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WO2010123083A1 - Polypeptide issu du virus de l'hépatite de la souris et/ou du polypeptide issu du virus sendai, coffret d'essai pour une infection par le virus de l'hépatite de la souris et/ou le virus sendai utilisant le polypeptide, et procédé de détection d'une infection par le virus de l'hépatite de la souris et/ou le virus sendai - Google Patents

Polypeptide issu du virus de l'hépatite de la souris et/ou du polypeptide issu du virus sendai, coffret d'essai pour une infection par le virus de l'hépatite de la souris et/ou le virus sendai utilisant le polypeptide, et procédé de détection d'une infection par le virus de l'hépatite de la souris et/ou le virus sendai Download PDF

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Publication number
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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Prior art keywords
mouse
virus
mouse hepatitis
hepatitis virus
amino acid
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English (en)
Japanese (ja)
Inventor
高志 安居院
宣哉 佐々木
大輔 鳥越
淳 浅野
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Hokkaido University NUC
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Hokkaido University NUC
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Priority claimed from JP2009104905A external-priority patent/JP2010254600A/ja
Priority claimed from JP2009104906A external-priority patent/JP2010254601A/ja
Application filed by Hokkaido University NUC filed Critical Hokkaido University NUC
Publication of WO2010123083A1 publication Critical patent/WO2010123083A1/fr
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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

L'invention porte sur un polypeptide issu d'un virus de l'hépatite de la souris et/ou un polypeptide issu d'un virus sendai qui a une spécificité élevée pour un anticorps anti-virus de l'hépatite de la souris et/ou un anticorps anti-virus sendai et une aptitude élevée de piégeage de l'anticorps, l'anticorps anti-virus de l'hépatite de la souris et/ou l'anticorps anti-virus sendai étant obtenus par une souris infectée par un virus de l'hépatite de la souris et/ou un rat et/ou une souris infecté par un virus sendai ; sur un coffret d'essai pour une infection par le virus de l'hépatite de la souris et/ou une infection par le virus sendai, qui utilise le polypeptide et peut détecter l'infection par le virus de l'hépatite de la souris et/ou l'infection par le virus sendai avec une sensibilité élevée ; et un procédé de détection de l'infection par le virus de l'hépatite de la souris et/ou de l'infection par le virus sendai. De façon spécifique, l'invention porte sur un polypeptide qui comprend une séquence spécifique d'acides aminés et qui peut se lier spécifiquement à l'anticorps anti-virus de l'hépatite de la souris obtenu par une souris infectée par un virus de l'hépatite de la souris et/ou un anticorps anti-virus sendai produit par un rat et/ou une souris infecté par un virus sendai.
PCT/JP2010/057188 2009-04-23 2010-04-22 Polypeptide issu du virus de l'hépatite de la souris et/ou du polypeptide issu du virus sendai, coffret d'essai pour une infection par le virus de l'hépatite de la souris et/ou le virus sendai utilisant le polypeptide, et procédé de détection d'une infection par le virus de l'hépatite de la souris et/ou le virus sendai Ceased WO2010123083A1 (fr)

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JP2009104906A JP2010254601A (ja) 2009-04-23 2009-04-23 マウス肝炎ウイルス由来ポリペプチドおよびこれを用いたマウス肝炎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の検出方法

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WO2014151265A1 (fr) * 2013-03-15 2014-09-25 The Trustees Of The University Of Pennsylvania Procédés et compositions pour stimuler une réponse immunitaire
CN103159833A (zh) * 2013-03-22 2013-06-19 中国医学科学院医学实验动物研究所 一种仙台病毒抗原肽及其在仙台病毒感染检测中的应用
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