗マウスＩｇＧ抗体（Ｊａｃｋｓｏｎ社製）を反応させ、化学発光により自己抗体が検出されるかどうかを検討した。検出されたバンドを、ソフトウェアＩｍａｇｅ　Ｌａｂ（Ｂｉｏ－Ｒａｄ社製）を用いて数値化し、これを抗ＳＦＰＱ抗体価とした。 (5) Detection of Anti-SFPQ Antibody by Western Blot The purified GST-SFPQ-His protein and the GST protein alone (control) were subjected to SDS-PAGE, and the protein fractionated in the gel was transferred to a PVDF membrane. After allowing the sample serum to react with this membrane, it was allowed to react with a horseradish peroxidase (HRP)-labeled anti-mouse IgG antibody (manufactured by Jackson) as a secondary antibody, and whether autoantibodies were detected by chemiluminescence was examined. The detected bands were quantified using software Image Lab (manufactured by Bio-Rad) and used as the anti-SFPQ antibody titer.
　＜結果＞
　表１に、ＳＦＰＱのエピトープとなるＳＦＰＱ３６－５０もしくはＳＦＰＱ４５２－４６６の合成ペプチドの配列を示す。なお、合成ペプチドのＮ末端にはシステインが付加されている。 <Results>
Table 1 shows the sequences of synthetic peptides of SFPQ36-50 or SFPQ452-466, which are epitopes of SFPQ. Cysteine is added to the N-terminus of the synthetic peptide.
Figure JPOXMLDOC01-appb-T000001
 　図３に、実施例３におけるＥＬＩＳＡによる抗ＳＦＰＱ抗体価、および、Ｗｅｓｔｅｎ
　Ｂｌｏｔによる、抗ＳＦＰＱ抗体価と、間接蛍光抗体法（ＩＦＡ）による抗ＳＩＴＨ－ＣＡＭＬ抗体価の検出結果（抗ＳＦＰＱ抗体価とＳＩＴＨ－ＣＡＭＬ抗体価の相関）との関係を示す。
Figure JPOXMLDOC01-appb-T000001
 Figure 3 shows the anti-SFPQ antibody titer by ELISA in Example 3, and Westen
The relationship between the anti-SFPQ antibody titer by Blot and the detection result of the anti-SITH-CAML antibody titer by the indirect fluorescent antibody method (IFA) (the correlation between the anti-SFPQ antibody titer and the SITH-CAML antibody titer) is shown.
　図３上図に、合成ペプチドを用いたＥＬＩＳＡによる抗ＳＦＰＱ抗体価の検出結果を示す。図３上図より明らかなように、ＳＦＰＱ３６－５０とＳＦＰＱ４５２－４６６のいずれのペプチドを抗原とした場合であっても、健常者と比較してうつ病患者の抗ＳＦＰＱ抗体量は有意に高いことが示された。＊＊＊＊Ｐ＜０．０００１。 The upper figure in Fig. 3 shows the results of anti-SFPQ antibody titer detection by ELISA using synthetic peptides. As is clear from the upper diagram of FIG. 3, the amount of anti-SFPQ antibody in depressed patients is significantly higher than in healthy subjects, regardless of whether the antigen is either SFPQ36-50 or SFPQ452-466. It has been shown. ***P<0.0001.
　図３左下図に、Ｗｅｓｔｅｒｎ　Ｂｌｏｔによる抗ＳＦＰＱ抗体価とＳＩＴＨ‐ＣＡＭＬ抗体価の相関プロットを示す。図３左下図より明らかなように、特に６０歳未満のヒトにおいて、抗ＳＦＰＱ抗体価とＳＩＴＨ－ＣＡＭＬ抗体価に相関関係が認められた。この結果は、ＳＩＴＨ－１タンパク質の発現を何度も繰り返している抗ＳＩＴＨ－ＣＡＭＬ抗体価の高いヒトほど抗ＳＦＰＱ抗体価が高いことを示している。 The lower left figure in Figure 3 shows the correlation plot between the anti-SFPQ antibody titer and the SITH-CAML antibody titer by Western Blot. As is clear from the lower left diagram of FIG. 3, a correlation was observed between the anti-SFPQ antibody titer and the SITH-CAML antibody titer, particularly in humans under the age of 60. This result indicates that the higher the anti-SITH-CAML antibody titer, the higher the anti-SFPQ antibody titer in a person who repeatedly expresses the SITH-1 protein.
　図３右下図にはＷｅｓｔｅｒｎ　Ｂｌｏｔによる抗ＳＦＰＱ抗体の年代別陽性率を示す。図３右下図より明らかなように、年代が高くなるほど抗ＳＦＰＱ抗体を保持する割合が高くなる傾向がある。このことから、抗ＳＦＰＱ抗体の保持率は老化に伴って上昇することが示された。 The lower right figure of Figure 3 shows the anti-SFPQ antibody positive rate by age group by Western Blot. As is clear from the lower right figure of FIG. 3, there is a tendency that the higher the age, the higher the ratio of holding anti-SFPQ antibodies. This indicated that the retention rate of the anti-SFPQ antibody increased with aging.
　〔実施例４〕
　ＳＦＰＱを免疫することによるうつ病関連症状の発生
　＜方法＞
　抗ＳＦＰＱ抗体を保持するマウスの表現型を解析するため、下記の方法でマウスに免疫し、行動試験、組織学的解析、および遺伝子発現解析を試みた。 [Example 4]
Occurrence of depression-related symptoms by immunization with SFPQ <Method>
In order to analyze the phenotype of mice carrying anti-SFPQ antibodies, mice were immunized as described below, and behavioral tests, histological analyses, and gene expression analyses were attempted.
　（１）マウスのＳＦＰＱ免疫
　まず、１ｍｇ／ｍＬのＫＬＨコンジュゲートＳＦＰＱ合成ペプチド溶液（ＳＦＰＱ３６－５０またはＳＦＰＱ４５２－４６６）と等量のＦｒｅｕｎｄＴｓ　Ｃｏｍｐｌｅｔｅ　Ａｄｊｕｖａｎｔ（富士フイルム和光純薬）を混合し、エマルジョン溶液を作製した。対照として、１ｍｇ／ｍＬのＫＬＨタンパク質溶液を用いたエマルジョン溶液を作製した。このエマルジョン溶液を４週齢のＣ５７ＢＬ／６雄マウスの皮下に４００μＬずつ（抗原投与量が０．２ｍｇ／匹となるよう）投与し（１回目の免疫）、ホームケージに戻して通常飼育した。 (1) SFPQ immunization of mice First, 1 mg/mL KLH conjugated SFPQ synthetic peptide solution (SFPQ36-50 or SFPQ452-466) and an equal amount of Freund T s Complete Adjuvant (Fujifilm Wako Pure Chemical) were mixed to form an emulsion. A solution was prepared. As a control, an emulsion solution was made using a 1 mg/mL KLH protein solution. This emulsion solution was subcutaneously administered to 4-week-old C57BL/6 male mice at 400 μL each (antigen dose was 0.2 mg/mouse) (first immunization), returned to their home cages and reared normally.
　１ｍｇ／ｍＬのＫＬＨコンジュゲートＳＦＰＱ合成ペプチド溶液（ＳＦＰＱ３６－５０またはＳＦＰＱ４５２－４６６）と等量のＦｒｅｕｎｄＴｓ　Ｉｎｃｏｍｐｌｅｔｅ　Ａｄｊｕｖａｎｔ（富士フイルム和光純薬）を混合して、エマルジョン溶液を作製し、このエマルジョン溶液を６週齢（上記１回目の免疫から２週間後）の前記マウス（１回目の免疫を行ったＣ５７ＢＬ／６雄マウス）の皮下に２００μＬずつ（抗原投与量が０．１ｍｇ／匹となるよう）投与し（２回目の免疫）、ホームケージに戻して通常飼育した。さらに２週間後、８週齢となった前記マウスについて、２回目の免疫と同様に前記エマルジョン溶液を投与し（３回目の免疫）、さらに１週間（９週齢となるまで）通常飼育した。これにより、計３回の免疫を実施した、ＳＦＰＱ免疫マウスを作製した。 A 1 mg/mL KLH conjugated SFPQ synthetic peptide solution (SFPQ36-50 or SFPQ452-466) and an equal amount of Freund T 's Incomplete Adjuvant (Fujifilm Wako Pure Chemical Industries) are mixed to prepare an emulsion solution. 200 μL each subcutaneously of the 6-week-old (2 weeks after the first immunization) mouse (C57BL/6 male mouse that was immunized for the first time) (antigen dose was 0.1 mg/animal) ) (second immunization), returned to their home cages and reared normally. Two weeks later, the 8-week-old mice were administered the emulsion solution in the same manner as the second immunization (third immunization), and were then fed normally for another week (until they reached the age of 9 weeks). In this way, SFPQ-immunized mice, which were immunized a total of three times, were produced.
　（２）マウスの自発運動量の解析
　作製したＳＦＰＱ免疫マウスの自発運動量を解析するために、Ｙ字迷路を用いた。１つのアームの先端にマウスを置き、１０分間自由に移動させ、各アームへの侵入回数を自発運動量とした。 (2) Analysis of locomotor activity of mice A Y-maze was used to analyze the locomotor activity of the prepared SFPQ-immunized mice. A mouse was placed on the tip of one arm and allowed to move freely for 10 minutes.
　（３）マウスの不安行動の解析（１）
　作製したＳＦＰＱ免疫マウスの不安行動を解析するために、高さ５００ｍｍの高架十字迷路を使用した。高架十字迷路の中央にＳＦＰＱ免疫マウスを置き、１０分間自由に移動させ、オープンアームに侵入した回数を計測した。高架十字迷路を使用した実験においては、この侵入回数が少ないほど不安行動を示すと考えられている。 (3) Analysis of anxiety behavior in mice (1)
An elevated plus maze with a height of 500 mm was used to analyze the anxiety behavior of generated SFPQ-immunized mice. SFPQ-immunized mice were placed in the center of the elevated plus maze, allowed to move freely for 10 minutes, and the number of open arm entries counted. In an experiment using an elevated plus maze, it is believed that the smaller the number of intrusions, the more anxious behavior is exhibited.
　（４）マウスの不安行動の解析（２）
　作製したＳＦＰＱ免疫マウスの不安行動を解析するために、ガラス玉覆い隠し試験を実施した。不透明なケージに約５０ｍｍの深さとなるよう床敷を敷き詰め、その上に１５個のガラス玉を均等に配置した。マウスをケージ内に置き、３０分間自由に探索させ、３０分後に２／３以上床敷で覆われているガラス玉の数を計測した。ガラス玉覆い隠し試験においては、床敷で覆われているガラス玉の数が多いほど不安行動を示すと考えられている。 (4) Analysis of anxiety behavior in mice (2)
To analyze the anxiety behavior of the generated SFPQ-immunized mice, a marble burying test was performed. An opaque cage was lined with bedding to a depth of about 50 mm, and 15 glass beads were evenly distributed on the bedding. Mice were placed in cages and allowed to explore freely for 30 minutes, after which the number of marbles covered by more than 2/3 of the bedding was counted after 30 minutes. In the marble occlusion test, it is believed that the greater the number of marbles covered with bedding, the more anxious behavior is exhibited.
　（５）マウスの脳の組織学的解析
　３回目のＳＦＰＱ免疫から２週間後の、１０週齢のＳＦＰＱ免疫マウスの脳を摘出して１０％中性ホルマリン溶液で固定した。固定化した脳をパラフィン包埋し、手綱（Ｂｒｅｇｍａ－１．７０ｍｍ）が観察できる位置で冠状切片（脳切片）を作製した。作製した脳切片について、脱パラフィン処理後に抗原賦活化処理を行い、抗コリンアセチルトランスフェラーゼ（ＣｈＡＴ）抗体（ａｂｃａｍ社製）で蛍光免疫染色した。 (5) Mouse Brain Histological Analysis Two weeks after the third SFPQ immunization, the brains of 10-week-old SFPQ-immunized mice were excised and fixed with a 10% neutral formalin solution. The fixed brain was embedded in paraffin, and a coronal section (brain section) was prepared at a position where the reins (Bregma-1.70 mm) could be observed. The prepared brain sections were subjected to antigen retrieval treatment after deparaffinization, and fluorescent immunostaining was performed with an anti-choline acetyltransferase (ChAT) antibody (manufactured by abcam).
　（６）マウスの脳における遺伝子発現解析
　３回目のＳＦＰＱ免疫から２週間後の、１０週齢のＳＦＰＱ免疫マウスの脳を摘出し、Ｂｕｆｆｅｒ　ＲＬＴ　（ＱＩＡＧＥＮ）を加えてホモジナイズすることで組織を溶解後、ＲＮｅａｓｙ　Ｍｉｎｉ　Ｋｉｔ（ＱＩＡＧＥＮ）でＲＮＡを精製した。精製ＲＮＡを鋳型にＰｒｉｍｅＳｃｒｉｐｔ　ＲＴ　ｒｅａｇｅｎｔ　Ｋｉｔ（タカラバイオ）を用いてｃＤＮＡを合成した。合成したｃＤＮＡを鋳型に、脳由来神経栄養因子ＢＤＮＦ、α７ニコチン性アセチルコリン受容体（Ｃｈｒｎａ７）、アポトーシス関連因子ＢａｘおよびＢｃｌ－２の遺伝子発現をＲＴ－ｑＰＣＲにより解析した。 (6) Gene expression analysis in mouse brain Two weeks after the third SFPQ immunization, the brain of a 10-week-old SFPQ-immunized mouse was excised, and Buffer RLT (QIAGEN) was added and homogenized to dissolve the tissue. , RNA was purified with the RNeasy Mini Kit (QIAGEN). Using the purified RNA as a template, cDNA was synthesized using PrimeScript RT reagent Kit (Takara Bio). Using the synthesized cDNA as a template, gene expressions of brain-derived neurotrophic factor BDNF, α7 nicotinic acetylcholine receptor (Chrna7), apoptosis-related factor Bax and Bcl-2 were analyzed by RT-qPCR.
　＜結果＞
　実施例４における、マウスの不安行動の解析結果、マウスの脳（手綱）の組織学的解析の結果を図４に、マウスの脳における遺伝子発現解析の結果を図５にそれぞれ示す。 <Results>
FIG. 4 shows the results of analysis of anxious behavior of mice, the results of histological analysis of mouse brains (bridle), and FIG. 5 shows the results of gene expression analysis in mouse brains in Example 4.
　図４左上図より明らかなように、ＳＦＰＱ免疫マウスはいずれもＹ字迷路（Ｙ－ｍａｚｅ）における自発運動量の低下が観察された。 As is clear from the upper left figure in Fig. 4, all SFPQ-immunized mice were observed to have reduced motor activity in the Y-maze.
　図４中央上図におよび図４右上図より明らかなように、ＳＦＰＱ抗体産生マウスはいずれも高架十字迷路（ＥＰＭ）でオープンアームの侵入回数が有意に少なく（図４中央上図）、また、ガラス玉覆い隠し試験（marble burying test:ＭＢＴ）でガラス玉を覆い隠した数が多かった（図４右上図）。従って、何れの試験においても、ＳＦＰＱ免疫マウスは不安様行動を示すことが示された。 As is clear from the upper center figure of FIG. 4 and the upper right figure of FIG. 4, the SFPQ antibody-producing mice had significantly fewer open arm invasions in the elevated plus maze (EPM) (upper center figure of FIG. 4). In the marble burying test (MBT), the number of burying glass marbles was large (Fig. 4, upper right). Therefore, both tests showed that SFPQ-immunized mice exhibited anxiety-like behavior.
　また、図４下図には、マウスの脳の組織学的解析結果として、手綱のコリンアセチルトランスフェラーゼ産生細胞を観察した写真およびコリン作動性ニューロンの数を示す。図４下図の各図より明らかなように、ＳＦＰＱ免疫マウスは、いずれも快感喪失に関与する内側手綱（ＭＨｂ）と、睡眠、概日リズム、報酬系および認知機能と関係する外側手綱（ＬＨｂ）においてコリンアセチルトランスフェラーゼ産生細胞の減少がみられることが示された。 In addition, the lower figure in Fig. 4 shows a photograph of observing choline acetyltransferase-producing cells in the habenula and the number of cholinergic neurons as the results of histological analysis of the mouse brain. As is clear from the lower diagrams of FIG. 4, SFPQ-immunized mice have medial habenula (MHb), which is involved in anhedonia, and lateral habenula (LHb), which is involved in sleep, circadian rhythm, reward system, and cognitive function. It was shown that there was a decrease in choline acetyltransferase-producing cells in
　さらに、図５より明らかなように、ＳＦＰＱ免疫マウスはいずれも脳の栄養因子であるＢＤＮＦと免疫抑制性受容体α７型ニコチン性アセチルコリンレセプター（α７　ｎＡＣｈＲ）の発現低下が観察される（図５左図および中図）。加えて、ＳＦＰＱ免疫マウスはいずれも嗅球細胞のアポトーシス亢進が観察された（図５右図）。 Furthermore, as is clear from Fig. 5, in both SFPQ-immunized mice, decreased expression of BDNF, a brain trophic factor, and immunosuppressive receptor α7-type nicotinic acetylcholine receptor (α7 nAChR) was observed (Fig. 5, left). and middle). In addition, enhanced apoptosis of olfactory bulb cells was observed in all SFPQ-immunized mice (Fig. 5, right).
　〔実施例５〕
　ＳＦＰＱを免疫することによる脳の老化現象の発生
　＜方法＞
　（１）マウスのＳＦＰＱ免疫
　まず、１ｍｇ／ｍｌのＫＬＨコンジュゲートＳＦＰＱ合成ペプチド溶液（ＳＦＰＱ３６－５０またはＳＦＰＱ４５２－４６６）と等量のＦｒｅｕｎｄＴｓ　Ｃｏｍｐｌｅｔｅ　Ａｄｊｕｖａｎｔ（富士フイルム和光純薬）を混合し、エマルジョン溶液を作製した。対照として、１ｍｇ／ｍｌのＫＬＨタンパク質溶液を用いたエマルジョン溶液を作製した。抗原投与量を０．２ｍｇ／匹とするため、このエマルジョン溶液を４週齢のＣ５７ＢＬ／６雄マウスの皮下に４００μＬずつ投与し（１回目の免疫）、ホームケージに戻して通常飼育した。 [Example 5]
Occurrence of brain aging phenomenon by immunization with SFPQ <Method>
(1) SFPQ immunization of mice First, a 1 mg/ml KLH conjugated SFPQ synthetic peptide solution (SFPQ36-50 or SFPQ452-466) and an equal amount of Freund T 's Complete Adjuvant (Fujifilm Wako Pure Chemical) were mixed to form an emulsion. A solution was prepared. As a control, an emulsion solution was prepared using a 1 mg/ml KLH protein solution. In order to adjust the dose of the antigen to 0.2 mg/mouse, 400 μL of this emulsion solution was subcutaneously administered to each 4-week-old C57BL/6 male mouse (first immunization), and the mice were returned to their home cages and reared normally.
　１ｍｇ／ｍＬのＫＬＨコンジュゲートＳＦＰＱ合成ペプチド溶液（ＳＦＰＱ３６－５０またはＳＦＰＱ４５２－４６６）と等量のＦｒｅｕｎｄＴｓ　Ｉｎｃｏｍｐｌｅｔｅ　Ａｄｊｕｖａｎｔ（富士フイルム和光純薬）を混合して、エマルジョン溶液を作製し、このエマルジョン溶液を６週齢（上記１回目の免疫から２週間後）の前記マウス（１回目の免疫を行ったＣ５７ＢＬ／６雄マウス）の皮下に２００μＬずつ（抗原投与量が０．１ｍｇ／匹となるよう）投与し（２回目の免疫）、ホームケージに戻して通常飼育した。さらに２週間後、８週齢となった前記マウスについて、２回目の免疫と同様に前記エマルジョン溶液を投与し（３回目の免疫）、さらに１週間（９週齢となるまで）通常飼育した。これにより、計３回の免疫を実施した、ＳＦＰＱ免疫マウスを作製した。 A 1 mg/mL KLH conjugated SFPQ synthetic peptide solution (SFPQ36-50 or SFPQ452-466) and an equal amount of Freund T 's Incomplete Adjuvant (Fujifilm Wako Pure Chemical Industries) are mixed to prepare an emulsion solution. 200 μL each subcutaneously of the 6-week-old (2 weeks after the first immunization) mouse (C57BL/6 male mouse that was immunized for the first time) (antigen dose was 0.1 mg/animal) ) (second immunization), returned to their home cages and reared normally. Two weeks later, the 8-week-old mice were administered the emulsion solution in the same manner as the second immunization (third immunization), and were then fed normally for another week (until they reached the age of 9 weeks). In this way, SFPQ-immunized mice, which were immunized a total of three times, were produced.
　（２）マウスの脳および嗅球における遺伝子発現解析
　実施例４の（６）の操作と同様の方法で脳および嗅球のｃＤＮＡを合成し、老化のマーカーであるエンドセリン１（Ｅｄｎ－１）の遺伝子発現をＲＴ－ｑＰＣＲにより解析した。 (2) Gene expression analysis in mouse brain and olfactory bulb cDNA was synthesized in the brain and olfactory bulb in the same manner as in Example 4 (6), and gene expression of endothelin 1 (Edn-1), an aging marker, was analyzed. was analyzed by RT-qPCR.
　（３）マウスの脳の組織学的解析
　作製したＳＦＰＱ免疫マウスについて、実施例４と同様の方法で、海馬（Ｂｒｅｇｍａ－１．７０ｍｍ）が観察できる位置で冠状切片（脳切片）を作製した。作製した脳切片について、脱パラフィン処理後に抗原賦活化処理を行い、抗ダブルコルチン（ＤＣＸ）抗体（ａｂｃａｍ社製）で蛍光免疫染色した。 (3) Histological Analysis of Mouse Brain A coronal section (brain section) was prepared in the same manner as in Example 4 from the prepared SFPQ-immunized mouse at a position where the hippocampus (Bregma-1.70 mm) could be observed. The prepared brain sections were subjected to antigen retrieval treatment after deparaffinization, and fluorescent immunostaining was performed with an anti-doublecortin (DCX) antibody (manufactured by abcam).
　（４）マウスの空間作業記憶の解析
　作製したＳＦＰＱ免疫マウスの空間作業記憶を解析するために、Ｙ字迷路を用いた。１つのアームの先端にマウスを置き、１０分間自由に移動させ、３回連続で異なるアームに侵入する割合を求めた。 (4) Analysis of Mouse Spatial Working Memory A Y-maze was used to analyze the spatial working memory of the prepared SFPQ-immunized mice. Mice were placed at the tip of one arm and allowed to move freely for 10 minutes, and the percentage of three consecutive intrusions into different arms was determined.
　（５）マウスの協調運動能力の解析
　作製したＳＦＰＱ免疫マウスの協調運動能力を解析するために、Ｒｏｔａｒｏｄ試験を用いた。具体的には、ＳＦＰＱ免疫マウスを４ｒｐｍで回転する棒の上に一定時間乗せ、慣れさせた後に１分間で４ｒｐｍから６０ｒｐｍまで回転速度を上昇させた際に、マウスが落下するまでの時間を計測した。 (5) Analysis of Coordinated Motor Ability of Mice Rotarod test was used to analyze the coordinated motor ability of the prepared SFPQ-immunized mice. Specifically, the SFPQ-immunized mouse was placed on a rod rotating at 4 rpm for a certain period of time, and after habituation, the rotation speed was increased from 4 rpm to 60 rpm in 1 minute, and the time until the mouse fell was measured. did.
　＜結果＞
　実施例５における、マウスの脳および嗅球における遺伝子発現解析の結果、および、マウスの脳（海馬）の組織学的解析の結果を図６に、マウスの空間作業記憶および協調運動能力の解析の結果を図７にそれぞれ示す。 <Results>
The results of gene expression analysis in the mouse brain and olfactory bulb and the results of histological analysis of the mouse brain (hippocampus) in Example 5 are shown in FIG. are shown in FIG. 7, respectively.
　図６上図に、ＳＦＰＱ免疫マウスの脳と嗅球における老化のマーカーであるＥｄｎ－１遺伝子の発現量を示す。図６上図より明らかなように、対照マウスと比較して、ＳＦＰＱ免疫マウスはいずれもＥｄｎ－１遺伝子の発現増加が確認された。 The upper figure in Fig. 6 shows the expression level of the Edn-1 gene, a marker of aging, in the brain and olfactory bulb of SFPQ-immunized mice. As is clear from the upper diagram of FIG. 6, increased expression of the Edn-1 gene was confirmed in all SFPQ-immunized mice compared with control mice.
　図６下図に、ＳＦＰＱ免疫マウスの海馬における神経新生マーカーであるＤＣＸ産生細胞を観察した写真およびＤＣＸ陽性細胞を計測したグラフを示す。図６下図の各図より明らかなように、ＳＦＰＱ免疫マウスはいずれも海馬における神経新生の低下が観察されることを示した。 The lower figure in Fig. 6 shows a photograph of observation of DCX-producing cells, which is a neurogenesis marker, in the hippocampus of SFPQ-immunized mice, and a graph of measurement of DCX-positive cells. As is clear from the lower diagrams of FIG. 6, all SFPQ-immunized mice showed decreased neurogenesis in the hippocampus.
　また、図７に、Ｙ字迷路（Ｙ－ｍａｚｅ）およびＲｏｔａｒｏｄ試験を用いたＳＦＰＱ免疫マウスの行動解析結果を示す。図７より明らかなように、ＳＦＰＱ免疫マウスはいずれも空間作業記憶が低下しており、また、協調運動の低下も観察された。従って、ＳＦＰＱ抗体産生マウスはいずれも老化様行動を示すことが示された。 In addition, FIG. 7 shows the behavioral analysis results of SFPQ-immunized mice using the Y-maze and Rotarod test. As is clear from FIG. 7, all SFPQ-immunized mice had impaired spatial working memory, and decreased coordination was also observed. Therefore, it was shown that all SFPQ antibody-producing mice exhibit senescence-like behavior.
　〔実施例６〕
　ＳＦＰＱを免疫することによる腸の老化現象の発生
　＜方法＞
　（１）マウスのＳＦＰＱ免疫
　まず、１ｍｇ／ｍＬのＫＬＨコンジュゲートＳＦＰＱ合成ペプチド溶液（ＳＦＰＱ３６－５０、ＳＦＰＱ４５２－４６６）と等量のアジュバントＴｉｔｅｒＭａｘ　Ｇｏｌｄ（ＴｉｔｅｒＭａｘ社）を混合し、エマルジョン溶液を作製した。対照として、１ｍｇ／ｍｌのＫＬＨタンパク質溶液を用いたエマルジョン溶液を作製した。抗原投与量が１２５μｇ／匹となるよう、このエマルジョン溶液を４週齢のＣ５７ＢＬ／６雄マウスの皮下に投与し（１回目の免疫）、ホームケージに戻して通常飼育した。１回目の免疫から４週間後に２回目の免疫として、抗原投与量が１２５μｇ／匹となるよう抗原溶液のみを前記マウスの皮下に投与した。さらに２回目の免疫から４週間後に３回目の免疫として、抗原投与量が１２５μｇ／匹となるよう抗原溶液のみを前記マウスの皮下に投与した。 [Example 6]
Occurrence of intestinal aging phenomenon by immunization with SFPQ <Method>
(1) SFPQ Immunization of Mice First, 1 mg/mL KLH-conjugated SFPQ synthetic peptide solutions (SFPQ36-50, SFPQ452-466) and an equal amount of adjuvant TiterMax Gold (TiterMax) were mixed to prepare an emulsion solution. As a control, an emulsion solution was prepared using a 1 mg/ml KLH protein solution. This emulsion solution was subcutaneously administered to 4-week-old C57BL/6 male mice (first immunization) so that the dose of antigen was 125 μg/mouse, and the mice were returned to their home cages and reared normally. Four weeks after the first immunization, as the second immunization, the antigen solution alone was subcutaneously administered to the mice so that the dose of the antigen was 125 μg/mouse. Further, 4 weeks after the second immunization, as the third immunization, only the antigen solution was subcutaneously administered to the mice so that the antigen dose was 125 μg/mouse.
　なお、以下の解析において、１回免疫のマウスとしては１回目の免疫から４週間後のマウスを、２回免疫のマウスとしては２回目の免疫から４週間後のマウスを、３回免疫のマウスとしては３回目の免疫から４週間後のマウスをそれぞれ使用した。尚、免疫期間中は２週間毎にマウスの体重を測定した。 In the following analysis, mice 4 weeks after the first immunization were used as single-immune mice, mice 4 weeks after the second immunization were used as mice with two immunizations, and three-time immunization mice were used. As the mice, mice 4 weeks after the third immunization were used. During the immunization period, the body weight of the mice was measured every two weeks.
　（２）マウスの小腸における遺伝子発現解析
　１回免疫のマウスと３回免疫のマウスの小腸を採取し、実施例４の（６）の操作と同様の方法でマウス小腸のｃＤＮＡを合成した。合成したｃＤＮＡを鋳型に幹細胞マーカーのＬｇｒ５とＳｏｘ２、老化マーカーのｃｙｃｌｉｎ－ｄｅｐｅｎｄｅｎｔ　ｋｉｎａｓｅ
　ｉｎｈｉｂｉｔｏｒ　２Ａ（Ｃｄｋｎ２ａ）とＥｄｎ－１の遺伝子発現をＲＴ－ｑＰＣＲにより解析した。 (2) Gene Expression Analysis in Small Intestines of Mouse Small intestines of mice immunized once and mice immunized three times were collected, and mouse small intestine cDNA was synthesized in the same manner as in Example 4 (6). Using the synthesized cDNA as a template, stem cell markers Lgr5 and Sox2, and senescence marker cyclin-dependent kinase
Gene expression of inhibitor 2A (Cdkn2a) and Edn-1 was analyzed by RT-qPCR.
　＜結果＞
　実施例６における、マウスの小腸における遺伝子発現解析の結果および免疫期間中のマウスの体重変化の測定結果を、図８に示す。 <Results>
FIG. 8 shows the results of gene expression analysis in the small intestine of mice and the measurement results of changes in body weight of mice during the immunization period in Example 6.
　図８上図に、１回免疫後に変化が観察された老化マーカーであるＣｄｋｎ２ａおよびＥｄｎ－１、ならびに、幹細胞マーカーであるＳｏｘ２の遺伝子発現解析結果を示す。図８上図より明らかなように、ＳＦＰＱ免疫マウスは対照マウスと比較してこれらの遺伝子発現量の上昇がみられた。 The upper diagram of FIG. 8 shows the gene expression analysis results of Cdkn2a and Edn-1, which are senescence markers, and Sox2, a stem cell marker, whose changes were observed after a single immunization. As is clear from the upper diagram of FIG. 8, the SFPQ-immunized mice exhibited elevated expression levels of these genes compared to the control mice.
　図８左下図に、３回免疫後に変化が観察された腸管の幹細胞マーカーであるＬｇｒ５の遺伝子発現解析結果を示す。図８左下図より明らかなように、ＳＦＰＱ免疫マウスはいずれも対照マウスと比較してＬｇｒ５の発現が低下していた。 The lower left figure in Figure 8 shows the gene expression analysis results of Lgr5, an intestinal stem cell marker, in which changes were observed after three immunizations. As is clear from the lower left diagram of FIG. 8, all SFPQ-immunized mice had reduced Lgr5 expression compared to control mice.
　また、図８右下図に示す通り、ＳＦＰＱ免疫マウスはいずれも対照マウスと比較して体重増加が抑制されていた。 In addition, as shown in the lower right diagram of FIG. 8, weight gain was suppressed in all SFPQ-immunized mice compared to control mice.
　これらの結果は、免疫期間の初期に抗ＳＦＰＱ抗体によって腸管の損傷が生じ、腸管の幹細胞の機能低下が長期間にわたって持続することを示唆している。 These results suggest that the anti-SFPQ antibody causes intestinal damage early in the immunization period, and that the dysfunction of intestinal stem cells persists over the long term.
　〔実施例７〕
　ＳＦＰＱを免疫することによる精巣の老化現象の発生
　＜方法＞
　実施例６と同様の方法でマウスのＳＦＰＱ免疫を行い、抗ＳＦＰＱ抗体を産生させたマウスである、ＳＦＰＱ免疫マウスを得た。その後、１回免疫のマウスと３回免疫のマウスの精巣を採取し、実施例４の（６）の操作と同様の方法でマウス精巣のｃＤＮＡを合成した。合成したｃＤＮＡを鋳型に幹細胞マーカーのＬｇｒ４、Ｌｇｒ５およびＳｏｘ２、そしてテストステロン産生マーカーのｓｔｅｒｏｉｄｏｇｅｎｉｃ　ａｃｕｔｅ　ｒｅｇｕｌａｔｏｒｙ　ｐｒｏｔｅｉｎ（ＳｔＡＲ）遺伝子発現をＲＴ－ｑＰＣＲにより解析した。 [Example 7]
Occurrence of testicular aging phenomenon by immunization with SFPQ <Method>
Mice were immunized with SFPQ in the same manner as in Example 6 to obtain SFPQ-immunized mice that produced anti-SFPQ antibodies. After that, the testis of the mouse immunized once and the mouse immunized three times was collected, and cDNA of the mouse testis was synthesized in the same manner as in the operation of Example 4 (6). Using the synthesized cDNA as a template, gene expressions of stem cell markers Lgr4, Lgr5 and Sox2 and testosterone production marker steroidogenic acute regulatory protein (StAR) gene expression were analyzed by RT-qPCR.
　さらにマウスの精巣の組織学的解析を行うために、１回免疫のマウスを使用し、実施例４の（５）の操作と同様の方法で、精巣切片を作製した。作製した精巣切片について、脱パラフィン処理後に抗原賦活化処理を行い、抗ライディッヒ細胞マーカーＨＳＤ３Ｂ１抗体（ａｂｃａｍ社）で蛍光免疫染色した。 Furthermore, in order to perform histological analysis of the mouse testis, a single immunized mouse was used, and a testis section was prepared in the same manner as in Example 4 (5). The prepared testis sections were subjected to antigen retrieval treatment after deparaffinization, and fluorescent immunostaining was performed with an anti-Leydig cell marker HSD3B1 antibody (abcam).
　＜結果＞
　実施例７における、マウスの精巣における遺伝子発現解析の結果を図９および図１０に、精巣切片の蛍光免疫染色の結果を図１０に、それぞれ示す。 <Results>
The results of gene expression analysis in mouse testis in Example 7 are shown in FIGS. 9 and 10, and the results of fluorescent immunostaining of testis sections are shown in FIG.
　図９には１回目のＳＦＰＱ免疫後４週間が経過したＳＦＰＱ免疫マウスの精巣における遺伝子発現の解析結果を示す。図９より明らかなように、ＳＦＰＱ免疫マウスにおいて、１回免疫で産生された抗ＳＦＰＱ抗体の影響で精巣の幹細胞マーカーであるＬｇｒ５と、テストステロン産生マーカーであるＳｔＡＲの低下がみられた。 Fig. 9 shows the analysis results of gene expression in the testis of SFPQ-immunized mice 4 weeks after the first SFPQ immunization. As is clear from FIG. 9, the testicular stem cell marker Lgr5 and the testosterone production marker StAR were decreased in the SFPQ-immunized mice due to the anti-SFPQ antibody produced by the single immunization.
　図１０上図には３回目のＳＦＰＱ免疫後４週間が経過したＳＦＰＱ免疫マウスの精巣における遺伝子発現の解析結果を示す。図１０上図より明らかなように、ＳＦＰＱ免疫マウスにおいて、３回免疫で産生された抗ＳＦＰＱ抗体の影響で、１回目の免疫時に確認された各マーカーの低下に加え、さらに幹細胞マーカーであるＬｇｒ４とＳｏｘ２の低下もすることが明らかになった。 The upper diagram of FIG. 10 shows the analysis results of gene expression in the testis of SFPQ-immunized mice 4 weeks after the third SFPQ immunization. As is clear from the upper diagram of FIG. 10, in the SFPQ-immunized mice, the effects of the anti-SFPQ antibody produced in the three immunizations reduced each marker confirmed in the first immunization. and Sox2 decreased.
　また、図１０下図に示すように、精巣のライディッヒ細胞数の低下も観察された。また、ＳＦＰＱ免疫マウスにおいては、鼠径ヘルニアの発生も観察された。これはテストステロン産生が低下した結果、２型コラーゲンの産生低下と低腹壁の構造異常が生じた結果と考えられる。 In addition, as shown in the lower figure of Fig. 10, a decrease in the number of Leydig cells in the testis was also observed. The development of inguinal hernia was also observed in SFPQ-immunized mice. This is considered to be the result of decreased production of type 2 collagen and structural abnormalities of the low abdominal wall as a result of decreased testosterone production.
　これの結果は、ＳＦＰＱ抗体が精巣の幹細胞の機能とテストステロン産生を低下させることを示唆している。 These results suggest that SFPQ antibodies reduce testicular stem cell function and testosterone production.
　〔実施例８〕
　ＳＦＰＱを免疫することによる皮膚の老化現象の発生
　＜方法＞
　実施例６と同様の方法でマウスのＳＦＰＱ免疫を行い、抗ＳＦＰＱ抗体を産生させたマウスである、ＳＦＰＱ免疫マウスを得た。その後、３回免疫のマウスの皮膚を採取し、実施例４の（６）の操作と同様の方法でマウス皮膚のｃＤＮＡを合成した。合成したｃＤＮＡを鋳型に幹細胞マーカーのＬｇｒ４、老化マーカーのＣｄｋｎ２ａ、炎症性サイトカインのＩＬ－１βとＩＬ－６、炎症の原因となるインフラマソームＮｌｒｐ３、およびアポトーシス関連因子のＢａｘとＢｃｌ－２の遺伝子発現をＲＴ－ｑＰＣＲにより解析した。 [Example 8]
Occurrence of skin aging phenomenon by immunization with SFPQ <Method>
Mice were immunized with SFPQ in the same manner as in Example 6 to obtain SFPQ-immunized mice that produced anti-SFPQ antibodies. After that, the skin of the mice immunized three times was collected, and the cDNA of the mouse skin was synthesized in the same manner as in the operation of Example 4 (6). Synthesized cDNA was used as a template for stem cell marker Lgr4, senescence marker Cdkn2a, inflammatory cytokines IL-1β and IL-6, inflammation-causing inflammasome Nlrp3, and apoptosis-related factors Bax and Bcl-2 genes. Expression was analyzed by RT-qPCR.
　さらに、ＳＦＰＱ免疫マウスの皮膚の組織学的解析を行うために、１回免疫のマウスを使用し、実施例４の（５）の操作と同様の方法で、皮膚切片を作製した。作製した皮膚切片について、脱パラフィン処理後に抗原賦活化処理を行い、毛包幹細胞マーカーＬｇｒ５抗体（ａｂｃａｍ社）および老化マーカーＣｄｋｎ２ａ抗体（ａｂｃａｍ社）で蛍光免疫染色した。 Furthermore, in order to perform histological analysis of the skin of SFPQ-immunized mice, single-immunized mice were used, and skin sections were prepared in the same manner as in Example 4 (5). The prepared skin sections were subjected to antigen retrieval treatment after deparaffinization, and fluorescent immunostaining was performed with a hair follicle stem cell marker Lgr5 antibody (abcam) and an aging marker Cdkn2a antibody (abcam).
　＜結果＞
　実施例８における、ＳＦＰＱ免疫マウスの皮膚における遺伝子発現解析の結果を図１１に、ＳＦＰＱ免疫マウスの皮膚の組織学的解析の結果を図１２に、それぞれ示す。 <Results>
FIG. 11 shows the results of gene expression analysis in the skin of SFPQ-immunized mice in Example 8, and FIG. 12 shows the results of histological analysis of the skin of SFPQ-immunized mice.
　図１１に、３回目のＳＦＰＱ免疫後４週間経過したＳＦＰＱ免疫マウスの皮膚における遺伝子発現解析の結果を示す。図１１より明らかなように、ＳＦＰＱ免疫マウスにおいては、抗ＳＦＰＱ抗体の影響で皮膚の幹細胞のマーカーであるＬｇｒ４の減少、老化マーカーＣｄｋｎ２ａの増加、炎症性サイトカインＩＬ－１βとＩＬ－６の増加、炎症の原因となるインフラマソームＮｌｒｐ３の増加が認められた。また、アポトーシスのマーカーであるＢａｘ／Ｂｃｌ－２比も増加していた。 FIG. 11 shows the results of gene expression analysis in the skin of SFPQ-immunized mice 4 weeks after the third SFPQ immunization. As is clear from FIG. 11, in SFPQ-immunized mice, under the influence of anti-SFPQ antibody, the cutaneous stem cell marker Lgr4 decreased, the senescence marker Cdkn2a increased, the inflammatory cytokines IL-1β and IL-6 increased, An increase in inflammasome Nlrp3, which causes inflammation, was observed. Also, the Bax/Bcl-2 ratio, a marker of apoptosis, was increased.
　図１２上図には、免疫作業中に観察された、ＳＦＰＱ４５２－４６６を認識する抗ＳＦＰＱ抗体を産生させたＳＦＰＱ免疫マウスの脱毛の様子を写真で示す。 The upper part of Fig. 12 shows a photograph of hair loss in SFPQ-immunized mice that produced anti-SFPQ antibodies that recognize SFPQ452-466, which was observed during the immunization procedure.
　図１２下図に、マウスの皮膚切片（毛包）の蛍光免疫染色による組織学的解析の結果を示す。図１２下図より明らかなように、ＳＦＰＱ免疫マウスにおいて、毛包の幹細胞であるＬｇｒ５陽性細胞の減少とＣｄｋｎ２ａ陽性細胞の増加がみられた。 The lower diagram of FIG. 12 shows the results of histological analysis by fluorescent immunostaining of mouse skin sections (hair follicles). As is clear from the lower diagram of FIG. 12, in the SFPQ-immunized mice, a decrease in Lgr5-positive cells, which are stem cells of hair follicles, and an increase in Cdkn2a-positive cells were observed.
　〔実施例９〕
　ＳＦＰＱを免疫することによる心臓の老化現象の発生
　＜方法＞
　実施例６と同様の方法でマウスのＳＦＰＱ免疫を行い、抗ＳＦＰＱ抗体を産生させたマウスである、ＳＦＰＱ免疫マウスを得た。その後、３回免疫のマウスの心臓を採取し、実施例４の（６）の操作と同様の方法でマウス心臓のｃＤＮＡを合成した。合成したｃＤＮＡを鋳型に老化マーカーＣｄｋｎ２ａ、炎症性サイトカインのＩＬ－１βとインフラマソームＮｌｒｐ３の遺伝子発現をＲＴ－ｑＰＣＲにより解析した。 [Example 9]
Occurrence of Cardiac Aging by Immunization with SFPQ <Method>
Mice were immunized with SFPQ in the same manner as in Example 6 to obtain SFPQ-immunized mice that produced anti-SFPQ antibodies. Thereafter, the hearts of the three-immunized mice were harvested, and the mouse heart cDNA was synthesized in the same manner as in Example 4 (6). Gene expressions of the senescence marker Cdkn2a, the inflammatory cytokine IL-1β, and the inflammasome Nlrp3 were analyzed by RT-qPCR using the synthesized cDNA as a template.
　＜結果＞
　実施例９における、ＳＦＰＱ免疫マウスの心臓における遺伝子発現解析の結果を図１３に示す。 <Results>
The results of gene expression analysis in the heart of SFPQ-immunized mice in Example 9 are shown in FIG.
　図１３は、３回目のＳＦＰＱ免疫後４週間経過したマウスの心臓における遺伝子発現解析の結果である。ｚ１３より明らかなように、ＳＦＰＱ免疫マウスにおいて、心臓の老化マーカーであるＣｄｋｎ２ａ、炎症性サイトカインであるＩＬ－１β、インフラマソームＮｌｒｐ３の発現上昇が観察された。 Fig. 13 shows the results of gene expression analysis in mouse hearts 4 weeks after the third SFPQ immunization. As is clear from z13, increased expression of cardiac senescence marker Cdkn2a, inflammatory cytokine IL-1β, and inflammasome Nlrp3 was observed in SFPQ-immunized mice.
　〔実施例１０〕
　ＳＦＰＱを免疫することによる肺の老化現象の発生
　＜方法＞
　実施例６と同様の方法でマウスのＳＦＰＱ免疫を行い、抗ＳＦＰＱ抗体を産生させたマウスである、ＳＦＰＱ免疫マウスを得た。その後、１回免疫のマウスと３回免疫のマウスの肺を採取し、実施例４の（６）の操作と同様の方法でマウス肺のｃＤＮＡを合成した。合成したｃＤＮＡを鋳型に老化マーカーのＥｄｎ－１、アポトーシス関連因子のＢａｘとＢｃｌ－２の遺伝子発現をＲＴ－ｑＰＣＲにより解析した。 [Example 10]
Occurrence of pulmonary aging phenomenon by immunization with SFPQ <Method>
Mice were immunized with SFPQ in the same manner as in Example 6 to obtain SFPQ-immunized mice that produced anti-SFPQ antibodies. After that, the lungs of the mouse immunized once and the mouse immunized three times were collected, and mouse lung cDNA was synthesized in the same manner as in Example 4 (6). Gene expressions of the senescence marker Edn-1 and the apoptosis-related factors Bax and Bcl-2 were analyzed by RT-qPCR using the synthesized cDNA as a template.
　＜結果＞
　実施例１０における、ＳＦＰＱ免疫マウスの肺における遺伝子発現解析の結果を図１４に示す。 <Results>
The results of gene expression analysis in the lungs of SFPQ-immunized mice in Example 10 are shown in FIG.
　図１４は、１回目のＳＦＰＱ免疫後４週間経過したマウスと３回目のＳＦＰＱ免疫後４週間経過したマウスの肺の遺伝子発現解析の結果である。図１４より明らかなように、ＳＦＰＱ免疫マウスにおいて、免疫回数が多くなるほど、すなわち、抗ＳＦＰＱ抗体量が増加するほど、肺の老化マーカーであるエンドセリンＥｄｎ１の発現量の増加とアポトーシス誘導のマーカーであるＢａｘ／Ｂｃｌ－２比の上昇が観察された。 Fig. 14 shows the results of gene expression analysis of the lungs of mice 4 weeks after the first SFPQ immunization and 4 weeks after the third SFPQ immunization. As is clear from FIG. 14, in SFPQ-immunized mice, the higher the number of immunizations, that is, the higher the amount of anti-SFPQ antibody, the higher the expression level of endothelin Edn1, a lung aging marker, and the marker of apoptosis induction. An increase in the Bax/Bcl-2 ratio was observed.
　〔実施例１１〕
　抗ＳＦＰＱ抗体のＴａｕタンパク質への影響
　＜方法＞
　Ｔａｕタンパク質のリン酸化の増加は老化や認知症と関係すると報告がなされている。そこで、神経細胞株Ｎｅｕｒｏ２ａへの抗ＳＦＰＱ抗体を添加し、抗ＳＦＰＱ抗体が存在するとＴａｕ遺伝子のスプライシングに影響を及ぼしてリン酸化が増加するかどうかを検討した。加えて、神経細胞株Ｎｅｕｒｏ２ａへの抗ＳＦＰＱ抗体添加がＴａｕタンパク質のリン酸化を促進するかどうかを検討した。 [Example 11]
Effect of anti-SFPQ antibody on Tau protein <Method>
Increased phosphorylation of Tau protein has been reported to be associated with aging and dementia. Therefore, an anti-SFPQ antibody was added to the neuronal cell line Neuro2a to examine whether the presence of the anti-SFPQ antibody affects Tau gene splicing and increases phosphorylation. In addition, we investigated whether the addition of anti-SFPQ antibody to the neuronal cell line Neuro2a promotes the phosphorylation of Tau protein.
　（１）抗ＳＦＰＱ抗血清の調製
　ウサギ抗ＳＦＰＱ抗血清を作製するために、免疫源として実施例３に記載の方法で、精製ＧＳＴ－ＳＦＰＱ－Ｈｉｓタンパク質、ならびに、ＫＬＨコンジュゲートした合成ペプチドＳＦＰＱ３６－５０およびＳＦＰＱ４５２－４６６を調製した。それぞれのタンパク質および合成ペプチドをウサギに免疫し、抗ＳＦＰＱ抗体を作製した。精製ＧＳＴ－ＳＦＰＱ－Ｈｉｓタンパク質を免疫した抗血清を抗ＳＦＰＱｆｕｌｌ抗血清、ＫＬＨコンジュゲートした合成ペプチドＳＦＰＱ３６－５０を免疫した抗血清を抗ＳＦＰＱ３６－５０抗血清、ＫＬＨコンジュゲートした合成ペプチドＳＦＰＱ４５２－４６６を免疫した抗血清を抗ＳＦＰＱ４５２－４６６抗血清とした。 (1) Preparation of anti-SFPQ antisera To generate rabbit anti-SFPQ antisera, the purified GST-SFPQ-His protein and the KLH-conjugated synthetic peptide SFPQ36- were used as immunogens by the method described in Example 3. 50 and SFPQ452-466 were prepared. Rabbits were immunized with each protein and synthetic peptide to generate anti-SFPQ antibodies. Antiserum immunized with purified GST-SFPQ-His protein was used as anti-SFPQfull antiserum, antiserum immunized with KLH-conjugated synthetic peptide SFPQ36-50 was used as anti-SFPQ36-50 antiserum, and KLH-conjugated synthetic peptide SFPQ452-466 was used as antiserum. The immunizing antiserum was designated as anti-SFPQ452-466 antiserum.
　（２）神経芽細胞腫Ｎｅｕｒｏ－２ａの抗ＳＦＰＱ抗体処理
　１０％　ＦＢＳ／　Ｍｉｎｉｍｕｍ　Ｅｓｓｅｎｔｉａｌ　Ｍｅｄｉｕｍ　Ｅａｇｌｅ（ＭＥＭ）培地（ＭＥＲＣＫ社製）で培養したＮｅｕｒｏ－２ａ細胞を１２ウェルプレートに播種後一晩培養し、プレートの底面に細胞を接着させた。その後培地を除去し、抗ＳＦＰＱ抗血清（抗ＳＦＰＱｆｕｌｌ抗血清、抗ＳＦＰＱ３６－５０抗血清、または、抗ＳＦＰＱ４５２－４６６抗血清）を５０倍希釈となるよう添加した０．５％　ＦＢＳ／ＭＥＭ培地を加えて４８時間培養した。この際、Ｎｅｕｒｏ－２ａの神経細胞への分化を誘導する試験区ではさらにレチノイン酸（ＲＡ）を終濃度２０μＭとなるように添加した。また、対照として免疫付加前のウサギ血清を使用した。 (2) Anti-SFPQ Antibody Treatment of Neuroblastoma Neuro-2a Neuro-2a cells cultured in 10% FBS/Minimum Essential Medium Eagle (MEM) medium (manufactured by MERCK) were seeded in a 12-well plate and cultured overnight. , which allowed the cells to adhere to the bottom of the plate. After that, the medium was removed, and 0.5% FBS/MEM medium added with anti-SFPQ antiserum (anti-SFPQfull antiserum, anti-SFPQ36-50 antiserum, or anti-SFPQ452-466 antiserum) at a 50-fold dilution was added. In addition, it was cultured for 48 hours. At this time, retinoic acid (RA) was further added to a final concentration of 20 μM in the test group for inducing differentiation of Neuro-2a into nerve cells. In addition, rabbit serum before immunization was used as a control.
　（３）Ｔａｕ遺伝子のスプライシングへの影響
　抗ＳＦＰＱ抗血清存在下で４８時間培養したＮｅｕｒｏ－２ａ細胞から培地を完全に除去し、Ｂｕｆｆｅｒ　ＲＬＴ（ＱＩＡＧＥＮ社製）を加えて細胞を溶解後、ＲＮｅａｓｙ
　Ｍｉｎｉ　Ｋｉｔ（ＱＩＡＧＥＮ社製）を用いてＲＮＡを精製した。得られた精製ＲＮＡを鋳型にＰｒｉｍｅＳｃｒｉｐｔ　ＲＴ　ｒｅａｇｅｎｔ　Ｋｉｔ（タカラバイオ写生）を用いてｃＤＮＡを合成した。合成したｃＤＮＡを鋳型に、Ｔａｕ遺伝子のｅｘｏｎ１０領域を増幅するプライマー（Ｆｏｒｗａｒｄ：ＴＣＣＣＣＣＴＡＡＧＴＣＡＣＣＡＴＣＡＧ（配列番号４），Ｒｅｖｅｒｓｅ：ＧＣＣＡＡＴＣＴＴＣＧＡＣＴＧＧＡＣＴＣ（配列番号５））を用いてＰＣＲ反応させた。ＰＣＲ反応後の溶液を１５％のポリアクリルアミドゲルｅ・パジェル（ＡＴＴＯ）にアプライし、電気泳動した。電気泳動後のポリアクリルアミドゲルはエチジウムブロマイドで染色され、ＵＶライト照射で検出されたＰＣＲ産物のバンドをソフトウェアＩｍａｇｅ　Ｌａｂ（Ｂｉｏ－Ｒａｄ社製）で数値化した。Ｔａｕのリン酸化が増幅するｅｘｏｎ１０を含むバンド（ｅｘｏｎ１０＋）の数値を、ｅｘｏｎ１０を含まないバンド（ｅｘｏｎ１０－）の数値で割った値を算出した。 (3) Effect on Tau gene splicing After completely removing the medium from Neuro-2a cells cultured for 48 hours in the presence of anti-SFPQ antiserum, adding Buffer RLT (manufactured by QIAGEN) to lyse the cells, RNeasy
RNA was purified using Mini Kit (manufactured by QIAGEN). Using the obtained purified RNA as a template, cDNA was synthesized using PrimeScript RT reagent Kit (Takara Bio-Shosei). Using the synthesized cDNA as a template, PCR reaction was performed using primers (Forward: TCCCCCTAAGTCACCATCAG (SEQ ID NO: 4), Reverse: GCCAATCTTCGACTGGACTC (SEQ ID NO: 5)) that amplify the exon 10 region of the Tau gene. The solution after the PCR reaction was applied to a 15% polyacrylamide gel e-pagel (ATTO) and subjected to electrophoresis. After electrophoresis, the polyacrylamide gel was stained with ethidium bromide, and the band of the PCR product detected by UV light irradiation was quantified using software Image Lab (manufactured by Bio-Rad). A value was calculated by dividing the numerical value of the band containing exon10 amplified by Tau phosphorylation (exon10+) by the numerical value of the band not containing exon10 (exon10−).
　（４）抗ＳＦＰＱ抗血清処理細胞の形態変化
　２０μＭ　ＲＡおよび抗ＳＦＰＱ抗血清存在下で４８時間培養したＮｅｕｒｏ－２ａ細胞から培地を完全に除去し、乾燥させた後、３％メタノール入りアセトン溶液で固定した。ＦＰＱへの抗ＳＦＰＱ抗体添加がＴａｕタンパク質のリン酸化を促進するかどうかを検討した。 (4) Morphological change of anti-SFPQ antiserum-treated cells Neuro-2a cells cultured for 48 hours in the presence of 20 μM RA and anti-SFPQ antiserum were completely removed of the medium, dried, and treated with a 3% methanol-containing acetone solution. Fixed. We investigated whether the addition of anti-SFPQ antibody to FPQ promotes the phosphorylation of Tau protein.
　さらに、神経細胞株Ｎｅｕｒｏ２ａへの抗ＳＦＰＱ抗体添加がＴａｕのリン酸化を増加していることを確認するために、抗ＳＦＰＱ存在下で培養したＮｅｕｒｏ２ａを抗リン酸化Ｔａｕ抗体（ＡＴ８）で蛍光免疫染色し、各細胞の蛍光強度をイメージアナライザーＡｒｒａｙＳｃａｎ　ＸＴ（ＴｈｅｒｍｏＦｉｓｈｅｒ）で測定した。また、同様にニューロンマーカーであるＭｉｃｒｏｔｕｂｕｌｅ－ａｓｓｏｃｉａｔｅｄ　ｐｒｏｔｅｉｎ　２（ＭＡＰ２）の抗体で蛍光免疫染色し、各細胞の細胞体サイズをイメージアナライザーＡｒｒａｙＳｃａｎ　ＸＴで測定した。 Furthermore, in order to confirm that the addition of anti-SFPQ antibody to neuronal cell line Neuro2a increases the phosphorylation of Tau, Neuro2a cultured in the presence of anti-SFPQ was immunofluorescently stained with an anti-phosphorylated Tau antibody (AT8). Then, the fluorescence intensity of each cell was measured with an image analyzer ArrayScan XT (ThermoFisher). Similarly, the cells were fluorescently immunostained with a neuron marker Microtubule-associated protein 2 (MAP2) antibody, and the cell body size of each cell was measured with an image analyzer ArrayScan XT.
　＜結果＞
　実施例１１におけるＰＣＲ産物の解析結果、Ｎｅｕｒｏ２ａ細胞の蛍光強度の測定結果、および、Ｎｅｕｒｏ２ａ細胞の細胞体サイズの測定結果を、それぞれ図１５に示す。 <Results>
FIG. 15 shows the results of PCR product analysis, the measurement results of the fluorescence intensity of Neuro2a cells, and the measurement results of the cell body size of Neuro2a cells in Example 11, respectively.
　ＰＣＲ産物の解析結果を図１５上図に示す。図１５左上図より明らかなように、抗ＳＦＰＱ抗体を神経細胞株Ｎｅｕｒｏ２ａに添加したところ、Ｔａｕ遺伝子から生じるｍＲＮＡのスプライシングが、翻訳の結果生じるＴａｕタンパク質がリン酸化されやすい形状（ｅｘｏｎ１０＋）のｍＲＮＡが多く産生されるように変化することが分かった。この検出されたバンドを数値化し、Ｅｘｏｎ１０－の単位定量あたりのＥｘｏｎ１０＋量を算出してグラフ化したものを図１５右上図に示す。図１５右上図より明らかなように、特に２０μＭ　ＲＡ存在下で神経細胞に分化誘導した際に抗ＳＦＰＱ抗体が存在すると、Ｔａｕ遺伝子のスプライシングバランスが崩壊し、リン酸化されやすい形状のＴａｕタンパク質の発現が促進されることが示唆された。 The analysis results of the PCR products are shown in the upper diagram of FIG. As is clear from the upper left diagram of FIG. 15, when the anti-SFPQ antibody was added to the neuronal cell line Neuro2a, splicing of mRNA generated from the Tau gene was suppressed, and mRNA in a form (exon10+) that facilitates phosphorylation of the Tau protein generated as a result of translation was generated. It was found that it changed to produce more. The detected band was quantified, and the amount of Exon10+ per unit determination of Exon10- was calculated and graphed, which is shown in the upper right figure of FIG. As is clear from the upper right figure of FIG. 15, the presence of anti-SFPQ antibody, especially when inducing differentiation into neurons in the presence of 20 μM RA, disrupts the splicing balance of the Tau gene, resulting in the expression of Tau protein in a form that is susceptible to phosphorylation. It was suggested that
　図１５左下図に、２０μＭ　ＲＡおよび抗ＳＦＰＱ抗体存在下のＮｅｕｒｏ２ａ細胞の染色結果を示す。図１５左下図より明らかなように、２０μＭ　ＲＡおよび抗ＳＦＰＱ抗体存在下のＮｅｕｒｏ２ａ細胞は、抗リン酸化Ｔａｕ抗体で有意に強く染色されることが示された。従って、抗ＳＦＰＱ抗体の存在は神経細胞においてリン酸化Ｔａｕタンパク質を増加することが示された。 The lower left diagram of FIG. 15 shows the results of staining Neuro2a cells in the presence of 20 μM RA and anti-SFPQ antibody. As is clear from the lower left diagram of FIG. 15, Neuro2a cells in the presence of 20 μM RA and anti-SFPQ antibody were shown to be significantly strongly stained with anti-phosphorylated Tau antibody. Therefore, the presence of anti-SFPQ antibody was shown to increase phosphorylated Tau protein in neuronal cells.
　図１５右下図に、２０μＭ　ＲＡおよび抗ＳＦＰＱ抗体存在下のＮｅｕｒｏ２ａ細胞の細胞体のサイズの変化を示す。図１５右下図より明らかなように、Ｔａｕタンパク質のリン酸化が促進される２０μＭ　ＲＡおよび抗ＳＦＰＱ抗体存在下のＮｅｕｒｏ２ａ細胞は、その毒性のために細胞や細胞核の膨隆が生じることが示された。 The lower right diagram of FIG. 15 shows changes in the size of Neuro2a cell bodies in the presence of 20 μM RA and anti-SFPQ antibody. As is clear from the lower right figure of FIG. 15, Neuro2a cells in the presence of 20 μM RA and anti-SFPQ antibody, which promote phosphorylation of Tau protein, showed swelling of cells and cell nuclei due to toxicity.
　〔実施例１２〕
　抗ＳＦＰＱ抗体の有無と神経幹細胞の生存との関係
　＜方法＞
　抗ＳＦＰＱ抗体の神経細胞分化への影響を調べるために、無血清フィーダーフリー培地Ｃｅｌｌａｒｔｉｓ　２ｉ　ｍＥＳ／ｉＰＳＣ　Ｃｕｌｔｕｒｅ　Ｍｅｄｉｕｍ（ＴａＫａＲａ）で培養したマウスＥＳ細胞Ｈ－１に、培養液中の濃度が２％となるよう３種類の抗ＳＦＰＱ抗血清（ＳＦＰＱｆｕｌｌ、ＳＦＰＱ３６－５０、ＳＦＰＱ４５２－４６６）のうちいずれか１つを添加し、そのまま２４時間もしくは４８時間培養した。対照としてＳＦＰＱで免疫する前のウサギ血清を使用した。その後、細胞から培地を完全に除去し、Ｂｕｆｆｅｒ　ＲＬＴ（ＱＩＡＧＥＮ社製）を加えて細胞を溶解後、ＲＮｅａｓｙ　Ｍｉｎｉ　Ｋｉｔ（ＱＩＡＧＥＮ社製）でＲＮＡを精製した。得られた精製ＲＮＡを鋳型にＰｒｉｍｅＳｃｒｉｐｔ　ＲＴ　ｒｅａｇｅｎｔ　Ｋｉｔ（タカラバイオ社製）を用いてｃＤＮＡを合成した。合成したｃＤＮＡを鋳型に、放射状グリア細胞マーカーＮｅｓｔｉｎ、神経芽細胞マーカーＤｏｕｂｌｅｃｏｒｔｉｎ（ＤＣＸ）、未成熟ニューロンマーカーＣａｌｒｅｔｉｎｉｎ、成熟ニューロンマーカーＣａｌｂｉｎｄｉｎの遺伝子発現を、ＲＴ－ｑＰＣＲにより解析した。 [Example 12]
Relationship between the presence or absence of anti-SFPQ antibody and survival of neural stem cells <Method>
In order to examine the effect of anti-SFPQ antibody on neuronal differentiation, mouse ES cell H-1 cultured in a serum-free feeder-free medium Cellartis 2i mES/iPSC Culture Medium (TaKaRa) was added at a concentration of 2% in the culture medium. Any one of the three types of anti-SFPQ antisera (SFPQfull, SFPQ36-50, SFPQ452-466) was added to the cells so that the cells were cultured for 24 hours or 48 hours. Rabbit serum before immunization with SFPQ was used as a control. Thereafter, the medium was completely removed from the cells, Buffer RLT (manufactured by QIAGEN) was added to lyse the cells, and RNA was purified using RNeasy Mini Kit (manufactured by QIAGEN). Using the obtained purified RNA as a template, cDNA was synthesized using PrimeScript RT reagent Kit (manufactured by Takara Bio Inc.). Using the synthesized cDNA as a template, gene expressions of radial glial cell marker Nestin, neuroblast marker Doublecortin (DCX), immature neuron marker Calretinin, and mature neuron marker Calbindin were analyzed by RT-qPCR.
　＜結果＞
　実施例１２における、神経細胞の遺伝子発現の解析の結果を図１６に示す。 <Results>
FIG. 16 shows the results of analysis of gene expression in neurons in Example 12.
　図１６上図に示すように、抗ＳＦＰＱ抗血清存在下２４時間後において、放射状グリア細胞マーカーであるＮｅｓｔｉｎおよび神経芽細胞マーカーＤＣＸの発現が低下していた。さらに、図１６下図に示すように、抗ＳＦＰＱ抗血清存在下４８時間後において、未成熟神経細胞マーカーであるＣａｌｒｅｔｉｎｉｎおよび、分化した成熟神経細胞のマーカーであるＣａｌｂｉｎｄｉｎの発現が低下していた。この結果は、抗ＳＦＰＱ抗体が神経細胞の分化を阻害することを示唆する。 As shown in the upper diagram of FIG. 16, the expression of the radial glial cell marker Nestin and the neuroblast marker DCX decreased after 24 hours in the presence of the anti-SFPQ antiserum. Furthermore, as shown in the lower diagram of FIG. 16, the expression of Calretinin, an immature neuron marker, and Calbindin, a differentiated mature neuron marker, decreased after 48 hours in the presence of anti-SFPQ antiserum. This result suggests that the anti-SFPQ antibody inhibits neuronal differentiation.
　〔実施例１３〕
　抗ＳＦＰＱ抗体の有無と未分化幹細胞の生存との関係
　＜方法＞
　抗ＳＦＰＱ抗体の神経細胞分化への影響を調べるために、無血清フィーダーフリー培地Ｃｅｌｌａｒｔｉｓ　２ｉ　ｍＥＳ／ｉＰＳＣ　Ｃｕｌｔｕｒｅ　Ｍｅｄｉｕｍ（ＴａＫａＲａ）で培養したマウスＥＳ細胞Ｈ－１に２％となるよう３種類の抗ＳＦＰＱ抗血清（ＳＦＰＱｆｕｌｌ、ＳＦＰＱ３６－５０、ＳＦＰＱ４５２－４６６）のうちいずれか１つを添加し、そのまま２４時間もしくは４８時間培養した。対照としてＳＦＰＱで免疫する前のウサギ血清を使用した。その後は実施例５と同様の方法で各細胞からｃＤＮＡを調製し、幹細胞マーカーＬｇｒ４、Ｌｇｒ５、Ｌｇｒ６、Ｓｏｘ２の遺伝子発現を、ＲＴ－ｑＰＣＲにより解析した。 [Example 13]
Relationship between presence or absence of anti-SFPQ antibody and survival of undifferentiated stem cells <Method>
In order to investigate the effect of anti-SFPQ antibodies on neuronal differentiation, three types of anti-SFPQ were added to mouse ES cells H-1 cultured in a serum-free feeder-free medium Cellartis 2i mES/iPSC Culture Medium (TaKaRa) at 2%. Any one of the antisera (SFPQfull, SFPQ36-50, SFPQ452-466) was added and cultured for 24 hours or 48 hours. Rabbit serum before immunization with SFPQ was used as a control. Thereafter, cDNA was prepared from each cell in the same manner as in Example 5, and gene expression of stem cell markers Lgr4, Lgr5, Lgr6 and Sox2 was analyzed by RT-qPCR.
　＜結果＞
　実施例１３における、Ｈ－１細胞の遺伝子発現の解析の結果を図１７および図１８に示す。 <Results>
The results of gene expression analysis of H-1 cells in Example 13 are shown in FIGS. 17 and 18. FIG.
　図１７上図および図１８上図に、抗ＳＦＰＱ抗血清添加２４時間後のＨ－１細胞の遺伝子発現を示し、図１７下図および図１８下図には、抗ＳＦＰＱ抗血清添加から４日後のＨ－１細胞の遺伝子発現を示す。図１７においては、左から幹細胞マーカーであるＬｇｒ４およびＬｇｒ５の遺伝子発現を、図１８においては、左から幹細胞マーカーであるＬｇｒ６およびＳｏｘ２の遺伝子発現を、それぞれ示す。 The upper figure in FIG. 17 and the upper figure in FIG. 18 show the gene expression of H-1 cells 24 hours after the addition of anti-SFPQ antiserum, and the lower figure in FIG. 17 and the lower figure in FIG. -1 cell gene expression. FIG. 17 shows the gene expression of stem cell markers Lgr4 and Lgr5 from the left, and FIG. 18 shows the gene expression of stem cell markers Lgr6 and Sox2 from the left.
　図１７および図１８より明らかなように、抗ＳＦＰＱ抗血清添加後短時間（２４ｈｒ）では、幹細胞マーカーの発現が亢進しており、幹細胞が一時的に増殖することが示唆された。しかしその後長時間経過（Ｄａｙ４）すると幹細胞マーカー（Ｌｇｒ４、Ｌｇｒ５、Ｌｇｒ６およびＳｏｘ２）の発現が低下し、幹細胞が枯渇することが示唆された。 As is clear from Figures 17 and 18, within a short time (24 hours) after the addition of the anti-SFPQ antiserum, the expression of stem cell markers was enhanced, suggesting that stem cells proliferate temporarily. However, after a long period of time (Day 4), the expression of stem cell markers (Lgr4, Lgr5, Lgr6 and Sox2) decreased, suggesting depletion of stem cells.
　〔実施例１４〕
　抗ＳＦＰＱ抗体を投与することによるＳＦＰＱ関連機能障害動物モデルの製造
　＜方法＞
　これまで実施してきたＳＦＰＱを免疫する方法では、抗ＳＦＰＱ抗体のみならず、細胞性免疫が惹起される可能性が残されている。従って、これまでに明らかにしてきたＳＦＰＱ免疫マウスの表現型が抗ＳＦＰＱ抗体によるものであると断定することができない。そこで、抗ＳＦＰＱ抗体をマウスの血中に直接投与することで、抗ＳＦＰＱ抗体により誘導される表現型を明らかにしようと下記手法を用いた。 [Example 14]
Production of SFPQ-related dysfunction animal model by administration of anti-SFPQ antibody <Method>
In the methods of immunizing against SFPQ that have been carried out so far, there is still the possibility that not only anti-SFPQ antibodies but also cell-mediated immunity will be induced. Therefore, it cannot be concluded that the phenotypes of SFPQ-immunized mice that have been clarified so far are due to anti-SFPQ antibodies. Therefore, the following method was used to clarify the phenotype induced by the anti-SFPQ antibody by directly administering the anti-SFPQ antibody into the blood of mice.
　（１）抗ＳＦＰＱマウスモノクローナル抗体の作製
　ＢＡＬＢ／ｃマウスにＫＬＨコンジュゲートＳＦＰＱ合成ペプチド溶液（ＳＦＰＱ３６－５０またはＳＦＰＱ４５２－４６６）をそれぞれ免疫し、脾臓から採取したＢ細胞とミエローマを細胞融合させ、ハイブリドーマ細胞を作製した。得られたハイブリドーマ細胞から抗原と反応し、なおかつＴａｕ遺伝子のスプライシングに影響を及ぼす抗体を産生する細胞を選別した。Ｐｒｉｓｔａｎｅを投与してから２週間経過したＢＡＬＢ／ｃマウスに、選別されたハイブリドーマ細胞を腹腔投与した後、腹水を採取して、抗ＳＦＰＱマウスモノクローナル抗体溶液とした。 (1) Preparation of anti-SFPQ mouse monoclonal antibody BALB/c mice were immunized with KLH-conjugated SFPQ synthetic peptide solutions (SFPQ36-50 or SFPQ452-466), respectively, and B cells and myeloma collected from the spleen were fused to form hybridomas. Cells were made. Cells that react with the antigen and produce antibodies that affect the splicing of the Tau gene were selected from the obtained hybridoma cells. Selected hybridoma cells were intraperitoneally administered to BALB/c mice two weeks after administration of Pristane, and ascites was collected to prepare an anti-SFPQ mouse monoclonal antibody solution.
　（２）抗ＳＦＰＱマウスモノクローナル抗体の尾静脈投与
　マウスの腹水から得られた抗ＳＦＰＱマウスモノクローナル抗体溶液を２００μＬ／匹となるように６週齢のＣ５７ＢＬ／６ＮＣｒＳｌｃ雄マウスの尾静脈に投与した。腹水の尾静脈投与は２週間毎に、計３回実施した。 (2) Tail Vein Administration of Anti-SFPQ Mouse Monoclonal Antibody An anti-SFPQ mouse monoclonal antibody solution obtained from mouse ascites was administered to the tail vein of 6-week-old C57BL/6NCrSlc male mice at a concentration of 200 μL/mouse. Ascites was administered to the tail vein every 2 weeks for a total of 3 times.
　（３）抗ＳＦＰＱマウスモノクローナル抗体尾静脈投与マウスの行動試験
　３回目の尾静脈投与から１週間後に実施例５の（５）の操作と同様の方法で、抗ＳＦＰＱマウスモノクローナル抗体尾静脈投与マウスの協調運動能力を測定した。 (3) Behavioral test of anti-SFPQ mouse monoclonal antibody tail vein-administered mice One week after the third tail vein administration, in the same manner as in Example 5 (5), anti-SFPQ mouse monoclonal antibody tail vein-administered mice were tested. Coordination was measured.
　（４）抗ＳＦＰＱウサギポリクローナル抗体の尾静脈投与
　実施例１１に記載の方法で得た、抗ＳＦＰＱｆｕｌｌ抗血清、抗ＳＦＰＱ３６－５０抗血清、または、抗ＳＦＰＱ４５２－４６６抗血清を６週齢のＣ５７ＢＬ／６ＮＣｒＳｌｃ雄マウスに２００μＬ／匹となるよう尾静脈投与した。 (4) Tail vein administration of anti-SFPQ rabbit polyclonal antibody Anti-SFPQ full antiserum, anti-SFPQ36-50 antiserum, or anti-SFPQ452-466 antiserum obtained by the method described in Example 11 was administered to 6-week-old C57BL/ 200 μL/mouse was administered to 6NCrSlc male mice through the tail vein.
　（５）抗ＳＦＰＱウサギポリクローナル抗体尾静脈投与マウスの行動試験
　尾静脈投与から１週間後、抗ＳＦＰＱウサギポリクローナル抗体尾静脈投与マウスの表現型を調べるために、うつ病様行動の評価に使用される強制水泳試験（ＦＳＴ）および尾懸垂試験（ＴＳＴ）を実施した。 (5) Behavioral test of anti-SFPQ rabbit polyclonal antibody tail vein administration mice One week after tail vein administration, to examine the phenotype of anti-SFPQ rabbit polyclonal antibody tail vein administration mice, it is used to evaluate depression-like behavior. A forced swim test (FST) and a tail suspension test (TST) were performed.
　具体的には、強制水泳試験の場合、深さ３００ｍｍの透明な強制水泳試験用の水槽に水を入れ、マウスを水槽に入れてから１０分間自由に泳がせた。その様子を側面から撮影し、強制水泳試験行動解析用ソフトＦｏｒｃｅｄＳｗｉｍＳｃａｎ（ＣｌｅｖｅｒＳｙｓ　Ｉｎｃ）で解析した。 Specifically, in the case of the forced swimming test, water was poured into a transparent water tank for the forced swimming test with a depth of 300 mm, and the mice were placed in the water tank and allowed to swim freely for 10 minutes. The behavior was photographed from the side and analyzed with Forced SwimScan (Clever Sys Inc.), software for behavioral analysis of the forced swimming test.
　尾懸垂試験の場合、マウスの尻尾を固定し、１０分間吊り下げ、その様子を録画し、画像解析ソフトＴａｉｌＳｕｓｐＳｃａｎ（ＣｌｅｖｅｒＳｙｓ　Ｉｎｃ）で解析し、無動時間を計測した。 In the case of the tail suspension test, the tail of the mouse was fixed, suspended for 10 minutes, recorded, analyzed with image analysis software TailSuspScan (CleverSys Inc), and immobility time was measured.
　（７）抗ＳＦＰＱウサギポリクローナル抗体尾静脈投与マウスの遺伝子発現解析
　抗ＳＦＰＱウサギポリクローナル抗体を３回尾静脈投与したマウスの脳を採取し、実施例４の（６）の操作と同様の方法でマウス脳のｃＤＮＡを合成した。合成したｃＤＮＡを鋳型に脳由来神経栄養因子ＢＤＮＦ、アポトーシス関連因子のＢａｘとＢｃｌ－２、および炎症抑制因子Ｚｆｐ３６の遺伝子発現をＲＴ－ｑＰＣＲにより解析した。 (7) Gene Expression Analysis of Anti-SFPQ Rabbit Polyclonal Antibody Tail Vein-Administered Mice Anti-SFPQ rabbit polyclonal antibody was administered three times to the tail vein of mice. Brain cDNA was synthesized. Gene expressions of brain-derived neurotrophic factor BDNF, apoptosis-related factors Bax and Bcl-2, and inflammation suppressor Zfp36 were analyzed by RT-qPCR using the synthesized cDNA as a template.
　＜結果＞
　実施例１４における、抗ＳＦＰＱマウスモノクローナル抗体尾静脈投与マウスの体重変化の測定結果および協調運動能力の測定結果を図１９に、抗ＳＦＰＱウサギポリクローナル抗体を尾静脈投与したマウスの行動試験の解析結果および抗ＳＦＰＱウサギポリクローナル抗体を尾静脈投与したマウスの脳における遺伝子発現解析の結果を図２０にそれぞれ示す。 <Results>
Figure 19 shows the measurement results of body weight change and the measurement results of coordinated motor ability of mice administered with anti-SFPQ mouse monoclonal antibody tail vein in Example 14. FIG. 20 shows the results of gene expression analysis in the brains of mice to which the anti-SFPQ rabbit polyclonal antibody was administered via the tail vein.
　図１９より明らかなように、抗ＳＦＰＱマウスモノクローナル抗体を投与したマウスは体重減少が誘導され、老化の指標である協調運動能力の低下が観察された。 As is clear from FIG. 19, weight loss was induced in mice administered anti-SFPQ mouse monoclonal antibody, and a decrease in coordination ability, an index of aging, was observed.
　図２０上図に、抗ＳＦＰＱウサギポリクローナル抗体を尾静脈投与したマウスの行動試験解析の結果を示す。図２０上図より明らかなように、抗ＳＦＰＱウサギポリクローナル抗体を投与したマウスはうつ病様行動の指標となる強制水泳試験におけるＦｌｏａｔ時間の上昇とＳｔｒｕｇｇｌｅ時間の短縮、および尾懸垂試験における無動時間の上昇が観察された。従って、抗ＳＦＰＱウサギポリクローナル抗体を投与したマウスはうつ病様行動を示すことが明らかになった。 The upper diagram of FIG. 20 shows the results of behavioral test analysis of mice to which anti-SFPQ rabbit polyclonal antibody was administered through the tail vein. As is clear from the upper diagram of FIG. 20, mice administered with anti-SFPQ rabbit polyclonal antibody increased Float time and shortened Struggle time in the forced swimming test, which are indicators of depression-like behavior, and immobility time in the tail suspension test. was observed. Therefore, it was revealed that mice administered with anti-SFPQ rabbit polyclonal antibody exhibited depression-like behavior.
　さらに図２０下図には、抗ＳＦＰＱウサギポリクローナル抗体を尾静脈投与したマウスの脳における遺伝子発現解析結果を示した。図２０下図より明らかなように、ＳＦＰＱウサギポリクローナル抗体を投与したマウスの脳では、うつ病で観察される脳由来神経栄養因子ＢＤＮＦの低下、アポトーシス誘導の指標であるＢａｘ／Ｂｃｌ－２比の上昇が観察され、脳内の炎症抑制因子Ｚｆｐ３６の発現低下も観察された。 Furthermore, the lower figure in FIG. 20 shows the results of gene expression analysis in the brain of mice to which anti-SFPQ rabbit polyclonal antibody was administered via the tail vein. As is clear from the lower figure of FIG. 20, in the brains of mice administered with the SFPQ rabbit polyclonal antibody, the brain-derived neurotrophic factor BDNF observed in depression decreased, and the Bax/Bcl-2 ratio, which is an index of apoptosis induction, increased. was observed, and a decrease in the expression of the inflammation suppressor Zfp36 in the brain was also observed.
　以上の結果から、ＳＦＰＱタンパク質または抗ＳＦＰＱ抗体をマウスに投与することによって新規のＳＦＰＱ関連機能障害モデルマウスを作製することができること、抗ＳＦＰＱ抗体の抗体価を測定することによってＳＦＰＱ関連機能障害の診断または検査のためのデータが取得できること、ならびに、ＳＦＰＱに対する自己免疫反応、特に抗ＳＦＰＱ抗体の存在が、老化、うつ病等を含む、ＳＦＰＱ関連機能障害の原因となっていることが示された。 From the above results, it is possible to prepare a novel SFPQ-related dysfunction model mouse by administering SFPQ protein or anti-SFPQ antibody to mice, and to diagnose SFPQ-related dysfunction by measuring the antibody titer of anti-SFPQ antibody. Alternatively, data can be obtained for testing and that autoimmune responses to SFPQ, particularly the presence of anti-SFPQ antibodies, are responsible for SFPQ-related dysfunction, including aging, depression, and the like.
　本発明の一実施形態によれば、うつ病と老化等に関与する自己免疫反応のデータの取得方法およびその利用を提供でき、うつ病と老化等の治療に好適に利用することができる。 According to one embodiment of the present invention, it is possible to provide a method for obtaining data on autoimmune reactions involved in depression, aging, etc. and its use, and it can be suitably used for treatment of depression, aging, etc.

Claims (8)

  1. 　被験体から分離した試料中の抗ＳＦＰＱ抗体の抗体価を測定する測定工程を含む、ＳＦＰＱ関連機能障害の診断または検査するためのデータの取得方法。 A method of obtaining data for diagnosing or testing SFPQ-related dysfunction, including a measurement step of measuring the antibody titer of an anti-SFPQ antibody in a sample isolated from a subject.
  2. 　前記ＳＦＰＱ関連機能障害は、脳、腸、精巣、皮膚、心臓および／または肺の老化と類似の機能障害である、請求項１に記載のデータの取得方法。 The method of acquiring data according to claim 1, wherein the SFPQ-related dysfunction is an aging-like dysfunction of the brain, intestine, testis, skin, heart and/or lungs.
  3. 　前記試料は、血液、血清、血漿、尿および唾液から選択される、請求項１または２に記載のデータの取得方法。 The data acquisition method according to claim 1 or 2, wherein the sample is selected from blood, serum, plasma, urine and saliva.
  4. 　以下の（ａ１）～（ａ３）、（ｂ１）～（ｂ３）、および、（ｃ１）～（ｃ３）から選択される少なくとも１つの何れかを含む、ＳＦＰＱ関連機能障害の診断薬：
    　（ａ１）配列番号１で表されるポリペプチド；
    　（ａ２）配列番号１で示されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換または付加されたアミノ酸配列からなるポリペプチド；
    　（ａ３）配列番号１で示されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列からなるポリペプチド；
    　（ｂ１）配列番号２または３で示されるアミノ酸配列からなるポリペプチド；
    　（ｂ２）配列番号２または３で示されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換または付加されたアミノ酸配列からなり、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；
    　（ｂ３）配列番号２または３で示されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列からなり、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；
    　（ｃ１）配列番号２または３で示されるアミノ酸配列を含み、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；
    　（ｃ２）配列番号２または３で示されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換または付加されたアミノ酸配列を含み、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；
    　（ｃ３）配列番号２または３で示されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含み、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド。     A diagnostic agent for SFPQ-related dysfunction, comprising at least one selected from the following (a1) to (a3), (b1) to (b3), and (c1) to (c3):
    (a1) a polypeptide represented by SEQ ID NO: 1;
    (a2) A polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 1;
    (a3) a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1;
    (b1) a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 2 or 3;
    (b2) a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 2 or 3, and having the ability to bind to an anti-SFPQ antibody;
    (b3) a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or 3 and having the ability to bind to an anti-SFPQ antibody;
    (c1) a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 2 or 3 and capable of binding to an anti-SFPQ antibody;
    (c2) A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 2 or 3, and having the ability to bind to an anti-SFPQ antibody;
    (c3) A polypeptide comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2 or 3 and having the ability to bind to an anti-SFPQ antibody.
  5. 　表面に、以下の（ａ１）～（ａ３）、（ｂ１）～（ｂ３）、および、（ｃ１）～（ｃ３）から選択される少なくとも１つを含む、血液浄化用担体：
    　（ａ１）配列番号１で表されるポリペプチド；
    　（ａ２）配列番号１で示されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換または付加されたアミノ酸配列からなるポリペプチド；
    　（ａ３）配列番号１で示されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列からなるポリペプチド；
    　（ｂ１）配列番号２または３で示されるアミノ酸配列からなるポリペプチド；
    　（ｂ２）配列番号２または３で示されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換または付加されたアミノ酸配列からなり、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；
    　（ｂ３）配列番号２または３で示されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列からなり、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；
    　（ｃ１）配列番号２または３で示されるアミノ酸配列を含み、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；
    　（ｃ２）配列番号２または３で示されるアミノ酸配列において、１もしくは数個のアミノ酸が欠失、置換または付加されたアミノ酸配列を含み、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド；
    　（ｃ３）配列番号２または３で示されるアミノ酸配列と９０％以上の同一性を有するアミノ酸配列を含み、かつ、抗ＳＦＰＱ抗体との結合能を有するポリペプチド。     A blood purification carrier comprising, on its surface, at least one selected from the following (a1) to (a3), (b1) to (b3), and (c1) to (c3):
    (a1) a polypeptide represented by SEQ ID NO: 1;
    (a2) A polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 1;
    (a3) a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1;
    (b1) a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 2 or 3;
    (b2) a polypeptide consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 2 or 3, and having the ability to bind to an anti-SFPQ antibody;
    (b3) a polypeptide consisting of an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2 or 3 and having the ability to bind to an anti-SFPQ antibody;
    (c1) a polypeptide comprising an amino acid sequence represented by SEQ ID NO: 2 or 3 and capable of binding to an anti-SFPQ antibody;
    (c2) A polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 2 or 3, and having the ability to bind to an anti-SFPQ antibody;
    (c3) A polypeptide comprising an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2 or 3 and having the ability to bind to an anti-SFPQ antibody.
  6. 　請求項５に記載の血液浄化用担体を備える、血液浄化カラム。 A blood purification column comprising the blood purification carrier according to claim 5.
  7. 　非ヒト哺乳動物にＳＦＰＱタンパク質または抗ＳＦＰＱ抗体を投与してなる、ＳＦＰＱ関連機能障害モデル動物。 A model animal of SFPQ-related dysfunction obtained by administering SFPQ protein or anti-SFPQ antibody to non-human mammals.
  8. 　非ヒト哺乳動物に、ＳＦＰＱタンパク質または抗ＳＦＰＱ抗体を投与する投与工程を含む、ＳＦＰＱ関連機能障害モデル動物の製造方法。 A method for producing an SFPQ-related dysfunction model animal, comprising an administration step of administering an SFPQ protein or an anti-SFPQ antibody to a non-human mammal.
PCT/JP2022/046880 2022-02-18 2022-12-20 Method for acquisition of data on autoimmune response involved in depression, aging and the like, and use of same WO2023157460A1 (en)

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