WO2010123083A1 - Polypeptide derived from mouse hepatitis virus and/or polypeptide derived from sendai virus, test kit for infection by mouse hepatitis virus and/or sendai virus using the polypeptide, and method for detecting infection by mouse hepatitis virus and/or sendai virus - Google Patents

Polypeptide derived from mouse hepatitis virus and/or polypeptide derived from sendai virus, test kit for infection by mouse hepatitis virus and/or sendai virus using the polypeptide, and method for detecting infection by mouse hepatitis virus and/or sendai virus Download PDF

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WO2010123083A1
WO2010123083A1 PCT/JP2010/057188 JP2010057188W WO2010123083A1 WO 2010123083 A1 WO2010123083 A1 WO 2010123083A1 JP 2010057188 W JP2010057188 W JP 2010057188W WO 2010123083 A1 WO2010123083 A1 WO 2010123083A1
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mouse
virus
mouse hepatitis
hepatitis virus
amino acid
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PCT/JP2010/057188
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French (fr)
Japanese (ja)
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高志 安居院
宣哉 佐々木
大輔 鳥越
淳 浅野
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国立大学法人北海道大学
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Priority claimed from JP2009104905A external-priority patent/JP2010254600A/en
Priority claimed from JP2009104906A external-priority patent/JP2010254601A/en
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Publication of WO2010123083A1 publication Critical patent/WO2010123083A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18811—Sendai virus
    • C12N2760/18822—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18811—Sendai virus
    • C12N2760/18834—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/085—Picornaviridae, e.g. coxsackie virus, echovirus, enterovirus
    • G01N2333/10—Hepatitis A virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/115—Paramyxoviridae, e.g. parainfluenza virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/18—Togaviridae; Flaviviridae
    • G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
    • G01N2333/186—Hepatitis C; Hepatitis NANB
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G01N2333/918—Carboxylic ester hydrolases (3.1.1)
    • G01N2333/92—Triglyceride splitting, e.g. by means of lipase

Definitions

  • the present invention provides a mouse hepatitis virus antigen epitope and / or amino acid sequence constituting a Sendai virus antigen epitope and / or a high identity with these amino acid sequences presented by mouse hepatitis virus nucleoprotein and / or Sendai virus nucleoprotein
  • a mouse hepatitis virus polypeptide that specifically binds to an anti-mouse hepatitis virus antibody and / or an anti-Sendai virus antibody produced by a rat and / or mouse infected with mouse hepatitis virus and / or Sendai virus comprising an amino acid sequence Sendai virus-derived polypeptide, mouse hepatitis virus infection and / or Sendai virus infection test kit using these, and mouse hepatitis virus infection or Sendai virus feeling Of a method for detecting.
  • Mouse hepatitis virus (Mouse hepatitis ; virus; MHV) is an RNA virus belonging to the genus Coronavirus of the Coronaviridae family, and the natural host is a mouse. Most cases are subclinical infections and grow in the liver and intestinal tract. Depending on the immunosuppressed state and young age, hepatic necrosis may spread throughout the liver and cause acute death. Several strains of murine hepatitis virus are also known to cause progressive demyelinating encephalitis in multiple sclerosis model mice. Therefore, attempts have been made to eliminate mouse hepatitis virus from breeding colonies by temporarily suspending breeding (Non-Patent Document 1).
  • Sendai virus (Sendai virus; SeV, or Hemagglutinating virus, Japan; HVJ) is an RNA virus belonging to the paramyxoviridae respirovirus genus, whose formal name is called mouse parainfluenza type I virus. Sendai virus infects rodents and rodents and causes respiratory diseases such as pneumonia.
  • the sensitivity is high in young mice, and inbred mouse C57BL / 6 (B6) It has been known for a long time that DBA / 2 has a high sensitivity while the sensitivity is relatively low. It is also known that rats can cause transient but severe rhinitis and pneumonia (Non-patent Document 2).
  • Mouse hepatitis virus is highly susceptible in mice
  • Sendai virus is highly susceptible in mice and rats
  • infection of laboratory animals impairs the reproducibility of experimental data.
  • periodic testing of Sendai virus infection in laboratory animal mice and rats is indispensable, and antibody detection methods, devices, kits, etc. have been developed for testing.
  • T. S. Golding et al. A method for detecting mouse hepatitis virus from mouse excreta using RT-PCR (Non-Patent Document 3), C.I. Lucas et al. Have proposed a quantitative immunofluorescence (QIF) method for detecting mouse blood antibodies against Sendai virus (Non-patent Document 4).
  • Japanese Patent Laid-Open No. 2000-131319 discloses an immunochromatographic method in which a substance capable of capturing a test antibody (pathogenic microorganism antigen) is immobilized on a support, and a colored particle labeled product of chicken egg yolk antibody specific for the pathogenic microorganism antigen.
  • Patent Document 1 Antibody test method and test kit (trade name: Monalyzer (R)) are disclosed (Patent Document 1), and Japanese Patent Publication No. 2007-532905 discloses a plurality of viral antigens or antiviral antibodies.
  • a biological sample is contacted with a microarray system that contains multiple subarrays capable of capturing the complex to form a complex, which is then contacted with a protein microarray system to detect captured viral antigens or antiviral antibodies.
  • Methods, microarray systems and kits are disclosed (patent text) 2).
  • Non-Patent Document 2 In the method using RT-PCR proposed in Non-Patent Document 2, it takes time to extract the mouse hepatitis virus gene and amplify the target gene, and it detects mouse hepatitis virus infection in a short time. Difficult to do.
  • the QIF method proposed in Non-Patent Document 4 or the detection methods and kits disclosed in Patent Document 1 and Patent Document 2 mouse hepatitis virus or Sendai virus itself is used as an antigen. Therefore, there are problems such as accuracy being different for each product, manufacturing cost, and many false positives.
  • the present invention has been made to solve such problems, and is an anti-mouse hepatitis virus produced by a mouse infected with mouse hepatitis virus and / or a rat infected with Sendai virus and / or an infected mouse.
  • the present inventors use a mouse hepatitis virus antigen epitope or a Sendai virus antigen epitope presented by mouse hepatitis virus nucleoprotein or Sendai virus nucleoprotein, and an immunoassay method, so that a conventional mouse hepatitis virus infection test kit or Sendai virus is used.
  • the inventors have found that mouse hepatitis virus infection or Sendai virus infection can be detected with high sensitivity as compared with infection test kits, and have completed the following inventions.
  • An anti-mouse hepatitis virus antibody and / or Sendai virus comprising one or more amino acid sequences selected from the group consisting of the following (a) to (d) and produced by a mouse infected with the mouse hepatitis virus: A polypeptide that specifically binds to an anti-Sendai virus antibody produced by infected rats and / or mice; (A) an amino acid sequence constituting a mouse hepatitis virus antigen epitope presented by the mouse hepatitis virus nucleoprotein, (B) an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence (a), (C) an amino acid sequence constituting a Sendai virus antigen epitope presented by Sendai virus nucleoprotein, (D) An amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence (c).
  • the amino acid sequence constituting the mouse hepatitis virus antigen epitope presented by the mouse hepatitis virus nucleoprotein is any amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 3 to 11. ) Polypeptide.
  • the amino acid sequence constituting the Sendai virus antigen epitope presented by the Sendai virus nucleoprotein is any amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 16 to 30, The described polypeptide.
  • a mouse hepatitis virus infection and / or Sendai virus infection test kit comprising the polypeptide according to any one of (1) to (3).
  • Mouse hepatitis virus infection performed by contacting the polypeptide according to any one of (1) to (3) with a specimen collected from a rat and / or mouse and determining the presence or absence of an antigen-antibody reaction And / or a method of detecting Sendai virus infection.
  • Mouse hepatitis virus-derived polypeptide and / or Sendai virus-derived polypeptide according to the present invention, mouse hepatitis virus infection and / or Sendai virus infection test kit using the same, and method for detecting mouse hepatitis virus infection and / or Sendai virus infection
  • mouse hepatitis virus infection or Sendai virus infection can be detected with high sensitivity.
  • Example 1 (1) a total of 446 kinds of polypeptides selected from the amino acid sequence of mouse hepatitis virus nucleoprotein consisting of 455 amino acids were prepared according to the SPOT synthesis method, and a schematic diagram showing a peptide array prepared using them. is there.
  • the serum of a mouse naturally infected with mouse hepatitis virus was chemiluminescent by immunochromatography, and the average value of the ratio of the luminescence intensity of each spot to the background Is a graph in which is plotted.
  • the vertical axis represents the average value of the luminescence intensity ratio
  • the horizontal axis represents the order from the N-terminus of the mouse hepatitis virus nucleoprotein amino acid sequence.
  • Example 2 (1) a total of 515 types of polypeptides selected from the amino acid sequence of Sendai virus nucleoprotein consisting of 524 amino acids were prepared according to the SPOT synthesis method, and a schematic diagram showing a peptide array prepared using them. .
  • the Sendai virus peptide array prepared in Example 2 (1) the serum before infection and the serum after infection of the same individual in BALB / c mice, C57BL / 6 (B6) mice and AKR mice were analyzed by immunochromatography.
  • Example 2 Using the Sendai virus peptide array prepared in Example 2 (1), the serum before and after infection of the same individual in C57BL / 6 (B6) mice was chemiluminescent by immunochromatography, and luminescence was confirmed. 3 is a table showing the ratio of luminescence intensity of each spot and the amino acid sequence. Using the Sendai virus peptide array prepared in Example 2 (1), the serum before infection and the serum after infection of the same individual in AKR mice were chemiluminescent by immunochromatography, and spots where luminescence was confirmed were extracted.
  • FIG. 4 is a table showing the ratio of light emission intensity of each spot, the amino acid sequence, and the like.
  • FIG. 4 is a table showing the ratio of light emission intensity of each spot, the amino acid sequence, and the like.
  • the pre-infection serum and the post-infection serum of the same individual in F344 rats were chemiluminescent by immunochromatography, and spots where luminescence was confirmed were extracted.
  • FIG. 4 is a table showing the ratio of light emission intensity of each spot, the amino acid sequence, and the like.
  • Example 3 (3) detection of anti-mouse hepatitis virus antibody for serum (normal mouse serum and infected mouse serum) with different serum concentrations was performed using the peptides of SEQ ID NO: 4 and SEQ ID NO: 11 by ELISA. This is a graph showing the results. The vertical axis represents the value of 450 nm absorbance (OD450), and the horizontal axis represents the dilution factor of serum as a specimen.
  • detection of anti-Sendai virus antibody for sera normal mouse serum and infected mouse serum
  • SEQ ID NO: 30 detection of anti-Sendai virus antibody for sera (normal mouse serum and infected mouse serum) with different serum concentrations was performed by ELISA using the peptides of SEQ ID NO: 17 and SEQ ID NO: 30. It is a graph which shows and shows the result.
  • the vertical axis represents the value of 450 nm absorbance (OD450), and the horizontal axis represents the dilution factor of serum as a specimen. It is a figure which shows the alignment with the amino acid sequence of a mouse
  • Antibody detection of experimentally infected mouse serum determined to be positive for infection using a commercially available Sendai virus infection determination kit was performed by ELISA using the peptides of SEQ ID NO: 17 and SEQ ID NO: 30. It is a graph which shows the result.
  • the vertical axis indicates the value of 450 nm absorbance (OD450), and the horizontal axis ( ⁇ ) and (+) indicate normal mouse serum and infected mouse serum, respectively.
  • the broken line in the figure indicates the value of OD450 obtained by adding 3 times the standard deviation of the OD value of normal mouse serum to the average value of OD value of normal mouse serum.
  • mouse hepatitis virus-derived polypeptide and / or Sendai virus-derived polypeptide mouse hepatitis virus infection and / or Sendai virus infection test kit using these, and mouse hepatitis virus infection and / or Sendai virus infection
  • the detection method will be described in detail.
  • the polypeptide according to the present invention comprises an anti-mouse hepatitis virus antibody produced by a mouse infected with murine hepatitis virus, comprising one or more amino acid sequences selected from the group consisting of the following (a) to (d): Binds specifically to anti-Sendai virus antibodies produced by rats and / or mice infected with Sendai virus; (A) an amino acid sequence constituting a mouse hepatitis virus antigen epitope presented by the mouse hepatitis virus nucleoprotein, (B) an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence (a), (C) an amino acid sequence constituting a Sendai virus antigen epitope presented by Sendai virus nucleoprotein, (D) An amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence (c).
  • mouse hepatitis virus antigen epitope selected from mouse hepatitis virus nucleoprotein or Sendai virus nucleoprotein selected from Sendai virus nucleoprotein in the present invention is presented by the amino acid sequence of mouse hepatitis virus nucleoprotein or Sendai virus nucleoprotein, Some are composed of straight-chain amino acids, and others are formed by amino acids located at sites distant from the primary structure forming higher-order structures.
  • epitopes in the present invention has the usual meaning of a site on an antigen recognized by an antibody, and can be used interchangeably with “antigenic determinant”.
  • Epitopes in the present invention are typically segments of amino acids that are a small part of the total protein and may be conformational or discontinuous as well as primary or continuous. That is, it is formed from amino acids encoded by adjacent or non-adjacent portions of the primary sequence arranged by protein folding.
  • a sequence of at least 3 amino acids in length is required, preferably at least 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 An amino acid, 19 amino acid, 20 amino acid long sequence is required.
  • the epitope is non-covalently bound to the binding site of the antibody that binds to it, that is, a paratope, through a hydrogen bond, an ionic bond, a hydrophobic bond, or a van der Waals bond.
  • the site of action on the epitope and the site of action on the paratope need to take a certain spatial arrangement, but by extending the chain length of the epitope in the present invention, Alternatively, by adding an amino acid, a three-dimensional structure (higher order structure) capable of binding to a paratope can be taken, and it may function as an epitope.
  • epitope identification is not particularly limited, and can be identified by any method known in the art.
  • the PEPSCAN method (Geysen et al., J. Immunol. Meth., 102, 259-274, 1987) and the SPOT synthesis method (R. Frank et al., Tetrahedron, 1992, 48, 9217).
  • W.R.G.Dostmann et al. Proc. Natl. Acad. Sci. USA, 2000, 97, 14772
  • the immunoassay that can be used in the present invention can be appropriately selected by those skilled in the art, and is not particularly limited. Methods, precipitation reactions, gel diffusion precipitation reactions, immunodiffusion assays, agglutination assays, complement binding assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and the like.
  • secondary antibodies used in these immunoassays can be appropriately selected by those skilled in the art, and are not particularly limited.
  • polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, Fab expression libraries And the like can be mentioned.
  • fragments produced by the Fab expression library include intact antibody fragments (Fab, F (ab ′) and F (ab ′) that retain binding activity to mouse hepatitis virus antigen and / or Sendai virus antigen. ) 2 fragments), single chain antibodies (scFv) containing the antigen binding site of the antibody, fusion proteins and other synthetic proteins (Bird et al., Science, Vol. 242, pages 423-426, 1988).
  • a polynucleotide encoding the epitope according to the present invention is selected, or StamppM. T.A.
  • a polynucleotide was synthesized from a full-length cDNA obtained according to the above-mentioned method (Stumppp MT, et al., J. Mol. Biol. 332 (2), 471-487, 2003), and the phage display method , The Bacteria two-hybrid method, the yeast two-hybrid method, the in vitro virus method, and the like.
  • the peptide array is obtained by chemically synthesizing the peptides according to the SPOT synthesis method.
  • amino acid sequences that can be identified by immunochromatography include amino acid sequences shown in SEQ ID NOs: 3 to 11 and SEQ ID NOs: 16 to 30.
  • “bond” is used interchangeably with “interact”, “react”, and “recognize”. Further, in the present invention, it is sufficient that it is clear that an antibody “specifically binds” to a specific antigen (immunogen) is reactive to the specific antigen.
  • the phrase “specifically reacts” with respect to the specific antigen includes not only reacting with other antigens at all, but also includes reacting with other antigens.
  • the “antigen” refers to an antigen that can elicit an immune response by administering it to a vertebrate, thereby promoting the production and release of an antibody that specifically binds to it. It has the above epitope.
  • the antigen in the present invention can be used interchangeably with “immunogen”, and subunit antigens and complexes in the state of being bound to antibodies are also included in the antigen in the present invention.
  • the antigen include peptides, polypeptides, proteins, lipoproteins, glycoproteins, nucleic acids, polysaccharides, lipopolysaccharides, lipids, etc.
  • peptides, polypeptides, proteins, etc. Lipoprotein and glycoprotein are preferred, and peptides, polypeptides and proteins are more preferred.
  • amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added the number of amino acids to be deleted, substituted, inserted and / or added is As long as the polypeptide having an amino acid sequence specifically binds to an anti-mouse hepatitis virus antibody produced by a mouse infected with mouse hepatitis virus and / or a rat infected with Sendai virus and / or an anti-Sendai virus antibody produced by mouse
  • an arbitrary number of 1 to 19, preferably 1 to 15, more preferably 1 to 10, further preferably 1 to 7, and still more preferably 1 to 5 may be mentioned. It can.
  • more amino acids may be substituted, inserted, and / or added as long as they encode the same or similar amino acid sequences.
  • all or at least the signal sequence of the amino acid sequence constituting the mouse hepatitis virus antigen epitope presented by the mouse hepatitis virus nucleoprotein and / or the Sendai virus antigen epitope presented by the Sendai virus nucleoprotein is excluded.
  • An anti-mouse hepatitis virus antibody produced by a mouse infected with mouse hepatitis virus and / or a rat infected with Sendai virus and / or an anti-sendai produced by mouse, comprising an amino acid sequence having a high identity with a part including the part Peptides that specifically bind to viral antibodies are included.
  • “high identity” refers to sequence identity of at least 50% or more, preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more.
  • the polypeptide according to the present invention specifically binds to an anti-mouse hepatitis virus antibody produced by a mouse infected with mouse hepatitis virus and / or a rat infected with Sendai virus and / or an anti-Sendai virus antibody produced by mouse.
  • a peptide consisting of an amino acid sequence having one or more conservative amino acid substitutions of these amino acid sequences is included.
  • a conservative amino acid substitution is a range that can generally be made without changing the physiological activity of the resulting molecule, that is, a conservative substitution (such as Watson et al., Molecular biology of Gene, etc.).
  • a conservative substitution such as Watson et al., Molecular biology of Gene, etc.
  • acidic amino acids of aspartic acid and glutamic acid basic amino acids of lysine, arginine and histidine
  • amino acids with similar side chains such as phenylalanine, tryptophan and tyrosine aromatic amino acids
  • acidic amino acids of aspartic acid and glutamic acid acidic amino acids of aspartic acid and glutamic acid; basic amino acids of lysine, arginine and histidine, aliphatic amino acids of glycine, alanine, valine, leucine, isoleucine, serine and threonine (classified as aliphatic-hydroxyamino acids of serine and threonine Can be classified); aromatic amino acids of phenylalanine, tyrosine and tryptophan; amides of asparagine and glutamine); sulfur-containing amino acids of cysteine and methionine.
  • the rat in the present invention is not particularly limited, it is generally used for animal experiments, such as SD rat, Fisher rat, Wistar rat, Wistar Hanover / Rcc rat, Wistar / ST rat, Donryu rat, BN rat, F344 rat, etc. Various strains of rats can be mentioned.
  • the mouse in the present invention is also not particularly limited.
  • A, AKR, BALB / c, C3H, C57BL / 6, DBA / 2, B6C3F1, BDF1, B6D2F1, and ICR are generally used in various animal experiments. Mention may be made of strains of mice.
  • rat salivary lacrimal adenitis virus As an RNA virus belonging to the genus Coronavirus belonging to the Coronaviridae family, rat salivary lacrimal adenitis virus (RAT sialadoadenitis coronavirus; SDAV) can be cited as a closely related mouse hepatitis virus. Since rat salivary lacrimal inflammation virus is similar in form and antigenicity to mouse hepatitis virus, the mouse hepatitis antigen epitope presented by mouse hepatitis virus nucleoprotein is not only anti-mouse hepatitis virus antibody but also anti-rat salivary gland It also recognizes lacrimal inflammation virus antibodies.
  • the mouse hepatitis virus-derived polypeptide according to the present invention specifically binds to an anti-mouse hepatitis virus antibody produced by a mouse infected with mouse hepatitis virus, and is also an anti-antigen produced by a rat infected with rat salivary lacrimal adenitis virus. Since it specifically binds to the rat salivary lacrimal inflammation virus antibody, it is also effective in detecting rat salivary lacrimal inflammation virus infection.
  • the present invention provides a kit containing the polypeptide according to the present invention.
  • the kit according to the present invention is a substance useful for carrying out a detection means for mouse hepatitis virus infection and / or Sendai virus infection, such as a secondary antibody, a labeling substance, or a substance or buffer useful for carrying out an immunological detection means. Etc. may be included as a component of the kit.
  • the present invention relates to mouse hepatitis virus infection and / or Sendai virus infection, which is carried out by contacting the polypeptide of the present invention with a subject collected from rats and / or mice to determine the presence or absence of an antigen-antibody reaction.
  • a detection method is provided.
  • the detection method for mouse hepatitis virus infection and / or Sendai virus infection according to the present invention includes an incubation step, a washing step, and the like unless the method for detecting mouse hepatitis virus infection and / or Sendai virus infection according to the present invention is impaired. You may go out.
  • the kit containing the polypeptide according to the present invention and the method for detecting mouse hepatitis virus infection and / or Sendai virus infection are used for periodic examination of mouse hepatitis virus infection and / or Sendai virus infection in laboratory animal mice and rats.
  • it can be used for testing of mice and rats that are falsely positive by a conventional mouse hepatitis virus infection and / or Sendai virus infection test kit or mouse hepatitis virus infection and / or Sendai virus infection test method. It is.
  • mouse hepatitis virus-derived polypeptide and / or Sendai virus-derived polypeptide mouse hepatitis virus infection and / or Sendai virus infection test kit using these, and mouse hepatitis virus infection and / or Sendai virus infection A detection method will be described based on examples. Note that the technical scope of the present invention is not limited to the features shown by these examples.
  • mice hepatitis virus epitope using mouse hepatitis virus peptide array (1) Preparation of mouse hepatitis virus peptide array
  • Mouse hepatitis virus nucleoprotein consisting of 455 amino acids (murine hepatitis virus NP, Accession number X00990; SEQ ID NO: A total of 446 polypeptides selected from the amino acid sequence of 1 and SEQ ID NO: 2) by shifting a 10-residue polypeptide one by one were synthesized by the SPOT synthesis method (R. Frank et al., Tetrahedron, 1992, No. 1). 48, page 9217, WR G. Dodmann et al., Proc. Natl. Acad.
  • mice hepatitis virus peptide array prepared in this Example (1) was added to 1% PBST-SM 50 mL prepared in this Example (2) [2-1]. After dipping and incubating at 4 ° C. overnight, the sample was further dipped in 20 mL of 1% PBST prepared in Example (2) [2-1] and washed by shaking at room temperature for 1 minute. The washed murine hepatitis virus peptide array was placed on a plastic film, and 5 mL each of 1% PBST-SM diluted serum was placed thereon, covered with the plastic film, and incubated at room temperature for 2 hours.
  • the mouse hepatitis virus peptide array from which the plastic film was removed after incubation was immersed in 20 mL of 1% PBST prepared in this Example (2) [2-1], washed by shaking for 15 minutes ⁇ 3 times at room temperature, and again After placing on a plastic film, 5 mL each of HRP-labeled anti-mouse IgG antibody (GE Healthcare Bio-Sciences) diluted 10,000 times with 1% PBST-SM prepared in this Example (2) [2-1] It was placed, covered again with a plastic film, and further incubated at room temperature for 2 hours. Thereafter, the plastic film was removed and the plate was immersed in 20 mL of 1% PBST prepared in Example (2) [2-1] and washed by shaking for 15 minutes ⁇ 3 times.
  • Table 1 shows the amino acid sequences obtained by extracting the spots on which the above was confirmed and their SEQ ID NOs.
  • FIG. 2 and Table 1 from the N-terminus of the mouse hepatitis virus NP amino acid sequence, the 1st to 14th (SEQ ID NO: 3), the 24th to 39th (SEQ ID NO: 4), the 49th to 64th ( SEQ ID NO: 5), 69th to 82nd (SEQ ID NO: 6), 240th to 253rd (SEQ ID NO: 7), 307th to 325th (SEQ ID NO: 8), 354 to 373rd (SEQ ID NO: 9) and the same It was confirmed that the luminescence intensity of each spot corresponding to the 381st to 393rd (SEQ ID NO: 10) was large in ratio to the luminescence intensity of these backgrounds.
  • the amino acid sequence of the spot having a ratio of 2 or more to the background luminescence intensity is 24th to 39th from the N-terminus (SEQ ID NO: 4), It was confirmed that they were positions 307 to 325 (SEQ ID NO: 8) and 354 to 373 (SEQ ID NO: 9).
  • Example 2 Identification of Sendai virus epitope using Sendai virus peptide array (1) Preparation of Sendai virus peptide array Sendai virus nucleoprotein consisting of 524 amino acids (Sendai virus NP, Accession number X00087; SEQ ID NO: 14 and SEQ ID NO: 15) ) By synthesizing a total of 515 kinds of polypeptides selected by shifting 10-residue polypeptides one by one from the amino acid sequence on the cellulose membrane in the same manner as in Example 1 (1), Sendai virus NP amino acid sequence N-terminal 1 to 10 (spot No. 1), 2 to 11 (spot No. 2), 3 to 12 (spot No. 3), followed by 515 to 524 (spot No. 515), total 51 Type 10 residue polypeptide chains to produce a Sendai virus peptide arrays arranged in order on a cellulose membrane. This is shown in FIG.
  • Example 2 Search for epitopes by Sendai virus peptide array Using the 1% PBST-SM diluted serum prepared in Example (2) [2-1], Example 1 (2) [2-2] and By the same method, IgG antibody specifically bound to the Sendai virus peptide array was detected and quantified.
  • FIG. 4 shows a plot of the average value of the ratio of the luminescence intensity of each spot between the serum before infection and the serum after infection of the same individual in BALB / c mice, C57BL / 6 (B6) mice and AKR mice.
  • C57BL / 6 (B6) mice, AKR mice, BN rats, and F344 rats, and spots where luminescence was confirmed were extracted.
  • FIGS. 5 to 9 show the ratio of the luminescence intensity and the amino acid sequence, respectively. These amino acid sequences and SEQ ID NOs are shown in Table 2.
  • positions 119 to 134 SEQ ID NO: 17
  • 144 to 158 SEQ ID NO: 18
  • 419 to 430 SEQ ID NO: 23
  • 457 to 471 SEQ ID NO: 25
  • 464 to 475 SEQ ID NO: 26
  • 472 to 487 SEQ ID NO: 27
  • 487 to 500 SEQ ID NO: 28
  • Example 3 Confirmation of mouse specificity for infection with mouse hepatitis virus NP epitope peptide
  • SEQ ID NO: 4 24th to 39th from the N-terminus of the NP amino acid sequence (SEQ ID NO: 4) and 357-372th from the N-terminus (RFDSTLPGFETIMKVL (R is arginine, F is phenylalanine, D is aspartic acid, S is serine, T is threonine, L is Leucine, P is proline, G is glycine, E is glutamic acid, I is isoleucine, M is methionine, K is lysine, and V is valine): SEQ ID NO: 11) The binding antibody was detected.
  • Tween-20 PBS solution Preparation of 0.5% Tween-20 PBS solution and blocking solution Each final solution concentration is 1.37 M NaCl, 27 mM KCl, 81 mM Na2HPO4, 15 mM KH2PO4 at a rate of 5 mL.
  • a 0.5% Tween-20 PBS solution (0.5% PBST) was prepared by adding Tween-20.
  • a blocking solution was prepared by adding bovine serum albumin (BSA) to this 0.5% PBST to a weight ratio of 0.5% and dissolving.
  • BSA bovine serum albumin
  • each well was washed three times with 200 ⁇ L of 0.5% PBST prepared in this Example (1), 200 ⁇ L of the serum prepared in this Example 2 (2) was added and incubated at 37 ° C. for 1 hour. Thereafter, each well was washed again with 200 ⁇ L of 0.5% PBST prepared in Example 2 (1) three times. 200 ⁇ L of HRP-labeled anti-mouse IgG antibody (GE Healthcare Bio-Sciences) diluted 10,000 times using the blocking solution prepared in Example 2 (1) was added to each well and incubated at 37 ° C. for 1 hour.
  • HRP-labeled anti-mouse IgG antibody diluted 10,000 times using the blocking solution prepared in Example 2 (1) was added to each well and incubated at 37 ° C. for 1 hour.
  • each well was washed 3 times with 200 ⁇ L of 0.5% PBST prepared in Example 2 (1), and 200 ⁇ L of a 1.5 mg / mL o-phenylenediamine ⁇ 2 hydrochloric acid (OPD) solution was added. Incubated for 10 minutes at 0 ° C. The color development reaction was stopped by adding 50 ⁇ L of 3.5 N sulfuric acid to each well, and the absorbance at 450 nm (OD450) was measured with an ELISA plate reader (Model 680 microplate reader; Bio-Rad Laboratories). The result is shown in FIG.
  • OPD o-phenylenediamine ⁇ 2 hydrochloric acid
  • the OD value of normal mouse serum is almost constant regardless of the concentration when any of the peptides of SEQ ID NO: 4 and SEQ ID NO: 11 is used as an antigen. It was confirmed that the OD value decreased with decreasing concentration. From the above results, it was shown that the peptides of SEQ ID NO: 4 and SEQ ID NO: 11 specifically bind to the antibody contained in the serum of mouse hepatitis virus-infected mice.
  • Example 4 Confirmation of infected mouse specificity of Sendai virus NP epitope peptide
  • the Sendai virus NP amino acid sequence was confirmed.
  • One individual's serum (infected mouse serum) on the 14th day after infection was collected.
  • Each of the collected sera was diluted 50 times, 100 times, 200 times, 400 times, 800 times, 1600 times, 3200 times and 6400 times with the blocking solution prepared in Example 3 (1), respectively.
  • a blocking solution-diluted serum having a concentration was prepared, and the antibody was detected by measuring the absorbance at 450 nm (OD450) in the same manner as in Example 3 (3). The result is shown in FIG.
  • Example 5 Detection of viral infection with mouse hepatitis virus NP epitope peptide Using the mouse hepatitis virus NP epitope identified in Example 1 (2) [2-3] and confirmed in Example 3 (3), We examined whether mouse hepatitis virus infection could be detected in experimentally infected mice that were determined to be positive for infection using a commercially available mouse hepatitis virus infection determination kit. Moreover, the sensitivity of the determination of mouse hepatitis virus infection was compared with a commercially available mouse hepatitis virus infection determination kit (Monilizer MHV; Wakamoto Pharmaceutical Co., Ltd.).
  • Example 3 (3) Detection by ELISA method
  • Example 3 (3) using the peptides of SEQ ID NO: 4 and SEQ ID NO: 11 used in Example 3 (3) and the blocking solution-diluted serum prepared in this Example (1)
  • the antibody was detected by measuring the absorbance at 450 nm (OD450) in the same manner as in (1).
  • Table 4 The results are shown in Table 4.
  • mice As shown in Table 4, for 8 normal mice, even when any peptide of SEQ ID NO: 4 and SEQ ID NO: 11 was used, all 8 individuals were determined to be negative. In addition, even when a commercially available mouse hepatitis virus infection determination kit (Monilizer MHV; Wakamoto Pharmaceutical Co., Ltd.) was used, it was determined to be negative in all 8 normal mice. On the other hand, for 14 individuals obtained from a population of mice naturally infected with mouse hepatitis virus, 10 individuals were determined to be positive and the remaining 4 individuals were determined to be negative regardless of which peptide was used.
  • Monilizer MHV Wakamoto Pharmaceutical Co., Ltd.
  • mice hepatitis virus infection determination kit (Monilizer MHV; Wakamoto Pharmaceutical Co., Ltd.) 8 individuals were determined to be positive, and the remaining 6 individuals were determined to be negative. Eight out of 10 individuals determined to be positive when using any of the peptides of SEQ ID NO: 4 and SEQ ID NO: 11 were determined to be positive by a commercially available mouse hepatitis virus infection determination kit (Monilizer MHV; Wakamoto Pharmaceutical Co., Ltd.). Matched 8 individuals. From the above results, it was shown that the peptides of SEQ ID NO: 4 and SEQ ID NO: 11 can determine mouse hepatitis virus infection with higher sensitivity than conventional mouse hepatitis virus infection determination kits.
  • Example 6 Detection of viral infection by Sendai virus NP epitope peptide Using the Sendai virus NP epitope identified in Example 1 (2) [2-3] and confirmed in Example 2 (3), commercially available We investigated whether Sendai virus infection could be detected in experimentally infected mice that were determined to be positive for infection using the Sendai virus infection determination kit.
  • Example 3 (2) Detection by ELISA method Example 3 (3) using the peptides of SEQ ID NO: 17 and SEQ ID NO: 30 used in Example 3 (3) and the blocking solution-diluted serum prepared in this Example (1) The antibody was detected by measuring the absorbance at 450 nm (OD450) in the same manner as in (1). The result is shown in FIG.
  • the OD value in the case of normal mouse serum is low in the case of using any of the peptides of SEQ ID NO: 17 and SEQ ID NO: 30 as the antigen, whereas the OD value in the case of infected mouse serum. The value was confirmed to be high.
  • Sendai virus infection determination kit (Monilizer HVJ) can be used when any peptide of SEQ ID NO: 17 and SEQ ID NO: 30 is used as an antigen. It was shown that all Sendai virus-infected mice determined to be positive for infection using Wakamoto Pharmaceutical Co., Ltd.) can be determined to be positive. From the above results, it was shown that the Sendai virus NP epitope peptides of SEQ ID NO: 17 and SEQ ID NO: 30 identified in Example 1 (2) can be used for detection of Sendai virus infection in mice.
  • FIG. 13 shows an alignment between the amino acid sequence of mouse hepatitis virus NP (455 amino acids, SEQ ID NO: 2) and the amino acid sequence of rat salivary gland adenitis virus NP (SEQ ID NO: 13).
  • the homology of the amino acid sequence of mouse hepatitis virus NP and rat salivary lacrimal adenitis virus NP is 93.4%.
  • the 38th glutamine from the N-terminal of rat salivary lacrimal inflammation virus NP is leucine in SEQ ID NO: 4. That the 59th threonine is serine in SEQ ID NO: 5, the 321st proline is alanine in SEQ ID NO: 8, and the 383rd alanine is aspartic acid in SEQ ID NO: 10.
  • the specificity to the anti-mouse hepatitis virus antibody produced by the mouse infected with the mouse hepatitis virus and / or the rat infected with Sendai virus and / or the anti-Sendai virus antibody produced by the mouse A peptide with high capture ability can be prepared, and mouse hepatitis virus infection and / or Sendai virus infection can be detected easily and with high sensitivity.
  • mouse hepatitis virus-derived polypeptide and / or Sendai virus-derived polypeptide according to the present invention, mouse hepatitis virus infection and / or Sendai virus infection test kit using these, and mouse hepatitis virus infection and / or Sendai virus infection
  • the detection method is not limited to the embodiment described above, and can be changed as appropriate.

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Abstract

Disclosed are: a polypeptide derived from a mouse hepatitis virus and/or a polypeptide derived from a sendai virus which has high specificity for an anti-mouse hepatitis virus antibody and/or an anti-sendai virus antibody and a high ability of trapping the antibody, wherein the anti-mouse hepatitis virus antibody and/or the anti-sendai virus antibody is produced by a mouse infected by a mouse hepatitis virus and/or a rat and/or a mouse infected by a sendai virus; a test kit for mouse hepatitis virus infection and/or sendai virus infection, which uses the polypeptide and can detect the mouse hepatitis virus infection and/or the sendai virus infection with high sensitivity; and a method for detecting mouse hepatitis virus infection and/or sendai virus infection. Specifically disclosed is a polypeptide which comprises a specific amino acid sequence, and which can bind specifically to an anti-mouse hepatitis virus antibody produced by a mouse infected by a mouse hepatitis virus and/or an anti-sendai virus antibody produced by a rat and/or a mouse infected by a sendai virus.

Description

マウス肝炎ウイルス由来ポリペプチドおよび／またはセンダイウイルス由来ポリペプチド、これらを用いたマウス肝炎ウイルス感染および／またはセンダイウイルス感染検査キット、ならびにマウス肝炎ウイルス感染および／またはセンダイウイルス感染の検出方法Mouse hepatitis virus-derived polypeptide and / or Sendai virus-derived polypeptide, mouse hepatitis virus infection and / or Sendai virus infection test kit using the same, and method for detecting mouse hepatitis virus infection and / or Sendai virus infection
　本発明は、マウス肝炎ウイルス核タンパク質および／またはセンダイウイルス核タンパク質により提示される、マウス肝炎ウイルス抗原エピトープおよび／またはセンダイウイルス抗原エピトープを構成するアミノ酸配列ならびに／もしくはこれらアミノ酸配列との同一性が高いアミノ酸配列からなる、マウス肝炎ウイルスおよび／またはセンダイウイルスに感染したラットならびに／もしくはマウスが産生する抗マウス肝炎ウイルス抗体および／または抗センダイウイルス抗体と特異的に結合するマウス肝炎ウイルスポリペプチドおよび／またはセンダイウイルス由来ポリペプチド、これらを用いたマウス肝炎ウイルス感染および／またはセンダイウイルス感染検査キット、ならびにマウス肝炎ウイルス感染もしくはセンダイウイルス感染の検出方法に関する。 The present invention provides a mouse hepatitis virus antigen epitope and / or amino acid sequence constituting a Sendai virus antigen epitope and / or a high identity with these amino acid sequences presented by mouse hepatitis virus nucleoprotein and / or Sendai virus nucleoprotein A mouse hepatitis virus polypeptide that specifically binds to an anti-mouse hepatitis virus antibody and / or an anti-Sendai virus antibody produced by a rat and / or mouse infected with mouse hepatitis virus and / or Sendai virus, comprising an amino acid sequence Sendai virus-derived polypeptide, mouse hepatitis virus infection and / or Sendai virus infection test kit using these, and mouse hepatitis virus infection or Sendai virus feeling Of a method for detecting.
　マウス肝炎ウイルス（Ｍｏｕｓｅ ｈｅｐａｔｉｔｉｓ ｖｉｒｕｓ；ＭＨＶ）とは、コロナウイルス科コロナウイルス属に属するＲＮＡウイルスであり、自然宿主はマウスである。不顕性感染である場合がほとんどであり、肝臓や腸管で増殖する。免疫抑制状態や若齢によっては肝壊死斑が肝臓全体に広がり、急性死亡する場合がある。また、マウス肝炎ウイルスのいくつかの株が、多発性硬化症のモデルマウスに対して進行性脱髄性脳炎を引き起こすことが知られている。そのため、繁殖を一時中断することにより繁殖コロニーからマウス肝炎ウイルスを排除する試みがなされている（非特許文献１）。 Mouse hepatitis virus (Mouse hepatitis ； virus; MHV) is an RNA virus belonging to the genus Coronavirus of the Coronaviridae family, and the natural host is a mouse. Most cases are subclinical infections and grow in the liver and intestinal tract. Depending on the immunosuppressed state and young age, hepatic necrosis may spread throughout the liver and cause acute death. Several strains of murine hepatitis virus are also known to cause progressive demyelinating encephalitis in multiple sclerosis model mice. Therefore, attempts have been made to eliminate mouse hepatitis virus from breeding colonies by temporarily suspending breeding (Non-Patent Document 1).
　一方、センダイウイルス（Ｓｅｎｄａｉ ｖｉｒｕｓ；ＳｅＶ、またはＨｅｍａｇｇｌｕｔｉｎａｔｉｎｇ ｖｉｒｕｓ ｏｆ Ｊａｐａｎ；ＨＶＪ）とは、正式名称をマウスパラインフルエンザＩ型ウイルスといい、パラミクソウイルス科レスピロウイルス属に属するＲＮＡウイルスである。センダイウイルスは齧歯類や兎類に感染し、肺炎などの呼吸器疾患を引き起こすが、マウスについては、幼若マウスにおいてその感受性が高いことや、近交系マウスＣ５７ＢＬ／６（Ｂ６）においては感受性が比較的低いのに対し、ＤＢＡ／２においては感受性が高いことなどが古くから知られている。また、ラットについても、一過性ではあるが重篤な鼻炎や肺炎を引き起こし得ることが知られている（非特許文献２）。 On the other hand, Sendai virus (Sendai virus; SeV, or Hemagglutinating virus, Japan; HVJ) is an RNA virus belonging to the paramyxoviridae respirovirus genus, whose formal name is called mouse parainfluenza type I virus. Sendai virus infects rodents and rodents and causes respiratory diseases such as pneumonia. In mice, the sensitivity is high in young mice, and inbred mouse C57BL / 6 (B6) It has been known for a long time that DBA / 2 has a high sensitivity while the sensitivity is relatively low. It is also known that rats can cause transient but severe rhinitis and pneumonia (Non-patent Document 2).
　マウス肝炎ウイルスはマウスにおいて感受性が高く、センダイウイルスはマウスやラットにおいて感受性が高いこと、さらに、実験動物の感染症は実験データの再現性を損ねることから、実験動物マウスに対するマウス肝炎ウイルス感染症、あるいは実験動物マウスやラットに対するセンダイウイルス感染症の定期的な検査が不可欠であり、検査のための抗体検出の方法や装置、キットなどが開発されている。 Mouse hepatitis virus is highly susceptible in mice, Sendai virus is highly susceptible in mice and rats, and further, infection of laboratory animals impairs the reproducibility of experimental data. Alternatively, periodic testing of Sendai virus infection in laboratory animal mice and rats is indispensable, and antibody detection methods, devices, kits, etc. have been developed for testing.
　例えば、Ｔ．Ｓ．Ｇｏｌｄｉｎｇらにより、ＲＴ－ＰＣＲを利用してマウス排泄物からマウス肝炎ウイルスを検出する方法が（非特許文献３）、Ｃ．Ｌｕｃａｓらにより、センダイウイルスに対するマウスの血中抗体を検出するための定量的免疫蛍光（ＱＩＦ）法が、それぞれ提案されている（非特許文献４）。また、特開２０００－１３１３１９号公報には、被検抗体を捕捉できる物質（病原微生物抗原）を支持体に固定したイムノクロマトグラフィ法に、病原微生物抗原に特異的な鶏卵卵黄抗体の着色粒子標識物を組み合わせて用いた抗体検査方法および検査用キット（商品名：モニライザ（Ｒ））が開示されており（特許文献１）、特表２００７－５３２９０５号公報には、複数のウイルス抗原あるいは抗ウイルス抗体を捕捉することができる複数のサブアレイを含むマイクロアレイシステムに生物学的サンプルを接触させて複合体を形成させ、これにタンパク質マイクロアレイシステムを接触させることにより、捕捉されたウイルス抗原あるいは抗ウイルス抗体を検出する方法、マイクロアレイシステムおよびキットが開示されている（特許文献２）。 For example, T. S. Golding et al., A method for detecting mouse hepatitis virus from mouse excreta using RT-PCR (Non-Patent Document 3), C.I. Lucas et al. Have proposed a quantitative immunofluorescence (QIF) method for detecting mouse blood antibodies against Sendai virus (Non-patent Document 4). Japanese Patent Laid-Open No. 2000-131319 discloses an immunochromatographic method in which a substance capable of capturing a test antibody (pathogenic microorganism antigen) is immobilized on a support, and a colored particle labeled product of chicken egg yolk antibody specific for the pathogenic microorganism antigen. Antibody test method and test kit (trade name: Monalyzer (R)) are disclosed (Patent Document 1), and Japanese Patent Publication No. 2007-532905 discloses a plurality of viral antigens or antiviral antibodies. A biological sample is contacted with a microarray system that contains multiple subarrays capable of capturing the complex to form a complex, which is then contacted with a protein microarray system to detect captured viral antigens or antiviral antibodies. Methods, microarray systems and kits are disclosed (patent text) 2).
特開２０００－１３１３１９号公報JP 2000-131319 A 特表２００７－５３２９０５号公報Special table 2007-532905 gazette
　しかしながら、非特許文献２において提案されているＲＴ－ＰＣＲを用いた方法では、マウス肝炎ウイルス遺伝子の抽出作業や標的遺伝子の増幅作業などの手間が掛かってしまい、マウス肝炎ウイルス感染を短時間で検出することが困難である。また、非特許文献４において提案されているＱＩＦ法、あるいは特許文献１および特許文献２において開示されている検出方法やキットなどにおいては、マウス肝炎ウイルスやセンダイウイルスそのものが抗原として用いられていることから、製品ごとに精度が異なること、製造コストがかかること、擬陽性が多いことなどの問題がある。 However, in the method using RT-PCR proposed in Non-Patent Document 2, it takes time to extract the mouse hepatitis virus gene and amplify the target gene, and it detects mouse hepatitis virus infection in a short time. Difficult to do. In addition, in the QIF method proposed in Non-Patent Document 4 or the detection methods and kits disclosed in Patent Document 1 and Patent Document 2, mouse hepatitis virus or Sendai virus itself is used as an antigen. Therefore, there are problems such as accuracy being different for each product, manufacturing cost, and many false positives.
　本発明は、このような問題点を解決するためになされたものであって、マウス肝炎ウイルスに感染したマウスおよび／またはセンダイウイルスに感染したラットならびに／もしくは感染したマウスが産生する抗マウス肝炎ウイルス抗体および／または抗センダイウイルス抗体に対する特異性や捕捉性の高いマウス肝炎ウイルス由来ポリペプチドおよび／またはセンダイウイルス由来ポリペプチド、これらを用いたマウス肝炎ウイルス感染および／またはセンダイウイルス感染を高感度で検出することができるマウス肝炎ウイルス感染および／またはセンダイウイルス感染検査キット、ならびにマウス肝炎ウイルス感染および／またはセンダイウイルス感染の検出方法を提供することを目的とする。 The present invention has been made to solve such problems, and is an anti-mouse hepatitis virus produced by a mouse infected with mouse hepatitis virus and / or a rat infected with Sendai virus and / or an infected mouse. Murine hepatitis virus-derived polypeptide and / or Sendai virus-derived polypeptide with high specificity and capture property against antibodies and / or anti-Sendai virus antibodies, mouse hepatitis virus infection and / or Sendai virus infection using these with high sensitivity It is an object of the present invention to provide a mouse hepatitis virus infection and / or Sendai virus infection test kit, and a detection method for mouse hepatitis virus infection and / or Sendai virus infection.
　本発明者らは、マウス肝炎ウイルス核タンパク質あるいはセンダイウイルス核タンパク質により提示されるマウス肝炎ウイルス抗原エピトープあるいはセンダイウイルス抗原エピトープとイムノアッセイ法とを用いることにより、従来のマウス肝炎ウイルス感染検査キットあるいはセンダイウイルス感染検査キットと比較してマウス肝炎ウイルス感染あるいはセンダイウイルス感染を高感度で検出することができることを見いだし、下記の各発明を完成した。 The present inventors use a mouse hepatitis virus antigen epitope or a Sendai virus antigen epitope presented by mouse hepatitis virus nucleoprotein or Sendai virus nucleoprotein, and an immunoassay method, so that a conventional mouse hepatitis virus infection test kit or Sendai virus is used. The inventors have found that mouse hepatitis virus infection or Sendai virus infection can be detected with high sensitivity as compared with infection test kits, and have completed the following inventions.
（１）以下の（ａ）～（ｄ）からなる群から選ばれる１または２以上のアミノ酸配列からなり、かつマウス肝炎ウイルスに感染したマウスが産生する抗マウス肝炎ウイルス抗体および／またはセンダイウイルスに感染したラットならびに／もしくはマウスが産生する抗センダイウイルス抗体と特異的に結合するポリペプチド；
（ａ）マウス肝炎ウイルス核タンパク質により提示されるマウス肝炎ウイルス抗原エピトープを構成するアミノ酸配列、
（ｂ）アミノ酸配列（ａ）において１個もしくは数個のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列、
（ｃ）センダイウイルス核タンパク質により提示されるセンダイウイルス抗原エピトープを構成するアミノ酸配列、
（ｄ）アミノ酸配列（ｃ）において１個もしくは数個のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列。 (1) An anti-mouse hepatitis virus antibody and / or Sendai virus comprising one or more amino acid sequences selected from the group consisting of the following (a) to (d) and produced by a mouse infected with the mouse hepatitis virus: A polypeptide that specifically binds to an anti-Sendai virus antibody produced by infected rats and / or mice;
(A) an amino acid sequence constituting a mouse hepatitis virus antigen epitope presented by the mouse hepatitis virus nucleoprotein,
(B) an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence (a),
(C) an amino acid sequence constituting a Sendai virus antigen epitope presented by Sendai virus nucleoprotein,
(D) An amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence (c).
（２）マウス肝炎ウイルス核タンパク質により提示されるマウス肝炎ウイルス抗原エピトープを構成するアミノ酸配列が配列番号３～１１に示されるアミノ酸配列からなる群から選択されるいずれかのアミノ酸配列である、（１）に記載のポリペプチド。 (2) The amino acid sequence constituting the mouse hepatitis virus antigen epitope presented by the mouse hepatitis virus nucleoprotein is any amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 3 to 11. ) Polypeptide.
（３）センダイウイルス核タンパク質により提示されるセンダイウイルス抗原エピトープを構成するアミノ酸配列が配列番号１６～３０に示されるアミノ酸配列からなる群から選択されるいずれかのアミノ酸配列である、（１）に記載のポリペプチド。 (3) The amino acid sequence constituting the Sendai virus antigen epitope presented by the Sendai virus nucleoprotein is any amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 16 to 30, The described polypeptide.
（４）（１）から（３）のいずれかに記載のポリペプチドを含む、マウス肝炎ウイルス感染および／またはセンダイウイルス感染検査キット。 (4) A mouse hepatitis virus infection and / or Sendai virus infection test kit comprising the polypeptide according to any one of (1) to (3).
（５）（１）から（３）のいずれかに記載のポリペプチドとラットおよび／またはマウスから採取した被検体とを接触させて抗原抗体反応の有無を判定することにより行う、マウス肝炎ウイルス感染および／またはセンダイウイルス感染の検出方法。 (5) Mouse hepatitis virus infection performed by contacting the polypeptide according to any one of (1) to (3) with a specimen collected from a rat and / or mouse and determining the presence or absence of an antigen-antibody reaction And / or a method of detecting Sendai virus infection.
　本発明に係るマウス肝炎ウイルス由来ポリペプチドおよび／またはセンダイウイルス由来ポリペプチド、これらを用いたマウス肝炎ウイルス感染および／またはセンダイウイルス感染検査キット、ならびにマウス肝炎ウイルス感染および／またはセンダイウイルス感染の検出方法によれば、マウス肝炎ウイルスに感染したマウスが産生する抗マウス肝炎ウイルス抗体および／またはセンダイウイルスに感染したラットならびに／もしくはマウスが産生する抗センダイウイルス抗体に対する特異性や捕捉性を高めることができ、従来のマウス肝炎ウイルス感染検査キットあるいはセンダイウイルス感染検査キットと比較して、マウス肝炎ウイルス感染あるいはセンダイウイルス感染を高感度で検出することができる。 Mouse hepatitis virus-derived polypeptide and / or Sendai virus-derived polypeptide according to the present invention, mouse hepatitis virus infection and / or Sendai virus infection test kit using the same, and method for detecting mouse hepatitis virus infection and / or Sendai virus infection According to the present invention, it is possible to increase the specificity and capture of anti-mouse hepatitis virus antibodies produced by mice infected with mouse hepatitis virus and / or anti-Sendai virus antibodies produced by rats and / or mice infected with Sendai virus. Compared with a conventional mouse hepatitis virus infection test kit or Sendai virus infection test kit, mouse hepatitis virus infection or Sendai virus infection can be detected with high sensitivity.
実施例１（１）において、４５５アミノ酸からなるマウス肝炎ウイルス核タンパク質のアミノ酸配列から選択した計４４６種類のポリペプチドをＳＰＯＴ合成法に従って作製し、これらを用いて作製したペプチドアレイを示す模式図である。In Example 1 (1), a total of 446 kinds of polypeptides selected from the amino acid sequence of mouse hepatitis virus nucleoprotein consisting of 455 amino acids were prepared according to the SPOT synthesis method, and a schematic diagram showing a peptide array prepared using them. is there. 実施例１（１）で作製したマウス肝炎ウイルスペプチドアレイを用いて、マウス肝炎ウイルスに自然感染したマウスの血清をイムノクロマトグラフィにより化学発光させ、各スポットの発光強度とバックグラウンドとの比の平均値をプロットしたグラフである。縦軸は発光強度の比の平均値を示し、横軸はマウス肝炎ウイルス核タンパク質アミノ酸配列のＮ末端からの順番を示す。Using the mouse hepatitis virus peptide array prepared in Example 1 (1), the serum of a mouse naturally infected with mouse hepatitis virus was chemiluminescent by immunochromatography, and the average value of the ratio of the luminescence intensity of each spot to the background Is a graph in which is plotted. The vertical axis represents the average value of the luminescence intensity ratio, and the horizontal axis represents the order from the N-terminus of the mouse hepatitis virus nucleoprotein amino acid sequence. 実施例２（１）において、５２４アミノ酸からなるセンダイウイルス核タンパク質のアミノ酸配列から選択した計５１５種類のポリペプチドをＳＰＯＴ合成法に従って作製し、これらを用いて作製したペプチドアレイを示す模式図である。In Example 2 (1), a total of 515 types of polypeptides selected from the amino acid sequence of Sendai virus nucleoprotein consisting of 524 amino acids were prepared according to the SPOT synthesis method, and a schematic diagram showing a peptide array prepared using them. . 実施例２（１）で作製したセンダイウイルスペプチドアレイを用いて、ＢＡＬＢ／ｃマウス、Ｃ５７ＢＬ／６（Ｂ６）マウスおよびＡＫＲマウスにおける同一個体の感染前の血清と感染後の血清とをイムノクロマトグラフィにより化学発光させ、各スポットの発光強度の比の平均値をプロットしたグラフである。縦軸は発光強度の比の平均値を示し、横軸はセンダイウイルス核タンパク質アミノ酸配列のＮ末端からの順番を示す。Using the Sendai virus peptide array prepared in Example 2 (1), the serum before infection and the serum after infection of the same individual in BALB / c mice, C57BL / 6 (B6) mice and AKR mice were analyzed by immunochromatography. It is the graph which chemiluminescent-emitted and plotted the average value of ratio of the emitted light intensity of each spot. The vertical axis represents the average value of the luminescence intensity ratio, and the horizontal axis represents the order from the N-terminus of the Sendai virus nucleoprotein amino acid sequence. 実施例２（１）で作製したセンダイウイルスペプチドアレイを用いて、ＢＡＬＢ／ｃマウスにおける同一個体の感染前の血清と感染後の血清とをイムノクロマトグラフィにより化学発光させ、発光が確認されたスポットを抽出し、各スポットの発光強度の比やアミノ酸配列などを表した表である。Using the Sendai virus peptide array prepared in Example 2 (1), the serum before and after infection of the same individual in BALB / c mice was chemiluminescentd by immunochromatography, and spots where luminescence was confirmed were detected. It is the table | surface which extracted and represented ratio of the luminescence intensity of each spot, an amino acid sequence, etc. 実施例２（１）で作製したセンダイウイルスペプチドアレイを用いて、Ｃ５７ＢＬ／６（Ｂ６）マウスにおける同一個体の感染前の血清と感染後の血清とをイムノクロマトグラフィにより化学発光させ、発光が確認されたスポットを抽出し、各スポットの発光強度の比やアミノ酸配列などを表した表である。Using the Sendai virus peptide array prepared in Example 2 (1), the serum before and after infection of the same individual in C57BL / 6 (B6) mice was chemiluminescent by immunochromatography, and luminescence was confirmed. 3 is a table showing the ratio of luminescence intensity of each spot and the amino acid sequence. 実施例２（１）で作製したセンダイウイルスペプチドアレイを用いて、ＡＫＲマウスにおける同一個体の感染前の血清と感染後の血清とをイムノクロマトグラフィにより化学発光させ、発光が確認されたスポットを抽出し、各スポットの発光強度の比やアミノ酸配列などを表した表である。Using the Sendai virus peptide array prepared in Example 2 (1), the serum before infection and the serum after infection of the same individual in AKR mice were chemiluminescent by immunochromatography, and spots where luminescence was confirmed were extracted. FIG. 4 is a table showing the ratio of light emission intensity of each spot, the amino acid sequence, and the like. 実施例２（１）で作製したセンダイウイルスペプチドアレイを用いて、ＢＮラットにおける同一個体の感染前の血清と感染後の血清とをイムノクロマトグラフィにより化学発光させ、発光が確認されたスポットを抽出し、各スポットの発光強度の比やアミノ酸配列などを表した表である。Using the Sendai virus peptide array prepared in Example 2 (1), the pre-infection serum and post-infection serum of the same individual in BN rats were chemiluminescent by immunochromatography, and spots where luminescence was confirmed were extracted. FIG. 4 is a table showing the ratio of light emission intensity of each spot, the amino acid sequence, and the like. 実施例２（１）で作製したセンダイウイルスペプチドアレイを用いて、Ｆ３４４ラットにおける同一個体の感染前の血清と感染後の血清とをイムノクロマトグラフィにより化学発光させ、発光が確認されたスポットを抽出し、各スポットの発光強度の比やアミノ酸配列などを表した表である。Using the Sendai virus peptide array prepared in Example 2 (1), the pre-infection serum and the post-infection serum of the same individual in F344 rats were chemiluminescent by immunochromatography, and spots where luminescence was confirmed were extracted. FIG. 4 is a table showing the ratio of light emission intensity of each spot, the amino acid sequence, and the like. 実施例３（３）において、血清の濃度を変化させた血清（正常マウス血清および感染マウス血清）についての抗マウス肝炎ウイルス抗体の検出を、配列番号４および配列番号１１のペプチドを用いてＥＬＩＳＡ法により行い、その結果を示すグラフである。縦軸は４５０ｎｍ吸光度（ＯＤ４５０）の値を示し、横軸は検体である血清の希釈倍率を示す。In Example 3 (3), detection of anti-mouse hepatitis virus antibody for serum (normal mouse serum and infected mouse serum) with different serum concentrations was performed using the peptides of SEQ ID NO: 4 and SEQ ID NO: 11 by ELISA. This is a graph showing the results. The vertical axis represents the value of 450 nm absorbance (OD450), and the horizontal axis represents the dilution factor of serum as a specimen. 実施例４（３）において、血清の濃度を変化させた血清（正常マウス血清および感染マウス血清）についての抗センダイウイルス抗体の検出を、配列番号１７および配列番号３０のペプチドを用いてＥＬＩＳＡ法により行い、その結果を示すグラフである。縦軸は４５０ｎｍ吸光度（ＯＤ４５０）の値を示し、横軸は検体である血清の希釈倍率を示す。In Example 4 (3), detection of anti-Sendai virus antibody for sera (normal mouse serum and infected mouse serum) with different serum concentrations was performed by ELISA using the peptides of SEQ ID NO: 17 and SEQ ID NO: 30. It is a graph which shows and shows the result. The vertical axis represents the value of 450 nm absorbance (OD450), and the horizontal axis represents the dilution factor of serum as a specimen. マウス肝炎ウイルス核タンパク質のアミノ酸配列と、ラット唾液腺涙腺炎ウイルス核タンパク質のアミノ酸配列とのアラインメントを示す図である。It is a figure which shows the alignment with the amino acid sequence of a mouse | mouth hepatitis virus nucleoprotein, and the amino acid sequence of a rat salivary gland ladenitis virus nucleoprotein. 市販のセンダイウイルス感染判定キットで感染陽性と判定された実験感染マウス血清（正常マウス血清および感染マウス血清）についての抗体の検出を、配列番号１７および配列番号３０のペプチドを用いてＥＬＩＳＡ法により行い、その結果を示すグラフである。縦軸は４５０ｎｍ吸光度（ＯＤ４５０）の値を示し、横軸の（－）、（＋）はそれぞれ正常マウス血清と感染マウス血清を示す。図中の破線は、正常マウス血清のＯＤ値の平均値に、正常マウス血清のＯＤ値の標準偏差の３倍の値を加えたＯＤ４５０の値を示す。Antibody detection of experimentally infected mouse serum (normal mouse serum and infected mouse serum) determined to be positive for infection using a commercially available Sendai virus infection determination kit was performed by ELISA using the peptides of SEQ ID NO: 17 and SEQ ID NO: 30. It is a graph which shows the result. The vertical axis indicates the value of 450 nm absorbance (OD450), and the horizontal axis (−) and (+) indicate normal mouse serum and infected mouse serum, respectively. The broken line in the figure indicates the value of OD450 obtained by adding 3 times the standard deviation of the OD value of normal mouse serum to the average value of OD value of normal mouse serum.
　以下、本発明に係るマウス肝炎ウイルス由来ポリペプチドおよび／またはセンダイウイルス由来ポリペプチド、これらを用いたマウス肝炎ウイルス感染および／またはセンダイウイルス感染検査キット、ならびにマウス肝炎ウイルス感染および／またはセンダイウイルス感染の検出方法について詳細に説明する。本発明に係るポリペプチドは、以下の（ａ）～（ｄ）からなる群から選ばれる１または２以上のアミノ酸配列からなり、かつマウス肝炎ウイルスに感染したマウスが産生する抗マウス肝炎ウイルス抗体および／またはセンダイウイルスに感染したラットならびに／もしくはマウスが産生する抗センダイウイルス抗体と特異的に結合する；
（ａ）マウス肝炎ウイルス核タンパク質により提示されるマウス肝炎ウイルス抗原エピトープを構成するアミノ酸配列、
（ｂ）アミノ酸配列（ａ）において１個もしくは数個のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列、
（ｃ）センダイウイルス核タンパク質により提示されるセンダイウイルス抗原エピトープを構成するアミノ酸配列、
（ｄ）アミノ酸配列（ｃ）において１個もしくは数個のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列。 Hereinafter, mouse hepatitis virus-derived polypeptide and / or Sendai virus-derived polypeptide, mouse hepatitis virus infection and / or Sendai virus infection test kit using these, and mouse hepatitis virus infection and / or Sendai virus infection The detection method will be described in detail. The polypeptide according to the present invention comprises an anti-mouse hepatitis virus antibody produced by a mouse infected with murine hepatitis virus, comprising one or more amino acid sequences selected from the group consisting of the following (a) to (d): Binds specifically to anti-Sendai virus antibodies produced by rats and / or mice infected with Sendai virus;
(A) an amino acid sequence constituting a mouse hepatitis virus antigen epitope presented by the mouse hepatitis virus nucleoprotein,
(B) an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence (a),
(C) an amino acid sequence constituting a Sendai virus antigen epitope presented by Sendai virus nucleoprotein,
(D) An amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence (c).
　本発明におけるマウス肝炎ウイルス核タンパク質から選択されるマウス肝炎ウイルス抗原エピトープまたはセンダイウイルス核タンパク質から選択されるセンダイウイルス抗原エピトープは、マウス肝炎ウイルス核タンパク質またはセンダイウイルス核タンパク質のアミノ酸配列により提示される、直鎖のアミノ酸で構成されるものと、一次構造上離れた部位にあるアミノ酸が高次構造を組むことによって生じるものとがある。 The mouse hepatitis virus antigen epitope selected from mouse hepatitis virus nucleoprotein or Sendai virus nucleoprotein selected from Sendai virus nucleoprotein in the present invention is presented by the amino acid sequence of mouse hepatitis virus nucleoprotein or Sendai virus nucleoprotein, Some are composed of straight-chain amino acids, and others are formed by amino acids located at sites distant from the primary structure forming higher-order structures.
　ここで、本発明における「エピトープ」とは、抗体によって認識される抗原上の部位の通常の意味を有し、「抗原決定基」と交換可能に用いることができる。本発明におけるエピトープは、典型的には、全タンパク質の小さな部分であるアミノ酸のセグメントであり、一次的すなわち連続的である場合のみならず、立体配座的すなわち不連続的である場合がある。すなわち、タンパク質のフォールディングによって並べられた一次配列の隣接部分または非隣接部分によってコードされるアミノ酸から形成されるものである。エピトープとして使用するためには、少なくとも３アミノ酸の長さの配列が必要であり、好ましくは、少なくとも１０アミノ酸、１１アミノ酸、１２アミノ酸、１３アミノ酸、１４アミノ酸、１５アミノ酸、１６アミノ酸、１７アミノ酸、１８アミノ酸、１９アミノ酸、２０アミノ酸の長さの配列が必要である。 Here, “epitope” in the present invention has the usual meaning of a site on an antigen recognized by an antibody, and can be used interchangeably with “antigenic determinant”. Epitopes in the present invention are typically segments of amino acids that are a small part of the total protein and may be conformational or discontinuous as well as primary or continuous. That is, it is formed from amino acids encoded by adjacent or non-adjacent portions of the primary sequence arranged by protein folding. For use as an epitope, a sequence of at least 3 amino acids in length is required, preferably at least 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 An amino acid, 19 amino acid, 20 amino acid long sequence is required.
　また、エピトープは、これに結合する抗体の結合部位すなわちパラトープと水素結合、イオン結合、疎水結合、ファンデルワールス結合により非共有的に結合する。この結合が成立するためには、エピトープ上の当該作用する部位とパラトープ上の当該作用する部位とが一定の空間配置を取る必要があるが、本発明におけるエピトープの鎖長を伸長させることにより、あるいはアミノ酸を付加させることにより、パラトープに結合可能な立体構造（高次構造）を取ることができ、エピトープとして機能する場合がある。 In addition, the epitope is non-covalently bound to the binding site of the antibody that binds to it, that is, a paratope, through a hydrogen bond, an ionic bond, a hydrophobic bond, or a van der Waals bond. In order for this binding to be established, the site of action on the epitope and the site of action on the paratope need to take a certain spatial arrangement, but by extending the chain length of the epitope in the present invention, Alternatively, by adding an amino acid, a three-dimensional structure (higher order structure) capable of binding to a paratope can be taken, and it may function as an epitope.
　本発明において、エピトープの同定は特に限定されず、当該技術分野に知られるあらゆる方法によって同定することができる。例えば、ＰＥＰＳＣＡＮ法（Ｇｅｙｓｅｎら、Ｊ．Ｉｍｍｕｎｏｌ．Ｍｅｔｈ．，第１０２巻、第２５９～２７４頁、１９８７年）やＳＰＯＴ合成法（Ｒ．Ｆｒａｎｋら、Ｔｅｔｒａｈｅｄｒｏｎ、１９９２年、第４８巻、第９２１７頁，Ｗ．Ｒ．Ｇ．Ｄｏｓｔｍａｎｎら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ、２０００年、第９７巻、１４７７２頁）に従い、ペプチドを化学合成あるいは遺伝子工学的に合成してペプチドアレイを作製し、イムノアッセイにより同定することができる。 In the present invention, epitope identification is not particularly limited, and can be identified by any method known in the art. For example, the PEPSCAN method (Geysen et al., J. Immunol. Meth., 102, 259-274, 1987) and the SPOT synthesis method (R. Frank et al., Tetrahedron, 1992, 48, 9217). , W.R.G.Dostmann et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 14772) to produce peptide arrays by chemically or genetically synthesizing peptides. Can be identified by immunoassay.
　本発明において用いることができるイムノアッセイは、当業者によって適宜選択することができ、特に限定されないが、例えば、ウェスタンブロット法、ラジオイムノアッセイ、ＥＬＩＳＡ（酵素結合免疫吸着測定法）、サンドイッチイムノアッセイ、免疫沈降測定法、沈降反応、ゲル拡散沈降反応、免疫拡散測定法、凝集測定法、補体結合測定法、免疫放射定量測定法、蛍光イムノアッセイ、プロテインＡイムノアッセイなどを挙げることができる。 The immunoassay that can be used in the present invention can be appropriately selected by those skilled in the art, and is not particularly limited. Methods, precipitation reactions, gel diffusion precipitation reactions, immunodiffusion assays, agglutination assays, complement binding assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and the like.
　また、これらイムノアッセイに用いられる二次抗体についても、当業者によって適宜選択することができ、特に限定されないが、例えば、ポリクローナル抗体、モノクローナル抗体、キメラ抗体、単鎖抗体、Ｆａｂフラグメント、Ｆａｂ発現ライブラリーによって産生されるフラグメントなどを挙げることができる。また、Ｆａｂ発現ライブラリーによって産生されるフラグメントには、マウス肝炎ウイルス抗原および／またはセンダイウイルス抗原に対する結合活性が保持されている完全な抗体のフラグメント（Ｆａｂ、Ｆ（ａｂ’）およびＦ（ａｂ’）２フラグメント）、抗体の抗原結合部位を含む単鎖抗体（ｓｃＦｖ）、融合タンパク質および他の合成タンパク質が含まれる（Ｂｉｒｄら、Ｓｃｉｅｎｃｅ、第２４２巻、第４２３～４２６頁、１９８８）。 Further, secondary antibodies used in these immunoassays can be appropriately selected by those skilled in the art, and are not particularly limited. For example, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, Fab expression libraries And the like can be mentioned. In addition, fragments produced by the Fab expression library include intact antibody fragments (Fab, F (ab ′) and F (ab ′) that retain binding activity to mouse hepatitis virus antigen and / or Sendai virus antigen. ) 2 fragments), single chain antibodies (scFv) containing the antigen binding site of the antibody, fusion proteins and other synthetic proteins (Bird et al., Science, Vol. 242, pages 423-426, 1988).
　なお、本発明に係るエピトープをコードするポリヌクレオチドを選択し、あるいは、Ｓｔｕｍｐｐ Ｍ．Ｔ．らの方法（Ｓｔｕｍｐｐ Ｍ．Ｔ．ら、Ｊ．Ｍｏｌ．Ｂｉｏｌ．第３３２（２）巻、第４７１～４８７頁、２００３）に従って得られた完全長ｃＤＮＡからポリヌクレオチドを合成して、ファージディスプレイ法、ｂａｃｔｅｒｉａ ｔｗｏ－ｈｙｂｒｉｄ法、ｙｅａｓｔ ｔｗｏ－ｈｙｂｒｉｄ法、ｉｎ ｖｉｔｒｏウイルス法等を用いて同定することもできる。 It should be noted that a polynucleotide encoding the epitope according to the present invention is selected, or StamppM. T.A. A polynucleotide was synthesized from a full-length cDNA obtained according to the above-mentioned method (Stumppp MT, et al., J. Mol. Biol. 332 (2), 471-487, 2003), and the phage display method , The Bacteria two-hybrid method, the yeast two-hybrid method, the in vitro virus method, and the like.
　マウス肝炎ウイルス核タンパク質により提示されるマウス肝炎ウイルス抗原エピトープおよび／またはセンダイウイルス核タンパク質により提示されるセンダイウイルス抗原エピトープを構成するアミノ酸配列のうち、ＳＰＯＴ合成法に従い、ペプチドを化学合成してペプチドアレイを作製し、イムノクロマトグラフィにより同定することのできるアミノ酸配列としては、配列番号３～１１や配列番号１６～３０に示されるアミノ酸配列を挙げることができる。 Among the amino acid sequences constituting the mouse hepatitis virus antigen epitope presented by the mouse hepatitis virus nucleoprotein and / or the Sendai virus antigen epitope presented by the Sendai virus nucleoprotein, the peptide array is obtained by chemically synthesizing the peptides according to the SPOT synthesis method. Examples of amino acid sequences that can be identified by immunochromatography include amino acid sequences shown in SEQ ID NOs: 3 to 11 and SEQ ID NOs: 16 to 30.
　なお、本発明において、「結合する」は、「相互作用する」、「反応する」、「認識する」と交換可能に用いられる。また、本発明において、抗体が、ある特定の抗原（免疫原）に対し「特異的に結合する」とは、前記特定抗原に対して反応性があることが明らかであればよい。前記特定抗原に対し「特異的に反応する」とは、他の抗原に全く反応しない場合も含むが、他の抗原に対し反応する場合も含む。 In the present invention, “bond” is used interchangeably with “interact”, “react”, and “recognize”. Further, in the present invention, it is sufficient that it is clear that an antibody “specifically binds” to a specific antigen (immunogen) is reactive to the specific antigen. The phrase “specifically reacts” with respect to the specific antigen includes not only reacting with other antigens at all, but also includes reacting with other antigens.
　本発明において「抗原」とは、脊椎動物にこれを投与することにより免疫応答を導き出すことができ、それによってこれと特異的に結合する抗体の産生、放出を促進させるものをいい、１または２以上のエピトープを有している。本発明における抗原は、「免疫原」と交換可能に用いることができ、サブユニット抗原や、抗体と結合した状態の複合体も本発明における抗原に含まれる。また、抗原としては、例えば、ペプチド、ポリペプチド、タンパク質、リポタンパク質、糖タンパク質、核酸、多糖類、リポ多糖類、脂質などを挙げることができるが、本発明においては、ペプチド、ポリペプチド、タンパク質、リポタンパク質、糖タンパク質であることが好ましく、ペプチド、ポリペプチド、タンパク質であることがより好ましい。 In the present invention, the “antigen” refers to an antigen that can elicit an immune response by administering it to a vertebrate, thereby promoting the production and release of an antibody that specifically binds to it. It has the above epitope. The antigen in the present invention can be used interchangeably with “immunogen”, and subunit antigens and complexes in the state of being bound to antibodies are also included in the antigen in the present invention. Examples of the antigen include peptides, polypeptides, proteins, lipoproteins, glycoproteins, nucleic acids, polysaccharides, lipopolysaccharides, lipids, etc. In the present invention, peptides, polypeptides, proteins, etc. Lipoprotein and glycoprotein are preferred, and peptides, polypeptides and proteins are more preferred.
　本発明において、「１個もしくは数個のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列」という場合の、欠失、置換、挿入および／または付加されるアミノ酸の個数は、そのアミノ酸配列を有するポリペプチドが、マウス肝炎ウイルスに感染したマウスが産生する抗マウス肝炎ウイルス抗体および／またはセンダイウイルスに感染したラットならびに／もしくはマウスが産生する抗センダイウイルス抗体と特異的に結合する限り特に限定されないが、例えば１～１９個、好ましくは１～１５個、より好ましくは１～１０個、さらに好ましくは１～７個、よりさらに好ましくは１～５個の任意の個数を挙げることができる。なお、同一あるいは性質の似たアミノ酸配列をコードするのであれば、さらに多くのアミノ酸が置換、挿入、および／または付加されてもよい。 In the present invention, in the case of “amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added”, the number of amino acids to be deleted, substituted, inserted and / or added is As long as the polypeptide having an amino acid sequence specifically binds to an anti-mouse hepatitis virus antibody produced by a mouse infected with mouse hepatitis virus and / or a rat infected with Sendai virus and / or an anti-Sendai virus antibody produced by mouse Although not particularly limited, for example, an arbitrary number of 1 to 19, preferably 1 to 15, more preferably 1 to 10, further preferably 1 to 7, and still more preferably 1 to 5 may be mentioned. it can. Further, more amino acids may be substituted, inserted, and / or added as long as they encode the same or similar amino acid sequences.
　すなわち、本発明においては、マウス肝炎ウイルス核タンパク質により提示されるマウス肝炎ウイルス抗原エピトープおよび／またはセンダイウイルス核タンパク質により提示されるセンダイウイルス抗原エピトープを構成するアミノ酸配列の全部または少なくともシグナル配列を除いた部分を含む一部と高い同一性を有するアミノ酸配列からなり、かつマウス肝炎ウイルスに感染したマウスが産生する抗マウス肝炎ウイルス抗体および／またはセンダイウイルスに感染したラットならびに／もしくはマウスが産生する抗センダイウイルス抗体と特異的に結合するペプチドが含まれる。ここにいう「高い同一性」とは、少なくとも５０％以上、好ましくは７０％以上、より好ましくは８０％以上、さらに好ましくは９０％以上、最も好ましくは９５％以上の配列の同一性を指す。 That is, in the present invention, all or at least the signal sequence of the amino acid sequence constituting the mouse hepatitis virus antigen epitope presented by the mouse hepatitis virus nucleoprotein and / or the Sendai virus antigen epitope presented by the Sendai virus nucleoprotein is excluded. An anti-mouse hepatitis virus antibody produced by a mouse infected with mouse hepatitis virus and / or a rat infected with Sendai virus and / or an anti-sendai produced by mouse, comprising an amino acid sequence having a high identity with a part including the part Peptides that specifically bind to viral antibodies are included. As used herein, “high identity” refers to sequence identity of at least 50% or more, preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more.
　本発明に係るポリペプチドには、マウス肝炎ウイルスに感染したマウスが産生する抗マウス肝炎ウイルス抗体および／またはセンダイウイルスに感染したラットならびに／もしくはマウスが産生する抗センダイウイルス抗体と特異的に結合する機能を有する限り、これらアミノ酸配列の１または複数の保存的アミノ酸置換を有するアミノ酸配列からなるペプチドが包含される。 The polypeptide according to the present invention specifically binds to an anti-mouse hepatitis virus antibody produced by a mouse infected with mouse hepatitis virus and / or a rat infected with Sendai virus and / or an anti-Sendai virus antibody produced by mouse. As long as it has a function, a peptide consisting of an amino acid sequence having one or more conservative amino acid substitutions of these amino acid sequences is included.
　本発明において、保存的アミノ酸置換とは、生じる分子の生理学的活性を変化させることなく一般的になされ得る範囲、すなわち保存的置換の範囲で認められるもの（Ｗａｔｓｏｎ ｅｔ ａｌ．，Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ ｏｆ Ｇｅｎｅなど）であり、例えば、アスパラギン酸およびグルタミン酸の酸性アミノ酸；リシン、アルギニンおよびヒスチジンの塩基性アミノ酸；アラニン、バリン、ロイシン、イソロイシン、プロリン、フェニルアラニン、メチオニンおよびトリプトファンの非極性アミノ酸；グリシン、アスパラギン、システイン、グルタミン、セリン、トレオニンおよびチロシンの極性無電荷側鎖アミノ酸；フェニルアラニン、トリプトファンおよびチロシンの芳香族アミノ酸といった側鎖に類似性のあるアミノ酸同士（アミノ酸のファミリー内部）で起こる置換を挙げることができる。同様に、アスパラギン酸およびグルタミン酸の酸性アミノ酸；リシン、アルギニンおよびヒスチジンの塩基性アミノ酸、グリシン、アラニン、バリン、ロイシン、イソロイシン、セリンおよびトレオニンの脂肪族アミノ酸（セリンおよびトレオニンの脂肪族－ヒドロキシアミノ酸と分類することもできる）；フェニルアラニン、チロシンおよびトリプトファンの芳香族アミノ酸；アスパラギンおよびグルタミンのアミド）；システインおよびメチオニンの含硫アミノ酸といった分類をすることができる。 In the present invention, a conservative amino acid substitution is a range that can generally be made without changing the physiological activity of the resulting molecule, that is, a conservative substitution (such as Watson et al., Molecular biology of Gene, etc.). For example, acidic amino acids of aspartic acid and glutamic acid; basic amino acids of lysine, arginine and histidine; nonpolar amino acids of alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan; glycine, asparagine, cysteine, Polar uncharged side chain amino acids of glutamine, serine, threonine and tyrosine; amino acids with similar side chains such as phenylalanine, tryptophan and tyrosine aromatic amino acids Substitutions that occur between acids (within the amino acid family) can be mentioned. Similarly, acidic amino acids of aspartic acid and glutamic acid; basic amino acids of lysine, arginine and histidine, aliphatic amino acids of glycine, alanine, valine, leucine, isoleucine, serine and threonine (classified as aliphatic-hydroxyamino acids of serine and threonine Can be classified); aromatic amino acids of phenylalanine, tyrosine and tryptophan; amides of asparagine and glutamine); sulfur-containing amino acids of cysteine and methionine.
　本発明におけるラットは特に限定されないが、例えば、ＳＤラット、Ｆｉｓｈｅｒラット、Ｗｉｓｔａｒラット、Ｗｉｓｔａｒ Ｈａｎｎｏｖｅｒ／Ｒｃｃラット、Ｗｉｓｔａｒ／ＳＴラット、Ｄｏｎｒｙｕラット、ＢＮラット、Ｆ３４４ラットなどの、一般に動物実験に使用される各種系統のラットを挙げることができる。 Although the rat in the present invention is not particularly limited, it is generally used for animal experiments, such as SD rat, Fisher rat, Wistar rat, Wistar Hanover / Rcc rat, Wistar / ST rat, Donryu rat, BN rat, F344 rat, etc. Various strains of rats can be mentioned.
　本発明におけるマウスもまた、特に限定されないが、例えば、Ａ、ＡＫＲ、ＢＡＬＢ／ｃ、Ｃ３Ｈ、Ｃ５７ＢＬ／６、ＤＢＡ／２、Ｂ６Ｃ３Ｆ１、ＢＤＦ１、Ｂ６Ｄ２Ｆ１、ＩＣＲなどの、一般に動物実験に使用される各種系統のマウスを挙げることができる。 The mouse in the present invention is also not particularly limited. For example, A, AKR, BALB / c, C3H, C57BL / 6, DBA / 2, B6C3F1, BDF1, B6D2F1, and ICR are generally used in various animal experiments. Mention may be made of strains of mice.
　なお、コロナウイルス科コロナウイルス属に属するＲＮＡウイルスであって、マウス肝炎ウイルスの近縁であるものとしてラット唾液腺涙腺炎ウイルス（Ｒａｔ ｓｉａｌｏｄａｃｒｙｏａｄｅｎｉｔｉｓ ｃｏｒｏｎａｖｉｒｕｓ；ＳＤＡＶ）を挙げることができる。ラット唾液腺涙腺炎ウイルスはマウス肝炎ウイルスと形態のみならず抗原性が類似しているため、マウス肝炎ウイルス核タンパク質により提示されるマウス肝炎抗原エピトープは、抗マウス肝炎ウイルス抗体のみならず、抗ラット唾液腺涙腺炎ウイルス抗体も認識する。すなわち、本発明に係るマウス肝炎ウイルス由来ポリペプチドは、マウス肝炎ウイルスに感染したマウスが産生する抗マウス肝炎ウイルス抗体と特異的に結合する他、ラット唾液腺涙腺炎ウイルスに感染したラットが産生する抗ラット唾液腺涙腺炎ウイルス抗体と特異的に結合することから、ラット唾液腺涙腺炎ウイルス感染の検出にも有効である。 As an RNA virus belonging to the genus Coronavirus belonging to the Coronaviridae family, rat salivary lacrimal adenitis virus (RAT sialadoadenitis coronavirus; SDAV) can be cited as a closely related mouse hepatitis virus. Since rat salivary lacrimal inflammation virus is similar in form and antigenicity to mouse hepatitis virus, the mouse hepatitis antigen epitope presented by mouse hepatitis virus nucleoprotein is not only anti-mouse hepatitis virus antibody but also anti-rat salivary gland It also recognizes lacrimal inflammation virus antibodies. That is, the mouse hepatitis virus-derived polypeptide according to the present invention specifically binds to an anti-mouse hepatitis virus antibody produced by a mouse infected with mouse hepatitis virus, and is also an anti-antigen produced by a rat infected with rat salivary lacrimal adenitis virus. Since it specifically binds to the rat salivary lacrimal inflammation virus antibody, it is also effective in detecting rat salivary lacrimal inflammation virus infection.
　次に、本発明は、本発明に係るポリペプチドを含むキットを提供する。本発明に係るキットは、二次抗体、標識物質その他の免疫学的検出手段の実施に有用な物質または緩衝液など、マウス肝炎ウイルス感染および／またはセンダイウイルス感染の検出手段の実施に有用な物質などをキットの構成物として含んでいてもよい。 Next, the present invention provides a kit containing the polypeptide according to the present invention. The kit according to the present invention is a substance useful for carrying out a detection means for mouse hepatitis virus infection and / or Sendai virus infection, such as a secondary antibody, a labeling substance, or a substance or buffer useful for carrying out an immunological detection means. Etc. may be included as a component of the kit.
　さらに本発明は、本発明に係るポリペプチドとラットおよび／またはマウスから採取した被検体とを接触させて抗原抗体反応の有無を判定することにより行う、マウス肝炎ウイルス感染および／またはセンダイウイルス感染の検出方法を提供する。本発明に係るマウス肝炎ウイルス感染および／またはセンダイウイルス感染の検出方法には、本発明に係るマウス肝炎ウイルス感染および／またはセンダイウイルス感染の検出方法を損なわない限り、インキュベート工程や洗浄工程などを含んでいてもよい。 Furthermore, the present invention relates to mouse hepatitis virus infection and / or Sendai virus infection, which is carried out by contacting the polypeptide of the present invention with a subject collected from rats and / or mice to determine the presence or absence of an antigen-antibody reaction. A detection method is provided. The detection method for mouse hepatitis virus infection and / or Sendai virus infection according to the present invention includes an incubation step, a washing step, and the like unless the method for detecting mouse hepatitis virus infection and / or Sendai virus infection according to the present invention is impaired. You may go out.
　本発明に係るポリペプチドを含むキットならびにマウス肝炎ウイルス感染および／またはセンダイウイルス感染の検出方法は、実験動物マウスやラットに対するマウス肝炎ウイルス感染症および／またはセンダイウイルス感染症の定期的な検査に用いることができる他、例えば、従来のマウス肝炎ウイルス感染および／またはセンダイウイルス感染検査キットやマウス肝炎ウイルス感染および／またはセンダイウイルス感染検査方法によって擬陽性とされたマウスやラットに対する検査に用いることなどが可能である。 The kit containing the polypeptide according to the present invention and the method for detecting mouse hepatitis virus infection and / or Sendai virus infection are used for periodic examination of mouse hepatitis virus infection and / or Sendai virus infection in laboratory animal mice and rats. In addition, for example, it can be used for testing of mice and rats that are falsely positive by a conventional mouse hepatitis virus infection and / or Sendai virus infection test kit or mouse hepatitis virus infection and / or Sendai virus infection test method. It is.
　以下、本発明に係るマウス肝炎ウイルス由来ポリペプチドおよび／またはセンダイウイルス由来ポリペプチド、これらを用いたマウス肝炎ウイルス感染および／またはセンダイウイルス感染検査キット、ならびにマウス肝炎ウイルス感染および／またはセンダイウイルス感染の検出方法について、実施例に基づいて説明する。なお、本発明の技術的範囲は、これらの実施例によって示される特徴に限定されない。 Hereinafter, mouse hepatitis virus-derived polypeptide and / or Sendai virus-derived polypeptide, mouse hepatitis virus infection and / or Sendai virus infection test kit using these, and mouse hepatitis virus infection and / or Sendai virus infection A detection method will be described based on examples. Note that the technical scope of the present invention is not limited to the features shown by these examples.
＜実施例１＞マウス肝炎ウイルスペプチドアレイを用いたマウス肝炎ウイルスエピトープの同定
（１）マウス肝炎ウイルスペプチドアレイの作製
　４５５アミノ酸からなるマウス肝炎ウイルス核タンパク質（マウス肝炎ウイルスＮＰ、Ａｃｃｅｓｓｉｏｎ ｎｕｍｂｅｒ Ｘ００９９０；配列番号１および配列番号２）のアミノ酸配列から、１０残基ポリペプチドを１残基ずつシフトさせることによって選択した計４４６種類のポリペプチドを、ＳＰＯＴ合成法（Ｒ．Ｆｒａｎｋら、Ｔｅｔｒａｈｅｄｒｏｎ、１９９２年、第４８巻、第９２１７頁，Ｗ．Ｒ．Ｇ．Ｄｏｓｔｍａｎｎら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ、２０００年、第９７巻、１４７７２頁）に従い、Ａｕｔｏ Ｓｐｏｔ Ｐｅｐｔｉｄｅ Ｓｙｎｔｈｅｓｉｚｅｒ ＡＳＰ－２２２（Ｉｎｔａｖｉｓ社）を用いてセルロース膜上に合成することにより、マウス肝炎ウイルスＮＰアミノ酸配列のＮ末端から１～１０番目（スポットＮｏ．１）、２～１１番目（スポットＮｏ．２）、３～１２番目（スポットＮｏ．３）と続いて４４６～４５５番目（スポットＮｏ．４４６）までの、計４４６種類の１０残基ポリペプチド鎖がセルロース膜上に順番に並んだマウス肝炎ウイルスペプチドアレイを作製した。これを図１に示す。 <Example 1> Identification of mouse hepatitis virus epitope using mouse hepatitis virus peptide array (1) Preparation of mouse hepatitis virus peptide array Mouse hepatitis virus nucleoprotein consisting of 455 amino acids (murine hepatitis virus NP, Accession number X00990; SEQ ID NO: A total of 446 polypeptides selected from the amino acid sequence of 1 and SEQ ID NO: 2) by shifting a 10-residue polypeptide one by one were synthesized by the SPOT synthesis method (R. Frank et al., Tetrahedron, 1992, No. 1). 48, page 9217, WR G. Dodmann et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 14772)) Auto Spot Peptide Synthesizer. By synthesizing on a cellulose membrane using ASP-222 (Intavis), 1st to 10th (spot No. 1), 2nd to 11th (spot No. 2) from the N-terminus of mouse hepatitis virus NP amino acid sequence A mouse hepatitis virus peptide in which a total of 446 types of 10-residue polypeptide chains from the 3rd to the 12th (spot No. 3) and subsequently the 446 to 455th (spot No. 446) are arranged in order on the cellulose membrane An array was made. This is shown in FIG.
（２）各種マウスにおけるマウス肝炎ウイルスエピトープの同定
　本実施例（１）で作製したマウス肝炎ウイルスペプチドアレイ、各種マウスの血清およびｈｏｒｓｅｒａｄｉｓｈ ｐｅｒｏｘｉｄａｓｅ（ＨＲＰ）標識抗マウスＩｇＧ抗体を用い、マウス肝炎ウイルスペプチドアレイに特異的に結合するＨＲＰ標識抗マウスＩｇＧをＥＬＩＳＡ法で検出することにより、エピトープを同定した。 (2) Identification of mouse hepatitis virus epitope in various mice Mouse hepatitis virus peptide array using mouse hepatitis virus peptide array prepared in Example (1), various mouse sera and horseradish peroxidase (HRP) -labeled anti-mouse IgG antibody Epitopes were identified by detecting HRP-labeled anti-mouse IgG that specifically binds to the ELISA by ELISA.
［２－１］１％Ｔｗｅｅｎ－２０ＰＢＳ溶液および１％Ｔｗｅｅｎ－２０ＰＢＳスキムミルク溶液の調製
　それぞれの最終濃度が、１．３７Ｍ ＮａＣｌ、２７ｍＭ ＫＣｌ、８１ｍＭ Ｎａ２ＨＰＯ４、１５ｍＭ ＫＨ２ＰＯ４となるように調製したＨｏｓｐｈａｔｅ ｂｕｆｆｅｒｅｄ ｓａｌｉｎｅ（ＰＢＳ）溶液１Ｌにつき、１０ｍＬの割合でＴｗｅｅｎ－２０を加えることにより、１％Ｔｗｅｅｎ－２０ＰＢＳ溶液（１％ＰＢＳＴ）を調製した。さらに、この１％ＰＢＳＴに重量比５％となるようスキムミルクを加えて溶解することにより、１％Ｔｗｅｅｎ－２０ＰＢＳスキムミルク溶液（１％ＰＢＳＴ－ＳＭ）を調製した。 [2-1] Preparation of 1% Tween-20 PBS Solution and 1% Tween-20 PBS Skimmed Milk Solution The phosphate buffered saline prepared to have final concentrations of 1.37 M NaCl, 27 mM KCl, 81 mM Na2HPO4, and 15 mM KH2PO4 ( A 1% Tween-20 PBS solution (1% PBST) was prepared by adding Tween-20 at a rate of 10 mL per 1 L of PBS) solution. Further, 1% Tween-20 PBS skim milk solution (1% PBST-SM) was prepared by adding and dissolving skim milk to this 1% PBST to a weight ratio of 5%.
［２－２］感染マウスの血清の調製
　マウス肝炎ウイルスに自然感染したマウスの集団から、市販のマウス肝炎ウイルス感染判定キット（モニライザＭＨＶ；わかもと製薬社）にて陽性を示した７個体を得、それらの血清を採取した。それぞれ採取した血清を、１％ＰＢＳＴ－ＳＭにて１０００倍に希釈することにより、１％ＰＢＳＴ－ＳＭ希釈血清を調製した。 [2-2] Preparation of Serum of Infected Mice From a population of mice naturally infected with mouse hepatitis virus, 7 individuals that showed a positive result with a commercially available mouse hepatitis virus infection determination kit (Monilizer MHV; Wakamoto Pharmaceutical Co., Ltd.) were obtained. Their sera were collected. Each collected serum was diluted 1000-fold with 1% PBST-SM to prepare 1% PBST-SM diluted serum.
［２－３］マウス肝炎ウイルスペプチドアレイによるエピトープの探索
　本実施例（１）で作製したマウス肝炎ウイルスペプチドアレイを、本実施例（２）［２－１］で調製した１％ＰＢＳＴ－ＳＭ５０ｍＬに浸漬させ、４℃下で一晩インキュベートした後、さらに本実施例（２）［２－１］で調製した１％ＰＢＳＴ２０ｍＬに浸漬させて、室温下で１分間振盪することにより洗浄した。この洗浄したマウス肝炎ウイルスペプチドアレイをプラスチックフィルム上に置き、１％ＰＢＳＴ－ＳＭ希釈血清をそれぞれ５ｍＬずつ乗せ、これをプラスチックフィルムで覆い、室温下で２時間インキュベートした。 [2-3] Search for epitope by mouse hepatitis virus peptide array The mouse hepatitis virus peptide array prepared in this Example (1) was added to 1% PBST-SM 50 mL prepared in this Example (2) [2-1]. After dipping and incubating at 4 ° C. overnight, the sample was further dipped in 20 mL of 1% PBST prepared in Example (2) [2-1] and washed by shaking at room temperature for 1 minute. The washed murine hepatitis virus peptide array was placed on a plastic film, and 5 mL each of 1% PBST-SM diluted serum was placed thereon, covered with the plastic film, and incubated at room temperature for 2 hours.
　インキュベート後にプラスチックフィルムを取り除いたマウス肝炎ウイルスペプチドアレイを本実施例（２）［２－１］で調製した１％ＰＢＳＴ２０ｍＬに浸漬させ、室温下で１５分×３回振盪することにより洗浄し、再度プラスチックフィルム上に置いた後、本実施例（２）［２－１］で調製した１％ＰＢＳＴ－ＳＭで１００００倍希釈したＨＲＰ標識抗マウスＩｇＧ抗体（ＧＥ Ｈｅａｌｔｈｃａｒｅ Ｂｉｏ－Ｓｃｉｅｎｃｅｓ社）をそれぞれ５ｍＬずつ乗せ、再度プラスチックフィルムで覆い、さらに室温下で２時間インキュベートした。その後、プラスチックフィルムを取り除き、本実施例（２）［２－１］で調製した１％ＰＢＳＴ２０ｍＬに浸漬させ、１５分×３回振盪することにより洗浄した。 The mouse hepatitis virus peptide array from which the plastic film was removed after incubation was immersed in 20 mL of 1% PBST prepared in this Example (2) [2-1], washed by shaking for 15 minutes × 3 times at room temperature, and again After placing on a plastic film, 5 mL each of HRP-labeled anti-mouse IgG antibody (GE Healthcare Bio-Sciences) diluted 10,000 times with 1% PBST-SM prepared in this Example (2) [2-1] It was placed, covered again with a plastic film, and further incubated at room temperature for 2 hours. Thereafter, the plastic film was removed and the plate was immersed in 20 mL of 1% PBST prepared in Example (2) [2-1] and washed by shaking for 15 minutes × 3 times.
　ＨＲＰ標識抗マウスＩｇＧを反応させたマウス肝炎ウイルスペプチドアレイを、ＥＣＬ Ａｄｖａｎｃｅ Ｗｅｓｔｅｒｎ Ｂｌｏｔｔｉｎｇ Ｄｅｔｅｃｔｉｏｎ Ｋｉｔ（ＧＥ Ｈｅａｌｔｈｃａｒｅ Ｂｉｏ－Ｓｃｉｅｎｃｅｓ社）を用いて化学発光させた後、さらにＬＡＳ－３０００（Ｆｕｊｉｆｉｌｍ社）を用いて、化学発光が確認された各スポット（各ポリペプチド）の検出、画像化および発光強度の数値化を行うことにより、マウス肝炎ウイルスペプチドアレイに特異的に結合したＩｇＧ抗体を検出して定量化した。 A mouse hepatitis virus peptide array reacted with HRP-labeled anti-mouse IgG was chemiluminescent using ECL Advanced Western Blotting Detection Kit (GE Healthcare Bio-Sciences), and further using LAS-3000 (Fujifilm). Detection and quantification of IgG antibody specifically bound to mouse hepatitis virus peptide array by detection, imaging and quantification of luminescence intensity of each spot (each polypeptide) in which chemiluminescence was confirmed .
　マウス肝炎ウイルスに自然感染したマウスの血清の、各スポットの発光強度とバックグラウンドとの比の平均値をプロットしたものを図２に示し、マウス肝炎ウイルスに自然感染したマウスの血清のうち、発光が確認されたスポットを抽出して得られたアミノ酸配列と、それらの配列番号とを表１に示す。 A plot of the average value of the ratio of the luminescence intensity of each spot to the background of the mouse serum naturally infected with mouse hepatitis virus is shown in FIG. Table 1 shows the amino acid sequences obtained by extracting the spots on which the above was confirmed and their SEQ ID NOs.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　図１、図２および表１に示すように、マウス肝炎ウイルスＮＰアミノ酸配列のＮ末端から１～１４番目（配列番号３）、同２４～３９番目（配列番号４）、同４９～６４番目（配列番号５）、同６９～８２番目（配列番号６）、同２４０～２５３番目（配列番号７）、同３０７～３２５番目（配列番号８）、同３５４～３７３番目（配列番号９）および同３８１～３９３番目（配列番号１０）に相当する各スポットの発光強度が、これらのバックグラウンドの発光強度との比で大きいことが確認された。また、このうち、マウス肝炎ウイルスに自然感染したマウス７個体すべてにおいて、バックグラウンドの発光強度との比が２以上であるスポットのアミノ酸配列は、Ｎ末端から２４～３９番目（配列番号４）、３０７～３２５番目（配列番号８）および３５４～３７３番目（配列番号９）であることが確認された。 As shown in FIG. 1, FIG. 2 and Table 1, from the N-terminus of the mouse hepatitis virus NP amino acid sequence, the 1st to 14th (SEQ ID NO: 3), the 24th to 39th (SEQ ID NO: 4), the 49th to 64th ( SEQ ID NO: 5), 69th to 82nd (SEQ ID NO: 6), 240th to 253rd (SEQ ID NO: 7), 307th to 325th (SEQ ID NO: 8), 354 to 373rd (SEQ ID NO: 9) and the same It was confirmed that the luminescence intensity of each spot corresponding to the 381st to 393rd (SEQ ID NO: 10) was large in ratio to the luminescence intensity of these backgrounds. Of these, in all 7 mice naturally infected with the mouse hepatitis virus, the amino acid sequence of the spot having a ratio of 2 or more to the background luminescence intensity is 24th to 39th from the N-terminus (SEQ ID NO: 4), It was confirmed that they were positions 307 to 325 (SEQ ID NO: 8) and 354 to 373 (SEQ ID NO: 9).
＜実施例２＞センダイウイルスペプチドアレイを用いたセンダイウイルスエピトープの同定
（１）センダイウイルスペプチドアレイの作製
　５２４アミノ酸からなるセンダイウイルス核タンパク質（センダイウイルスＮＰ、Ａｃｃｅｓｓｉｏｎ ｎｕｍｂｅｒ Ｘ０００８７；配列番号１４および配列番号１５）のアミノ酸配列から、１０残基ポリペプチドを１残基ずつシフトさせることによって選択した計５１５種類のポリペプチドを、実施例１（１）と同様の方法によりセルロース膜上に合成することにより、センダイウイルスＮＰアミノ酸配列のＮ末端から１～１０番目（スポットＮｏ．１）、２～１１番目（スポットＮｏ．２）、３～１２番目（スポットＮｏ．３）と続いて５１５～５２４番目（スポットＮｏ．５１５）までの、計５１５種類の１０残基ポリペプチド鎖がセルロース膜上に順番に並んだセンダイウイルスペプチドアレイを作製した。これを図３に示す。 <Example 2> Identification of Sendai virus epitope using Sendai virus peptide array (1) Preparation of Sendai virus peptide array Sendai virus nucleoprotein consisting of 524 amino acids (Sendai virus NP, Accession number X00087; SEQ ID NO: 14 and SEQ ID NO: 15) ) By synthesizing a total of 515 kinds of polypeptides selected by shifting 10-residue polypeptides one by one from the amino acid sequence on the cellulose membrane in the same manner as in Example 1 (1), Sendai virus NP amino acid sequence N-terminal 1 to 10 (spot No. 1), 2 to 11 (spot No. 2), 3 to 12 (spot No. 3), followed by 515 to 524 (spot No. 515), total 51 Type 10 residue polypeptide chains to produce a Sendai virus peptide arrays arranged in order on a cellulose membrane. This is shown in FIG.
（２）各種マウスおよびラットにおけるセンダイウイルスエピトープの同定
　本実施例（１）で作製したセンダイウイルスペプチドアレイ、各種マウスおよびラットの血清およびＨＲＰ標識抗マウスＩｇＧ抗体を用い、実施例１（２）と同様の方法により、エピトープを同定した。 (2) Identification of Sendai virus epitopes in various mice and rats Using the Sendai virus peptide array prepared in Example (1), various mouse and rat sera and HRP-labeled anti-mouse IgG antibody, Example 1 (2) and Epitopes were identified by similar methods.
［２－１］各種マウスおよびラットの血清の調製
　６個体のＢＡＬＢ／ｃマウス、６個体のＣ５７ＢＬ／６（Ｂ６）マウス、５個体のＡＫＲマウス、５個体のＢＮラットおよび４個体のＦ３４４ラット（日本エスエルシー社）の各個体の血清（正常マウス血清）を採取した後、センダイウイルスＭＮ株（１×１０２ ＴＣＩＤ５０）をそれぞれ実験感染させ、市販のセンダイウイルス感染判定キット（モニライザＨＶＪ；わかもと製薬社）にて感染を確認し、感染後１４日目の血清（感染マウス血清）を採取した。それぞれ採取した血清を実施例１（２）［２－１］で調製した１％ＰＢＳＴ－ＳＭにて１０００倍に希釈することにより、１％ＰＢＳＴ－ＳＭ希釈血清を調製した。 [2-1] Serum preparation of various mice and rats 6 BALB / c mice, 6 C57BL / 6 (B6) mice, 5 AKR mice, 5 BN rats and 4 F344 rats ( After collecting serum (normal mouse serum) of each individual of Japan SLC, Inc., each was experimentally infected with Sendai virus MN strain (1 × 102 TCID50), and a commercially available Sendai virus infection determination kit (Monilizer HVJ; Wakamoto Pharmaceutical Co., Ltd.) ), And the serum (infected mouse serum) 14 days after infection was collected. Each collected serum was diluted 1000-fold with 1% PBST-SM prepared in Example 1 (2) [2-1] to prepare 1% PBST-SM diluted serum.
［２－２］センダイウイルスペプチドアレイによるエピトープの探索
　本実施例（２）［２－１］で調製した１％ＰＢＳＴ－ＳＭ希釈血清を用いて、実施例１（２）［２－２］と同様の方法により、センダイウイルスペプチドアレイに特異的に結合したＩｇＧ抗体を検出して定量化した。 [2-2] Search for epitopes by Sendai virus peptide array Using the 1% PBST-SM diluted serum prepared in Example (2) [2-1], Example 1 (2) [2-2] and By the same method, IgG antibody specifically bound to the Sendai virus peptide array was detected and quantified.
　ＢＡＬＢ／ｃマウス、Ｃ５７ＢＬ／６（Ｂ６）マウスおよびＡＫＲマウスにおける同一個体の感染前の血清と感染後の血清との、各スポットの発光強度の比の平均値をプロットしたものを図４に示し、ＢＡＬＢ／ｃマウス、Ｃ５７ＢＬ／６（Ｂ６）マウス、ＡＫＲマウス、ＢＮラットおよびＦ３４４ラットにおける同一個体の感染前の血清と感染後の血清のうち、発光が確認されたスポットを抽出し、各スポットの発光強度の比やアミノ酸配列などを表したものをそれぞれ図５～図９に示す。また、これらアミノ酸配列と配列番号とを表２に示す。 FIG. 4 shows a plot of the average value of the ratio of the luminescence intensity of each spot between the serum before infection and the serum after infection of the same individual in BALB / c mice, C57BL / 6 (B6) mice and AKR mice. Of sera before and after infection of the same individual in BALB / c mice, C57BL / 6 (B6) mice, AKR mice, BN rats, and F344 rats, and spots where luminescence was confirmed were extracted. FIGS. 5 to 9 show the ratio of the luminescence intensity and the amino acid sequence, respectively. These amino acid sequences and SEQ ID NOs are shown in Table 2.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　図４～図９に示すように、センダイウイルスＮＰアミノ酸配列のＮ末端から１１９～１３４番目（配列番号１７）、同１４４～１５８番目（配列番号１８）、同４１９～４３０番目（配列番号２３）、同４５７～４７１番目（配列番号２５）、同４６４～４７５番目（配列番号２６）、同４７２～４８７番目（配列番号２７）および同４８７～５００番目（配列番号２８）に相当する各スポットの発光強度の比が、各個体および各系統に共通して大きいことが確認された。また、センダイウイルスＮＰアミノ酸配列のＮ末端から５０３～５１６番目（配列番号２９）に相当するスポットの発光強度の比が、ＢＮラットおよびＦ３４４ラットに共通して大きいことが確認された。以上の結果から、センダイウイルスＮＰアミノ酸配列のＮ末端から１１９～１３４番目（配列番号１７）、同１４４～１５８番目（配列番号１８）、同４１９～４３０番目（配列番号２３）、同４５７～４７１番目（配列番号２５）、同４６４～４７５番目（配列番号２６）、同４７２～４８７番目（配列番号２７）および同４８７～５００番目（配列番号２８）がセンダイウイルスＮＰのエピトープであることが示された。また、センダイウイルスＮＰアミノ酸配列のＮ末端から５０３～５１６番目（配列番号２９）はラットに特異的に抗原性を有するセンダイウイルスＮＰのエピトープであることが示された。以上の結果をまとめて表３に示す。 As shown in FIGS. 4 to 9, positions 119 to 134 (SEQ ID NO: 17), 144 to 158 (SEQ ID NO: 18), and 419 to 430 (SEQ ID NO: 23) from the N-terminus of the Sendai virus NP amino acid sequence. 457 to 471 (SEQ ID NO: 25), 464 to 475 (SEQ ID NO: 26), 472 to 487 (SEQ ID NO: 27), and 487 to 500 (SEQ ID NO: 28). It was confirmed that the ratio of luminescence intensity was large for each individual and each line. It was also confirmed that the ratio of the luminescence intensity of the spots corresponding to the 503th to 516th positions (SEQ ID NO: 29) from the N-terminal of the Sendai virus NP amino acid sequence was large in both the BN rat and the F344 rat. From the above results, from the N-terminal of the Sendai virus NP amino acid sequence, 119 to 134th (SEQ ID NO: 17), 144 to 158th (SEQ ID NO: 18), 419 to 430 (SEQ ID NO: 23), 457 to 471 (SEQ ID NO: 25), 464 to 475 (SEQ ID NO: 26), 472 to 487 (SEQ ID NO: 27), and 487 to 500 (SEQ ID NO: 28) are epitopes of Sendai virus NP. It was done. Further, it was shown that the 503 to 516th positions (SEQ ID NO: 29) from the N-terminal of the Sendai virus NP amino acid sequence are epitopes of Sendai virus NP having antigenicity specifically in rats. The above results are summarized in Table 3.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
＜実施例３＞マウス肝炎ウイルスＮＰエピトープペプチドの感染マウス特異性の確認
　実施例１（２）［２－３］で同定したマウス肝炎ウイルスＮＰエピトープの感染マウス特異性を確認するため、マウス肝炎ウイルスＮＰアミノ酸配列のＮ末端から２４～３９番目（配列番号４）およびＮ末端から３５７～３７２番目（ＲＦＤＳＴＬＰＧＦＥＴＩＭＫＶＬ（Ｒはアルギニン、Ｆはフェニルアラニン、Ｄはアスパラギン酸、Ｓはセリン、Ｔはトレオニン、Ｌはロイシン、Ｐはプロリン、Ｇはグリシン、Ｅはグルタミン酸、Ｉはイソロイシン、Ｍはメチオニン、Ｋはリシン、Ｖはバリンを示す）：配列番号１１）のペプチドを化学合成し、ＥＬＩＳＡ法により、これらに結合する抗体の検出を行った。 <Example 3> Confirmation of mouse specificity for infection with mouse hepatitis virus NP epitope peptide In order to confirm the specificity of mouse infection with mouse hepatitis virus NP epitope identified in Example 1 (2) [2-3] 24th to 39th from the N-terminus of the NP amino acid sequence (SEQ ID NO: 4) and 357-372th from the N-terminus (RFDSTLPGFETIMKVL (R is arginine, F is phenylalanine, D is aspartic acid, S is serine, T is threonine, L is Leucine, P is proline, G is glycine, E is glutamic acid, I is isoleucine, M is methionine, K is lysine, and V is valine): SEQ ID NO: 11) The binding antibody was detected.
（１）０．５％Ｔｗｅｅｎ－２０ＰＢＳ溶液およびブロッキング溶液の調製
　それぞれの最終濃度が、１．３７Ｍ ＮａＣｌ、２７ｍＭ ＫＣｌ、８１ｍＭ Ｎａ２ＨＰＯ４、１５ｍＭ ＫＨ２ＰＯ４となるように調製したＰＢＳ溶液１Ｌにつき、５ｍＬの割合でＴｗｅｅｎ－２０を加えることにより、０．５％Ｔｗｅｅｎ－２０ＰＢＳ溶液（０．５％ＰＢＳＴ）を調製した。さらに、この０．５％ＰＢＳＴに重量比０．５％となるようウシ血清アルブミン（ＢＳＡ）を加えて溶解することにより、ブロッキング液を調製した。 (1) Preparation of 0.5% Tween-20 PBS solution and blocking solution Each final solution concentration is 1.37 M NaCl, 27 mM KCl, 81 mM Na2HPO4, 15 mM KH2PO4 at a rate of 5 mL. A 0.5% Tween-20 PBS solution (0.5% PBST) was prepared by adding Tween-20. Furthermore, a blocking solution was prepared by adding bovine serum albumin (BSA) to this 0.5% PBST to a weight ratio of 0.5% and dissolving.
（２）感染マウスの血清の調製
　マウス肝炎ウイルスに自然感染したマウスの集団から、市販のマウス肝炎ウイルス感染判定キット（モニライザＭＨＶ；わかもと製薬社）にて陽性を示した１４個体を得、それらの血清（感染マウス血清）と、マウス肝炎ウイルスに感染していない８個体の血清（正常マウス血清）とを採取した。それぞれ採取した血清を本実施例（１）で調製したブロッキング液にて、それぞれ５０倍、１００倍、２００倍、４００倍、８００倍、１６００倍、３２００倍および６４００倍に希釈することにより、各濃度のブロッキング液希釈血清を調製した。 (2) Preparation of Serum of Infected Mice From the population of mice naturally infected with mouse hepatitis virus, 14 individuals that showed a positive result with a commercially available mouse hepatitis virus infection determination kit (Monilizer MHV; Wakamoto Pharmaceutical Co., Ltd.) were obtained. Serum (infected mouse serum) and serum of 8 individuals not infected with mouse hepatitis virus (normal mouse serum) were collected. Each of the collected sera was diluted 50 times, 100 times, 200 times, 400 times, 800 times, 1600 times, 3200 times and 6400 times with the blocking solution prepared in this Example (1), respectively. Concentration blocking solution diluted serum was prepared.
（３）ＥＬＩＳＡ法による抗体の検出
　配列番号４および配列番号１１のペプチドを０．１Ｍ Ｎａ２ＣＯ３に溶解し、０．５μＭに調製した。これを９６ウェル平底プレート（ＢＤ Ｂｉｏｓｃｉｅｎｃｅ社）に２００μＬずつ加え、４℃下で一晩インキュベートした後、本実施例（１）で調製した０．５％ＰＢＳＴ ２００μＬで各ウェルを３回洗浄し、本実施例（１）で調製したブロッキング液２００μＬを各ウェルに加え、さらに３７℃下で１時間インキュベートした。次いで、各ウェルを本実施例（１）で調製した０．５％ＰＢＳＴ ２００μＬで３回洗浄し、本実施例２（２）で調製した血清を２００μＬずつ加え、３７℃下で１時間インキュベートした後、再び各ウェルを本実施例２（１）で調製した０．５％ＰＢＳＴ ２００μＬで３回洗浄した。本実施例２（１）で調製したブロッキング液を用いて１００００倍に希釈したＨＲＰ標識抗マウスＩｇＧ抗体（ＧＥ　Ｈｅａｌｔｈｃａｒｅ　Ｂｉｏ－Ｓｃｉｅｎｃｅｓ社）を各ウェルに２００μＬずつ加え、３７℃下で１時間インキュベートした後、各ウェルを本実施例２（１）で調製した０．５％ＰＢＳＴ ２００μＬで３回洗浄し、１．５ｍｇ／ｍＬのｏ－フェニレンジアミン・２塩酸（ＯＰＤ）溶液を２００μＬずつ加え、３７℃下で１０分間インキュベートした。各ウェルに３．５Ｎ硫酸を５０μＬずつ加えて発色反応を停止させ、ＥＬＩＳＡプレートリーダー（モデル６８０マイクロプレートリーダー；バイオ・ラッド　ラボラトリーズ社）で４５０ｎｍ吸光度（ＯＤ４５０）を測定した。その結果を図１０に示す。 (3) Detection of antibody by ELISA method The peptides of SEQ ID NO: 4 and SEQ ID NO: 11 were dissolved in 0.1 M Na2CO3 to prepare 0.5 μM. 200 μL of this was added to a 96-well flat bottom plate (BD Bioscience) and incubated overnight at 4 ° C., and then each well was washed three times with 200 μL of 0.5% PBST prepared in this Example (1). 200 μL of the blocking solution prepared in this Example (1) was added to each well, and further incubated at 37 ° C. for 1 hour. Next, each well was washed three times with 200 μL of 0.5% PBST prepared in this Example (1), 200 μL of the serum prepared in this Example 2 (2) was added and incubated at 37 ° C. for 1 hour. Thereafter, each well was washed again with 200 μL of 0.5% PBST prepared in Example 2 (1) three times. 200 μL of HRP-labeled anti-mouse IgG antibody (GE Healthcare Bio-Sciences) diluted 10,000 times using the blocking solution prepared in Example 2 (1) was added to each well and incubated at 37 ° C. for 1 hour. Thereafter, each well was washed 3 times with 200 μL of 0.5% PBST prepared in Example 2 (1), and 200 μL of a 1.5 mg / mL o-phenylenediamine · 2 hydrochloric acid (OPD) solution was added. Incubated for 10 minutes at 0 ° C. The color development reaction was stopped by adding 50 μL of 3.5 N sulfuric acid to each well, and the absorbance at 450 nm (OD450) was measured with an ELISA plate reader (Model 680 microplate reader; Bio-Rad Laboratories). The result is shown in FIG.
　図１０に示すように、配列番号４および配列番号１１のいずれのペプチドを抗原として用いた場合でも、正常マウス血清のＯＤ値は濃度にかかわらずほぼ一定の値であるのに対し、感染マウス血清のＯＤ値は濃度の低下に伴い低下することが確認された。以上の結果より、配列番号４および配列番号１１のペプチドは、マウス肝炎ウイルス感染マウスの血清に含まれる抗体に特異的に結合することが示された。 As shown in FIG. 10, the OD value of normal mouse serum is almost constant regardless of the concentration when any of the peptides of SEQ ID NO: 4 and SEQ ID NO: 11 is used as an antigen. It was confirmed that the OD value decreased with decreasing concentration. From the above results, it was shown that the peptides of SEQ ID NO: 4 and SEQ ID NO: 11 specifically bind to the antibody contained in the serum of mouse hepatitis virus-infected mice.
＜実施例４＞センダイウイルスＮＰエピトープペプチドの感染マウス特異性の確認
　実施例２（２）［２－２］で同定したセンダイウイルスＮＰエピトープの感染マウス特異性を確認するため、センダイウイルスＮＰアミノ酸配列のＮ末端から１１９～１３４番目（配列番号１７）およびＮ末端から４５８～４７３番目｛ＦＶＴＬＨＧＡＥＲＬＥＥＥＴＮＤ（Ｆはフェニルアラニン、Ｖはバリン、Ｔはトレオニン、Ｌはロイシン、Ｈはヒスチジン、Ｇはグリシン、Ａはアラニン、Ｅはグルタミン酸、Ｒはアルギニン、Ｎはアスパラギン、Ｄはアスパラギン酸を示す）：配列番号３０｝のペプチドを化学合成し、ＥＬＩＳＡ法により、これらに結合する抗体の検出を行った。 <Example 4> Confirmation of infected mouse specificity of Sendai virus NP epitope peptide In order to confirm the specificity of Sendai virus NP epitope identified in Example 2 (2) [2-2], the Sendai virus NP amino acid sequence was confirmed. 119-134 from the N-terminus (SEQ ID NO: 17) and 458-473 from the N-terminus {FVTLHGAERLEEEETND (F is phenylalanine, V is valine, T is threonine, L is leucine, H is histidine, G is glycine, and A is Alanine, E is glutamic acid, R is arginine, N is asparagine, and D is aspartic acid): SEQ ID NO: 30} was chemically synthesized, and antibodies bound to these were detected by ELISA.
　Ｃ５７ＢＬ／６（Ｂ６）マウス（日本エスエルシー社）１個体の血清（正常マウス血清）と、センダイウイルスＭＮ株（１ｘ１０２ ＴＣＩＤ５０）を実験感染させたＣ５７ＢＬ／６（Ｂ６）マウス（財団法人実験動物中央研究所ＩＣＬＡＳモニタリングセンターより譲受）１個体の感染後１４日目の血清（感染マウス血清）とを採取した。それぞれ採取した血清を実施例３（１）で調製したブロッキング液にて、それぞれ５０倍、１００倍、２００倍、４００倍、８００倍、１６００倍、３２００倍および６４００倍に希釈することにより、各濃度のブロッキング液希釈血清を調製し、実施例３（３）と同様の方法により、４５０ｎｍ吸光度（ＯＤ４５０）を測定することによって抗体を検出した。その結果を図１１に示す。 C57BL / 6 (B6) mouse (Japan SLC, Inc.) C57BL / 6 (B6) mouse (laboratory animal center) experimentally infected with one individual's serum (normal mouse serum) and Sendai virus MN strain (1 × 10 2 TCID50) (Acquired from the Institute ICLAS Monitoring Center) One individual's serum (infected mouse serum) on the 14th day after infection was collected. Each of the collected sera was diluted 50 times, 100 times, 200 times, 400 times, 800 times, 1600 times, 3200 times and 6400 times with the blocking solution prepared in Example 3 (1), respectively. A blocking solution-diluted serum having a concentration was prepared, and the antibody was detected by measuring the absorbance at 450 nm (OD450) in the same manner as in Example 3 (3). The result is shown in FIG.
　図１１に示すように、配列番号１７および配列番号３０のいずれのペプチドを抗原として用いた場合でも、正常マウス血清のＯＤ値は濃度にかかわらずほぼ一定の値であるのに対し、感染マウス血清のＯＤ値は濃度の低下に伴い低下することが確認された。以上の結果より、配列番号１７および配列番号３０のペプチドは、センダイウイルス感染マウスの血清に含まれる抗体に特異的に結合することが示された。 As shown in FIG. 11, even when any peptide of SEQ ID NO: 17 and SEQ ID NO: 30 is used as an antigen, the OD value of normal mouse serum is almost constant regardless of the concentration, whereas infected mouse serum It was confirmed that the OD value decreased with decreasing concentration. From the above results, it was shown that the peptides of SEQ ID NO: 17 and SEQ ID NO: 30 specifically bind to antibodies contained in the serum of Sendai virus-infected mice.
＜実施例５＞マウス肝炎ウイルスＮＰエピトープペプチドによるウイルス感染の検出
　実施例１（２）［２－３］で同定し、実施例３（３）で確認されたマウス肝炎ウイルスＮＰエピトープを用いて、市販のマウス肝炎ウイルス感染判定キットで感染陽性と判定された実験感染マウスについて、マウス肝炎ウイルス感染の検出が可能かどうかを検討した。また、マウス肝炎ウイルス感染の判定の感度について、市販のマウス肝炎ウイルス感染判定キット（モニライザＭＨＶ；わかもと製薬社）との比較を行った。 <Example 5> Detection of viral infection with mouse hepatitis virus NP epitope peptide Using the mouse hepatitis virus NP epitope identified in Example 1 (2) [2-3] and confirmed in Example 3 (3), We examined whether mouse hepatitis virus infection could be detected in experimentally infected mice that were determined to be positive for infection using a commercially available mouse hepatitis virus infection determination kit. Moreover, the sensitivity of the determination of mouse hepatitis virus infection was compared with a commercially available mouse hepatitis virus infection determination kit (Monilizer MHV; Wakamoto Pharmaceutical Co., Ltd.).
（１）マウスの血清の調製
　マウス肝炎ウイルスに自然感染したマウスの集団から得られた１４個体の血清と、マウス肝炎ウイルスに感染していない８個体の血清を採取した。それぞれ採取した血清を実施例２（１）で調製したブロッキング液を用いて、それぞれ５０倍に希釈することによりブロッキング液希釈血清を調製した。 (1) Preparation of mouse sera 14 sera obtained from a group of mice naturally infected with mouse hepatitis virus and 8 sera not infected with mouse hepatitis virus were collected. Each of the collected sera was diluted 50 times with the blocking solution prepared in Example 2 (1) to prepare a blocking solution diluted serum.
（２）ＥＬＩＳＡ法による検出
　実施例３（３）で用いた配列番号４および配列番号１１のペプチドと、本実施例（１）で調製したブロッキング液希釈血清とを用いて、実施例３（３）と同様の方法により、４５０ｎｍ吸光度（ＯＤ４５０）を測定することによって抗体を検出した。その結果を表４に示す。 (2) Detection by ELISA method Example 3 (3) using the peptides of SEQ ID NO: 4 and SEQ ID NO: 11 used in Example 3 (3) and the blocking solution-diluted serum prepared in this Example (1) The antibody was detected by measuring the absorbance at 450 nm (OD450) in the same manner as in (1). The results are shown in Table 4.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　表４に示すように、正常マウス８個体については、配列番号４および配列番号１１のいずれのペプチドを用いた場合でも、８個体すべてにおいて陰性と判定された。また、市販のマウス肝炎ウイルス感染判定キット（モニライザＭＨＶ；わかもと製薬社）を用いた場合でも、正常マウス８個体すべてにおいて陰性と判定された。一方、マウス肝炎ウイルスに自然感染したマウスの集団から得られた１４個体については、いずれのペプチドを用いた場合でも、１０個体が陽性と判定され、残り４個体が陰性と判定されたが、市販のマウス肝炎ウイルス感染判定キット（モニライザＭＨＶ；わかもと製薬社）を用いた場合では、８個体が陽性と判定され、残り６個体が陰性と判定された。配列番号４および配列番号１１のいずれのペプチドを用いた場合に陽性と判定された１０個体のうちの８個体は、市販のマウス肝炎ウイルス感染判定キット（モニライザＭＨＶ；わかもと製薬社）で陽性と判定された８個体と一致した。以上の結果より、配列番号４および配列番号１１のペプチドは従来のマウス肝炎ウイルス感染判定キットよりも高感度にマウス肝炎ウイルス感染の判定を行うことができることが示された。 As shown in Table 4, for 8 normal mice, even when any peptide of SEQ ID NO: 4 and SEQ ID NO: 11 was used, all 8 individuals were determined to be negative. In addition, even when a commercially available mouse hepatitis virus infection determination kit (Monilizer MHV; Wakamoto Pharmaceutical Co., Ltd.) was used, it was determined to be negative in all 8 normal mice. On the other hand, for 14 individuals obtained from a population of mice naturally infected with mouse hepatitis virus, 10 individuals were determined to be positive and the remaining 4 individuals were determined to be negative regardless of which peptide was used. When using the mouse hepatitis virus infection determination kit (Monilizer MHV; Wakamoto Pharmaceutical Co., Ltd.), 8 individuals were determined to be positive, and the remaining 6 individuals were determined to be negative. Eight out of 10 individuals determined to be positive when using any of the peptides of SEQ ID NO: 4 and SEQ ID NO: 11 were determined to be positive by a commercially available mouse hepatitis virus infection determination kit (Monilizer MHV; Wakamoto Pharmaceutical Co., Ltd.). Matched 8 individuals. From the above results, it was shown that the peptides of SEQ ID NO: 4 and SEQ ID NO: 11 can determine mouse hepatitis virus infection with higher sensitivity than conventional mouse hepatitis virus infection determination kits.
＜実施例６＞センダイウイルスＮＰエピトープペプチドによるウイルス感染の検出
　実施例１（２）［２－３］で同定し、実施例２（３）で確認されたセンダイウイルスＮＰエピトープを用いて、市販のセンダイウイルス感染判定キットで感染陽性と判定された実験感染マウスについて、センダイウイルス感染の検出が可能かどうかを検討した。 <Example 6> Detection of viral infection by Sendai virus NP epitope peptide Using the Sendai virus NP epitope identified in Example 1 (2) [2-3] and confirmed in Example 2 (3), commercially available We investigated whether Sendai virus infection could be detected in experimentally infected mice that were determined to be positive for infection using the Sendai virus infection determination kit.
（１）マウスの血清の調製
　ＢＡＬＢ／ｃマウス、Ｃ５７ＢＬ／６（Ｂ６）マウスおよびＡＫＲマウスの各４個体（日本エスエルシー社）、合計１２個体の血清（正常マウス血清）と、センダイウイルスＭＮ株（１ｘ１０２ ＴＣＩＤ５０）を実験感染させるとともに市販のセンダイウイルス感染判定キット（モニライザＨＶＪ；わかもと製薬社）を用いてそれぞれ感染陽性と判定した６個体のＢＡＬＢ／ｃマウス、３個体のＣ５７ＢＬ／６（Ｂ６）マウスおよび５個体のＡＫＲマウス、合計１４個体の感染後１４日目の血清（感染マウス血清）とを採取した。これらの血清を実施例２（１）で調製したブロッキング液を用いて、それぞれ５０倍に希釈することによりブロッキング液希釈血清を調製した。 (1) Preparation of mouse serum 4 BALB / c mice, 4 C57BL / 6 (B6) mice and 4 AKR mice each (Japan SLC), a total of 12 sera (normal mouse serum), Sendai virus MN strain 6 BALB / c mice, 3 C57BL / 6 (B6), each of which was experimentally infected with (1 × 102 TCID50) and determined to be positive for infection using a commercially available Sendai virus infection determination kit (Monilizer HVJ; Wakamoto Pharmaceutical Co., Ltd.). A total of 14 mice and 5 sera of AKR mice were collected with 14 days post-infection serum (infected mouse serum). These serums were diluted 50-fold with the blocking solution prepared in Example 2 (1) to prepare blocking solution-diluted sera.
（２）ＥＬＩＳＡ法による検出
　実施例３（３）で用いた配列番号１７および配列番号３０のペプチドと、本実施例（１）で調製したブロッキング液希釈血清とを用いて、実施例３（３）と同様の方法により、４５０ｎｍ吸光度（ＯＤ４５０）を測定することによって抗体を検出した。その結果を図１２に示す。 (2) Detection by ELISA method Example 3 (3) using the peptides of SEQ ID NO: 17 and SEQ ID NO: 30 used in Example 3 (3) and the blocking solution-diluted serum prepared in this Example (1) The antibody was detected by measuring the absorbance at 450 nm (OD450) in the same manner as in (1). The result is shown in FIG.
　図１２に示すように、配列番号１７および配列番号３０のいずれのペプチドを抗原として用いた場合でも、正常マウス血清の場合のＯＤ値は低値であるのに対し、感染マウス血清の場合のＯＤ値は高値であることが確認された。また、正常マウス血清のＯＤ値の平均値に、正常マウス血清のＯＤ値の標準偏差の３倍の値（３ＳＤ）を加えた値を、感染陰性と感染陽性との境界値とみなした場合でも、感染マウス血清の場合のＯＤ値のすべてがこの境界値を上回ることから、配列番号１７および配列番号３０のいずれのペプチドを抗原として用いた場合においても、市販のセンダイウイルス感染判定キット（モニライザＨＶＪ；わかもと製薬社）を用いて感染陽性と判定したセンダイウイルス感染マウスのすべてにおいて陽性と判定することができることが示された。以上の結果より、実施例１（２）で同定した配列番号１７および配列番号３０のセンダイウイルスＮＰエピトープペプチドは、マウスのセンダイウイルス感染の検出に使用可能であることが示された。 As shown in FIG. 12, the OD value in the case of normal mouse serum is low in the case of using any of the peptides of SEQ ID NO: 17 and SEQ ID NO: 30 as the antigen, whereas the OD value in the case of infected mouse serum. The value was confirmed to be high. Even when a value obtained by adding a value (3SD) three times the standard deviation of the OD value of normal mouse serum to the average value of OD value of normal mouse serum is regarded as a boundary value between infection negative and infection positive Since all of the OD values in the case of infected mouse sera exceed this boundary value, a commercially available Sendai virus infection determination kit (Monilizer HVJ) can be used when any peptide of SEQ ID NO: 17 and SEQ ID NO: 30 is used as an antigen. It was shown that all Sendai virus-infected mice determined to be positive for infection using Wakamoto Pharmaceutical Co., Ltd.) can be determined to be positive. From the above results, it was shown that the Sendai virus NP epitope peptides of SEQ ID NO: 17 and SEQ ID NO: 30 identified in Example 1 (2) can be used for detection of Sendai virus infection in mice.
＜比較例＞マウス肝炎ウイルス核タンパク質アミノ酸配列とラット唾液腺涙腺炎ウイルス核タンパク質アミノ酸配列との比較
　ラット唾液腺涙腺炎ウイルス核タンパク質（ラット唾液腺涙腺炎ウイルスＮＰ、Ａｃｃｅｓｓｉｏｎ ｎｕｍｂｅｒ Ｄ１０７６０；配列番号１２および配列番号１３）は４５４アミノ酸からなる。ここで、マウス肝炎ウイルスＮＰのアミノ酸配列（４５５アミノ酸、配列番号２）と、このラット唾液腺涙腺炎ウイルスＮＰのアミノ酸配列（配列番号１３）とのアラインメントを図１３に示す。 <Comparative example> Comparison between mouse hepatitis virus nucleoprotein amino acid sequence and rat salivary lacrimal adenitis virus nucleoprotein amino acid sequence Rat salivary lacrimal inflammation virus nucleoprotein (rat salivary lacrimal inflammation virus NP, Accession number D10760; SEQ ID NO: 12 and SEQ ID NO: 13) ) Consists of 454 amino acids. Here, FIG. 13 shows an alignment between the amino acid sequence of mouse hepatitis virus NP (455 amino acids, SEQ ID NO: 2) and the amino acid sequence of rat salivary gland adenitis virus NP (SEQ ID NO: 13).
　図１３に示すように、マウス肝炎ウイルスＮＰとラット唾液腺涙腺炎ウイルスＮＰとのアミノ酸配列の相同性は９３．４％であることが分かる。特に、ラット唾液腺涙腺炎ウイルスＮＰのアミノ酸配列と配列番号３～配列番号１１に示されるアミノ酸配列とを比較すると、ラット唾液腺涙腺炎ウイルスＮＰのＮ末端から３８番目のグルタミンが配列番号４においてはロイシンであること、同５９番目のトレオニンが配列番号５においてはセリンであること、同３２１番目のプロリンが配列番号８においてはアラニンであること、同３８３番目のアラニンが配列番号１０においてはアスパラギン酸あることのみ相違していることが分かる。このことから、配列番号３～配列番号１１に示されるアミノ酸配列からなるペプチドは、抗マウスＩｇＧ抗体（抗マウス肝炎ウイルス抗体）のみならず、抗ラット唾液腺涙腺炎ウイルス抗体を認識することが示された。 As shown in FIG. 13, it can be seen that the homology of the amino acid sequence of mouse hepatitis virus NP and rat salivary lacrimal adenitis virus NP is 93.4%. In particular, when comparing the amino acid sequence of rat salivary lacrimal inflammation virus NP with the amino acid sequences shown in SEQ ID NO: 3 to SEQ ID NO: 11, the 38th glutamine from the N-terminal of rat salivary lacrimal inflammation virus NP is leucine in SEQ ID NO: 4. That the 59th threonine is serine in SEQ ID NO: 5, the 321st proline is alanine in SEQ ID NO: 8, and the 383rd alanine is aspartic acid in SEQ ID NO: 10. It can be seen that only this is different. This indicates that the peptide consisting of the amino acid sequence shown in SEQ ID NO: 3 to SEQ ID NO: 11 recognizes not only anti-mouse IgG antibody (anti-mouse hepatitis virus antibody) but also anti-rat salivary lacrimal adenitis virus antibody. It was.
　以上のような本実施例によれば、マウス肝炎ウイルスに感染したマウスが産生する抗マウス肝炎ウイルス抗体および／またはセンダイウイルスに感染したラットならびに／もしくはマウスが産生する抗センダイウイルス抗体に対する特異性や捕捉性の高いペプチドを作製することができ、かつ、マウス肝炎ウイルス感染および／またはセンダイウイルス感染を簡便かつ高感度で検出することができる。 According to the present Example as described above, the specificity to the anti-mouse hepatitis virus antibody produced by the mouse infected with the mouse hepatitis virus and / or the rat infected with Sendai virus and / or the anti-Sendai virus antibody produced by the mouse, A peptide with high capture ability can be prepared, and mouse hepatitis virus infection and / or Sendai virus infection can be detected easily and with high sensitivity.
　なお、本発明に係るマウス肝炎ウイルス由来ポリペプチドおよび／またはセンダイウイルス由来ポリペプチド、これらを用いたマウス肝炎ウイルス感染および／またはセンダイウイルス感染検査キット、ならびにマウス肝炎ウイルス感染および／またはセンダイウイルス感染の検出方法は、前述した実施形態に限定されるものではなく、適宜変更することができる。 In addition, the mouse hepatitis virus-derived polypeptide and / or Sendai virus-derived polypeptide according to the present invention, mouse hepatitis virus infection and / or Sendai virus infection test kit using these, and mouse hepatitis virus infection and / or Sendai virus infection The detection method is not limited to the embodiment described above, and can be changed as appropriate.

Claims (5)

  1. 　以下の（ａ）～（ｄ）からなる群から選ばれる１または２以上のアミノ酸配列からなり、かつマウス肝炎ウイルスに感染したマウスが産生する抗マウス肝炎ウイルス抗体および／またはセンダイウイルスに感染したラットならびに／もしくはマウスが産生する抗センダイウイルス抗体と特異的に結合するポリペプチド；
    （ａ）マウス肝炎ウイルス核タンパク質により提示されるマウス肝炎ウイルス抗原エピトープを構成するアミノ酸配列、
    （ｂ）アミノ酸配列（ａ）において１個もしくは数個のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列、
    （ｃ）センダイウイルス核タンパク質により提示されるセンダイウイルス抗原エピトープを構成するアミノ酸配列、
    （ｄ）アミノ酸配列（ｃ）において１個もしくは数個のアミノ酸が欠失、置換、挿入および／または付加されたアミノ酸配列。     Rats infected with anti-mouse hepatitis virus antibody and / or Sendai virus, which consists of one or more amino acid sequences selected from the group consisting of the following (a) to (d) and produced by mice infected with mouse hepatitis virus And / or a polypeptide that specifically binds to an anti-Sendai virus antibody produced by a mouse;
    (A) an amino acid sequence constituting a mouse hepatitis virus antigen epitope presented by the mouse hepatitis virus nucleoprotein,
    (B) an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence (a),
    (C) an amino acid sequence constituting a Sendai virus antigen epitope presented by Sendai virus nucleoprotein,
    (D) An amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence (c).
  2. 　マウス肝炎ウイルス核タンパク質により提示されるマウス肝炎ウイルス抗原エピトープを構成するアミノ酸配列が配列番号３～１１に示されるアミノ酸配列からなる群から選択されるいずれかのアミノ酸配列である、請求項１に記載のポリペプチド。 The amino acid sequence constituting the mouse hepatitis virus antigen epitope presented by the mouse hepatitis virus nucleoprotein is any amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 3 to 11. Polypeptide.
  3. 　センダイウイルス核タンパク質により提示されるセンダイウイルス抗原エピトープを構成するアミノ酸配列が配列番号１６～３０に示されるアミノ酸配列からなる群から選択されるいずれかのアミノ酸配列である、請求項１に記載のポリペプチド。 The polyamino acid according to claim 1, wherein the amino acid sequence constituting the Sendai virus antigen epitope presented by the Sendai virus nucleoprotein is any amino acid sequence selected from the group consisting of the amino acid sequences shown in SEQ ID NOs: 16 to 30. peptide.
  4. 　請求項１から請求項３のいずれかに記載のポリペプチドを含む、マウス肝炎ウイルス感染および／またはセンダイウイルス感染検査キット。 A test kit for mouse hepatitis virus infection and / or Sendai virus infection, comprising the polypeptide according to any one of claims 1 to 3.
  5. 　請求項１から請求項３のいずれかに記載のポリペプチドとラットおよび／またはマウスから採取した被検体とを接触させて抗原抗体反応の有無を判定することにより行う、マウス肝炎ウイルス感染および／またはセンダイウイルス感染の検出方法。 4. A mouse hepatitis virus infection and / or carried out by contacting the polypeptide according to any one of claims 1 to 3 with a subject collected from rats and / or mice and determining the presence or absence of an antigen-antibody reaction. How to detect Sendai virus infection.
PCT/JP2010/057188 2009-04-23 2010-04-22 Polypeptide derived from mouse hepatitis virus and/or polypeptide derived from sendai virus, test kit for infection by mouse hepatitis virus and/or sendai virus using the polypeptide, and method for detecting infection by mouse hepatitis virus and/or sendai virus WO2010123083A1 (en)

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