WO2022071492A1 - Fusion protein for suppressing influenza virus, and pharmaceutical composition containing same - Google Patents

Fusion protein for suppressing influenza virus, and pharmaceutical composition containing same Download PDF

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WO2022071492A1
WO2022071492A1 PCT/JP2021/036149 JP2021036149W WO2022071492A1 WO 2022071492 A1 WO2022071492 A1 WO 2022071492A1 JP 2021036149 W JP2021036149 W JP 2021036149W WO 2022071492 A1 WO2022071492 A1 WO 2022071492A1
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fusion protein
amino acid
protein
seq
deficient
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PCT/JP2021/036149
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French (fr)
Japanese (ja)
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賢二 関川
文雄 高岩
統 小林
寛 小宮山
一朗 小原
栄虎 山村
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キリンホールディングス株式会社
株式会社グリーンバイオメッド
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • 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
    • C07K14/08—RNA viruses
    • C07K14/11—Orthomyxoviridae, e.g. influenza virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the present invention relates to a fusion protein for suppressing influenza virus and a pharmaceutical composition containing the same.
  • influenza virus the HA antigen present in the outer membrane of influenza virus particles is marketed as a seasonal influenza virus type A HA split vaccine.
  • Influenza virus HA gene undergoes genetic exchange between subtypes A, and mutations in the base sequence cause antigenic variation, but mutations are concentrated in the head of the three-dimensional structure of HA protein. Since the HA head is the major neutralizing antibody epitope, the HA split vaccines and virus inactivated vaccines on the market have little effect on new mutants.
  • the stem region has few mutations but low immunogenicity, so it has not been used as a vaccine antigen for subtype A.
  • Non-Patent Document 1 An anti-stem antibody capable of neutralizing types 1 and 2 was induced in animals immunized with HA in which the head region of the HA gene was deleted. Since then, the development of a universal vaccine using a stem antigen has been promoted in the world (Non-Patent Documents 3 to 6). On the other hand, since the monomeric antigen protein has low immunogenicity, the weak point of the HA split vaccine is that it is ineffective.
  • Non-Patent Documents 7-9 It has been reported that the fusion protein of HA and ferritin enhances the immunogenicity of HA by forming a multimer by the oligomerization activity of ferritin (Non-Patent Documents 10 and 11).
  • the host immune response with the influenza virus HA split vaccine and inactivated vaccine is humoral immunity that neutralizes the virus primarily by inducing antibodies to the HA antigen, while another that destroys infected cells and suppresses the spread of virus infection.
  • the immune response is cell-mediated immunity.
  • the influenza virus antigen (CTL epitope) that binds to the major histocompatibility complex antigen class 1 molecule of infected cells and is recognized by the T cell receptor of cytotoxic T cells (CTL) is the matrix (M1) protein and nucleocapsid (NP). It has been reported to be found in proteins (Non-Patent Documents 12-14).
  • Non-Patent Documents 15 and 16 For the development status of influenza virus type A vaccine, refer to Non-Patent Documents 17 and 18.
  • the present inventors obtained a protein having excellent characteristics by further deleting the amino acid sequence at a specific site in the head-deficient HA-M1 fusion protein designed based on the HA and M1 of influenza virus.
  • the present invention is based on this finding.
  • the present invention provides a head-deficient HA-M1 fusion protein having excellent characteristics, and a pharmaceutical composition containing the same.
  • the present invention includes the following inventions.
  • the HA protein has a protein having an amino acid sequence represented by the 18th to 566 amino acid residues in SEQ ID NO: 1, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence, and has an amino acid sequence.
  • the M1 protein has a protein having an amino acid sequence represented by the 1st to 252nd amino acid residues in SEQ ID NO: 5, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence, and , A protein that functions as an M1 protein derived from influenza virus.
  • At least the amino acid residues in the regions corresponding to the 59th to 335th amino acid residues and the 529th to 554th amino acid residues in SEQ ID NO: 1 are deleted, and A partial defect in which at least the amino acid residues in the regions corresponding to the 18th to 50th amino acid residues, the 340th to 528th amino acid residues in SEQ ID NO: 1 and the 1st to 252nd amino acid residues in SEQ ID NO: 5 are retained.
  • HA-M1 fusion protein At least the amino acid residues in the regions corresponding to the 59th to 335th amino acid residues and the 529th to 554th amino acid residues in SEQ ID NO: 1 are deleted, and A partial defect in which at least the amino acid residues in the regions corresponding to the 18th to 50th amino acid residues, the 340th to 528th amino acid residues in SEQ ID NO: 1 and the 1st to 252nd amino acid residues in SEQ ID NO: 5 are retained.
  • the partially deficient HA-M1 fusion protein according to any one of (1) to (3) above which comprises the HA protein and the M1 protein in this order from the N-terminal to the C-terminal.
  • the linker is a GSG linker, GSGSG linker, GSGSGSGS linker, GSAGSA linker, or GGGGGSGGGGGSGGGS linker.
  • An expression vector comprising the nucleic acid molecule according to (7) above.
  • a transformant comprising the nucleic acid molecule described in (7) above or the expression vector described in (8) above.
  • a method for producing a partially deficient HA-M1 fusion protein which comprises culturing or growing the transformant according to (9) or (10) above.
  • a pharmaceutical comprising the partially defective HA-M1 fusion protein according to any one of (1) to (6), the nucleic acid molecule according to (7), or the expression vector according to (8).
  • Composition (13) The pharmaceutical composition according to (12) above, for preventing or treating an influenza virus infection.
  • the pharmaceutical composition according to (12) above for use as a vaccine against influenza virus.
  • the partially defective HA-M1 fusion protein according to any one of (1) to (6), the nucleic acid molecule according to (7), or the expression vector according to (8) is administered to the subject.
  • the partially defective HA-M1 fusion protein according to any one of (1) to (6), the nucleic acid molecule according to (7), or the expression vector according to (8) is administered to the subject.
  • a method of inducing a protective immune response against an influenza virus in the subject is administered.
  • a head-deficient HA-M1 fusion protein having excellent characteristics is provided.
  • the fusion protein of the present invention can prevent or treat an influenza virus infection.
  • the fusion protein of the present invention is advantageous in that it can be produced in transformed rice and yeast.
  • FIG. 1 is a diagram showing the structure of an expression vector of a head-deficient HA-M1 fusion protein in rice.
  • FIG. 2 is a photograph showing the results of electrophoresis of proteins in inecalus and culture medium expressing the head-deficient HA-M1 fusion protein gene.
  • FIG. 3 is a diagram showing an outline of the improved head-deficient HA-M1 fusion protein.
  • FIG. 4 is a photograph showing the results of western blotting of Inekals expressing the improved head-deficient HA-M1 protein.
  • FIG. 5 is a diagram showing an outline of a re-improved head-deficient HA-M1 fusion protein rice expression vector.
  • FIG. 6 is a photograph showing the expression of the re-improved head-deficient HA-M1 fusion protein in seeds.
  • FIG. 7 is a diagram showing an outline of vector construction for expression of the re-improved head-deficient HA-M1 fusion protein D in rice callus.
  • FIG. 8 is a photograph showing the results of SDS-PAGE and Western blotting of 6 independent lines of transformants by Vector A and 5 independent lines of transformants by Vector B.
  • FIG. 9 is a photograph showing the results of Western blotting of 5 independent lines of the transformant by Vector B, 4 independent lines of the transformant by Vector Hei, and 5 independent lines of the transformant by Vector Ding. be.
  • FIG. 10 is a diagram showing the structures of the plasmids for yeast expression of the head-deficient HA-M1 fusion protein and the improved head-deficient HA-M1 fusion protein B.
  • FIG. 11 is a photograph showing the expression of the head-deficient HA-M1 fusion protein and the improved head-deficient HA-M1 fusion protein B by Saccharomyces cerevisiae.
  • FIG. 12 is a diagram showing an outline of a vector for expressing various head-deficient fusion proteins in yeast.
  • FIG. 13 is a photograph showing the expression of various head-deficient fusion proteins by yeast.
  • FIG. 14 is a diagram showing an outline of a drug efficacy test using influenza virus-infected mice in Example 7.
  • FIG. 15-1 is a diagram showing the results of a drug efficacy test in influenza virus-infected mice using the improved fusion protein.
  • FIG. 15-2 is a diagram showing the results of a drug efficacy test in influenza virus-infected mice using the improved fusion protein.
  • FIG. 16 is a diagram showing an outline of a drug efficacy test using influenza virus-infected mice in Example 8.
  • FIG. 17-1 is a diagram showing the results of a drug efficacy test in influenza virus-infected mice using the improved fusion protein and the re-improved fusion protein.
  • FIG. 17-2 is a diagram showing the results of a drug efficacy test in influenza virus-infected mice using the improved fusion protein and the re-improved fusion protein.
  • the fusion protein of the present invention is a partially deficient HA-M1 fusion protein in which an amino acid residue in a specific region is deleted in a fusion protein of an HA protein derived from influenza virus and an M1 protein.
  • influenza virus examples include type A, type B, type C, and the like, and any of these may be used, but type A influenza virus is preferable.
  • the HA protein is a protein having an amino acid sequence represented by the 18th to 566 amino acid residues in SEQ ID NO: 1, or at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence.
  • Amino acid sequences other than the amino acid sequences represented by the 18th to 566 amino acid residues in SEQ ID NO: 1 replace amino acid residues in the original amino acid sequence with amino acid residues having the same or similar properties (conservative). It can be mutated by substitution).
  • the M1 protein is a protein having an amino acid sequence represented by the 1st to 252nd amino acid residues in SEQ ID NO: 5, or at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence.
  • Amino acid sequences other than the amino acid sequences represented by the 1st to 252nd amino acid residues in SEQ ID NO: 5 replace amino acid residues in the original amino acid sequence with amino acid residues having the same or similar properties (conservative). It can be mutated by substitution).
  • sequence identity between amino acid sequences can be calculated using a known sequence comparison program such as BLAST. In these programs, the parameters can be changed according to the purpose, but the default parameters may be used.
  • amino acid residues in the regions corresponding to the 59th to 335th amino acid residues and the 529 to 554 amino acid residues in SEQ ID NO: 1 are deleted.
  • amino acid residue in the region corresponding to refers not only to the amino acid residue itself in the specified region in a specific amino acid sequence, but also to the amino acid in the region at the same position in another amino acid sequence. It is also used to mean a residue. That is, the HA protein and M1 protein in the present invention are not only proteins having a specific amino acid sequence (18th to 566 amino acid residues in SEQ ID NO: 1 and 1st to 252 amino acid residues in SEQ ID NO: 5, respectively).
  • proteins having an amino acid sequence having 90% or more sequence identity include proteins having an amino acid sequence having 90% or more sequence identity with these. Therefore, when a protein having an amino acid sequence having a specific sequence identity different from the original amino acid sequence is used in the present invention, the amino acid residue in the region at the same position as the region specified in the original amino acid sequence is used. The group will be missing (or retained).
  • the 59th to 335th amino acid residues, the 51st to 335th amino acid residues, the 59th to 339th amino acid residues, or the 51st to 51st amino acid residues in SEQ ID NO: 1 are used.
  • the amino acid residue in the region corresponding to the 339 amino acid residue is missing.
  • the amino acid residue in the region corresponding to the 555 to 566 amino acid residue in SEQ ID NO: 1 is deleted.
  • the amino acid residue in the region corresponding to the 18th to 50th amino acid residues in SEQ ID NO: 1, the 340th to 528 amino acid residues, and the 1st to 252nd amino acid residues in SEQ ID NO: 5 is at least retained.
  • the amino acid residues in the regions corresponding to the 51st to 339th amino acid residues and the 529th to 554th amino acid residues in SEQ ID NO: 1 are deleted. Moreover, other amino acid residues are retained.
  • An example of the amino acid sequence of this fusion protein is shown in SEQ ID NO: 61 (where the GSGSG linker is inserted in place of the missing 51-339 amino acid residue), and the nucleotide sequence encoding this is shown in SEQ ID NO: 12.
  • amino acid residues in the regions corresponding to amino acid residues 51 to 335 and amino acids 529 to 554 in SEQ ID NO: 1 are deleted. Moreover, other amino acid residues are retained.
  • An example of the amino acid sequence of this fusion protein is shown in SEQ ID NO: 62 (where the GSG linker is inserted in place of the missing 51-335 amino acid residues), and the nucleotide sequence encoding this is shown in SEQ ID NO: 14.
  • the amino acid residues in the regions corresponding to the 59th to 339th amino acid residues and the 529 to 554th amino acid residues in SEQ ID NO: 1 are deleted. Moreover, other amino acid residues are retained.
  • An example of the amino acid sequence of this fusion protein is shown in SEQ ID NO: 63 (where the GSGSG linker is inserted in place of the missing 59-339 amino acid residue), and the nucleotide sequence encoding this is shown in SEQ ID NO: 15.
  • amino acid residues in the regions corresponding to amino acid residues 59 to 335 and amino acids 529 to 554 in SEQ ID NO: 1 are deleted. Moreover, other amino acid residues are retained.
  • An example of the amino acid sequence of this fusion protein is shown in SEQ ID NO: 64 (where a GSG linker is inserted in place of the missing 59-335 amino acid residue), and the nucleotide sequence encoding this is shown in SEQ ID NO: 26.
  • An example of the amino acid sequence of this fusion protein is SEQ ID NO: 65 (GSG linker is inserted in place of the missing 51-335 amino acid residue, and GSAGSA linker is inserted in place of the missing 555-566 amino acid residue. ), And the nucleotide sequence encoding this is shown in SEQ ID NO: 25.
  • An example of the amino acid sequence of this fusion protein is SEQ ID NO: 66 (GSG linker is inserted in place of the missing 59-335 amino acid residue, and GSAGSA linker is inserted in place of the missing 555-566 amino acid residue. ), And the nucleotide sequence encoding this is shown in SEQ ID NO: 27.
  • the HA protein and the M1 protein may be contained in this order from the N-terminal to the C-terminal, or may be contained in the reverse order, but preferably in this order. include.
  • a linker may be inserted in a region lacking an amino acid residue. Further, in the fusion protein of the present invention, a linker may be inserted at the fusion site of the partially defective HA protein and the M1 protein.
  • the length of the linker (linker peptide) is not particularly limited as long as the fusion protein of the present invention has an inhibitory effect on influenza virus, but is usually 1 to 100 amino acids, preferably 1 to 50 amino acids, and more preferably 1 to 25 amino acids. , More preferably set to 1 to 15 amino acids.
  • linker peptide examples include a linker peptide in which a flexible peptide having no secondary structure, which is composed of a peptide bond of glycine, serine, etc., is linked in a plurality of tandems, and more specifically. , (GGGGS) n (eg, GGGGGSGGGGSGGGGS linker, etc.), GSG linker, GSGSG linker, GSGSGSGS linker, GSAGSA linker and the like.
  • the fusion protein of the present invention may have an additional sequence at its N-terminal and / or C-terminal.
  • the length of the additional sequence is not particularly limited as long as the fusion protein of the present invention has an inhibitory effect on influenza virus, but is usually 1 to 100 amino acids, preferably 1 to 50 amino acids, more preferably 1 to 25 amino acids, still more preferable. Is 1 to 15 amino acids.
  • the additional sequence can be a tag sequence for facilitating purification or detection of the fusion protein of the invention.
  • the type of tag sequence is not particularly limited, but consists of, for example, FLAG (Hopp, T.P. et al., BioTechnology (1988) 6, 1204-1210) and 6 to 10 His (histidine) residues. 6 ⁇ His-10 ⁇ His, human c-myc fragment, ⁇ -tubulin fragment, B-tag, Protein C fragment, GST (glutathione-S-transferase), ⁇ -galactosidase, MBP (maltose binding protein) S Examples include tags, T7 tags, Strep tags, Nus tags, Trx tags, GFP tags and the like.
  • a signal sequence that can be actuated in the host expressing the fusion protein of the present invention for example, a secretory signal, an endoplasmic reticulum mooring signal (for example, KDEL sequence) and the like can also be preferably used.
  • the fusion protein of the present invention can be produced by using a well-known method for producing a recombinant protein.
  • a nucleic acid molecule preferably DNA
  • an expression vector is prepared by incorporating the nucleic acid molecule (preferably DNA) into an appropriate vector
  • the nucleic acid molecule or the expression vector is incorporated into a host for transformation.
  • the fusion protein of the present invention can be produced by producing a transformant and culturing or growing the transformant.
  • the host used to generate the transformant may be any host available for recombinant production of the protein, eg, bacteria such as Escherichia coli, fungi such as yeast, plant cells or plants, animal cells or Examples include animals.
  • the host is a yeast (eg, Saccharomyces cerevisiae ) or a plant (eg, rice).
  • the transformant may be prepared by introducing an expression vector into a host cell, or by incorporating a nucleic acid molecule into the genome of the host cell by homologous recombination.
  • the expression vector can be selected to function appropriately in that host, depending on the host used.
  • the nucleic acid molecule encoding the fusion protein of the present invention can be configured to contain many codons that are frequently used in the host to be used.
  • expression control sequences such as promoters, enhancers, and terminators that function appropriately in the host may be arranged before and after the coding sequence.
  • the pharmaceutical composition of the present invention comprises the fusion protein of the present invention.
  • a pharmaceutical composition can be obtained by formulating the fusion protein of the present invention according to a conventional method.
  • the pharmaceutical composition of the present invention may contain the nucleic acid molecule of the present invention encoding the fusion protein of the present invention or the expression vector of the present invention, and such a pharmaceutical composition may also be produced according to conventional means. can.
  • the pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include, for example, excipients such as sucrose, starch, mannit, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, cellulose, methylcellulose, hydroxypropylcellulose, gelatin, Arabia.
  • Excipients such as rubber, polyethylene glycol, sucrose, starch, disintegrants such as starch, carboxymethyl cellulose, hydroxypropyl starch, sodium-glycol-starch, sodium hydrogen carbonate, calcium phosphate, calcium citrate, magnesium stearate, aerodil, talc , Lubricants such as sodium lauryl sulfate, fragrances such as citric acid, menthol, glycyrrhizin / ammonium salt, glycine, orange powder, preservatives such as sodium benzoate, sodium hydrogen sulfite, methylparaben, propylparaben, citric acid, citrate Stabilizers such as sodium citrate and acetic acid, suspending agents such as methyl cellulose, polyvinylpyrrolidone and aluminum stearate, dispersants such as surfactants, diluents such as water and physiological saline, cocoa butter, polyethylene glycol, white kerosene and the like.
  • the pharmaceutical composition of the present invention may further contain an adjuvant in order to enhance the immune response-inducing effect of the fusion protein of the present invention.
  • an adjuvant include, but are not limited to, aluminum hydroxide, complete Freund's adjuvant, incomplete Freund's adjuvant, Bordetella pertussis adjuvant, poly (I: C), CpG-DNA, 2', 3'-cGAMP and the like.
  • composition of the present invention is provided as a dosage form suitable for oral or parenteral administration (preferably parenteral administration).
  • composition for parenteral administration for example, an injection, a nasal administration (nasal drop, nasal spray, etc.), a suppository, etc. are used, and the injection is an intravenous injection, a subcutaneous injection, a skin. Dosage forms such as internal injection, intramuscular injection, and drip injection may be included.
  • Such injections and nasal administrations can be prepared according to known methods.
  • the fusion protein of the present invention can be prepared by dissolving or suspending it in a sterile aqueous solvent usually used for injections.
  • aqueous solvent for injection for example, distilled water; physiological saline; a phosphate buffer solution, a carbonate buffer solution, a Tris buffer solution, a citrate buffer solution, a buffer solution such as an acetate buffer solution, or the like can be used.
  • the pH of such an aqueous solvent is 5 to 10, preferably 6 to 8.
  • the prepared injection solution or nasal administration solution is a prefilled type that is filled in an appropriate ampoule or vial, or is previously filled in a syringe or a nasal drop drug injector.
  • a powder formulation of the fusion protein of the present invention can be obtained. It can also be prepared.
  • the fusion protein of the present invention can be used by storing it in a powder state and dispersing the powder in an aqueous solvent for injection or nasal administration at the time of use.
  • the content of the fusion protein of the present invention in the pharmaceutical composition is usually about 0.1 to 100% by mass, preferably about 1 to 99% by mass, more preferably about 10 to 90% by mass, based on the whole pharmaceutical composition. ..
  • the fusion protein of the present invention can be used for the prevention or treatment of influenza virus infections. That is, by administering the fusion protein of the present invention or the pharmaceutical composition of the present invention to an animal (including animals in general, but especially mammals such as primates such as humans and mammals such as rodents such as mice), in the animals. , Can prevent or treat influenza virus infections.
  • the fusion protein of the invention comprises an antigen or fragment thereof containing a B cell epitope conserved between subtypes of the virus and an antigen or fragment thereof containing a T cell epitope conserved between the subtypes of the virus.
  • influenza virus infection by inducing a humoral and cellular immunity against influenza virus, i.e., by inducing a protective immune response in the mammal, by administration to a mammal).
  • influenza virus infection by inducing a humoral and cellular immunity against influenza virus, i.e., by inducing a protective immune response in the mammal, by administration to a mammal).
  • the fusion protein of the present invention or the pharmaceutical composition of the present invention may be administered to animals that may be infected with influenza virus (particularly, primates such as humans and mammals such as rodents such as mice).
  • the target influenza virus is influenza A virus.
  • the fusion protein of the invention or the pharmaceutical composition of the invention can be shown to be effective across a wide range of subtype A influenza viruses, including seasonal influenza virus and the expected highly pathogenic pandemic influenza virus.
  • a fusion protein of the invention for use in therapy, for the prevention or treatment of influenza virus infections, or for use as a vaccine against influenza virus.
  • the nucleic acid molecule of the invention or the expression vector of the invention for use in therapy, for the prevention or treatment of influenza virus infections, or for use as a vaccine against influenza virus. Will be done.
  • a method of preventing or treating an influenza virus infection in a subject comprising administering to the subject (including humans) the fusion protein of the invention.
  • a method of inducing a protective immune response against influenza virus in a subject is provided.
  • the prevention or treatment of an influenza virus infection in a subject comprising administering to the subject (including humans) the nucleic acid molecule of the invention or the expression vector of the invention.
  • Methods, or methods of inducing a protective immune response against influenza virus in the subject are also provided.
  • the use of fusion proteins of the invention is provided in the manufacture of agents for the prevention or treatment of influenza virus infections, or in the production of agents for use as vaccines against influenza virus. Will be done.
  • the nucleic acid molecule of the present invention or the expression vector of the present invention in the production of a drug for preventing or treating an infectious disease of influenza virus, or in the production of a drug for use as a vaccine against influenza virus. Use of is also provided.
  • SEQ ID NO: 1 Michigan strain HA amino acid sequence
  • SEQ ID NO: 2 Headless HA
  • SEQ ID NO: 3 with headless HA linker
  • SEQ ID NO: 4 Secretory signal
  • SEQ ID NO: 5 Michigan strain M1
  • SEQ ID NO: 6 HIS tag
  • SEQ ID NO: 7 HA-M1 for rice expression, no linker. ORF from the 10th A to the 1731th A.
  • SEQ ID NO: 8 HA-M1 for rice expression, with linker. ORF from the 10th A to the 1776th A.
  • SEQ ID NO: 9 Rice ⁇ -amylase 3D promoter
  • SEQ ID NO: 10 Rice ⁇ -amylase 3D terminator
  • SEQ ID NO: 11 GSGSGSGS Linker
  • SEQ ID NO: 12 Improved head defect HA-M1 fusion protein A, HA51-339.
  • 10th A ORF to the 1578th A.
  • SEQ ID NO: 13 GSG linker
  • SEQ ID NO: 14 Improved head defect HA-M1 fusion protein B, HA51-335.
  • SEQ ID NO: 15 Improved head defect HA-M1 fusion protein C, HA59-339. ORF to 10th A to 1602nd A.
  • SEQ ID NO: 16 A / SWINE / IL / 00685/2005 (H1N1) M1 SEQ ID NO: 17: With HA-M1 linker for wheat cell-free system. ORF from 1st G to 1,692th A.
  • SEQ ID NO: 18 Improved head defect HA-M1 for wheat cell-free system. ORF in 1st G to 1,515th A.
  • SEQ ID NO: 19 Head defect HA-M1 fusion protein
  • SEQ ID NO: 20 Improved head defect HA-M1 fusion protein
  • SEQ ID NO: 24 GSAGSA linker
  • SEQ ID NO: 25 Re-improved head defect HA-M1 fusion protein B + KDEL. ORF from the 10th A to the 1,581th A.
  • SEQ ID NO: 26 Improved head defect HA-M1 fusion protein D + KDEL. ORF from the 10th A to the 1623th A.
  • SEQ ID NO: 27 Re-improved head defect HA-M1 fusion protein D + KDEL. ORF from 1st A to 1605th A.
  • SEQ ID NO: 28 Glb-1 promoter SEQ ID NO: 29: Glb-1 terminator SEQ ID NO: 30: Rice Ubi1 promoter SEQ ID NO: 31: NOS terminator SEQ ID NO: 32: RAP 3rd intron SEQ ID NO: 33: GluABC SEQ ID NO: 34: Complementary strand of GluABC SEQ ID NO: 35: Pro13K16K SEQ ID NO: 36: Complementary strand of Pro13K16K SEQ ID NO: 37: TKIWVER3_1 SEQ ID NO: 38: TKIWVER3_2 SEQ ID NO: 39: TKIWVER3_3 SEQ ID NO: 40: TKIWVER3_4 SEQ ID NO: 41: Re-improved head defect HA-M1 fusion protein SEQ ID NO: 42: FLAG tag SEQ ID NO: 43: MFA1 secretory signal SEQ ID NO: 44: GSAGSA linker SEQ ID NO: 45: expression gene of pYHAM1-15.
  • SEQ ID NO: 46 Expression gene of pYHAM1-16. ORF from the 10th A to the 1,812th A.
  • SEQ ID NO: 47 Expression gene of pYHAM1-1. ORF to the 10th A to 1,671st A.
  • SEQ ID NO: 48 Expression gene of pYHAM1-2. ORF from the 10th A to the 1,635th A.
  • SEQ ID NO: 49 GGGGS linker
  • SEQ ID NO: 50 Glb-1 secretion signal
  • SEQ ID NO: 53 Re-improved head-deficient HA-M1 fusion protein on the vector instep.
  • SEQ ID NO: 54 Re-improved head-deficient HA-M1 fusion protein loaded on Vector B. ORF from the 10th A to the 1665th A.
  • SEQ ID NO: 55 Code region of HA335-M1 for the re-improved head defect HA-M1 fusion protein. The first A to ORF.
  • SEQ ID NO: 56 Code region of HA335-M1 of the re-improved head defect HA-M1 fusion protein. Codon modified version. The first A to ORF.
  • SEQ ID NO: 57 Re-improved head-deficient HA-M1 fusion protein loaded on Vector Hei.
  • SEQ ID NO: 58 Re-improved head-deficient HA-M1 fusion protein mounted on Vector Ding. ORF from the 10th A to the 1590th A.
  • SEQ ID NO: 59 Head-deficient HA-M1 fusion protein (N-terminal rice ⁇ -amylase 3D secretory signal, C-terminal 6xHis tag removed from the amino acid sequence translated from SEQ ID NO: 7)
  • SEQ ID NO: 60 Head-deficient HA-M1 fusion protein with linker (N-terminal rice ⁇ -amylase 3D secretory signal, C-terminal 6xHis tag removed from the amino acid sequence translated from SEQ ID NO: 8)
  • SEQ ID NO: 61 Improved head-
  • Example 1 Expression of head-deficient HA-M1 fusion protein in rice 1.
  • Construction of plasmid for rice expression of head-deficient HA-M1 fusion protein A) Design of head-deficient HA-M1 fusion protein and head-deficient HA-M1 fusion protein with linker (residues 76 to 308 and 529 to 554 in SEQ ID NO: 1 are missing)
  • SEQ ID NO: 1 From (SEQ ID NO: 1), a sequence consisting of 17 amino acids from the 1st methionine to the 17th alanine corresponding to the secretory signal, and the 529th methionine to the 554th methionine corresponding to the transmembrane region.
  • a head-deficient HA was obtained in which the sequence consisting of 26 amino acids up to the above was deleted, and the sequence consisting of 233 amino acids from the 76th glycine to the 308th proline in the head was deleted (SEQ ID NO: 2).
  • the head-deficient HA with a linker (SEQ ID NO: 3) in which the sequence consisting of 233 amino acids from the deleted 76th glycine to the 308th proline of the head-deficient HA was replaced with the GS linker shown in SEQ ID NO: 49. was designed.
  • a sequence was designed and designated as a head-deficient HA-M1 fusion protein (SEQ ID NO: 59) or a linker-equipped head-deficient HA-M1 fusion protein (SEQ ID NO: 60).
  • This rice ⁇ -amylase 3D secretory signal, head-deficient HA-M1 fusion protein or head-deficient HA-M1 fusion protein with linker, 6 ⁇ His tag (SEQ ID NO: 6) is directed from the N-terminus to the C-terminus.
  • An amino acid sequence arranged in this order was designed and used as a secretory signal / 6 ⁇ His-tagged head-deficient HA-M1 fusion protein or a secretory signal / 6 ⁇ His-tagged head-deficient fusion protein.
  • the DNA base sequence was obtained from the database of The National Center for Biotechnology Information. As shown in FIG. 1, the ⁇ -amylase 3D gene promoter of about 2 kb of rice, the 5'-UTR derived from the ⁇ -amylase 3D gene of rice, the nucleotide encoding the secretory signal of ⁇ -amylase 3D of rice, and the head.
  • the sequence for amplification of each part is 25 mer so that each part of the defective HA-M1 fusion protein gene and the ⁇ -amylase 3D gene terminator of about 0.5 kb of rice can be connected in this order from the 5'side.
  • Primers were designed so that the overlap sequence for ligation was 15 mer, and each part was amplified using KOD FX Neo (Toyo Spinning Co., Ltd.) according to the attached protocol. After purifying all these parts with the illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare), all the parts and pUC18 digested with SmaI (Takara Bio) are mixed so that they have an equimolar ratio. Then, using the In-Fusion (R) HD Cloning Kit (Clontech), they were ligated according to the attached protocol to obtain Escherichia coli transformants.
  • KOD FX Neo Toyo Spinning Co., Ltd.
  • the target head-deficient HA-M1 fusion protein expression cassette (base which is the base sequence of SEQ ID NOs: 7, 9 and 10 or base which is the base sequence of SEQ ID NOs: 8, 9 and 10) was inserted. Clone with plasmid was selected by sequencing.
  • the target head-deficient HA-M1 fusion protein expression cassette was amplified by PCR using Ex-Taq (Takara Bio) and pKS221MCS (Wakasa et al., 2006, Plant Biotechnol. J. 4: 499). -510) was subcloned. Transfer of the head-deficient HA-M1 fusion protein expression cassette to the rice transformation vector p35SHPTAg7-GW (Wakasa et al., 2006, Plant Biotechnol. J. 4: 499-510) was performed using Gateway TM cloning technology. carried out.
  • Gateway (R) LR between the attL1 and attL2 sequences subcloned across the head-deficient HA-M1 fusion protein expression cassette subcloned into pKS221MCS and the attR1 and attR2 sequences present at p35SHPTAg7-GW, respectively. Recombination was performed by the chronaseTM II enzyme mix (Invitrogen) according to the attached protocol, and the head-deficient HA-M1 fusion protein expression cassette was transferred to the rice transformation vector p35SHPTAg7-GW. It was confirmed by sequencing that the prepared plasmid had the desired shape and sequence. In this way, a plasmid for expressing the head-deficient HA-M1 fusion protein in rice was constructed (Fig. 1).
  • FIG. 2 The results of SDS-PAGE of four independent strains of rice transformant callus expressing the head-deficient HA-M1 fusion protein are shown in FIG. In FIG. 2, the contents of each lane are as follows. Lane 1: A culture medium obtained by culturing transformant strain No. 1 expressing the head-deficient HA-M1 fusion protein in a medium containing no sucrose. Lane 2: A culture medium in which transformant lineage No. 2 expressing the head-deficient HA-M1 fusion protein was cultured in a medium containing no sucrose. Lane 3: A culture medium in which transformant lineage No.
  • Lane 4 A culture medium in which transformant lineage No. 4 expressing the head-deficient HA-M1 fusion protein was cultured in a medium containing no sucrose.
  • Lane 5 A culture medium in which transformant lineage No. 1 expressing the head-deficient HA-M1 fusion protein was cultured in a medium containing sucrose.
  • Lane 6 A culture medium in which transformant lineage No. 2 expressing the head-deficient HA-M1 fusion protein was cultured in a medium containing sucrose.
  • Lane 7 A culture medium in which transformant lineage No.
  • Lane 8 A culture medium in which transformant lineage No. 4 expressing the head-deficient HA-M1 fusion protein was cultured in a medium containing sucrose.
  • Lane 9 Cell mass extract obtained by culturing transformant lineage No. 1 expressing the head-deficient HA-M1 fusion protein in a medium containing no sucrose.
  • Lane 10 Cell mass extract obtained by culturing transformant lineage No. 2 expressing the head-deficient HA-M1 fusion protein in a medium containing no sucrose.
  • Lane 11 Cell mass extract obtained by culturing transformant lineage No.
  • Lane 12 Cell mass extract obtained by culturing transformant lineage No. 4 expressing the head-deficient HA-M1 fusion protein in a medium containing no sucrose.
  • Lane 13 Cell mass extract obtained by culturing transformant lineage No. 1 expressing the head-deficient HA-M1 fusion protein in a medium containing sucrose.
  • Lane 14 Cell mass extract obtained by culturing transformant lineage No. 2 expressing the head-deficient HA-M1 fusion protein in a medium containing sucrose.
  • Lane 15 Cell mass extract obtained by culturing transformant lineage No.
  • Lane 16 Cell mass extract obtained by culturing transformant lineage No. 4 expressing the head-deficient HA-M1 fusion protein in a medium containing sucrose.
  • Example 2 Production of improved head-deficient HA-M1 fusion protein by rice 1.
  • Construction of a plasmid for rice expression of the improved head-deficient HA-M1 fusion protein As described above, the head-deficient HA-M1 fusion protein was not expressed in rice, so improvement was performed. The outline is shown in FIG.
  • A) Design of improved head defect HA-M1 fusion protein (I) Improved head defect HA-M1 fusion protein A (defective residues 51 to 339 and residues 529 to 554 in SEQ ID NO: 1) Based on the head-deficient HA-M1 fusion protein gene shown in SEQ ID NO: 7, glutamic acid encoded by codons from 184th guanine to 186th guanine to 349th cytosine to 351st adenin.
  • the amino acid sequence of the improved head-deficient HA-M1 fusion protein A with a linker from which the secretory signal 6xHis tag has been removed is SEQ ID NO: 61.
  • Improved head defect HA-M1 fusion protein B lacking a sequence consisting of 52 amino acids up to codon-encoded leucine, with a linker encoded by SEQ ID NO: 13 inserted (improved head defect with linker)
  • a nucleotide (SEQ ID NO: 14) encoding HA-M1 fusion protein B) was designed.
  • the amino acid sequence of the improved head-deficient HA-M1 fusion protein B with a linker from which the secretory signal 6xHis tag has been removed is SEQ ID NO: 62.
  • Improved head defect HA-M1 fusion protein C lacking a sequence consisting of 48 amino acids up to codon-encoded proline, with a linker encoded by SEQ ID NO: 67 inserted (improved head defect with linker)
  • a nucleotide encoding HA-M1 fusion protein C (SEQ ID NO: 15) was designed.
  • the amino acid sequence of the improved head-deficient HA-M1 fusion protein C with linker from which the secretory signal 6xHis tag was removed is SEQ ID NO: 63. Become.
  • Improved head defect HA-M1 fusion protein D lacking a sequence consisting of 44 amino acids up to codon-encoded leucine, with a linker encoded by SEQ ID NO: 13 inserted (improved head defect with linker)
  • a nucleotide encoding the HA-M1 fusion protein D) was designed.
  • the amino acid sequence of the improved head-deficient HA-M1 fusion protein D with a linker from which the secretory signal 6xHis tag has been removed is SEQ ID NO: 64.
  • PCR was performed using a plasmid for expressing the head-deficient HA-M1 fusion protein in rice as a template, using KOD-FX Neo (Toyobo), and according to the attached protocol.
  • the PCR product was electrophoresed on a 0.3% agarose gel, the amplified band was stained with ethidium bromide, and the target band was excised under UV irradiation. This was purified with illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare), and then cyclized using In-Fusion (R) HD Cloning Kit (Clontech) according to the attached protocol.
  • the extracted protein was subjected to SDS-PAGE and Western blotting. Detection was performed by anti-His antibody.
  • the results of SDS-PAGE and Western blotting of 10 independent strains of rice transformant callus expressing the improved head-deficient HA-M1 fusion protein A and the improved head-deficient HA-M1 fusion protein B are shown. Shown in 4. In FIG. 4, the contents of each lane are as follows. Lane 1: Cell mass extract of transformant lineage No. 1 expressing the improved head-deficient HA-M1 fusion protein A. Lane 2: Cell mass extract of transformant lineage No. 2 expressing the improved head-deficient HA-M1 fusion protein A. Lane 3: Cell mass extract of transformant lineage No.
  • Lane 4 Cell mass extract of transformant lineage No. 4 expressing the improved head defect HA-M1 fusion protein A.
  • Lane 5 Cell mass extract of transformant lineage No. 5 expressing the improved head-deficient HA-M1 fusion protein A.
  • Lane 6 Cell mass extract of transformant lineage No. 6 expressing the improved head defect HA-M1 fusion protein A.
  • Lane 7 Cell mass extract of transformant lineage number 7 expressing the improved head-deficient HA-M1 fusion protein A.
  • Lane 8 Cell mass extract of transformant lineage No. 8 expressing the improved head defect HA-M1 fusion protein A.
  • Lane 9 Cell mass extract of transformant lineage No.
  • Lane 10 Cell mass extract of transformant lineage No. 10 expressing the improved head defect HA-M1 fusion protein A.
  • Lane 11 Cell mass extract of transformant lineage No. 1 expressing the improved head-deficient HA-M1 fusion protein B.
  • Lane 12 Cell mass extract of transformant lineage No. 2 expressing the improved head-deficient HA-M1 fusion protein B.
  • Lane 13 Cell mass extract of transformant lineage No. 3 expressing improved head defect HA-M1 fusion protein B.
  • Lane 14 Cell mass extract of transformant lineage No. 4 expressing the improved head defect HA-M1 fusion protein B.
  • Lane 15 Cell mass extract of transformant lineage No.
  • Lane 16 Cell mass extract of transformant lineage No. 6 expressing the improved head defect HA-M1 fusion protein B.
  • Lane 17 Cell mass extract of transformant lineage number 7 expressing the improved head defect HA-M1 fusion protein B.
  • Lane 18 Cell mass extract of transformant lineage No. 8 expressing improved head defect HA-M1 fusion protein B.
  • Lane 19 Cell mass extract of transformant lineage number 9 expressing the improved head defect HA-M1 fusion protein B.
  • Lane 20 Cell mass extract of transformant lineage No. 10 expressing improved head defect HA-M1 fusion protein B.
  • Example 3 Production of re-improved head defect HA-M1 fusion protein by rice 1.
  • nucleotide sequence encoding the Glb-1 secretion signal Substituted with the nucleotide sequence encoding the Glb-1 secretion signal, and further linked the nucleotide sequence encoding the KDEL sequence, which is the vesicle mooring signal, downstream of the sequence encoding the 6xHis tag at the C-terminal (SEQ ID NO: 22).
  • the nucleotide sequence encoding the improved head defect fusion protein B + KDEL was designed (SEQ ID NO: 23).
  • For the improved head defect fusion protein B + KDEL 36 from 775th timine to 810th timine of SEQ ID NO: 23, which encodes a cytoplasmic tail consisting of a 12-amino acid sequence present at the C-terminal of HA.
  • a nucleotide sequence encoding the re-improved head-deficient fusion protein B + KDEL was designed by removing the base sequence and inserting a nucleotide sequence (SEQ ID NO: 24) encoding a linker of GSAGSA having a flexible structure therein (SEQ ID NO: 24). SEQ ID NO: 25).
  • SEQ ID NO: 65 The sequence of the re-improved head defect fusion protein B not linked with the secretory signal, 6xHis tag and KDEL was designated as SEQ ID NO: 65.
  • the sequence consisting of 44 amino acids up to Leucine was deleted, the amino acid sequence encoded by the linker encoded by SEQ ID NO: 13 was inserted (improved head-deficient HA-M1 fusion protein D), and the C-terminal 6xHis tag was added.
  • a nucleotide encoding the improved head-deficient HA-M1 fusion protein D + KDEL (SEQ ID NO: 22), in which a nucleotide sequence encoding the KDEL sequence, which is a vesicle mooring signal, is ligated downstream of the sequence encoding 26) was designed.
  • the improved head-deficient fusion protein D the 36-base sequence from 799th timine to 834th timine of SEQ ID NO: 26, which encodes a cytoplasmic tail consisting of a 12-amino acid sequence, located at the C-terminal of HA.
  • FIG. 5 shows an outline of the construction of a vector for expression in rice. From the database of National Center for Biotechnology Information, the nucleotide sequences of the promoter (SEQ ID NO: 28) and terminator (SEQ ID NO: 29) of the Globulin-1 gene (Glb-1) of rice were obtained. Then, a glutelin expression-suppressing cassette and a prolamin expression-suppressing cassette, which are structures for causing two kinds of RNA interference, having the structure shown in FIG. 5 were designed.
  • a promoter (SEQ ID NO: 30) of about 2 kb of the Ubi1 gene of rice and pRI 101-AN (Takara).
  • a part of the glutelin A, B, and C genes (sequence) is sandwiched between the NOS terminator (SEQ ID NO: 31) derived from bio) and the 3rd intron (RAP 3rd intron, SEQ ID NO: 32) of the rice aspartic promoter.
  • SEQ ID NO: 34 a sequence complementary to the sequence was arranged was designed and named as a glutelin expression-suppressing cassette.
  • the promoter of about 2 kb of the Ubi1 gene of rice (SEQ ID NO: 28) and pRI 101-AN (Takarabio)
  • the 3rd intron (RAP 3rd intron, SEQ ID NO: 32) of the rice aspartic protease is sandwiched between the terminators of the NOS from which it is derived (SEQ ID NO: 29), and a partial sequence of the prolamin gene of 13 KD and 16 KD (SEQ ID NO: 35) is sandwiched.
  • a sequence in which a sequence complementary to the sequence (SEQ ID NO: 36) was arranged was designed and named as a promoter expression inhibitory cassette.
  • These two expression-suppressing cassettes were synthesized by PCR using Ex-Taq (Takara Bio), and the entry vectors pKS4-MCS and pKS221MCS (both Wakasa et al., 2006, Plant Biotechnol. J. 4: 499). Subcloned into -510).
  • an expression cassette of the improved or re-improved head defect fusion protein B or D was synthesized by PCR using Ex-Taq (Takara Bio), and the entry vector pK Subcloned into S2-3MCS (detailed in Wakasa et al., 2006, Plant Biotechnol. J. 4: 499-510). Then, the above three types of cassettes were transferred from the obtained three types of entry vectors to the rice transformation vector p35SHPTAg7-43GW (Wakasa et al., 2006, Plant Biotechnol. J. 4: 499-510) using Gateway TM cloning technology. Transferred.
  • the attL4 and attR1 sequences subcloned into pKS4-MCS sandwiching the gluterin expression-suppressing cassette the attL1 and attL2 sequences subcloned into pKS221MCS, and the attL1 and attL2 sequences, pKS2-3MCS.
  • the attR2 and attL3 sequences that are present across the expression cassette of the improved or re-improved head defect fusion protein B or D subcloned into and the attR4 and attR3 sequences that are present in the rice transformation vector p35SHPTAg7-43GW.
  • AttL1 and attR1, attL2 and attR2, attL3 and attR3, attL4 and attR4 sequences are recombined by Gateway (R) LR clonase TM II enzyme mix (invitrogen) according to the attached protocol, and three cassettes are used.
  • Gateway (R) LR clonase TM II enzyme mix invitrogen
  • three cassettes are used.
  • FIG. 5 As a result, as shown in FIG. 5, of Glbpro: HA52-335 (-linker), Glbpro: HA52-335 (+ linker), Glbpro: HA59-335 (-linker), Glbpro: HA59-335 (+ linker), Four types of plasmids were constructed.
  • urea-SDS buffer 50 mM Tris-HCl pH 6.8, 8 M Urea, 4% SDS, 5% 2-mercaptoethanol,
  • 20% Glycerol 20% Glycerol
  • FIG. 6 the contents of each lane are as follows in order from the left.
  • Glbpro HA52-335-M1 (-linker) transformant lineage # 02, # 03, # 06, # 12, # 18 in Fig. 5.
  • Glbpro HA59-335-M1 (-linker) transformant lineage # 02, # 04, # 16, # 21, # 23 in FIG.
  • Glbpro HA52-335-M1 (+ linker) transformant lineage # 03, # 08, # 14, # 19, # 25 in Fig. 5.
  • Glbpro HA59-335-M1 (+ linker) transformant lineage # 01, # 04, # 05, # 18 in Fig. 5.
  • the difference in the head defect region of HA did not bring about a large difference in the expression level of the head defect fusion protein.
  • removal of the cytoplasmic tail consisting of the 12 amino acid sequence present at the C-terminal of HA and / or insertion of a linker of GSAGSA having a flexible structure has been shown to have the effect of increasing the expression level of the head-deficient fusion protein. was done.
  • FIG. 7 shows an outline of the construction of rice callus expression vectors.
  • the Glb-1 promoter is replaced with the Ubi promoter, and the nucleotide sequence encoding the Glb-1 secretion signal is obtained.
  • the nucleotide sequence encoding 3xFLAG of SEQ ID NO: 52 was inserted in frame between the nucleotide sequences encoding the N-terminal of HA, and a vector instep having the gene coding region described in SEQ ID NO: 53 was constructed.
  • Vector B The nucleotide sequence encoding KDEL was removed from Vector A, and Vector B having the gene coding region set forth in SEQ ID NO: 54 was constructed.
  • the collected callus was frozen in liquid nitrogen, pulverized with a multi-bead shocker (registered trademark, Yasui Instrument), and the protein was extracted with a urea-SDS buffer.
  • the extracted protein was subjected to SDS-PAGE and Western blotting. Detection was performed by anti-FLAG antibody.
  • FIG. 8 The results of SDS-PAGE and Western blotting of 6 independent strains of transformants by Vector A and 5 independent strains of transformants by Vector B are shown in FIG. In FIG. 8, the contents of each lane are as follows. Lanes 1-6: Expression of HA-M1 fusion protein of rice callus expressing vector instep (each lane is an independent clone). Lanes 7-11: Expression of HA-M1 fusion protein of rice callus expressing Vector B (each lane is an independent clone).
  • the expression level of the transformant by the vector B expressing the re-improved head defect HA-M1 fusion protein D to which KDEL was not given is the transformant by the vector A to which KDEL was given. It was shown that the addition of KDEL tended to suppress the expression of the head-deficient HA-M1 fusion protein.
  • FIG. 9 shows the results of Western blotting of 5 independent lines of the transformant by Vector B, 4 independent lines of the transformant by Vector Hei, and 5 independent lines of the transformant by Vector Ding.
  • the contents of each lane are as follows. Lanes 1-5: Expression of HA-M1 fusion protein of rice callus expressing Vector B (each lane is an independent clone). Lanes 6-10: Expression of HA-M1 fusion protein of rice callus expressing Vector Hei (each lane is an independent clone). Lanes 11-14: Expression of the HA-M1 fusion protein of rice callus expressing Vector Ding (each lane is an independent clone).
  • the expression level of the transformant by the vector ⁇ expressing the re-improved head defect fusion protein D in which the codon is changed is significantly different from the expression level of the transformant by the vector B. It was shown that the change of the codon of the gene expressing the head-deficient HA-M1 fusion protein without changing the frequency of codon use does not have a great effect on the expression level.
  • the expression level of the transformant by the vector Ding expressing the re-improved head defect fusion protein D which does not have the Glb-1 secretory signal in the head is the trait by the vector ⁇ which has the Glb-1 secretory signal. It tends to be lower than that of the transformant, and it was shown that the addition of the secretory signal of Glb-1 has the effect of promoting the expression of the head-deficient HA-M1 fusion protein.
  • Example 4 Synthesis of antigen protein for animal testing 1 Construction of a plasmid for antigen protein synthesis, which is a head-deficient HA-M1 fusion protein and an improved head-deficient HA-M1 fusion protein.
  • a plasmid for antigen protein synthesis which is a head-deficient HA-M1 fusion protein and an improved head-deficient HA-M1 fusion protein.
  • the amino acid sequence of M1 was changed to the amino acid sequence of the M1 protein of the (A / swine / IL / 00685/2005 (H1N1)) strain (registration number: ACM17279.1, SEQ ID NO: 16).
  • a nucleotide sequence was designed in which the codons were returned to the codons of the original virus for HA and M1, that is, the sequence of registration number MK622940.1 for HA and the codon of registration number FJ638301.1 for M1 (sequence). Number 17).
  • a plasmid containing a nucleotide sequence encoding an improved head-deficient HA-M1 fusion protein In order to synthesize an improved head-deficient HA-M1 fusion protein in a wheat cell-free protein synthesis system, the following is based on pKBac1199. Modified. Regarding the nucleotide sequence set forth in SEQ ID NO: 17, the nucleotide sequence from the 100th guanine to the 300th guanine was deleted, and the sequence set forth in SEQ ID NO: 11 was inserted therein to design the nucleotide sequence of SEQ ID NO: 18.
  • pEU-E01-MCS CellFree Science
  • pKBac1201 a plasmid for synthetic systems
  • the obtained plasmid was cloned into Escherichia coli DH5 ⁇ (Toyobo Co., Ltd.) and prepared in large quantities using the QIAGEN plasmid Maxi Kit (Qiagen Co., Ltd.).
  • WEPRO7240H CellFree Science
  • WEPRO7240 CellFree Science
  • HA-M1 head-deficient HA-M1 protein
  • WEPRO7240 CellFree Science
  • HA-M1 fusion protein Centrifuge the synthesized protein (total fraction) (21,600xG, 4 ° C, 10 minutes), wash the precipitated fraction twice with a translation buffer for wheat cell-free protein synthesis (CellFree Sciences, Inc.), and then wheat. It was suspended in a translation buffer for a cell-free protein synthesis system to a concentration of about 1 mg / mL.
  • the sequence of the head-deficient HA-M1 fusion protein was SEQ ID NO: 19, and the sequence of the improved head-deficient HA-M1 fusion protein was SEQ ID NO: 20.
  • a plasmid for antigen protein synthesis which is a re-improved head-deficient HA-M1 fusion protein
  • a plasmid containing a nucleotide sequence encoding the re-improved head-deficient HA-M1 fusion protein The following vector preparation for synthesizing the re-improved head-deficient HA-M1 fusion protein in a wheat cell-free protein synthesis system It was carried out as follows.
  • PCR is performed using two primers pointing outward from the region encoding the 12 amino acid sequence of the C-terminal of the HA to be deleted. To prepare a fragment of the ring-opened vector lacking this region.
  • this amino acid sequence is encoded, and at both ends, the two ends of the cyclized vector sequence and 15 mer.
  • Two oligonucleotides having a homologous sequence of No. 2 and having a complementary strand relationship with each other were synthesized to prepare an annealed fragment. These two prepared fragments were ligated using an In-Fusion cloning kit to complete a plasmid.
  • TKIWver3_1 SEQ ID NO: 37
  • TKIWver3_2 SEQ ID NO: 38
  • TKIWver3_3 SEQ ID NO: 39
  • TKIWver3_4 SEQ ID NO: 40
  • PCR was performed using the plasmid pKBac1201 as a template, TKIWver3_1 and TKIWver3_2 as primer sets, and KODOne (Toyo Spinning Co., Ltd.) according to the attached protocol. Purified and recovered with andGelBandPurificationKit (GE Healthcare).
  • mix 40 ⁇ L of TKIWver3_3 and TKIWver3_4 (each prepared as an aqueous solution with a concentration of 0.1 nmol / ⁇ L), boil for 1 minute on water in a pot, and then keep the water in the pot at room temperature. I left it until I returned to, and urged annealing.
  • the obtained fragments were concentrated by ethanol precipitation according to a conventional method.
  • the two types of fragments thus obtained were cyclized using an In-Fusion cloning kit and transformed with Escherichia coli DH5 ⁇ (Toyobo). It was confirmed by sequencing that the plasmid thus prepared was constructed as intended, and the name was pKBac1211.
  • the obtained plasmid was cloned into Escherichia coli DH5 ⁇ (Toyobo) and prepared using QIAGEN Plasmamid MaxiKit (Qiagen).
  • Example 5 Expression of head-deficient HA-M1 fusion protein and improved head-deficient HA-M1 fusion protein B in yeast Head-deficient HA-M1 fusion protein and improved head-deficient HA-M1 fusion protein B ( For B) prepared in the above rice example, expression was attempted by the budding yeast Saccharomyces cerevisiae .
  • the skeletal vector, pYES2 (Invitrogen), was digested with SpeI (New England Biolabs) and NotI (New England Biolabs), electrophoresed on a 1% agarose gel, stained with ethidium bromide, and then the band of the vector was formed. It was cut out under UV irradiation.
  • the TDH3 promoter, head-deficient HA-M1 fusion protein or improved head-deficient HA-M1 fusion protein B is encoded between the SpeI and NotI sites of the yeast plasmid pYES2 from the 5'side.
  • the nucleotides to be subjected to were ligated in this order using Ligation High ver.2 (Toyobo). Sequencing confirmed that the plasmid produced by ligation had the desired shape and sequence.
  • plasmids for expressing the head-deficient HA-M1 fusion protein and the improved head-deficient HA-M1 fusion protein B in yeast were constructed and named pKBac1207 and pKBac1208, respectively. These plasmids are multicopy, of the type that autonomously replicates outside the nucleus of yeast, and carry the URA3 gene, which complements the host's uracil requirement as a selectable marker.
  • yeast is collected by centrifugation and in the presence of 0.1 mL extraction buffer (50 mM Tris-HCl pH 6.8, 8 M Urea, 4% SDS, 50 mM DTT, 20% Glycerol). Cells were disrupted by vigorous stirring with glass beads (diameter: 425-600 ⁇ m, sigma). Cell extracts were collected by centrifugation and tested on SDS-PAGE and Western blotting using Atto's system. Detection was performed by anti-His antibody.
  • extraction buffer 50 mM Tris-HCl pH 6.8, 8 M Urea, 4% SDS, 50 mM DTT, 20% Glycerol.
  • the expression level of the improved head-deficient HA-M1 fusion protein B was higher than the expression level of the head-deficient HA-M1 fusion protein. From this, it was confirmed that the expression level of the head-deficient HA-M1 fusion protein is increased by expanding the head-deficient region of HA not only when rice is used as the host but also when yeast is used as the host. did it. It was
  • Saccharomycetale Saccharomycetale, Saccharomycetale, Saccharomycetale, Saccharomycetale, Saccharomycetale, Saccharomycetale, Saccharomycetale, Saccharomycetale, Saccharomycetale, Saccharomycetale, Saccharomycetale, Saccharomycetale, Saccharomycetale, H. Since there was an effect, it was shown that the expansion of the head-deficient region of HA is effective in increasing the expression level of the head-deficient HA-M1 fusion protein, which is common in both the plant and fungal fields.
  • Example 6 Expression of re-improved head-deficient HA-M1 fusion protein by yeast 1.
  • Design of fusion protein In order to investigate the effect of modification of the HA portion of the head-deficient HA-M1 fusion protein on the expression level in yeast, a new series of expression vectors was constructed as outlined in FIG. That is, based on the vector used for the expression of the head-deficient HA-M1 fusion protein, the selectable marker gene in yeast was replaced with a G418 resistance cassette consisting of a G418 resistance gene sandwiched between the PGK1 promoter and PGK1 terminator from URA3. ..
  • HA heads there are two types of HA heads, one in which the same region as the head-deficient HA-M1 fusion protein is deleted and the other in which the same region as the improved head-deficient HA-M1 fusion protein is deleted.
  • a total of 4 types of genes are synthesized by combining two types of terminals, one with the region encoding the 12-amino acid sequence at the C-terminal, which is the same as the re-improved head-deficient HA-M1 fusion protein, and the other without the deletion. rice field.
  • these genes have a nucleotide sequence in which 3 copies of the FLAG tag-encoding nucleotide sequence (SEQ ID NO: 42) are tandemly linked to the 5'side of the nucleotide sequence encoding each fusion protein, and further to the 5'side.
  • the nucleotide sequence encoding the secretory signal of the MFA1 gene (SEQ ID NO: 43) and the Hind III sequence were added to the 5'side thereof.
  • a nucleotide sequence (SEQ ID NO: 44) encoding an amino acid sequence consisting of GSAGSA was inserted between HA and M1. In this way, four types of fusion proteins were designed (SEQ ID NOs: 45-48).
  • URA3 gene which is a selectable marker for pKBac1207
  • the URA3 gene was obtained by PCR using the plasmid pZNEO described in the paper by Yamano et al. (J. Biotechnol., 32: 173-178, 1994) as a template, and an In- Substituting by Fusion cloning gave pKBac1207NEO.
  • the DNA fragment encoding the secretory signal of the MFA1 gene was obtained by PCR using the chromosomal DNA of BY4741 (Funakoshi) as a template.
  • a DNA fragment in which 3 copies of the nucleotide sequence encoding the FLAG tag were tandemly linked was obtained by artificial synthesis.
  • the DNA fragment encoding the fusion protein is amplified by KOD One (Toyobo) in combination with PCR using pKBac1199, pKBac1201 and pKBac1211 as templates, and finally all the fragments are cloned into pKBac1207NEO.
  • KOD One Toyobo
  • pYHAM1-1, pYHAM1-2, pYHAM1-15, and pYHAM1-16 were obtained. Sequencing confirmed that the nucleotide sequence encoding the fusion protein to be expressed was correct.
  • yeast After culturing, yeast is collected by centrifugation, and glass beads (diameter) are present in the presence of 0.1 mL extraction buffer (50 mM Tris-HCl pH 6.8, 8 M Urea, 4% SDS, 50 mM DTT, 20% Glycerol). : 425-600 ⁇ m, Sigma) was vigorously stirred to disrupt the cells. Cell extracts were collected by centrifugation and tested on SDS-PAGE and Western blotting using Atto's system. Detection was performed by anti-His antibody.
  • extraction buffer 50 mM Tris-HCl pH 6.8, 8 M Urea, 4% SDS, 50 mM DTT, 20% Glycerol. : 425-600 ⁇ m, Sigma
  • each lane contains as follows. 1: pYHAM1-15, 2: pYHAM1-16, 3: pYHAM1-1, 4: pYHAM1-2. From the comparison between the strains introduced with pYHAM1-15 and pYHAM1-16 and the strains introduced with pYHAM1-1 and pYHAM1-2, the expression level of the head-deficient HA-M1 fusion protein was expanded by expanding the head-deficient region of HA. Was confirmed to increase.
  • the expression level of the head-deficient HA-M1 fusion protein was further increased by improving the fusion protein.
  • the expression level of the head-deficient HA-M1 fusion protein is deleted in the sprouting yeast of the family Saccharomycetale, Saccharomycetale, and the phylum Saccharomycetale of the family Saccharomycetale.
  • Example 7 Drug efficacy test using influenza-infected mice (improved type. Subcutaneous / nasal) 1. 1. Outline of the test In order to confirm the inhibitory effect of the test substance on influenza virus, an antigen protein (test substance) was administered to mice in advance (subcutaneous administration or nasal administration), and the reaction to influenza virus was examined. The outline is shown in FIG. The test group was 12 groups (Table 1).
  • Test method A Test substance The improved head-deficient HA-M1 fusion protein prepared in Example 6 was used as an antigen protein. Improved version; HA51-335 fusion protein B (deficient in residues 51-335 and 529-554 in SEQ ID NO: 1)
  • mice BALB / c, females, and 4-week-old animals (at the start of the test) (purchased from Charles River Japan and used after acclimatization) were used.
  • the animals were bred in an environment of 5 animals / cage, room temperature 24 ⁇ 3 ° C, humidity 50 ⁇ 20%, ventilation 10-25 times / hour, and lighting 12 hours.
  • the feed was fed by free intake of MF (Oriental Yeast Co., Ltd.). Groups were grouped based on body weight at the end of acclimation (at the start of the test). The number of animals was 10 per group.
  • Influenza virus Influenza virus is H1N1 (strain name: A / PR / 8/34, ATCC. No .: VR-1469, BSL: 2, virus titer: 1.6 ⁇ 10 8 TCID 50 / mL) and H3N2 (strain). Name: A / Port Chalmers / 1/73, ATCC. No .: VR-810, BSL: 2, virus titer: 1.3 ⁇ 10 7 TCID 50 / mL), both viruses have been reported (Nakano).
  • the above-mentioned administration solution was subcutaneously administered at 0.2 mL per mouse, twice at 7-day intervals, for a total of 0.4 mL (25 ⁇ g / head for mice in the high-dose group, for a total of 50 ⁇ g / head).
  • the low-dose group of mice received 2.5 ⁇ g / head of protein at a time, for a total of 5 ⁇ g / head).
  • the above administration solution was nasally administered at 50 ⁇ L per mouse, twice at 7-day intervals, for a total of 100 ⁇ L (12.5 ⁇ g / head for mice in the high-dose group, for a total of 25 ⁇ g / head).
  • Mice in the low-dose group received 1.25 ⁇ g / head of protein at a time, for a total of 2.5 ⁇ g / head). It was
  • mice 14 days (Day-14), 7 days (Day-7), virus inoculation day (Day 0), 3 days (Day 3), 7 days (Day 7) after virus inoculation to grasp the condition of the evaluated mice. ), 10 days later (Day 10), and 14 days later (Day 14).
  • life and death of mice and the general condition were also evaluated during the period from 14 days before the virus inoculation date to 14 days after the virus inoculation date.
  • the measurement data for each test group obtained as test data are described as mean ⁇ standard deviation.
  • the weight loss of the 3rd group peaked (14.5 ⁇ 0.9g), and the body weights of the 1st and 2nd groups at this time were 17.4 ⁇ 2.3g and 20.4 ⁇ 1.2g, respectively. Met.
  • the suppression of weight loss was significantly different in the 1st group on the 3rd, 7th and 14th days after the inoculation, and in the 2nd group on the 3rd to 14th days after the inoculation, compared with the 3rd group (Vehicle group). That is, the suppression of weight loss was observed in the improved administration group as compared with the vehicle group.
  • the weight loss of the 6th group peaked (14.9 ⁇ 1.2g), and at this time, it was 20.4 ⁇ 1.3g and 21.1 ⁇ 1.4g in the 4th and 5th groups, respectively. ..
  • Coarse coat was observed in many individuals in groups 7 and 8, but recovered afterwards except for dead individuals, and recovered from the 11th day after inoculation in the 7th group and from the 9th day in the 8th group. Coarse coat was observed in all cases in group 9, and most of the individuals recovered on the day of the end of observation (14th day of inoculation). That is, it was confirmed that the improved administration group had at least less coarsening of the coat or faster recovery.
  • the weight loss of the 9th group peaked (17.2 ⁇ 1.0g), and at this time, it was 17.9 ⁇ 1.3g and 18.9 ⁇ 1.3g in the 7th and 8th groups, respectively.
  • Coarse coat was observed in about half of the individuals in the 10th and 11th groups, but most of the individuals recovered on the 9th to 11th days except the dead individuals. In the 12th group (vehicle group), coarse stiffness of the coat was observed in all cases, and most of the individuals recovered on the 13th day after inoculation. That is, it was confirmed that the improved administration group had at least less coarsening of the coat or faster recovery.
  • the weight loss of the 12th group peaked (17.0 ⁇ 2.7g), and at this time, it was 19.2 ⁇ 2.4g and 20.6 ⁇ 1.2g in the 10th and 11th groups, respectively. ..
  • the suppression of weight loss was significantly different between the 12 groups (vehicle group) and the 11 groups (improved 1.25 ⁇ g / head administration group) on the 10th and 14th days after inoculation. That is, the improved administration group showed suppression of weight loss as compared with the vehicle group.
  • mice treated with the improved antigen protein have acquired resistance to influenza virus (H1N1 type and H3N2 type) as compared with the vehicle group not treated with the antigen protein. Therefore, it was shown that the improved antigenic protein is useful as a vaccine capable of imparting cross-immunity between influenza A virus subspecies.
  • Example 8 Drug efficacy test using influenza virus-infected mice (improved / re-improved. Nasal) 1. 1. Outline of the test In order to confirm the inhibitory effect of the test substance on influenza virus, an antigen protein (test substance) was administered nasally to mice in advance, and the reaction to influenza virus was examined. The outline is shown in FIG. The test group was 14 groups (Table 2).
  • Test substance The following three head-deficient HA-M1 fusion proteins prepared in Example 4 were used as antigen proteins.
  • Original type Original fusion protein (deficient in residues 76-308 and 529-554 in SEQ ID NO: 1) Improved version; HA51-335 fusion protein B (deficient in residues 51-335 and 529-554 in SEQ ID NO: 1) Re-improved; HA51-335, 555-566 Fusion Protein B (deficient in residues 51-335, 529-554 and 555-566 in SEQ ID NO: 1)
  • mice BALB / c, females, and 6-week-old animals (at the start of the test) (purchased from Charles River Japan and used after acclimatization) were used.
  • the animals were bred in an environment of 5 animals / cage, room temperature 24 ⁇ 3 ° C, humidity 50 ⁇ 20%, ventilation 10-25 times / hour, and lighting 12 hours.
  • the feed was fed by free intake of MF (Oriental Yeast Co., Ltd.). Groups were grouped based on body weight at the end of acclimation (at the start of the test). The number of animals was 10 per group.
  • Influenza virus Influenza virus is H1N1 (strain name: A / PR / 8/34, ATCC. No .: VR-1469, BSL: 2, virus titer: 1.6 ⁇ 10 8 TCID 50 / mL) and H3N2 (strain). Name: A / Port Chalmers / 1/73, ATCC. No .: VR-810, BSL: 2, virus titer: 1.3 ⁇ 10 7 TCID 50 / mL), both viruses have been reported (Nakano).
  • the administration method of antigen protein is as follows. That is, the original suspension, the improved suspension, or the re-improved suspension prepared to 250 ⁇ g / mL with physiological saline was used as a high-dose group administration solution, and further diluted 10-fold with physiological saline. Was used as a low-dose group administration solution. In addition, the physiological saline solution was used as the vehicle administration solution.
  • the above administration solution was nasally administered at 50 ⁇ L per mouse, twice at 7-day intervals, for a total of 100 ⁇ L (12.5 ⁇ g / head for mice in the high-dose group, for a total of 25 ⁇ g / head).
  • Mice in the low-dose group received 1.25 ⁇ g / head of protein at a time, for a total of 2.5 ⁇ g / head).
  • mice 14 days (Day-14), 7 days (Day-7), virus inoculation day (Day 0), 3 days (Day 3), 7 days (Day 7) after virus inoculation to grasp the condition of the evaluated mice. ), 10 days later (Day 10), and 14 days later (Day 14).
  • life and death of mice and the general condition were also evaluated during the period from 14 days before the virus inoculation date to 14 days after the virus inoculation date.
  • the measurement data for each test group obtained as test data are described as mean ⁇ standard deviation.
  • mice nasally administered with the antigenic protein of the present invention acquired resistance to influenza virus (H1N1 type and H3N2 type) as compared with the group not administered with the antigenic protein. Therefore, it has been shown that the antigenic protein of the present invention is useful as a vaccine capable of imparting cross-immunity between influenza A virus subspecies.
  • Example 9 Influenza virus neutralizing antibody test using test substance-administered mice 1. 1. Outline of the test A test was conducted to confirm that an antibody having an activity of neutralizing influenza virus was produced in the blood of mice to which the test substance was administered. Antigen protein (test substance) was administered to mice in advance (nasal administration), and the reaction to influenza virus was examined. The test group was 9 groups (see Table 3 below).
  • Test substance (I) Test substance The following antigen proteins prepared according to Example 4 were used. That is, each protein synthesized using the wheat cell-free protein synthesis system is recovered as a precipitate by centrifugation at 15,000 rpm for 10 minutes at 4 ° C, and then the physiological saline solution (Otsuka Pharmaceutical Factory) is adjusted to 500 ⁇ g / mL. It was suspended in the water and stored in an ultra-low temperature bath at -80 ° C until it was used for the experiment. Improved: improved head-deficient HA-M1 chimeric protein B (SEQ ID NO: 20); and re-improved: re-improved head-deficient HA-M1 chimeric protein (SEQ ID NO: 41).
  • the improved head-deficient HA-M1 chimeric protein B and the re-improved head-deficient HA-M1 chimeric protein are gently inverted and mixed after thawing, and the protein is diffused into the suspension by ultrasonic treatment (protein).
  • Undiluted solution the dose per animal is protein undiluted solution 40 ⁇ L + Adj A 5 ⁇ L + physiological saline solution 5 ⁇ L (+ Adj A group), protein undiluted solution 40 ⁇ L + Adj B 10 ⁇ L (+ Adj B group) , Or protein stock solution 40 ⁇ L + physiological saline solution 10 ⁇ L (Adj-free group) was prepared (Table 3).
  • Adj A (2', 3'-cGAMP (InvivoGen)
  • Adj B Poly (I: C) (InvivoGen)
  • Adj B is prepared by adding physiological saline and gently mixing by pipetting to a concentration of 1 ⁇ g / ⁇ L to promote annealing. After keeping warm in the range of 70 ° C. for 10 minutes, it was allowed to stand at room temperature for 1 hour before use.
  • Adj A and Adj B were treated as described above and subjected to administration together with the protein stock solution as described in (i) above or as a test solution in which only the adjuvant was mixed with a physiological saline solution.
  • a control test group in which only physiological saline was administered was also provided.
  • mice were bred using autoclave-sterilized heat-resistant polysulfon cages (207W x 365D x 140H mm, TECNIPLAST Ltd) with five mice per cage.
  • the animals were bred under the conditions of room temperature 24 ⁇ 3 ° C, humidity 50 ⁇ 20%, ventilation (10 to 25 times / hour), and lighting for 12 hours (8:00 to 20:00).
  • the feed was MF (Oriental Yeast Co., Ltd.) as a free intake.
  • autoclave-sterilized city water was used as free intake.
  • Influenza virus Influenza virus is H1N1 (strain name: A / PR / 8/34, ATCC. No .: VR-1469, BSL: 2, virus titer: 1.6 ⁇ 10 8 TCID50 / mL) and H3N2 (strain name). : A / Port Chalmers / 1/73, ATCC.
  • test substance was administered nasally at 50 ⁇ L / body using a pipette under isoflurane inhalation anesthesia twice at weekly intervals (day 0, 7).
  • the collected serum is allowed to stand at 56 ° C for 30 minutes before use, and then in a 96-well plate, DMEM medium for measurement (BSA 0.2% (w / v), penicillin 100 units / mL, streptomycin 100 ⁇ g / mL, Dilute to 10, 20, 40, 80, 160, 320, 640, 1280, 2560, 5120, 10240, 20480-fold (50 ⁇ L / hole) with fungizone 0.5 ⁇ g / mL, trypsin 1.0 ⁇ g / mL).
  • a virus solution (10 2 TCID 50/50 ⁇ L / hole) prepared in DMEM medium for measurement was added to each diluted solution, and the mixture was allowed to stand at 37 ° C. for 30 minutes (serum / virus mixed solution).
  • DMEM medium for culture containing 10% FBS, penicillin 100 units / mL, streptomycin 100 ⁇ g / mL
  • DMEM medium for culture containing 10% FBS, penicillin 100 units / mL, streptomycin 100 ⁇ g / mL
  • PBS was added one by one and washed, and then 0.1 mL of serum / virus mixture was inoculated on a 24-well plate.
  • 0.1 mL of medium (cell control) and 0.1 mL of virus / medium equivalent mixture (virus control) were inoculated for 6 holes each. After allowing to stand at 34 ° C for 1 hour, 0.5 mL of the medium was added, and the cells were cultured for about 2 to 3 days to determine the presence or absence of viral replication.
  • the reciprocal of the maximum dilution ratio of serum that completely suppresses viral growth was defined as the neutralizing antibody titer.
  • the geometric mean and 95% confidence interval were calculated.
  • the significance test was performed by using JMP (SAS Institute Japan) and calculating the p-value by Dunnett's test method. When the p-value was calculated, if the neutralizing antibody titer was less than 10, it was read as 10 and calculated.
  • Table 4 shows the neutralizing antibody titers of H1N1 against influenza A virus in the serum of mice to which the test substance was administered.
  • the improved head-deficient HA-M1 chimeric protein B (groups 1, 2, and 3) and the re-improved head-deficient HA-M1 chimeric protein (groups 4, 5, and 6) were used as administration substances.
  • the growth of the virus was still suppressed at a dilution ratio of 40 to 160 times with or without an adjuvant.
  • the improved head-deficient HA-M1 chimeric protein B and the re-improved head-deficient HA-M1 chimeric protein were administered to treated mice by producing an antibody that neutralizes the H1N1 subtype virus. , Showed to confer resistance to this virus.
  • Table 5 shows the neutralizing antibody titers of H3N2 against influenza A virus in the serum of mice to which the test substance was administered.
  • the improved head-deficient HA-M1 chimeric protein B (groups 1, 2, and 3) and the re-improved head-deficient HA-M1 chimeric protein (groups 4, 5, and 6) were used as administration substances.
  • the growth of the virus was still suppressed at a dilution ratio of 40 to 160 times with or without an adjuvant.
  • the improved head-deficient HA-M1 chimeric protein B and the re-improved head-deficient HA-M1 chimeric protein were administered to treated mice by producing an antibody that neutralizes the H3N2 subtype virus. , Showed to confer resistance to this virus.

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Abstract

Disclosed is a fusion protein of an influenza-derived HA protein and an M1 protein, the fusion protein being a partially-deleted HA-M1 fusion protein in which an amino acid residue in a specific region has been deleted. This partially-deleted HA-M1 fusion protein has excellent characteristics.

Description

インフルエンザウイルス抑制用の融合タンパク質およびこれを含む医薬組成物Fusion protein for controlling influenza virus and pharmaceutical composition containing it 関連出願の参照Reference of related application
　本特許出願は、先に出願された日本国における特許出願である特願２０２０－１６６１８８号（出願日：２０２０年９月３０日）に基づく優先権の主張を伴うものである。この先の特許出願における全開示内容は、引用することにより本明細書の一部とされる。 This patent application is accompanied by a priority claim based on Japanese Patent Application No. 2020-166188 (filed from September 30, 2020), which is a previously filed patent application in Japan. All disclosures in future patent applications are hereby incorporated by reference.
発明の背景Background of the invention
技術分野
　本発明は、インフルエンザウイルス抑制用の融合タンパク質、およびこれを含む医薬組成物に関する。 The present invention relates to a fusion protein for suppressing influenza virus and a pharmaceutical composition containing the same.
背景技術
　抗原変異を繰り返すウイルスや血清型サブタイプが複数存在するウイルスに対する種々のワクチンの開発が行われてきた。例えば、インフルエンザウイルスの場合、インフルエンザウイルス粒子の外皮膜に存在するHA抗原が、季節性インフルエンザウイルスA型HAスプリットワクチンとして上市されている。インフルエンザウイルスHA遺伝子は、A亜型間で遺伝的交換が起こり、また塩基配列の突然変異により抗原変異が生じるが、HAタンパク質3次元構造の頭部に変異が集中して生じる。HAの頭部が主要な中和抗体エピトープのため、上市されているHAスプリットワクチンおよびウイルス不活化ワクチンは新しい変異株に対し効果がほとんどない。一方、ステム領域は変異が少ないが免疫原性が低いためA亜型のワクチン抗原としての利用は行われていなかった。大阪大学微生物病研究所の奥野らは1993年に初めてインフルエンザウイルスA1型株とA2型株に交差して中和できる抗ステム抗体が誘導されることを報告した（非特許文献１）。また、同グループは1996年HA遺伝子の頭部領域を欠損させたHAで免疫した動物で1型および2型を中和できる抗ステム抗体が誘導されることを報告した（非特許文献２）。以来、世界でステム抗原による万能ワクチンの開発が進められている（非特許文献３～６）。一方、単量体（モノマー）抗原タンパク質は免疫原性が低いため、HAスプリットワクチンは効果が低いことが弱点となっている。免疫原性と抗原構造との関係をM.F.Bachmannらは解析し、高度に組織化（多量体化、オリゴマー化）された抗原は免疫原性が高く、メモリーB細胞の誘導も行えることから、ワクチン抗原は組織化されることが重要であることを報告した（非特許文献７～９）。HAとフェリチンの融合タンパク質は、フェリチンのオリゴマー化活性により多量体を形成することによりHAの免疫原性が強化されることが報告された（非特許文献１０および１１）。インフルエンザウイルスHAスプリットワクチンおよび不活化ワクチンによる宿主免疫応答は、主としてHA抗原に対する抗体の誘導によりウイルスを中和する液性免疫であるが、一方、感染細胞を破壊しウイルス感染拡大を押さえるもう一つの免疫応答は細胞性免疫である。現在市販されているインフルエンザウイルスワクチンには細胞性免疫誘導能がほとんど無い。感染細胞の主要組織適合抗原クラス1分子に結合し細胞障害性T細胞(CTL)のT細胞受容体で認識されるインフルエンザウイルス抗原（CTLエピトープ)が、マトリックス(M1)タンパク質及びヌクレオキャプシド(NP)タンパク質にあることが報告されている（非特許文献１２～１４）。マトリックス(M1)タンパク質とヌクレオキャプシド(NP)タンパク質は、A亜型間でアミノ酸配列が保存されているので、A亜型間で交差細胞性免疫が成立する。M1タンパク質は重合体（オリゴマー）形成能を有し、また、NPと結合する（非特許文献１５および１６）。インフルエンザウイルスA型ワクチンの開発状況は、非特許文献１７および１８を参照のこと。 Background Technology Various vaccines have been developed against viruses that repeat antigenic variation and viruses that have multiple serotype subtypes. For example, in the case of influenza virus, the HA antigen present in the outer membrane of influenza virus particles is marketed as a seasonal influenza virus type A HA split vaccine. Influenza virus HA gene undergoes genetic exchange between subtypes A, and mutations in the base sequence cause antigenic variation, but mutations are concentrated in the head of the three-dimensional structure of HA protein. Since the HA head is the major neutralizing antibody epitope, the HA split vaccines and virus inactivated vaccines on the market have little effect on new mutants. On the other hand, the stem region has few mutations but low immunogenicity, so it has not been used as a vaccine antigen for subtype A. Okuno et al. Of the Institute for Microbial Diseases, Osaka University reported for the first time in 1993 that an anti-stem antibody that could cross and neutralize influenza virus A1 and A2 strains was induced (Non-Patent Document 1). In 1996, the same group reported that an anti-stem antibody capable of neutralizing types 1 and 2 was induced in animals immunized with HA in which the head region of the HA gene was deleted (Non-Patent Document 2). Since then, the development of a universal vaccine using a stem antigen has been promoted in the world (Non-Patent Documents 3 to 6). On the other hand, since the monomeric antigen protein has low immunogenicity, the weak point of the HA split vaccine is that it is ineffective. MFBachmann et al. Analyzed the relationship between immunogenicity and antigen structure, and highly organized (multimerized, oligomerized) antigens are highly immunogenic and can also induce memory B cells, so vaccine antigens. Reported that it was important to be organized (Non-Patent Documents 7-9). It has been reported that the fusion protein of HA and ferritin enhances the immunogenicity of HA by forming a multimer by the oligomerization activity of ferritin (Non-Patent Documents 10 and 11). The host immune response with the influenza virus HA split vaccine and inactivated vaccine is humoral immunity that neutralizes the virus primarily by inducing antibodies to the HA antigen, while another that destroys infected cells and suppresses the spread of virus infection. The immune response is cell-mediated immunity. Currently available influenza virus vaccines have little ability to induce cell-mediated immunity. The influenza virus antigen (CTL epitope) that binds to the major histocompatibility complex antigen class 1 molecule of infected cells and is recognized by the T cell receptor of cytotoxic T cells (CTL) is the matrix (M1) protein and nucleocapsid (NP). It has been reported to be found in proteins (Non-Patent Documents 12-14). Since the amino acid sequences of the matrix (M1) protein and the nucleocapsid (NP) protein are conserved between the A subtypes, cross-cell-mediated immunity is established between the A subtypes. M1 protein has the ability to form polymers (oligomers) and binds to NP (Non-Patent Documents 15 and 16). For the development status of influenza virus type A vaccine, refer to Non-Patent Documents 17 and 18.
　このような状況下において、頭部欠損ＨＡ－Ｍ１融合タンパク質が、２種のインフルエンザウイルスに対して、抗体価、ＣＴＬおよび生存率を改善することが報告されている（特許文献１）。しかし、抗体価、ＣＴＬおよび生存率などのワクチンとしての特性において、さらには生産効率などにおいて、未だ改良の余地がある。 Under such circumstances, it has been reported that the head-deficient HA-M1 fusion protein improves the antibody titer, CTL and survival rate against two influenza viruses (Patent Document 1). However, there is still room for improvement in vaccine characteristics such as antibody titer, CTL and survival rate, and in terms of production efficiency and the like.
国際公開第２０１９／１２４５５７号International Publication No. 2019/124557
　本発明者らは、インフルエンザウイルスのＨＡおよびＭ１に基づいて設計された頭部欠損ＨＡ－Ｍ１融合タンパク質において、さらに特定部位のアミノ酸配列を欠損させることにより、優れた特徴を有するタンパク質を得た。本発明はこの知見に基づくものである。 The present inventors obtained a protein having excellent characteristics by further deleting the amino acid sequence at a specific site in the head-deficient HA-M1 fusion protein designed based on the HA and M1 of influenza virus. The present invention is based on this finding.
　従って、本発明は、優れた特徴を有する頭部欠損ＨＡ－Ｍ１融合タンパク質、およびこれを含む医薬組成物を提供する。 Therefore, the present invention provides a head-deficient HA-M1 fusion protein having excellent characteristics, and a pharmaceutical composition containing the same.
　本発明は、以下の発明を包含する。
（１）インフルエンザウイルスに由来するＨＡタンパク質とＭ１タンパク質との融合タンパク質において、特定の領域のアミノ酸残基が欠損してなる部分欠損ＨＡ－Ｍ１融合タンパク質であって、
　前記ＨＡタンパク質が、配列番号１中の第１８～５６６アミノ酸残基で表されるアミノ酸配列を有するタンパク質、または該アミノ酸配列に対して少なくとも９０％の配列同一性を有するアミノ酸配列を有し、かつ、インフルエンザウイルス由来のＨＡタンパク質として機能するタンパク質であり、
　前記Ｍ１タンパク質が、配列番号５中の第１～２５２アミノ酸残基で表されるアミノ酸配列を有するタンパク質、または該アミノ酸配列に対して少なくとも９０％の配列同一性を有するアミノ酸配列を有し、かつ、インフルエンザウイルス由来のＭ１タンパク質として機能するタンパク質であり、
　配列番号１中の第５９～３３５アミノ酸残基および第５２９～５５４アミノ酸残基に相当する領域のアミノ酸残基が少なくとも欠損しており、かつ、
　配列番号１中の第１８～５０アミノ酸残基、第３４０～５２８アミノ酸残基および配列番号５中の第１～２５２アミノ酸残基に相当する領域のアミノ酸残基が少なくとも保持されている、部分欠損ＨＡ－Ｍ１融合タンパク質。
（２）配列番号１中の第５９～３３５アミノ酸残基、第５１～３３５アミノ酸残基、第５９～３３９アミノ酸残基、または第５１～３３９アミノ酸残基に相当する領域のアミノ酸残基が欠損している、前記（１）に記載の部分欠損ＨＡ－Ｍ１融合タンパク質。
（３）配列番号１中の第５５５～５６６アミノ酸残基に相当する領域のアミノ酸残基が欠損している、前記（１）または（２）に記載の部分欠損ＨＡ－Ｍ１融合タンパク質。
（４）前記ＨＡタンパク質と前記Ｍ１タンパク質とを、Ｎ末端からＣ末端に向けてこの順で含んでなる、前記（１）～（３）のいずれかに記載の部分欠損ＨＡ－Ｍ１融合タンパク質。
（５）欠損した領域および／または部分欠損ＨＡタンパク質とＭ１タンパク質の融合部位にリンカーが挿入されている、前記（１）～（４）のいずれかに記載の部分欠損ＨＡ－Ｍ１融合タンパク質。
（６）前記リンカーが、ＧＳＧリンカー、ＧＳＧＳＧリンカー、ＧＳＧＳＧＳＧＳリンカー、ＧＳＡＧＳＡリンカー、またはＧＧＧＧＳＧＧＧＧＳＧＧＧＧＳリンカーである、前記（５）に記載の部分欠損ＨＡ－Ｍ１融合タンパク質。
（７）前記（１）～（６）のいずれかに記載の部分欠損ＨＡ－Ｍ１融合タンパク質をコードする、核酸分子。
（８）前記（７）に記載の核酸分子を含んでなる、発現ベクター。
（９）前記（７）に記載の核酸分子または前記（８）に記載の発現ベクターを含んでなる、形質転換体。
（１０）前記形質転換体がイネまたは酵母である、前記（９）に記載の形質転換体。
（１１）前記（９）または（１０）に記載の形質転換体を培養または育成することを含んでなる、部分欠損ＨＡ－Ｍ１融合タンパク質を製造する方法。
（１２）前記（１）～（６）のいずれかに記載の部分欠損ＨＡ－Ｍ１融合タンパク質、前記（７）に記載の核酸分子または前記（８）に記載の発現ベクターを含んでなる、医薬組成物。
（１３）インフルエンザウイルスの感染症を予防または治療するための、前記（１２）に記載の医薬組成物。
（１４）インフルエンザウイルスに対するワクチンとして用いるための、前記（１２）に記載の医薬組成物。
（１５）前記インフルエンザウイルスがインフルエンザウイルスＡ型である、前記（１３）または（１４）に記載の医薬組成物。
（１６）療法に用いるための、前記（１）～（６）のいずれかに記載の部分欠損ＨＡ－Ｍ１融合タンパク質、前記（７）に記載の核酸分子または前記（８）に記載の発現ベクター。
（１７）インフルエンザウイルスの感染症を予防または治療するための、前記（１６）に記載の部分欠損ＨＡ－Ｍ１融合タンパク質、核酸分子または発現ベクター。
（１８）インフルエンザウイルスに対するワクチンとして用いるための、前記（１６）に記載の部分欠損ＨＡ－Ｍ１融合タンパク質、核酸分子または発現ベクター。
（１９）前記インフルエンザウイルスがインフルエンザウイルスＡ型である、前記（１７）または（１８）に記載の部分欠損ＨＡ－Ｍ１融合タンパク質、核酸分子または発現ベクター。
（２０）前記（１）～（６）のいずれかに記載の部分欠損ＨＡ－Ｍ１融合タンパク質、前記（７）に記載の核酸分子または前記（８）に記載の発現ベクターを被験体に投与することを含んでなる、該被験体におけるインフルエンザウイルスの感染症を予防または治療する方法。
（２１）前記（１）～（６）のいずれかに記載の部分欠損ＨＡ－Ｍ１融合タンパク質、前記（７）に記載の核酸分子または前記（８）に記載の発現ベクターを被験体に投与することを含んでなる、該被験体においてインフルエンザウイルスに対する防御免疫反応を誘導する方法。
（２２）前記インフルエンザウイルスがインフルエンザウイルスＡ型である、前記（２０）または（２１）に記載の方法。
（２３）アジュバントをさらに含んでなる、前記（１２）～（１５）のいずれかに記載の医薬組成物。
（２４）アジュバントと組み合わせて用いるための、前記（１６）～（１９）のいずれかに記載の部分欠損ＨＡ－Ｍ１融合タンパク質、核酸分子または発現ベクター。
（２５）前記部分欠損ＨＡ－Ｍ１融合タンパク質、核酸分子または発現ベクターが、アジュバントとともに投与される、前記（２０）～（２２）のいずれかに記載の方法。 The present invention includes the following inventions.
(1) A partially deficient HA-M1 fusion protein in which an amino acid residue in a specific region is deleted in a fusion protein of an HA protein derived from influenza virus and an M1 protein.
The HA protein has a protein having an amino acid sequence represented by the 18th to 566 amino acid residues in SEQ ID NO: 1, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence, and has an amino acid sequence. , A protein that functions as an HA protein derived from influenza virus,
The M1 protein has a protein having an amino acid sequence represented by the 1st to 252nd amino acid residues in SEQ ID NO: 5, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence, and , A protein that functions as an M1 protein derived from influenza virus.
At least the amino acid residues in the regions corresponding to the 59th to 335th amino acid residues and the 529th to 554th amino acid residues in SEQ ID NO: 1 are deleted, and
A partial defect in which at least the amino acid residues in the regions corresponding to the 18th to 50th amino acid residues, the 340th to 528th amino acid residues in SEQ ID NO: 1 and the 1st to 252nd amino acid residues in SEQ ID NO: 5 are retained. HA-M1 fusion protein.
(2) Amino acid residues in the region corresponding to the 59th to 335th amino acid residues, the 51st to 335th amino acid residues, the 59th to 339th amino acid residues, or the 51st to 339th amino acid residues in SEQ ID NO: 1 are deleted. The partially deficient HA-M1 fusion protein according to (1) above.
(3) The partially deficient HA-M1 fusion protein according to (1) or (2) above, wherein the amino acid residue in the region corresponding to the 555 to 566 amino acid residues in SEQ ID NO: 1 is deficient.
(4) The partially deficient HA-M1 fusion protein according to any one of (1) to (3) above, which comprises the HA protein and the M1 protein in this order from the N-terminal to the C-terminal.
(5) The partially deficient HA-M1 fusion protein according to any one of (1) to (4) above, wherein the linker is inserted into the deficient region and / or the fusion site of the partially deficient HA protein and the M1 protein.
(6) The partially deficient HA-M1 fusion protein according to (5) above, wherein the linker is a GSG linker, GSGSG linker, GSGSGSGS linker, GSAGSA linker, or GGGGGSGGGGGSGGGS linker.
(7) A nucleic acid molecule encoding the partially deficient HA-M1 fusion protein according to any one of (1) to (6) above.
(8) An expression vector comprising the nucleic acid molecule according to (7) above.
(9) A transformant comprising the nucleic acid molecule described in (7) above or the expression vector described in (8) above.
(10) The transformant according to (9) above, wherein the transformant is rice or yeast.
(11) A method for producing a partially deficient HA-M1 fusion protein, which comprises culturing or growing the transformant according to (9) or (10) above.
(12) A pharmaceutical comprising the partially defective HA-M1 fusion protein according to any one of (1) to (6), the nucleic acid molecule according to (7), or the expression vector according to (8). Composition.
(13) The pharmaceutical composition according to (12) above, for preventing or treating an influenza virus infection.
(14) The pharmaceutical composition according to (12) above, for use as a vaccine against influenza virus.
(15) The pharmaceutical composition according to (13) or (14) above, wherein the influenza virus is influenza virus type A.
(16) The partially defective HA-M1 fusion protein according to any one of (1) to (6), the nucleic acid molecule according to (7), or the expression vector according to (8) for use in therapy. ..
(17) The partially deficient HA-M1 fusion protein, nucleic acid molecule or expression vector according to (16) above, for preventing or treating an influenza virus infection.
(18) The partially deficient HA-M1 fusion protein, nucleic acid molecule or expression vector according to (16) above, for use as a vaccine against influenza virus.
(19) The partially defective HA-M1 fusion protein, nucleic acid molecule or expression vector according to (17) or (18) above, wherein the influenza virus is influenza virus type A.
(20) The partially defective HA-M1 fusion protein according to any one of (1) to (6), the nucleic acid molecule according to (7), or the expression vector according to (8) is administered to the subject. A method of preventing or treating an influenza virus infection in the subject, comprising the above.
(21) The partially defective HA-M1 fusion protein according to any one of (1) to (6), the nucleic acid molecule according to (7), or the expression vector according to (8) is administered to the subject. A method of inducing a protective immune response against an influenza virus in the subject.
(22) The method according to (20) or (21) above, wherein the influenza virus is influenza virus type A.
(23) The pharmaceutical composition according to any one of (12) to (15) above, further comprising an adjuvant.
(24) The partially defective HA-M1 fusion protein, nucleic acid molecule or expression vector according to any one of (16) to (19) above, for use in combination with an adjuvant.
(25) The method according to any one of (20) to (22) above, wherein the partially defective HA-M1 fusion protein, nucleic acid molecule or expression vector is administered together with an adjuvant.
　本発明によれば、優れた特徴を有する頭部欠損ＨＡ－Ｍ１融合タンパク質が提供される。本発明の融合タンパク質により、インフルエンザウイルスの感染症を予防または治療することができる。特に、本発明の融合タンパク質は、形質転換されたイネや酵母において製造できる点で有利である。 According to the present invention, a head-deficient HA-M1 fusion protein having excellent characteristics is provided. The fusion protein of the present invention can prevent or treat an influenza virus infection. In particular, the fusion protein of the present invention is advantageous in that it can be produced in transformed rice and yeast.
図１は、頭部欠損HA-M1融合タンパク質のイネでの発現ベクターの構造を示す図である。FIG. 1 is a diagram showing the structure of an expression vector of a head-deficient HA-M1 fusion protein in rice. 図２は、頭部欠損HA-M1融合タンパク質遺伝子を発現するイネカルスおよび培養液のタンパク質の電気泳動を行った結果を示す写真である。FIG. 2 is a photograph showing the results of electrophoresis of proteins in inecalus and culture medium expressing the head-deficient HA-M1 fusion protein gene. 図３は、改良型頭部欠損HA-M1融合タンパク質の概略を示す図である。FIG. 3 is a diagram showing an outline of the improved head-deficient HA-M1 fusion protein. 図４は、改良型頭部欠損HA-M1タンパク質を発現するイネカルスのウエスタンブロティングを行った結果を示す写真である。FIG. 4 is a photograph showing the results of western blotting of Inekals expressing the improved head-deficient HA-M1 protein. 図５は、再改良型頭部欠損HA-M1融合タンパク質イネ発現ベクターの概略を示す図である。FIG. 5 is a diagram showing an outline of a re-improved head-deficient HA-M1 fusion protein rice expression vector. 図６は、再改良型頭部欠損HA-M1融合タンパク質の種子での発現を示す写真である。FIG. 6 is a photograph showing the expression of the re-improved head-deficient HA-M1 fusion protein in seeds. 図７は、再改良型頭部欠損HA-M1融合タンパク質Ｄの、イネのカルスでの発現用ベクター構築の概略を示す図である。FIG. 7 is a diagram showing an outline of vector construction for expression of the re-improved head-deficient HA-M1 fusion protein D in rice callus. 図８は、ベクター甲による形質転換体の独立した6系統と、ベクター乙による形質転換体の独立した5系統の、SDS-PAGEおよびウエスタンブロッティングの結果を示す写真である。FIG. 8 is a photograph showing the results of SDS-PAGE and Western blotting of 6 independent lines of transformants by Vector A and 5 independent lines of transformants by Vector B. 図９は、ベクター乙による形質転換体の独立した5系統と、ベクター丙による形質転換体の独立した4系統、ベクター丁による形質転換体の独立した5系統の、ウエスタンブロッティングの結果を示す写真である。FIG. 9 is a photograph showing the results of Western blotting of 5 independent lines of the transformant by Vector B, 4 independent lines of the transformant by Vector Hei, and 5 independent lines of the transformant by Vector Ding. be. 図１０は、頭部欠損HA-M1融合タンパク質および改良型頭部欠損HA-M1融合タンパク質Ｂの酵母発現用プラスミドの構造を示す図である。FIG. 10 is a diagram showing the structures of the plasmids for yeast expression of the head-deficient HA-M1 fusion protein and the improved head-deficient HA-M1 fusion protein B. 図１１は、頭部欠損HA-M1融合タンパク質および改良型頭部欠損HA-M1融合タンパク質Ｂの出芽酵母による発現を示す写真である。FIG. 11 is a photograph showing the expression of the head-deficient HA-M1 fusion protein and the improved head-deficient HA-M1 fusion protein B by Saccharomyces cerevisiae. 図１２は、各種頭部欠損融合タンパク質を酵母で発現させるためのベクターの概略を示す図である。FIG. 12 is a diagram showing an outline of a vector for expressing various head-deficient fusion proteins in yeast. 図１３は、酵母による各種頭部欠損融合タンパク質の発現を示す写真である。FIG. 13 is a photograph showing the expression of various head-deficient fusion proteins by yeast. 図１４は、実施例７におけるインフルエンザウイルス感染マウスによる薬効試験の概要を示す図である。FIG. 14 is a diagram showing an outline of a drug efficacy test using influenza virus-infected mice in Example 7. 図１５－１は、改良型融合タンパク質を用いたインフルエンザウイルス感染マウスにおける薬効試験の結果を示す図である。FIG. 15-1 is a diagram showing the results of a drug efficacy test in influenza virus-infected mice using the improved fusion protein. 図１５－２は、改良型融合タンパク質を用いたインフルエンザウイルス感染マウスにおける薬効試験の結果を示す図である。FIG. 15-2 is a diagram showing the results of a drug efficacy test in influenza virus-infected mice using the improved fusion protein. 図１６は、実施例８におけるインフルエンザウイルス感染マウスによる薬効試験の概要を示す図である。FIG. 16 is a diagram showing an outline of a drug efficacy test using influenza virus-infected mice in Example 8. 図１７－１は、改良型融合タンパク質および再改良型融合タンパク質を用いたインフルエンザウイルス感染マウスにおける薬効試験の結果を示す図である。FIG. 17-1 is a diagram showing the results of a drug efficacy test in influenza virus-infected mice using the improved fusion protein and the re-improved fusion protein. 図１７－２は、改良型融合タンパク質および再改良型融合タンパク質を用いたインフルエンザウイルス感染マウスにおける薬効試験の結果を示す図である。FIG. 17-2 is a diagram showing the results of a drug efficacy test in influenza virus-infected mice using the improved fusion protein and the re-improved fusion protein.
発明の具体的説明Specific description of the invention
１．融合タンパク質
　本発明の融合タンパク質は、インフルエンザウイルスに由来するＨＡタンパク質とＭ１タンパク質との融合タンパク質において、特定の領域のアミノ酸残基が欠損してなる部分欠損ＨＡ－Ｍ１融合タンパク質である。 1. 1. Fusion protein The fusion protein of the present invention is a partially deficient HA-M1 fusion protein in which an amino acid residue in a specific region is deleted in a fusion protein of an HA protein derived from influenza virus and an M1 protein.
　本発明において、インフルエンザウイルスとしては、Ａ型、Ｂ型、Ｃ型等が挙げられ、これらのいずれであってもよいが、好ましくはＡ型インフルエンザウイルスである。 In the present invention, examples of the influenza virus include type A, type B, type C, and the like, and any of these may be used, but type A influenza virus is preferable.
　前記ＨＡタンパク質は、配列番号１中の第１８～５６６アミノ酸残基で表されるアミノ酸配列を有するタンパク質、または該アミノ酸配列に対して少なくとも９０％、少なくとも９１％、少なくとも９２％、少なくとも９３％、少なくとも９４％、少なくとも９５％、少なくとも９６％、少なくとも９７％、少なくとも９８％、または少なくとも９９％の配列同一性を有するアミノ酸配列を有し、かつ、インフルエンザウイルス由来のＨＡタンパク質として機能するタンパク質である。配列番号１中の第１８～５６６アミノ酸残基で表されるアミノ酸配列以外のアミノ酸配列は、元のアミノ酸配列におけるアミノ酸残基を、同一または類似の性質を有するアミノ酸残基への置換（保存的置換）により変異させたものとすることができる。 The HA protein is a protein having an amino acid sequence represented by the 18th to 566 amino acid residues in SEQ ID NO: 1, or at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence. A protein having an amino acid sequence having at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity and functioning as an HA protein derived from influenza virus. .. Amino acid sequences other than the amino acid sequences represented by the 18th to 566 amino acid residues in SEQ ID NO: 1 replace amino acid residues in the original amino acid sequence with amino acid residues having the same or similar properties (conservative). It can be mutated by substitution).
　前記Ｍ１タンパク質は、配列番号５中の第１～２５２アミノ酸残基で表されるアミノ酸配列を有するタンパク質、または該アミノ酸配列に対して少なくとも９０％、少なくとも９１％、少なくとも９２％、少なくとも９３％、少なくとも９４％、少なくとも９５％、少なくとも９６％、少なくとも９７％、少なくとも９８％、または少なくとも９９％の配列同一性を有するアミノ酸配列を有し、かつ、インフルエンザウイルス由来のＭ１タンパク質として機能するタンパク質である。配列番号５中の第１～２５２アミノ酸残基で表されるアミノ酸配列以外のアミノ酸配列は、元のアミノ酸配列におけるアミノ酸残基を、同一または類似の性質を有するアミノ酸残基への置換（保存的置換）により変異させたものとすることができる。 The M1 protein is a protein having an amino acid sequence represented by the 1st to 252nd amino acid residues in SEQ ID NO: 5, or at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence. A protein having an amino acid sequence having at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity and functioning as an M1 protein derived from influenza virus. .. Amino acid sequences other than the amino acid sequences represented by the 1st to 252nd amino acid residues in SEQ ID NO: 5 replace amino acid residues in the original amino acid sequence with amino acid residues having the same or similar properties (conservative). It can be mutated by substitution).
　アミノ酸配列どうしの配列同一性は、ＢＬＡＳＴ等の公知の配列比較用プログラムを用いて算出することができる。これらのプログラムでは、目的に応じてパラメーターを変更することもできるが、デフォルトのパラメーターのまま使用してもよい。 The sequence identity between amino acid sequences can be calculated using a known sequence comparison program such as BLAST. In these programs, the parameters can be changed according to the purpose, but the default parameters may be used.
　本発明の融合タンパク質では、配列番号１中の第５９～３３５アミノ酸残基および第５２９～５５４アミノ酸残基に相当する領域のアミノ酸残基が少なくとも欠損している。本発明において、「に相当する領域のアミノ酸残基」との用語は、特定のアミノ酸配列中において指定された領域のアミノ酸残基そのものだけでなく、他のアミノ酸配列中における同じ位置の領域のアミノ酸残基をも意味するために用いられる。つまり、本発明におけるＨＡタンパク質およびＭ１タンパク質は、特定のアミノ酸配列（それぞれ配列番号１中の第１８～５６６アミノ酸残基および配列番号５中の第１～２５２アミノ酸残基）を有するタンパク質だけでなく、これらと９０％以上の配列同一性を有するアミノ酸配列を有するタンパク質をも包含している。よって、本発明において元のアミノ酸配列とは異なる、特定の配列同一性を有するアミノ酸配列を有するタンパク質が用いられる場合には、元のアミノ酸配列において指定された領域と同等の位置の領域のアミノ酸残基が欠損（または保持）されることになる。 In the fusion protein of the present invention, at least the amino acid residues in the regions corresponding to the 59th to 335th amino acid residues and the 529 to 554 amino acid residues in SEQ ID NO: 1 are deleted. In the present invention, the term "amino acid residue in the region corresponding to" refers not only to the amino acid residue itself in the specified region in a specific amino acid sequence, but also to the amino acid in the region at the same position in another amino acid sequence. It is also used to mean a residue. That is, the HA protein and M1 protein in the present invention are not only proteins having a specific amino acid sequence (18th to 566 amino acid residues in SEQ ID NO: 1 and 1st to 252 amino acid residues in SEQ ID NO: 5, respectively). , Also include proteins having an amino acid sequence having 90% or more sequence identity with these. Therefore, when a protein having an amino acid sequence having a specific sequence identity different from the original amino acid sequence is used in the present invention, the amino acid residue in the region at the same position as the region specified in the original amino acid sequence is used. The group will be missing (or retained).
　本発明の好ましい実施態様によれば、本発明の融合タンパク質では、配列番号１中の第５９～３３５アミノ酸残基、第５１～３３５アミノ酸残基、第５９～３３９アミノ酸残基、または第５１～３３９アミノ酸残基に相当する領域のアミノ酸残基が欠損している。 According to a preferred embodiment of the present invention, in the fusion protein of the present invention, the 59th to 335th amino acid residues, the 51st to 335th amino acid residues, the 59th to 339th amino acid residues, or the 51st to 51st amino acid residues in SEQ ID NO: 1 are used. The amino acid residue in the region corresponding to the 339 amino acid residue is missing.
　本発明の好ましい実施態様によれば、本発明の融合タンパク質では、上述の欠損に加えて、配列番号１中の第５５５～５６６アミノ酸残基に相当する領域のアミノ酸残基が欠損している。 According to a preferred embodiment of the present invention, in the fusion protein of the present invention, in addition to the above-mentioned deletion, the amino acid residue in the region corresponding to the 555 to 566 amino acid residue in SEQ ID NO: 1 is deleted.
　一方で、本発明の融合タンパク質では、配列番号１中の第１８～５０アミノ酸残基、第３４０～５２８アミノ酸残基および配列番号５中の第１～２５２アミノ酸残基に相当する領域のアミノ酸残基が少なくとも保持されている。 On the other hand, in the fusion protein of the present invention, the amino acid residue in the region corresponding to the 18th to 50th amino acid residues in SEQ ID NO: 1, the 340th to 528 amino acid residues, and the 1st to 252nd amino acid residues in SEQ ID NO: 5 The group is at least retained.
　本発明の一つの実施態様によれば、本発明の融合タンパク質において、配列番号１中の第５１～３３９アミノ酸残基および第５２９～５５４アミノ酸残基に相当する領域のアミノ酸残基が欠損し、かつ、その他のアミノ酸残基が保持されている。この融合タンパク質のアミノ酸配列の例を配列番号６１（欠損した第５１～３３９アミノ酸残基に代えて、ＧＳＧＳＧリンカーが挿入されている）に示し、これをコードするヌクレオチド配列を配列番号１２に示す。 According to one embodiment of the present invention, in the fusion protein of the present invention, the amino acid residues in the regions corresponding to the 51st to 339th amino acid residues and the 529th to 554th amino acid residues in SEQ ID NO: 1 are deleted. Moreover, other amino acid residues are retained. An example of the amino acid sequence of this fusion protein is shown in SEQ ID NO: 61 (where the GSGSG linker is inserted in place of the missing 51-339 amino acid residue), and the nucleotide sequence encoding this is shown in SEQ ID NO: 12.
　本発明の他の実施態様によれば、本発明の融合タンパク質において、配列番号１中の第５１～３３５アミノ酸残基および第５２９～５５４アミノ酸残基に相当する領域のアミノ酸残基が欠損し、かつ、その他のアミノ酸残基が保持されている。この融合タンパク質のアミノ酸配列の例を配列番号６２（欠損した第５１～３３５アミノ酸残基に代えて、ＧＳＧリンカーが挿入されている）に示し、これをコードするヌクレオチド配列を配列番号１４に示す。 According to another embodiment of the present invention, in the fusion protein of the present invention, amino acid residues in the regions corresponding to amino acid residues 51 to 335 and amino acids 529 to 554 in SEQ ID NO: 1 are deleted. Moreover, other amino acid residues are retained. An example of the amino acid sequence of this fusion protein is shown in SEQ ID NO: 62 (where the GSG linker is inserted in place of the missing 51-335 amino acid residues), and the nucleotide sequence encoding this is shown in SEQ ID NO: 14.
　本発明の他の実施態様によれば、本発明の融合タンパク質において、配列番号１中の第５９～３３９アミノ酸残基および第５２９～５５４アミノ酸残基に相当する領域のアミノ酸残基が欠損し、かつ、その他のアミノ酸残基が保持されている。この融合タンパク質のアミノ酸配列の例を配列番号６３（欠損した第５９～３３９アミノ酸残基に代えて、ＧＳＧＳＧリンカーが挿入されている）に示し、これをコードするヌクレオチド配列を配列番号１５に示す。 According to another embodiment of the present invention, in the fusion protein of the present invention, the amino acid residues in the regions corresponding to the 59th to 339th amino acid residues and the 529 to 554th amino acid residues in SEQ ID NO: 1 are deleted. Moreover, other amino acid residues are retained. An example of the amino acid sequence of this fusion protein is shown in SEQ ID NO: 63 (where the GSGSG linker is inserted in place of the missing 59-339 amino acid residue), and the nucleotide sequence encoding this is shown in SEQ ID NO: 15.
　本発明の他の実施態様によれば、本発明の融合タンパク質において、配列番号１中の第５９～３３５アミノ酸残基および第５２９～５５４アミノ酸残基に相当する領域のアミノ酸残基が欠損し、かつ、その他のアミノ酸残基が保持されている。この融合タンパク質のアミノ酸配列の例を配列番号６４（欠損した第５９～３３５アミノ酸残基に代えて、ＧＳＧリンカーが挿入されている）に示し、これをコードするヌクレオチド配列を配列番号２６に示す。 According to another embodiment of the present invention, in the fusion protein of the present invention, amino acid residues in the regions corresponding to amino acid residues 59 to 335 and amino acids 529 to 554 in SEQ ID NO: 1 are deleted. Moreover, other amino acid residues are retained. An example of the amino acid sequence of this fusion protein is shown in SEQ ID NO: 64 (where a GSG linker is inserted in place of the missing 59-335 amino acid residue), and the nucleotide sequence encoding this is shown in SEQ ID NO: 26.
　本発明の他の実施態様によれば、本発明の融合タンパク質において、配列番号１中の第５１～３３５アミノ酸残基、第５２９～５５４アミノ酸残基および第５５５～５６６アミノ酸残基に相当する領域のアミノ酸残基が欠損し、かつ、その他のアミノ酸残基が保持されている。この融合タンパク質のアミノ酸配列の例を配列番号６５（欠損した第５１～３３５アミノ酸残基に代えてＧＳＧリンカーが挿入され、欠損した第５５５～５６６アミノ酸残基に代えてＧＳＡＧＳＡリンカーが挿入されている）に示し、これをコードするヌクレオチド配列を配列番号２５に示す。 According to another embodiment of the present invention, in the fusion protein of the present invention, the regions corresponding to the 51st to 335th amino acid residues, the 529th to 554th amino acid residues and the 555th to 566th amino acid residues in SEQ ID NO: 1. Amino acid residue is deleted and other amino acid residues are retained. An example of the amino acid sequence of this fusion protein is SEQ ID NO: 65 (GSG linker is inserted in place of the missing 51-335 amino acid residue, and GSAGSA linker is inserted in place of the missing 555-566 amino acid residue. ), And the nucleotide sequence encoding this is shown in SEQ ID NO: 25.
　本発明の他の実施態様によれば、本発明の融合タンパク質において、配列番号１中の第５９～３３５アミノ酸残基、第５２９～５５４アミノ酸残基および第５５５～５６６アミノ酸残基に相当する領域のアミノ酸残基が欠損し、かつ、その他のアミノ酸残基が保持されている。この融合タンパク質のアミノ酸配列の例を配列番号６６（欠損した第５９～３３５アミノ酸残基に代えてＧＳＧリンカーが挿入され、欠損した第５５５～５６６アミノ酸残基に代えてＧＳＡＧＳＡリンカーが挿入されている）に示し、これをコードするヌクレオチド配列を配列番号２７に示す。 According to another embodiment of the present invention, in the fusion protein of the present invention, the regions corresponding to the 59th to 335th amino acid residues, the 529th to 554th amino acid residues and the 555th to 566th amino acid residues in SEQ ID NO: 1. Amino acid residue is deleted and other amino acid residues are retained. An example of the amino acid sequence of this fusion protein is SEQ ID NO: 66 (GSG linker is inserted in place of the missing 59-335 amino acid residue, and GSAGSA linker is inserted in place of the missing 555-566 amino acid residue. ), And the nucleotide sequence encoding this is shown in SEQ ID NO: 27.
　本発明の融合タンパク質において、前記ＨＡタンパク質と前記Ｍ１タンパク質とは、Ｎ末端からＣ末端に向けてこの順で含まれてもよく、逆の順で含まれてもよいが、好ましくはこの順で含まれている。 In the fusion protein of the present invention, the HA protein and the M1 protein may be contained in this order from the N-terminal to the C-terminal, or may be contained in the reverse order, but preferably in this order. include.
　本発明の融合タンパク質において、アミノ酸残基が欠損した領域にリンカーを挿入してもよい。また、本発明の融合タンパク質において、部分欠損ＨＡタンパク質とＭ１タンパク質の融合部位にリンカーを挿入してもよい。リンカー（リンカーペプチド）の長さは、本発明の融合タンパク質がインフルエンザウイルスに対する抑制効果を有する限り特に限定されないが、通常、1～100アミノ酸、好ましくは1～50アミノ酸、より好ましくは1～25アミノ酸、さらに好ましくは1～15アミノ酸に設定するのが好ましい。リンカーペプチドの例としては、グリシンやセリン等がペプチド結合して構成された、二次構造を有さないフレキシブルなペプチドが、複数個タンデムに連結されたリンカーペプチドが挙げられ、さらに具体的には、(ＧＧＧＧＳ)n（例えばＧＧＧＧＳＧＧＧＧＳＧＧＧＧＳリンカーなど）、ＧＳＧリンカー、ＧＳＧＳＧリンカー、ＧＳＧＳＧＳＧＳリンカー、ＧＳＡＧＳＡリンカー等を挙げることができる。 In the fusion protein of the present invention, a linker may be inserted in a region lacking an amino acid residue. Further, in the fusion protein of the present invention, a linker may be inserted at the fusion site of the partially defective HA protein and the M1 protein. The length of the linker (linker peptide) is not particularly limited as long as the fusion protein of the present invention has an inhibitory effect on influenza virus, but is usually 1 to 100 amino acids, preferably 1 to 50 amino acids, and more preferably 1 to 25 amino acids. , More preferably set to 1 to 15 amino acids. Examples of the linker peptide include a linker peptide in which a flexible peptide having no secondary structure, which is composed of a peptide bond of glycine, serine, etc., is linked in a plurality of tandems, and more specifically. , (GGGGS) n (eg, GGGGGSGGGGSGGGGS linker, etc.), GSG linker, GSGSG linker, GSGSGSGS linker, GSAGSA linker and the like.
　本発明の融合タンパク質は、そのＮ末端および／またはＣ末端に、付加配列を有していてもよい。付加配列の長さは、本発明の融合タンパク質がインフルエンザウイルスに対する抑制効果を有する限り特に限定されないが、通常、1～100アミノ酸、好ましくは1～50アミノ酸、より好ましくは1～25アミノ酸、さらに好ましくは1～15アミノ酸である。 The fusion protein of the present invention may have an additional sequence at its N-terminal and / or C-terminal. The length of the additional sequence is not particularly limited as long as the fusion protein of the present invention has an inhibitory effect on influenza virus, but is usually 1 to 100 amino acids, preferably 1 to 50 amino acids, more preferably 1 to 25 amino acids, still more preferable. Is 1 to 15 amino acids.
　本発明の一つの実施態様において、前記付加配列は、本発明の融合タンパク質の精製や検出を容易にするためのタグ配列であり得る。タグ配列の種類としては、特に限定されないが、例えば、FLAG（Hopp, T. P. et al.， BioTechnology （1988） 6, 1204-1210）、6～10個のHis（ヒスチジン）残基からなる6×His～10×His、ヒトc-mycの断片、α-tubulinの断片、B-tag、Protein Cの断片、GST（グルタチオン－S－トランスフェラーゼ）、β－ガラクトシダーゼ、MBP（マルトース結合タンパク質）Sタグ、T7タグ、Strepタグ、Nusタグ、Trxタグ、GFPタグ等が挙げられる。また、付加配列としては、本発明の融合タンパク質を発現させる宿主において作動可能なシグナル配列、例えば、分泌シグナル、小胞体係留シグナル（例えばKDEL配列）なども好適に用いることができる。 In one embodiment of the invention, the additional sequence can be a tag sequence for facilitating purification or detection of the fusion protein of the invention. The type of tag sequence is not particularly limited, but consists of, for example, FLAG (Hopp, T.P. et al., BioTechnology (1988) 6, 1204-1210) and 6 to 10 His (histidine) residues. 6 × His-10 × His, human c-myc fragment, α-tubulin fragment, B-tag, Protein C fragment, GST (glutathione-S-transferase), β-galactosidase, MBP (maltose binding protein) S Examples include tags, T7 tags, Strep tags, Nus tags, Trx tags, GFP tags and the like. Further, as the addition sequence, a signal sequence that can be actuated in the host expressing the fusion protein of the present invention, for example, a secretory signal, an endoplasmic reticulum mooring signal (for example, KDEL sequence) and the like can also be preferably used.
　本発明の融合タンパク質は、周知の組換えタンパク質産生方法を用いることにより製造することができる。例えば、本発明の融合タンパク質をコードする核酸分子（好ましくはＤＮＡ）を作製し、これを適切なベクターに組み込むことにより発現ベクターを作製し、該核酸分子または該発現ベクターを宿主に組み込むことにより形質転換体を作製し、この形質転換体を培養または育成することにより、本発明の融合タンパク質を製造することができる。これらの核酸分子、発現ベクターおよび形質転換体もまた、それぞれ本発明の態様をなす。 The fusion protein of the present invention can be produced by using a well-known method for producing a recombinant protein. For example, a nucleic acid molecule (preferably DNA) encoding the fusion protein of the present invention is prepared, an expression vector is prepared by incorporating the nucleic acid molecule (preferably DNA) into an appropriate vector, and the nucleic acid molecule or the expression vector is incorporated into a host for transformation. The fusion protein of the present invention can be produced by producing a transformant and culturing or growing the transformant. These nucleic acid molecules, expression vectors and transformants also form aspects of the invention, respectively.
　形質転換体の作製に用いられる宿主は、タンパク質の組換え生産に利用可能ないずれの宿主であってもよいが、例えば、大腸菌などの細菌、酵母などの真菌、植物細胞もしくは植物、動物細胞もしくは動物などが挙げられる。本発明の好ましい実施態様によれば、前記宿主は、酵母（例えばSaccharomyces cerevisiae）または植物（例えばイネ）とされる。形質転換体は、発現ベクターを宿主細胞に導入することにより作製してもよいし、相同組換えにより宿主細胞のゲノム中に核酸分子を組み込むことにより作製してもよい。 The host used to generate the transformant may be any host available for recombinant production of the protein, eg, bacteria such as Escherichia coli, fungi such as yeast, plant cells or plants, animal cells or Examples include animals. According to a preferred embodiment of the invention, the host is a yeast (eg, Saccharomyces cerevisiae ) or a plant (eg, rice). The transformant may be prepared by introducing an expression vector into a host cell, or by incorporating a nucleic acid molecule into the genome of the host cell by homologous recombination.
　発現ベクターは、使用する宿主に応じて、その宿主において適切に機能するように選択することができる。また、本発明の融合タンパク質をコードする核酸分子は、使用する宿主において使用頻度の高いコドンを多く含むように構成することもできる。さらに、使用する宿主に応じて、その宿主において適切に機能するプロモーター、エンハンサー、ターミネーター等の発現調節配列を、コード配列の前後に配置してもよい。 The expression vector can be selected to function appropriately in that host, depending on the host used. In addition, the nucleic acid molecule encoding the fusion protein of the present invention can be configured to contain many codons that are frequently used in the host to be used. Furthermore, depending on the host used, expression control sequences such as promoters, enhancers, and terminators that function appropriately in the host may be arranged before and after the coding sequence.
２．医薬組成物
　本発明の医薬組成物は、本発明の融合タンパク質を含む。このような医薬組成物は、本発明の融合タンパク質を、常套手段に従って製剤化することにより得ることができる。あるいは、本発明の医薬組成物は、本発明の融合タンパク質をコードする本発明の核酸分子や本発明の発現ベクターを含んでもよく、このような医薬組成物もまた、常套手段に従って製造することができる。 2. 2. Pharmaceutical Composition The pharmaceutical composition of the present invention comprises the fusion protein of the present invention. Such a pharmaceutical composition can be obtained by formulating the fusion protein of the present invention according to a conventional method. Alternatively, the pharmaceutical composition of the present invention may contain the nucleic acid molecule of the present invention encoding the fusion protein of the present invention or the expression vector of the present invention, and such a pharmaceutical composition may also be produced according to conventional means. can.
　本発明の医薬組成物は、医薬上許容される担体をさらに含んでいてもよい。医薬上許容される担体としては、例えば、ショ糖、デンプン、マンニット、ソルビット、乳糖、グルコース、セルロース、タルク、リン酸カルシウム、炭酸カルシウム等の賦形剤、セルロース、メチルセルロース、ヒドロキシプロピルセルロース、ゼラチン、アラビアゴム、ポリエチレングリコール、ショ糖、デンプン等の結合剤、デンプン、カルボキシメチルセルロース、ヒドロキシプロピルスターチ、ナトリウム－グリコール－スターチ、炭酸水素ナトリウム、リン酸カルシウム、クエン酸カルシウム等の崩壊剤、ステアリン酸マグネシウム、エアロジル、タルク、ラウリル硫酸ナトリウム等の滑沢剤、クエン酸、メントール、グリチルリチン・アンモニウム塩、グリシン、オレンジ粉等の芳香剤、安息香酸ナトリウム、亜硫酸水素ナトリウム、メチルパラベン、プロピルパラベン等の保存剤、クエン酸、クエン酸ナトリウム、酢酸等の安定剤、メチルセルロース、ポリビニルピロリドン、ステアリン酸アルミニウム等の懸濁剤、界面活性剤等の分散剤、水、生理食塩液等の希釈剤、カカオ脂、ポリエチレングリコール、白灯油等のベースワックスなどが挙げられるが、それらに限定されるものではない。 The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, excipients such as sucrose, starch, mannit, sorbit, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, cellulose, methylcellulose, hydroxypropylcellulose, gelatin, Arabia. Excipients such as rubber, polyethylene glycol, sucrose, starch, disintegrants such as starch, carboxymethyl cellulose, hydroxypropyl starch, sodium-glycol-starch, sodium hydrogen carbonate, calcium phosphate, calcium citrate, magnesium stearate, aerodil, talc , Lubricants such as sodium lauryl sulfate, fragrances such as citric acid, menthol, glycyrrhizin / ammonium salt, glycine, orange powder, preservatives such as sodium benzoate, sodium hydrogen sulfite, methylparaben, propylparaben, citric acid, citrate Stabilizers such as sodium citrate and acetic acid, suspending agents such as methyl cellulose, polyvinylpyrrolidone and aluminum stearate, dispersants such as surfactants, diluents such as water and physiological saline, cocoa butter, polyethylene glycol, white kerosene and the like. Base wax, etc., but is not limited to them.
　本発明の医薬組成物は、本発明の融合タンパク質の免疫反応誘導効果を増強するため、アジュバントをさらに含有してもよい。アジュバントとしては、水酸化アルミニウム、完全フロイントアジュバント、不完全フロイントアジュバント、百日咳菌アジュバント、ポリ（Ｉ：Ｃ）、ＣｐＧ－ＤＮＡ、２’，３’－ｃＧＡＭＰ等が挙げられるが、これらに限定されない。 The pharmaceutical composition of the present invention may further contain an adjuvant in order to enhance the immune response-inducing effect of the fusion protein of the present invention. Examples of the adjuvant include, but are not limited to, aluminum hydroxide, complete Freund's adjuvant, incomplete Freund's adjuvant, Bordetella pertussis adjuvant, poly (I: C), CpG-DNA, 2', 3'-cGAMP and the like.
　本発明の医薬組成物は、経口又は非経口投与（好ましくは非経口投与）に適する剤形として提供される。 The pharmaceutical composition of the present invention is provided as a dosage form suitable for oral or parenteral administration (preferably parenteral administration).
　非経口投与のための組成物としては、例えば、注射剤、経鼻投与剤（点鼻液、点鼻スプレー等）、坐剤等が用いられ、注射剤は静脈注射剤、皮下注射剤、皮内注射剤、筋肉注射剤、点滴注射剤等の剤形を包含してもよい。このような注射剤や経鼻投与剤は、公知の方法に従って調製できる。注射剤の調製方法としては、例えば、本発明の融合タンパク質を通常注射剤に用いられる無菌の水性溶媒に溶解又は懸濁することによって調製できる。注射用の水性溶媒としては、例えば、蒸留水；生理的食塩水；リン酸緩衝液、炭酸緩衝液、トリス緩衝液、クエン酸緩衝液、酢酸緩衝液等の緩衝液等が使用できる。このような水性溶媒のpHは5～10が挙げられ、好ましくは6～8である。調製された注射液や経鼻投与液は、適当なアンプルやバイアルに充填されたり、あらかじめ注射器や点鼻用医薬品注入器に充填されたプレフィルド型とすることが好ましい。 As the composition for parenteral administration, for example, an injection, a nasal administration (nasal drop, nasal spray, etc.), a suppository, etc. are used, and the injection is an intravenous injection, a subcutaneous injection, a skin. Dosage forms such as internal injection, intramuscular injection, and drip injection may be included. Such injections and nasal administrations can be prepared according to known methods. As a method for preparing an injection, for example, the fusion protein of the present invention can be prepared by dissolving or suspending it in a sterile aqueous solvent usually used for injections. As the aqueous solvent for injection, for example, distilled water; physiological saline; a phosphate buffer solution, a carbonate buffer solution, a Tris buffer solution, a citrate buffer solution, a buffer solution such as an acetate buffer solution, or the like can be used. The pH of such an aqueous solvent is 5 to 10, preferably 6 to 8. It is preferable that the prepared injection solution or nasal administration solution is a prefilled type that is filled in an appropriate ampoule or vial, or is previously filled in a syringe or a nasal drop drug injector.
　また、本発明の融合タンパク質の溶液又は懸濁液を、医薬上許容可能な担体とともに又は単独で真空乾燥、凍結乾燥、噴霧乾燥等の処理に付すことにより、本発明の融合タンパク質の粉末製剤を調製することもできる。本発明の融合タンパク質を粉末状態で保存し、使用時に該粉末を注射用や経鼻投与用の水性溶媒で分散することにより、使用に供することができる。 Further, by subjecting the solution or suspension of the fusion protein of the present invention to a treatment such as vacuum drying, freeze drying, spray drying, etc. together with a pharmaceutically acceptable carrier or alone, a powder formulation of the fusion protein of the present invention can be obtained. It can also be prepared. The fusion protein of the present invention can be used by storing it in a powder state and dispersing the powder in an aqueous solvent for injection or nasal administration at the time of use.
　医薬組成物中の本発明の融合タンパク質の含有量は、通常、医薬組成物全体の約0.1～100質量％、好ましくは約1～99質量％、さらに好ましくは約10～90質量％程度である。 The content of the fusion protein of the present invention in the pharmaceutical composition is usually about 0.1 to 100% by mass, preferably about 1 to 99% by mass, more preferably about 10 to 90% by mass, based on the whole pharmaceutical composition. ..
３．医薬用途
　本発明の融合タンパク質は、インフルエンザウイルス感染症の予防または治療に用いることができる。つまり、本発明の融合タンパク質または本発明の医薬組成物を動物（動物全般を含むが、とくにヒト等の霊長類、マウス等のげっ歯類等の哺乳動物）に投与することにより、該動物において、インフルエンザウイルス感染症を予防又は治療することができる。本発明の融合タンパク質は、ウイルスのサブタイプ間で保存されたＢ細胞エピトープを含有する抗原又はその断片と、該ウイルスのサブタイプ間で保存されたＴ細胞エピトープを含有する抗原又はその断片を含むことにより、変異体ウイルスや広範なサブタイプに対して有効な交差反応性を有する液性免疫反応（抗体（好適には、中和抗体）産生）及び細胞性免疫反応（ＣＴＬ増殖）を誘導すると考えられるので、様々なサブタイプのウイルスに交差して有効なワクチンとして、該ウイルスの感染症を予防又は治療することができる。 3. 3. Pharmaceutical Use The fusion protein of the present invention can be used for the prevention or treatment of influenza virus infections. That is, by administering the fusion protein of the present invention or the pharmaceutical composition of the present invention to an animal (including animals in general, but especially mammals such as primates such as humans and mammals such as rodents such as mice), in the animals. , Can prevent or treat influenza virus infections. The fusion protein of the invention comprises an antigen or fragment thereof containing a B cell epitope conserved between subtypes of the virus and an antigen or fragment thereof containing a T cell epitope conserved between the subtypes of the virus. This induces a humoral immune response (antibody (preferably neutralizing antibody) production) and a cellular immune response (CTL proliferation) that have cross-reactivity that is effective against mutant viruses and a wide range of subtypes. As conceivable, it is possible to prevent or treat an infectious disease of the virus as an effective vaccine across various subtypes of the virus.
　例えば、本発明の融合タンパク質または本発明の医薬組成物を、インフルエンザウイルス感染症の患者や、インフルエンザウイルスに感染する可能性がある動物（特に、ヒト等の霊長類、マウス等のげっ歯類等哺乳動物）に投与することにより、投与を受けた対象において、インフルエンザウイルスに対する液性免疫反応および細胞性免疫を誘導し、すなわち、該哺乳動物の防御免疫反応を誘導することにより、インフルエンザウイルス感染症を予防又は治療することができる。また、本発明の融合タンパク質または本発明の医薬組成物を、インフルエンザウイルスに感染する可能性がある動物（特に、ヒト等の霊長類、マウス等のげっ歯類等の哺乳動物）に投与することにより、投与を受けた対象において、インフルエンザウイルスに対する液性免疫反応および細胞性免疫を誘導し、すなわち、該哺乳動物の防御免疫反応を誘導することにより、インフルエンザウイルスに感染するリスクを低減させることができる。 For example, patients with influenza virus infection or animals that may be infected with influenza virus (particularly primates such as humans, rodents such as mice, etc.) using the fusion protein of the present invention or the pharmaceutical composition of the present invention. Influenza virus infection by inducing a humoral and cellular immunity against influenza virus, i.e., by inducing a protective immune response in the mammal, by administration to a mammal). Can be prevented or treated. In addition, the fusion protein of the present invention or the pharmaceutical composition of the present invention may be administered to animals that may be infected with influenza virus (particularly, primates such as humans and mammals such as rodents such as mice). By inducing a humoral and cell-mediated immunity against influenza virus, that is, by inducing a protective immune response in the mammal, the risk of infection with influenza virus can be reduced. can.
　本発明の好ましい実施態様によれば、目的とするインフルエンザウイルスはＡ型インフルエンザウイルスとされる。本発明の融合タンパク質または本発明の医薬組成物は、季節性インフルエンザウイルスおよび予想される高病原性パンデミックインフルエンザウイルスを含む広範囲のＡ亜型インフルエンザウイルスに交差して有効性を示すことができる。 According to a preferred embodiment of the present invention, the target influenza virus is influenza A virus. The fusion protein of the invention or the pharmaceutical composition of the invention can be shown to be effective across a wide range of subtype A influenza viruses, including seasonal influenza virus and the expected highly pathogenic pandemic influenza virus.
　本発明の他の態様によれば、療法に用いるための、インフルエンザウイルスの感染症を予防または治療するための、あるいはインフルエンザウイルスに対するワクチンとして用いるための、本発明の融合タンパク質が提供される。また、この態様によれば、療法に用いるための、インフルエンザウイルスの感染症を予防または治療するための、あるいはインフルエンザウイルスに対するワクチンとして用いるための、本発明の核酸分子または本発明の発現ベクターも提供される。 According to another aspect of the invention, there is provided a fusion protein of the invention for use in therapy, for the prevention or treatment of influenza virus infections, or for use as a vaccine against influenza virus. Also provided according to this aspect is the nucleic acid molecule of the invention or the expression vector of the invention for use in therapy, for the prevention or treatment of influenza virus infections, or for use as a vaccine against influenza virus. Will be done.
　本発明の他の態様によれば、本発明の融合タンパク質を被験体（ヒトを含む）に投与することを含んでなる、該被験体におけるインフルエンザウイルスの感染症を予防または治療する方法、あるいは該被験体においてインフルエンザウイルスに対する防御免疫反応を誘導する方法が提供される。また、この態様によれば、本発明の核酸分子または本発明の発現ベクターを被験体（ヒトを含む）に投与することを含んでなる、該被験体におけるインフルエンザウイルスの感染症を予防または治療する方法、あるいは該被験体においてインフルエンザウイルスに対する防御免疫反応を誘導する方法も提供される。 According to another aspect of the invention, a method of preventing or treating an influenza virus infection in a subject, comprising administering to the subject (including humans) the fusion protein of the invention. A method of inducing a protective immune response against influenza virus in a subject is provided. Also according to this aspect, the prevention or treatment of an influenza virus infection in a subject, comprising administering to the subject (including humans) the nucleic acid molecule of the invention or the expression vector of the invention. Methods, or methods of inducing a protective immune response against influenza virus in the subject, are also provided.
　本発明の他の態様によれば、インフルエンザウイルスの感染症を予防または治療するための薬剤の製造における、あるいはインフルエンザウイルスに対するワクチンとして用いるための薬剤の製造における、本発明の融合タンパク質の使用が提供される。また、この態様によれば、インフルエンザウイルスの感染症を予防または治療するための薬剤の製造における、あるいはインフルエンザウイルスに対するワクチンとして用いるための薬剤の製造における、本発明の核酸分子または本発明の発現ベクターの使用も提供される。 According to another aspect of the invention, the use of fusion proteins of the invention is provided in the manufacture of agents for the prevention or treatment of influenza virus infections, or in the production of agents for use as vaccines against influenza virus. Will be done. Further, according to this aspect, the nucleic acid molecule of the present invention or the expression vector of the present invention in the production of a drug for preventing or treating an infectious disease of influenza virus, or in the production of a drug for use as a vaccine against influenza virus. Use of is also provided.
４．配列表に含まれる配列の説明
配列番号１：ミシガン株HAアミノ酸配列
配列番号２：ヘッドレスHA
配列番号３：ヘッドレスHAリンカー付
配列番号４：分泌シグナル
配列番号５：ミシガン株M1
配列番号６：HISタグ
配列番号７：イネ発現用HA-M1、リンカーなし。10番目のA～1731番目のAにORF。
配列番号８：イネ発現用HA-M1、リンカーあり。10番目のA～1776番目のAにORF。
配列番号９：イネα-アミラーゼ3Dプロモーター
配列番号１０：イネα-アミラーゼ3Dターミネーター
配列番号１１：GSGSGSGSリンカー
配列番号１２：改良型頭部欠損HA-M1融合タンパク質A、HA51-339。10番目のA～1578番目のAにORF。
配列番号１３：GSGリンカー
配列番号１４：改良型頭部欠損HA-M1融合タンパク質B、HA51-335。10番目のA～1584番目のAにORF。
配列番号１５：改良型頭部欠損HA-M1融合タンパク質C、HA59-339。10番目のA～1602番目のAにORF。
配列番号１６：A/SWINE/IL/00685/2005(H1N1)　M1
配列番号１７：小麦無細胞系用　HA-M1 リンカー付。1番目のG～1,692番目のAにORF。
配列番号１８：小麦無細胞系用　改良型頭部欠損HA-M1。1番目のG～1,515番目のAにORF。
配列番号１９：頭部欠損HA-M1融合タンパク質
配列番号２０：改良型頭部欠損HA-M1融合タンパク質
配列番号２１：TDH3プロモーター
配列番号２２：KDELシグナル
配列番号２３：改良型頭部欠損HA-M1融合タンパク質B+KDEL。10番目のA～1599番目のAにORF。
配列番号２４：GSAGSAリンカー
配列番号２５：再改良型頭部欠損HA-M1融合タンパク質B+KDEL。10番目のA～1,581番目のAにORF。
配列番号２６：改良型頭部欠損HA-M1融合タンパク質D+KDEL。10番目のA～1623番目のAにORF。
配列番号２７：再改良型頭部欠損HA-M1融合タンパク質D+KDEL。1番目のA～1605番目のAにORF。
配列番号２８：Glb-1プロモーター
配列番号２９：Glb-1 ターミネーター
配列番号３０：イネUbi1 プロモーター
配列番号３１：NOSターミネーター
配列番号３２：RAP 3rd intron
配列番号３３：GluABC
配列番号３４：GluABCの相補鎖
配列番号３５：Pro13K16K
配列番号３６：Pro13K16Kの相補鎖
配列番号３７：TKIWVER3_1
配列番号３８：TKIWVER3_2
配列番号３９：TKIWVER3_3
配列番号４０：TKIWVER3_4
配列番号４１：再改良型頭部欠損HA-M1融合タンパク質
配列番号４２：FLAGタグ
配列番号４３：MFA1分泌シグナル
配列番号４４：GSAGSAリンカー
配列番号４５：pYHAM1-15の発現遺伝子。10番目のＡ～1,848番目のＡにORF。
配列番号４６：pYHAM1-16の発現遺伝子。10番目のＡ～1,812番目のＡにORF。
配列番号４７：pYHAM1-1の発現遺伝子。10番目のＡ～1,671番目のＡにORF。
配列番号４８：pYHAM1-2の発現遺伝子。10番目のＡ～1,635番目のＡにORF。
配列番号４９：GGGGSリンカー
配列番号５０：Glb-1の分泌シグナル
配列番号５１：トウモロコシのユビキチンプロモーター
配列番号５２：3xFLAG
配列番号５３：ベクター甲に搭載の再改良型頭部欠損HA-M1融合タンパク質。10番目のA～1677番目のAにORF。
配列番号５４：ベクター乙に搭載の再改良型頭部欠損HA-M1融合タンパク質。10番目のA～1665番目のAにORF。
配列番号５５：再改良型頭部欠損HA-M1融合タンパク質のHA335-M1のコード領域。1番目のAからORF。
配列番号５６：再改良型頭部欠損HA-M1融合タンパク質のHA335-M1のコード領域。コドン改変バージョン。1番目のAからORF。
配列番号５７：ベクター丙に搭載の再改良型頭部欠損HA-M1融合タンパク質。10番目のA～1665番目のAにORF。
配列番号５８：ベクター丁に搭載の再改良型頭部欠損HA-M1融合タンパク質。10番目のA～1590番目のAにORF。
配列番号５９：頭部欠損HA-M1融合タンパク質（配列番号７を翻訳したアミノ酸配列から、N末端のイネのα-アミラーゼ3Dの分泌シグナル、C末端の6xHisタグを除去）
配列番号６０：リンカー付頭部欠損HA-M1融合タンパク質（配列番号８を翻訳したアミノ酸配列から、N末端のイネのα-アミラーゼ3Dの分泌シグナル、C末端の6xHisタグを除去）
配列番号６１：改良型頭部欠損HA-M1融合タンパク質A（配列番号１２を翻訳したアミノ酸配列から、N末端のイネのα-アミラーゼ3Dの分泌シグナル、C末端の6xHisタグを除去）
配列番号６２：改良型頭部欠損HA-M1融合タンパク質B（配列番号１４を翻訳したアミノ酸配列から、N末端のイネのα-アミラーゼ3Dの分泌シグナル、C末端の6xHisタグを除去）
配列番号６３：改良型頭部欠損HA-M1融合タンパク質C（配列番号１５を翻訳したアミノ酸配列から、N末端のイネのα-アミラーゼ3Dの分泌シグナル、C末端の6xHisタグを除去）
配列番号６４：改良型頭部欠損HA-M1融合タンパク質D（配列番号２６を翻訳したアミノ酸配列から、N末端のイネのGlb-1の分泌シグナル、C末端の6xHisタグおよびKDELシグナルを除去）
配列番号６５：再改良型頭部欠損HA-M1融合タンパク質B（配列番号２５を翻訳したアミノ酸配列から、N末端のイネのGlb-1の分泌シグナル、C末端の6xHisタグおよびKDELシグナルを除去）
配列番号６６：再改良型頭部欠損HA-M1融合タンパク質D（配列番号２７を翻訳したアミノ酸配列から、N末端のイネのGlb-1の分泌シグナル、C末端の6xHisタグおよびKDELシグナルを除去）
配列番号６７：GSGSGリンカー 4. Description of the sequence contained in the sequence listing <br /> SEQ ID NO: 1: Michigan strain HA amino acid sequence SEQ ID NO: 2: Headless HA
SEQ ID NO: 3: with headless HA linker SEQ ID NO: 4: Secretory signal SEQ ID NO: 5: Michigan strain M1
SEQ ID NO: 6: HIS tag SEQ ID NO: 7: HA-M1 for rice expression, no linker. ORF from the 10th A to the 1731th A.
SEQ ID NO: 8: HA-M1 for rice expression, with linker. ORF from the 10th A to the 1776th A.
SEQ ID NO: 9: Rice α-amylase 3D promoter SEQ ID NO: 10: Rice α-amylase 3D terminator SEQ ID NO: 11: GSGSGSGS Linker SEQ ID NO: 12: Improved head defect HA-M1 fusion protein A, HA51-339. 10th A ORF to the 1578th A.
SEQ ID NO: 13: GSG linker SEQ ID NO: 14: Improved head defect HA-M1 fusion protein B, HA51-335. ORF to 10th A to 1584th A.
SEQ ID NO: 15: Improved head defect HA-M1 fusion protein C, HA59-339. ORF to 10th A to 1602nd A.
SEQ ID NO: 16: A / SWINE / IL / 00685/2005 (H1N1) M1
SEQ ID NO: 17: With HA-M1 linker for wheat cell-free system. ORF from 1st G to 1,692th A.
SEQ ID NO: 18: Improved head defect HA-M1 for wheat cell-free system. ORF in 1st G to 1,515th A.
SEQ ID NO: 19: Head defect HA-M1 fusion protein SEQ ID NO: 20: Improved head defect HA-M1 fusion protein SEQ ID NO: 21: TDH3 promoter SEQ ID NO: 22: KDEL signal SEQ ID NO: 23: Improved head defect HA-M1 Fusion protein B + KDEL. ORF from the 10th A to the 1599th A.
SEQ ID NO: 24: GSAGSA linker SEQ ID NO: 25: Re-improved head defect HA-M1 fusion protein B + KDEL. ORF from the 10th A to the 1,581th A.
SEQ ID NO: 26: Improved head defect HA-M1 fusion protein D + KDEL. ORF from the 10th A to the 1623th A.
SEQ ID NO: 27: Re-improved head defect HA-M1 fusion protein D + KDEL. ORF from 1st A to 1605th A.
SEQ ID NO: 28: Glb-1 promoter SEQ ID NO: 29: Glb-1 terminator SEQ ID NO: 30: Rice Ubi1 promoter SEQ ID NO: 31: NOS terminator SEQ ID NO: 32: RAP 3rd intron
SEQ ID NO: 33: GluABC
SEQ ID NO: 34: Complementary strand of GluABC SEQ ID NO: 35: Pro13K16K
SEQ ID NO: 36: Complementary strand of Pro13K16K SEQ ID NO: 37: TKIWVER3_1
SEQ ID NO: 38: TKIWVER3_2
SEQ ID NO: 39: TKIWVER3_3
SEQ ID NO: 40: TKIWVER3_4
SEQ ID NO: 41: Re-improved head defect HA-M1 fusion protein SEQ ID NO: 42: FLAG tag SEQ ID NO: 43: MFA1 secretory signal SEQ ID NO: 44: GSAGSA linker SEQ ID NO: 45: expression gene of pYHAM1-15. ORF to the 10th A to 1,848th A.
SEQ ID NO: 46: Expression gene of pYHAM1-16. ORF from the 10th A to the 1,812th A.
SEQ ID NO: 47: Expression gene of pYHAM1-1. ORF to the 10th A to 1,671st A.
SEQ ID NO: 48: Expression gene of pYHAM1-2. ORF from the 10th A to the 1,635th A.
SEQ ID NO: 49: GGGGS linker SEQ ID NO: 50: Glb-1 secretion signal SEQ ID NO: 51: Corn ubiquitin promoter SEQ ID NO: 52: 3xFLAG
SEQ ID NO: 53: Re-improved head-deficient HA-M1 fusion protein on the vector instep. ORF from the 10th A to the 1677th A.
SEQ ID NO: 54: Re-improved head-deficient HA-M1 fusion protein loaded on Vector B. ORF from the 10th A to the 1665th A.
SEQ ID NO: 55: Code region of HA335-M1 for the re-improved head defect HA-M1 fusion protein. The first A to ORF.
SEQ ID NO: 56: Code region of HA335-M1 of the re-improved head defect HA-M1 fusion protein. Codon modified version. The first A to ORF.
SEQ ID NO: 57: Re-improved head-deficient HA-M1 fusion protein loaded on Vector Hei. ORF from the 10th A to the 1665th A.
SEQ ID NO: 58: Re-improved head-deficient HA-M1 fusion protein mounted on Vector Ding. ORF from the 10th A to the 1590th A.
SEQ ID NO: 59: Head-deficient HA-M1 fusion protein (N-terminal rice α-amylase 3D secretory signal, C-terminal 6xHis tag removed from the amino acid sequence translated from SEQ ID NO: 7)
SEQ ID NO: 60: Head-deficient HA-M1 fusion protein with linker (N-terminal rice α-amylase 3D secretory signal, C-terminal 6xHis tag removed from the amino acid sequence translated from SEQ ID NO: 8)
SEQ ID NO: 61: Improved head-deficient HA-M1 fusion protein A (N-terminal rice α-amylase 3D secretory signal, C-terminal 6xHis tag removed from the amino acid sequence translated from SEQ ID NO: 12)
SEQ ID NO: 62: Improved head-deficient HA-M1 fusion protein B (N-terminal rice α-amylase 3D secretory signal, C-terminal 6xHis tag removed from the amino acid sequence translated from SEQ ID NO: 14)
SEQ ID NO: 63: Improved head-deficient HA-M1 fusion protein C (N-terminal rice α-amylase 3D secretory signal, C-terminal 6xHis tag removed from the amino acid sequence translated from SEQ ID NO: 15)
SEQ ID NO: 64: Improved head-deficient HA-M1 fusion protein D (N-terminal rice Glb-1 secretory signal, C-terminal 6xHis tag and KDEL signal removed from the amino acid sequence translated from SEQ ID NO: 26)
SEQ ID NO: 65: Re-improved head-deficient HA-M1 fusion protein B (N-terminal rice Glb-1 secretory signal, C-terminal 6xHis tag and KDEL signal removed from the amino acid sequence translated from SEQ ID NO: 25)
SEQ ID NO: 66: Re-improved head-deficient HA-M1 fusion protein D (N-terminal rice Glb-1 secretory signal, C-terminal 6xHis tag and KDEL signal removed from the amino acid sequence translated from SEQ ID NO: 27)
SEQ ID NO: 67: GSGSG linker
　以下の実施例において本発明を更に具体的に説明するが、本発明はこれらの例によってなんら限定されるものではない。 The present invention will be described in more detail in the following examples, but the present invention is not limited to these examples.
実施例１：頭部欠損HA-M1融合タンパク質のイネでの発現
１．頭部欠損HA-M1融合タンパク質のイネ発現用プラスミドの構築
ア）頭部欠損HA-M1融合タンパク質及びリンカー付き頭部欠損HA-M1融合タンパク質の設計（配列番号１中の第７６～３０８番残基および第５２９～５５４番残基が欠損）
（i）頭部欠損HA-M1融合タンパク質及びリンカー付き頭部欠損HA-M1融合タンパク質
　インフルエンザウイルス（A/Michigan/45/2015(H1N1)）のヘマグルチニン（HA）のアミノ酸配列（登録番号：APC60198.1）（配列番号１）から、分泌シグナルに相当する、１番目のメチオニンから１７番目のアラニンまでの１７アミノ酸よりなる配列、および、膜貫通領域に相当する、５２９番目のグルタミンから５５４番目のメチオニンまでの２６アミノ酸よりなる配列を欠損させ、さらに頭部の76番目のグリシンから308番目のプロリンまでの２３３アミノ酸よりなる配列を欠損させた、頭部欠損HAを得た（配列番号２）。 Example 1: Expression of head-deficient HA-M1 fusion protein in rice
1. 1. Construction of plasmid for rice expression of head-deficient HA-M1 fusion protein
A) Design of head-deficient HA-M1 fusion protein and head-deficient HA-M1 fusion protein with linker (residues 76 to 308 and 529 to 554 in SEQ ID NO: 1 are missing)
(I) Head-deficient HA-M1 fusion protein and head-deficient HA-M1 fusion protein with linker Amino acid sequence of hemagglutinine (HA) of influenza virus (A / Michigan / 45/2015 (H1N1)) (registration number: APC60198. 1) From (SEQ ID NO: 1), a sequence consisting of 17 amino acids from the 1st methionine to the 17th alanine corresponding to the secretory signal, and the 529th methionine to the 554th methionine corresponding to the transmembrane region. A head-deficient HA was obtained in which the sequence consisting of 26 amino acids up to the above was deleted, and the sequence consisting of 233 amino acids from the 76th glycine to the 308th proline in the head was deleted (SEQ ID NO: 2).
　また、頭部欠損HAの、欠損させた76番目のグリシンから308番目のプロリンまでの２３３アミノ酸よりなる配列を、配列番号４９に示すGSリンカーに置換したリンカー付頭部欠損HA（配列番号３）をデザインした。 Further, the head-deficient HA with a linker (SEQ ID NO: 3) in which the sequence consisting of 233 amino acids from the deleted 76th glycine to the 308th proline of the head-deficient HA was replaced with the GS linker shown in SEQ ID NO: 49. Was designed.
　頭部欠損HAまたはリンカー付頭部欠損HAのC末端に、インフルエンザウイルス（A/Michigan/45/2015(H1N1)）のマトリックスタンパク質（登録番号：APC60201.1）（配列番号５）を配置したアミノ酸配列をデザインし、これを頭部欠損HA-M1融合タンパク質（配列番号５９）またはリンカー付頭部欠損HA-M1融合タンパク質（配列番号６０）とした。 Amino acid in which a matrix protein (registration number: APC60201.1) (SEQ ID NO: 5) of influenza virus (A / Michigan / 45/2015 (H1N1)) is placed at the C-terminal of head-deficient HA or head-deficient HA with linker. A sequence was designed and designated as a head-deficient HA-M1 fusion protein (SEQ ID NO: 59) or a linker-equipped head-deficient HA-M1 fusion protein (SEQ ID NO: 60).
（ii）分泌シグナル等の考慮
　さらに、National Center for Biotechnology Informationのデータベースより取得したイネのα-アミラーゼ3Dのアミノ酸配列を入手し（登録番号：AAA33895.1）、G. von Heijneの方法（Nuc. Acids Res., 14: 4683-4690, 1986）にて分泌シグナルの予測を行ない、１番目のメチオニンから２５番目のアラニンに至る２５アミノ酸よりなる配列を、イネのα-アミラーゼ3Dの分泌シグナルとした（配列番号４）。ただし、元のアミノ酸配列の二番目のリシンは、イネでより一般的な二番目のアミノ酸残基であるグリシンに置換した。このイネのα-アミラーゼ3Dの分泌シグナル、頭部欠損HA-M1融合タンパク質またはリンカー付頭部欠損HA-M1融合タンパク質、6×Hisタグ（配列番号６）を、N末端からC末端に向けてこの順で並べたアミノ酸配列をデザインし、これを分泌シグナル・6×Hisタグ付頭部欠損HA-M1融合タンパク質または分泌シグナル・6×Hisタグ付リンカー付頭部欠損融合タンパク質とした。 (Ii) Consideration of secretory signals, etc. Furthermore, the amino acid sequence of α-amylase 3D of rice obtained from the database of National Center for Biotechnology Information was obtained (registration number: AAA33895.1), and the method of G. von Heijne (Nuc. Acids Res., 14: 4683-4690, 1986) predicted the secretory signal, and the sequence consisting of 25 amino acids from the 1st methionine to the 25th alanine was used as the secretory signal of α-amylase 3D in rice. (SEQ ID NO: 4). However, the second lysine in the original amino acid sequence was replaced with glycine, the second amino acid residue more common in rice. This rice α-amylase 3D secretory signal, head-deficient HA-M1 fusion protein or head-deficient HA-M1 fusion protein with linker, 6 × His tag (SEQ ID NO: 6) is directed from the N-terminus to the C-terminus. An amino acid sequence arranged in this order was designed and used as a secretory signal / 6 × His-tagged head-deficient HA-M1 fusion protein or a secretory signal / 6 × His-tagged head-deficient fusion protein.
イ）プラスミドの構築
　GenScript社に委託し、同社プログラム"OptimumGene"にて、分泌シグナル・6×Hisタグ付頭部欠損HA-M1融合タンパク質および分泌シグナル・6×Hisタグ付リンカー付頭部欠損融合タンパク質をコードする、コドン使用頻度をイネに最適化したヌクレオチドをデザインし、その遺伝子を全合成した（配列番号７、８）。イネのα-アミラーゼ3D遺伝子プロモーターおよびイネのα-アミラーゼ3D遺伝子に由来する5'-UTRの合計約2kbのヌクレオチド配列（配列番号９）、イネの約0.5kbのα-アミラーゼ3D遺伝子ターミネーター（配列番号１０）について、DNA塩基配列をThe National Center for Biotechnology Informationのデータベースより取得した。図１に示すように、イネの約2kbのα-アミラーゼ3D遺伝子プロモーターおよびイネのα-アミラーゼ3D遺伝子に由来する5'-UTR、イネのα-アミラーゼ3Dの分泌シグナルをコードするヌクレオチド、頭部欠損HA-M1融合タンパク質遺伝子、イネの約0.５kbのα-アミラーゼ3D遺伝子ターミネーターの各パーツを、5'側からこの順で連結できるように、各パーツの増幅のための配列が25 mer、連結のためのオーバーラップの配列が15 merとなるようにプライマーを設計し、KOD FX Neo（東洋紡）を用い、その添付プロトコールに従って各パーツの増幅を行なった。これらすべてのパーツを、illustra GFX PCR DNA and Gel Band Purification Kit（GE ヘルスケア）で精製した後、すべてのパーツと、SmaI（タカラバイオ）で消化したpUC18とが、等モル比となるように混合し、In-Fusion(R) HD Cloning Kit（Clontech）を用い、その添付プロトコールに従って連結し大腸菌形質転換体を得た。得られた形質転換体より、目的の頭部欠損HA-M1融合タンパク質発現カセット（配列番号７、９及び１０の塩基配列である塩基又は配列番号８、９及び１０である塩基）が挿入されたプラスミドを持つクローンを、シーケンシングにより選抜した。 B ) Construction of plasmid Outsourced to GenScript, secretory signal 6 × His-tagged head defect HA-M1 fusion protein and secretory signal 6 × His-tagged head defect fusion with linker We designed a nucleotide that encodes a protein and optimized the frequency of codon use for rice, and synthesized the gene completely (SEQ ID NOs: 7 and 8). Nucleotide sequence of about 2 kb in total of α-amylase 3D gene promoter of rice and 5'-UTR derived from α-amylase 3D gene of rice (SEQ ID NO: 9), α-amylase 3D gene terminator of about 0.5 kb of rice (sequence) For No. 10), the DNA base sequence was obtained from the database of The National Center for Biotechnology Information. As shown in FIG. 1, the α-amylase 3D gene promoter of about 2 kb of rice, the 5'-UTR derived from the α-amylase 3D gene of rice, the nucleotide encoding the secretory signal of α-amylase 3D of rice, and the head. The sequence for amplification of each part is 25 mer so that each part of the defective HA-M1 fusion protein gene and the α-amylase 3D gene terminator of about 0.5 kb of rice can be connected in this order from the 5'side. Primers were designed so that the overlap sequence for ligation was 15 mer, and each part was amplified using KOD FX Neo (Toyo Spinning Co., Ltd.) according to the attached protocol. After purifying all these parts with the illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare), all the parts and pUC18 digested with SmaI (Takara Bio) are mixed so that they have an equimolar ratio. Then, using the In-Fusion (R) HD Cloning Kit (Clontech), they were ligated according to the attached protocol to obtain Escherichia coli transformants. From the obtained transformant, the target head-deficient HA-M1 fusion protein expression cassette (base which is the base sequence of SEQ ID NOs: 7, 9 and 10 or base which is the base sequence of SEQ ID NOs: 8, 9 and 10) was inserted. Clone with plasmid was selected by sequencing.
　得られたプラスミドより、目的の頭部欠損HA-M1融合タンパク質発現カセットを、Ex-Taq（タカラバイオ）を用いたＰＣＲにより増幅し、pKS221MCS（Wakasaら、2006、Plant Biotechnol. J. 4: 499-510）へのサブクローニングを行なった。イネ形質転換用ベクターp35SHPTAg7-GW（Wakasaら、2006、Plant Biotechnol. J. 4: 499-510）への、頭部欠損HA-M1融合タンパク質発現カセットの移し替えは、GatewayTMクローニング技術を用いて実施した。すなわち、pKS221MCSにサブクローニングされた頭部欠損HA-M1融合タンパク質発現カセットを挟んで存在するattL1およびattL2の配列と、p35SHPTAg7-GWに存在するattR1およびattR2の配列の間でそれぞれ、Gateway(R)LR クロナーゼTMII 酵素ミックス（インビトロジェン）により、その添付プロトコールに従って組換えを起こさせ、頭部欠損HA-M1融合タンパク質発現カセットを、イネ形質転換用ベクターp35SHPTAg7-GWに移し替えた。製成したプラスミドが、目的の形および配列となっていることを、シーケンシングによって確認した。このようにして、イネで頭部欠損HA-M1融合タンパク質を発現させるためのプラスミドを構築した（図１）。 From the obtained plasmid, the target head-deficient HA-M1 fusion protein expression cassette was amplified by PCR using Ex-Taq (Takara Bio) and pKS221MCS (Wakasa et al., 2006, Plant Biotechnol. J. 4: 499). -510) was subcloned. Transfer of the head-deficient HA-M1 fusion protein expression cassette to the rice transformation vector p35SHPTAg7-GW (Wakasa et al., 2006, Plant Biotechnol. J. 4: 499-510) was performed using Gateway TM cloning technology. carried out. That is, Gateway (R) LR between the attL1 and attL2 sequences subcloned across the head-deficient HA-M1 fusion protein expression cassette subcloned into pKS221MCS and the attR1 and attR2 sequences present at p35SHPTAg7-GW, respectively. Recombination was performed by the chronaseTM II enzyme mix (Invitrogen) according to the attached protocol, and the head-deficient HA-M1 fusion protein expression cassette was transferred to the rice transformation vector p35SHPTAg7-GW. It was confirmed by sequencing that the prepared plasmid had the desired shape and sequence. In this way, a plasmid for expressing the head-deficient HA-M1 fusion protein in rice was constructed (Fig. 1).
２．イネ形質転換体作製と頭部欠損HA-M1融合タンパク質の生産能評価
ア）イネ形質転換体を用いた生産
　完成したプラスミドにより、アグロバクテリウム法にてイネ（品種：キタアケ（Kitaake）。種子胚由来カルス）の形質転換を実施し各プラスミドにつき20-25個体の独立したハイグロマイシンB（50 μg/mL）に耐性の形質転換体カルスを得た。イネ形質転換体カルスは、2回のハイグロマイシンＢによる選抜後、3%のスクロースを含む、または含まない、N6D液体培地に置床し、28℃、暗黒下で、3日間の振とう培養（80 rpm）を行なった。所定時間の培養後、細胞（カルス）と培地を分離した。 2. 2. Production of rice transformants and evaluation of production capacity of HA-M1 fusion protein with head defect
A) Production using rice transformants The completed plasmids were transformed with rice (cultivar: Kitaake . Seed embryo-derived curls) by the Agrobacterium method, and 20-25 individuals were independent for each plasmid. A transformant plasmid resistant to Hyglomycin B (50 μg / mL) was obtained. Rice transformant callus was selected with hygromycin B twice and then placed in N6D liquid medium with or without 3% sucrose and shake-cultured at 28 ° C. in the dark for 3 days (80). rpm) was performed. After culturing for a predetermined time, cells (callus) and medium were separated.
イ）タンパク質の分析
　培地の3倍量のアセトンを入れ、-20℃で１時間の処理後、13500rpmで10分間遠心し、沈殿物から尿素-SDS バッファー（50 mM Tris-HCl pH 6.8, 8 M Urea, 4 % SDS, 5 % 2-mercaptoethanol, 20 % Glycerol）でタンパク質を抽出した。細胞内のタンパク質の分析に関しては、カルスを液体窒素で凍結し、マルチビーズショッカー（登録商標。安井器械）で粉砕後、尿素-SDS バッファーでタンパク質を抽出した。 B ) Add 3 times the amount of acetone as the protein analysis medium, treat at -20 ° C for 1 hour, centrifuge at 13500 rpm for 10 minutes, and use urea-SDS buffer (50 mM Tris-HCl pH 6.8, 8 M) from the precipitate. Protein was extracted with Urea, 4% SDS, 5% 2-mercaptoethanol, 20% Glycerol). For intracellular protein analysis, callus was frozen in liquid nitrogen, pulverized with a multi-bead shocker (registered trademark, Yasui Instrument), and then the protein was extracted with a urea-SDS buffer.
　抽出したタンパク質は、SDS-PAGEおよびウエスタンブロッティングに供試した。検出は、抗His抗体によって行なった。頭部欠損HA-M1融合タンパク質を発現させたイネ形質転換体カルスの、独立した4系統のSDS-PAGEの結果を図２に示す。図２において、各レーンの内容は次の通りである。レーン1：頭部欠損HA-M1融合タンパク質を発現させた形質転換体系統番号1を、スクロースを含まない培地で培養した培養液。レーン2：頭部欠損HA-M1融合タンパク質を発現させた形質転換体系統番号2を、スクロースを含まない培地で培養した培養液。レーン3：頭部欠損HA-M1融合タンパク質を発現させた形質転換体系統番号3を、スクロースを含まない培地で培養した培養液。レーン4：頭部欠損HA-M1融合タンパク質を発現させた形質転換体系統番号4を、スクロースを含まない培地で培養した培養液。レーン5：頭部欠損HA-M1融合タンパク質を発現させた形質転換体系統番号1を、スクロースを含む培地で培養した培養液。レーン6：頭部欠損HA-M1融合タンパク質を発現させた形質転換体系統番号2を、スクロースを含む培地で培養した培養液。レーン7：頭部欠損HA-M1融合タンパク質を発現させた形質転換体系統番号3を、スクロースを含む培地で培養した培養液。レーン8：頭部欠損HA-M1融合タンパク質を発現させた形質転換体系統番号4を、スクロースを含む培地で培養した培養液。レーン9：頭部欠損HA-M1融合タンパク質を発現させた形質転換体系統番号1を、スクロースを含まない培地で培養した細胞塊抽出物。レーン10：頭部欠損HA-M1融合タンパク質を発現させた形質転換体系統番号2を、スクロースを含まない培地で培養した細胞塊抽出物。レーン11：頭部欠損HA-M1融合タンパク質を発現させた形質転換体系統番号3を、スクロースを含まない培地で培養した細胞塊抽出物。レーン12：頭部欠損HA-M1融合タンパク質を発現させた形質転換体系統番号4を、スクロースを含まない培地で培養した細胞塊抽出物。レーン13：頭部欠損HA-M1融合タンパク質を発現させた形質転換体系統番号1を、スクロースを含む培地で培養した細胞塊抽出物。レーン14：頭部欠損HA-M1融合タンパク質を発現させた形質転換体系統番号2を、スクロースを含む培地で培養した細胞塊抽出物。レーン15：頭部欠損HA-M1融合タンパク質を発現させた形質転換体系統番号3を、スクロースを含む培地で培養した細胞塊抽出物。レーン16：頭部欠損HA-M1融合タンパク質を発現させた形質転換体系統番号4を、スクロースを含む培地で培養した細胞塊抽出物。 The extracted protein was subjected to SDS-PAGE and Western blotting. Detection was performed by anti-His antibody. The results of SDS-PAGE of four independent strains of rice transformant callus expressing the head-deficient HA-M1 fusion protein are shown in FIG. In FIG. 2, the contents of each lane are as follows. Lane 1: A culture medium obtained by culturing transformant strain No. 1 expressing the head-deficient HA-M1 fusion protein in a medium containing no sucrose. Lane 2: A culture medium in which transformant lineage No. 2 expressing the head-deficient HA-M1 fusion protein was cultured in a medium containing no sucrose. Lane 3: A culture medium in which transformant lineage No. 3 expressing the head-deficient HA-M1 fusion protein was cultured in a medium containing no sucrose. Lane 4: A culture medium in which transformant lineage No. 4 expressing the head-deficient HA-M1 fusion protein was cultured in a medium containing no sucrose. Lane 5: A culture medium in which transformant lineage No. 1 expressing the head-deficient HA-M1 fusion protein was cultured in a medium containing sucrose. Lane 6: A culture medium in which transformant lineage No. 2 expressing the head-deficient HA-M1 fusion protein was cultured in a medium containing sucrose. Lane 7: A culture medium in which transformant lineage No. 3 expressing the head-deficient HA-M1 fusion protein was cultured in a medium containing sucrose. Lane 8: A culture medium in which transformant lineage No. 4 expressing the head-deficient HA-M1 fusion protein was cultured in a medium containing sucrose. Lane 9: Cell mass extract obtained by culturing transformant lineage No. 1 expressing the head-deficient HA-M1 fusion protein in a medium containing no sucrose. Lane 10: Cell mass extract obtained by culturing transformant lineage No. 2 expressing the head-deficient HA-M1 fusion protein in a medium containing no sucrose. Lane 11: Cell mass extract obtained by culturing transformant lineage No. 3 expressing the head-deficient HA-M1 fusion protein in a medium containing no sucrose. Lane 12: Cell mass extract obtained by culturing transformant lineage No. 4 expressing the head-deficient HA-M1 fusion protein in a medium containing no sucrose. Lane 13: Cell mass extract obtained by culturing transformant lineage No. 1 expressing the head-deficient HA-M1 fusion protein in a medium containing sucrose. Lane 14: Cell mass extract obtained by culturing transformant lineage No. 2 expressing the head-deficient HA-M1 fusion protein in a medium containing sucrose. Lane 15: Cell mass extract obtained by culturing transformant lineage No. 3 expressing the head-deficient HA-M1 fusion protein in a medium containing sucrose. Lane 16: Cell mass extract obtained by culturing transformant lineage No. 4 expressing the head-deficient HA-M1 fusion protein in a medium containing sucrose.
　イネ形質転換体のカルスを上記条件（暗黒下で3日間、振とう培養）で培養すると、スクロースを含まない培地では、α-アミラーゼが培地中に分泌されるのが観察された。しかしながら、ウエスタンブロッティングの結果、目的の頭部欠損HA-M1融合タンパク質およびリンカー付頭部欠損融合タンパク質は、培地中にも細胞抽出物にも、検出されなかった（データは示していない）。 When callus of rice transformant was cultured under the above conditions (shaking culture for 3 days in the dark), α-amylase was observed to be secreted into the medium in a medium containing no sucrose. However, as a result of Western blotting, the target head-deficient HA-M1 fusion protein and the linker-equipped head-deficient fusion protein were not detected in the medium or in the cell extract (data not shown).
実施例２：改良型頭部欠損HA-M1融合タンパク質のイネによる生産
１．改良型頭部欠損HA-M1融合タンパク質のイネ発現用プラスミドの構築
　上述のように、頭部欠損HA-M1融合タンパク質がイネで発現しなかったので、改良を行なった。その概略を、図３に示す。 Example 2: Production of improved head-deficient HA-M1 fusion protein by rice
1. 1. Construction of a plasmid for rice expression of the improved head-deficient HA-M1 fusion protein As described above, the head-deficient HA-M1 fusion protein was not expressed in rice, so improvement was performed. The outline is shown in FIG.
ア）改良型頭部欠損HA-M1融合タンパク質のデザイン
（i）改良型頭部欠損HA-M1融合タンパク質Ａ（配列番号１中の第５１～３３９番残基および第５２９～５５４番残基が欠損）
　配列番号７に示された頭部欠損HA-M1融合タンパク質遺伝子を元に、１８４番目のグアニンから１８６番目のグアニンまでのコドンにコードされたグルタミン酸から、３４９番目のシトシンから３５１番目のアデニンまでのコドンにコードされたプロリンまでの５６アミノ酸よりなる配列を欠損させた改良型頭部欠損HA-M1融合タンパク質Ａに、配列番号６７にコードされたリンカーにコードされたアミノ酸配列を挿入したタンパク質（リンカー付き改良型頭部欠損HA-M1融合タンパク質Ａ）をコードするヌクレオチド（配列番号１２）をデザインした。分泌シグナル・6xHisタグを除去したリンカー付改良型頭部欠損HA-M1融合タンパク質Ａのアミノ酸配列は、配列番号６１となる。 A) Design of improved head defect HA-M1 fusion protein
(I) Improved head defect HA-M1 fusion protein A (defective residues 51 to 339 and residues 529 to 554 in SEQ ID NO: 1)
Based on the head-deficient HA-M1 fusion protein gene shown in SEQ ID NO: 7, glutamic acid encoded by codons from 184th guanine to 186th guanine to 349th cytosine to 351st adenin. A protein in which the amino acid sequence encoded by the linker encoded by SEQ ID NO: 67 is inserted into the improved head-deficient HA-M1 fusion protein A in which the sequence consisting of 56 amino acids up to the codon-encoded proline is deleted (linker). A nucleotide (SEQ ID NO: 12) encoding an improved head defect HA-M1 fusion protein A) was designed. The amino acid sequence of the improved head-deficient HA-M1 fusion protein A with a linker from which the secretory signal 6xHis tag has been removed is SEQ ID NO: 61.
（ii）改良型頭部欠損HA-M1融合タンパク質Ｂ（配列番号１中の第５１～３３５番残基および第５２９～５５４番残基が欠損）
　配列番号７に示された頭部欠損HA-M1融合タンパク質遺伝子を元に、１８４番目のグアニンから１８６番目のグアニンまでのコドンにコードされたグルタミン酸から、３３７番目のシトシンから３３９番目のグアニンまでのコドンにコードされたロイシンまでの５２アミノ酸よりなる配列を欠損させた改良型頭部欠損HA-M1融合タンパク質Ｂに、配列番号１３にコードされたリンカーを挿入したタンパク質（リンカー付き改良型頭部欠損HA-M1融合タンパク質Ｂ）をコードするヌクレオチド（配列番号１４）をデザインした。分泌シグナル・6xHisタグを除去したリンカー付改良型頭部欠損HA-M1融合タンパク質Ｂのアミノ酸配列は、配列番号６２となる。 (Ii) Improved head defect HA-M1 fusion protein B (defective residues 51 to 335 and residues 529 to 554 in SEQ ID NO: 1)
Based on the head-deficient HA-M1 fusion protein gene shown in SEQ ID NO: 7, glutamic acid encoded by codons from 184th guanine to 186th guanine to 337th cytosine to 339th guanine. Improved head defect HA-M1 fusion protein B lacking a sequence consisting of 52 amino acids up to codon-encoded leucine, with a linker encoded by SEQ ID NO: 13 inserted (improved head defect with linker) A nucleotide (SEQ ID NO: 14) encoding HA-M1 fusion protein B) was designed. The amino acid sequence of the improved head-deficient HA-M1 fusion protein B with a linker from which the secretory signal 6xHis tag has been removed is SEQ ID NO: 62.
（iii）改良型頭部欠損HA-M1融合タンパク質Ｃ（配列番号１中の第５９～３３９番残基および第５２９～５５４番残基が欠損）
　配列番号７に示された頭部欠損HA-M1融合タンパク質遺伝子を元に、２０８番目のチミンから２１０番目のシトシンまでのコドンにコードされたシステインから、３４９番目のシトシンから３５１番目のアデニンまでのコドンにコードされたプロリンまでの４８アミノ酸よりなる配列を欠損させた改良型頭部欠損HA-M1融合タンパク質Ｃに、配列番号６７にコードされたリンカーを挿入したタンパク質（リンカー付き改良型頭部欠損HA-M1融合タンパク質Ｃをコードするヌクレオチド（配列番号１５）を、デザインした。分泌シグナル・6xHisタグを除去したリンカー付改良型頭部欠損HA-M1融合タンパク質Ｃのアミノ酸配列は、配列番号６３となる。 (Iii) Improved head defect HA-M1 fusion protein C (residues 59 to 339 and residues 529 to 554 in SEQ ID NO: 1 are deleted)
Based on the head-deficient HA-M1 fusion protein gene shown in SEQ ID NO: 7, from the cysteine encoded by the codon from the 208th timine to the 210th cytosine to the 349th cytosine to the 351st adenin. Improved head defect HA-M1 fusion protein C lacking a sequence consisting of 48 amino acids up to codon-encoded proline, with a linker encoded by SEQ ID NO: 67 inserted (improved head defect with linker) A nucleotide encoding HA-M1 fusion protein C (SEQ ID NO: 15) was designed. The amino acid sequence of the improved head-deficient HA-M1 fusion protein C with linker from which the secretory signal 6xHis tag was removed is SEQ ID NO: 63. Become.
（iv）改良型頭部欠損HA-M1融合タンパク質Ｄ（配列番号１中の第５９～３３５番残基および第５２９～５５４番残基が欠損）
　配列番号７に示された頭部欠損HA-M1融合タンパク質遺伝子を元に、２０８番目のチミンから２１０番目のシトシンまでのコドンにコードされたシステインから、３３７番目のシトシンから３３９番目のグアニンまでのコドンにコードされたロイシンまでの４４アミノ酸よりなる配列を欠損させた改良型頭部欠損HA-M1融合タンパク質Ｄに、配列番号１３にコードされたリンカーを挿入したタンパク質（リンカー付き改良型頭部欠損HA-M1融合タンパク質Ｄ）をコードするヌクレオチドを、デザインした。分泌シグナル・6xHisタグを除去したリンカー付改良型頭部欠損HA-M1融合タンパク質Ｄのアミノ酸配列は、配列番号６４となる。 (Iv) Improved head defect HA-M1 fusion protein D (defective residues 59 to 335 and residues 529 to 554 in SEQ ID NO: 1)
Based on the head-deficient HA-M1 fusion protein gene shown in SEQ ID NO: 7, from the cysteine encoded by the codon from the 208th timine to the 210th citosine to the 337th citosine to the 339th guanine. Improved head defect HA-M1 fusion protein D lacking a sequence consisting of 44 amino acids up to codon-encoded leucine, with a linker encoded by SEQ ID NO: 13 inserted (improved head defect with linker) A nucleotide encoding the HA-M1 fusion protein D) was designed. The amino acid sequence of the improved head-deficient HA-M1 fusion protein D with a linker from which the secretory signal 6xHis tag has been removed is SEQ ID NO: 64.
イ）プラスミドの構築
　リンカー付き改良型頭部欠損HA-M1融合タンパク質Ａ、Ｂ、Ｃ３種の遺伝子を含むプラスミドを増幅した。 B ) Construction of plasmid A plasmid containing the genes of the improved head-deficient HA-M1 fusion proteins A, B, and C with a linker was amplified.
　すなわち、頭部欠損HA-M1融合タンパク質のイネでの発現用プラスミド（実施例１に記載）を元に、このプラスミド中の頭部欠損HA-M1融合タンパク質をコードする領域の、欠損を拡大したい領域の外側に向けて、両側とも35 merの配列に、ひとつは5’側にリンカー配列をコードするヌクレオチドを付与したプライマー、もうひとつは5’側にもう片方のプライマーの5’側より15 merの配列と相補関係にある配列を付与したプライマーを設計した。このプライマーを使用し、頭部欠損HA-M1融合タンパク質のイネでの発現用プラスミドを鋳型に、KOD-FX Neo（東洋紡）を用いて、その添付プロトコールに従って、PCRを実施した。PCR産物を、0.3% アガロースゲルにて電気泳動し、増幅されたバンドを臭化エチジウムで染色後、目的のバンドをUV照射下で切り出した。これを、illustra GFX PCR DNA and Gel Band Purification Kit（GE ヘルスケア）で精製した後、In-Fusion(R) HD Cloning Kit（Clontech）を用い、その添付プロトコールに従って環状化した。 That is, based on the plasmid for expressing the head-deficient HA-M1 fusion protein in rice (described in Example 1), we would like to expand the defect in the region encoding the head-deficient HA-M1 fusion protein in this plasmid. Toward the outside of the region, a primer with a sequence of 35 mer on both sides, one with a nucleotide encoding the linker sequence on the 5'side, and the other on the 5'side 15 mer from the 5'side of the other primer. We designed a primer with a sequence complementary to the sequence of. Using this primer, PCR was performed using a plasmid for expressing the head-deficient HA-M1 fusion protein in rice as a template, using KOD-FX Neo (Toyobo), and according to the attached protocol. The PCR product was electrophoresed on a 0.3% agarose gel, the amplified band was stained with ethidium bromide, and the target band was excised under UV irradiation. This was purified with illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare), and then cyclized using In-Fusion (R) HD Cloning Kit (Clontech) according to the attached protocol.
　連結によって製成したプラスミドが、目的の形および配列となっていることを、シーケンシングによって確認した。このようにして、イネで3種の改良型頭部欠損HA-M1融合タンパク質を発現させるためのプラスミドが構築された。 It was confirmed by sequencing that the plasmid produced by ligation had the desired shape and sequence. In this way, a plasmid for expressing three improved head-deficient HA-M1 fusion proteins in rice was constructed.
２．イネ形質転換体作製と改良型頭部欠損HA-M1融合タンパク質の生産能評価
ア）イネ形質転換体による生産
　完成した3種のプラスミドにより、アグロバクテリウム法にてイネ（品種：キタアケ(Kitaake)。種子胚由来カルス）の形質転換を実施し各プラスミドにつき20-25個体の独立したハイグロマイシンＢ 50 μg/mL）に耐性のイネ形質転換体カルスを得た。イネ形質転換体カルスは、2回のハイグロマイシンＢによる選抜後、3%のスクロースを含む、または含まない、N6D液体培地に置床し、28℃、暗黒下で、3日間の振とう培養（80 rpm）を行なった。所定時間の培養後、細胞（カルス）と培地を分離した。 2. 2. Production of rice transformants and evaluation of productivity of improved head-deficient HA-M1 fusion protein
A) Production by rice transformants Rice (cultivar: Kitaake . Seed embryo-derived curls) was transformed by the Agrobacterium method using the completed 3 types of plasmids, and 20-25 individuals were used for each plasmid. A rice transformant plasmid resistant to independent hyglomycin B (50 μg / mL) was obtained. Rice transformant callus was selected with hygromycin B twice and then placed in N6D liquid medium with or without 3% sucrose and shake-cultured at 28 ° C. in the dark for 3 days (80). rpm) was performed. After culturing for a predetermined time, cells (callus) and medium were separated.
イ）タンパク質の分析
　培地に分泌されたタンパク質の分析に関しては、培地容積の3倍量のアセトンを入れて混合し、-20℃で１時間の放置後、13500rpmで10分間遠心し、沈殿物から尿素-SDS バッファーでタンパク質を抽出した。細胞内のタンパク質の分析に関しては、カルスを液体窒素で凍結し、マルチビーズショッカー（登録商標。安井器械）で粉砕後、尿素-SDS バッファーでタンパク質を抽出した。 B) Protein analysis For analysis of proteins secreted into the medium, add 3 times the volume of the medium, mix, leave at -20 ° C for 1 hour, centrifuge at 13500 rpm for 10 minutes, and use the precipitate. Protein was extracted with urea-SDS buffer. For intracellular protein analysis, callus was frozen in liquid nitrogen, pulverized with a multi-bead shocker (registered trademark, Yasui Instrument), and then the protein was extracted with a urea-SDS buffer.
　抽出したタンパク質は、SDS-PAGEおよびウエスタンブロッティングに供試した。検出は、抗His抗体によって行なった。改良型頭部欠損HA-M1融合タンパク質Ａおよび改良型頭部欠損HA-M1融合タンパク質Ｂを発現させたイネ形質転換体カルスの、それぞれ独立した10系統のSDS-PAGEおよびウエスタンブロッティングの結果を図４に示す。図４において、各レーンの内容は次の通りである。レーン1：改良型頭部欠損HA-M1融合タンパク質Ａを発現させた形質転換体系統番号1の細胞塊抽出物。レーン2：改良型頭部欠損HA-M1融合タンパク質Ａを発現させた形質転換体系統番号2の細胞塊抽出物。レーン3：改良型頭部欠損HA-M1融合タンパク質Ａを発現させた形質転換体系統番号3の細胞塊抽出物。レーン4：改良型頭部欠損HA-M1融合タンパク質Ａを発現させた形質転換体系統番号4の細胞塊抽出物。レーン5：改良型頭部欠損HA-M1融合タンパク質Ａを発現させた形質転換体系統番号5の細胞塊抽出物。レーン6：改良型頭部欠損HA-M1融合タンパク質Ａを発現させた形質転換体系統番号6の細胞塊抽出物。レーン7：改良型頭部欠損HA-M1融合タンパク質Ａを発現させた形質転換体系統番号7の細胞塊抽出物。レーン8：改良型頭部欠損HA-M1融合タンパク質Ａを発現させた形質転換体系統番号8の細胞塊抽出物。レーン9：改良型頭部欠損HA-M1融合タンパク質Ａを発現させた形質転換体系統番号9の細胞塊抽出物。レーン10：改良型頭部欠損HA-M1融合タンパク質Ａを発現させた形質転換体系統番号10の細胞塊抽出物。レーン11：改良型頭部欠損HA-M1融合タンパク質Ｂを発現させた形質転換体系統番号1の細胞塊抽出物。レーン12：改良型頭部欠損HA-M1融合タンパク質Ｂを発現させた形質転換体系統番号2の細胞塊抽出物。レーン13：改良型頭部欠損HA-M1融合タンパク質Bを発現させた形質転換体系統番号3の細胞塊抽出物。レーン14：改良型頭部欠損HA-M1融合タンパク質Ｂを発現させた形質転換体系統番号4の細胞塊抽出物。レーン15：改良型頭部欠損HA-M1融合タンパク質Ｂを発現させた形質転換体系統番号5の細胞塊抽出物。レーン16：改良型頭部欠損HA-M1融合タンパク質Ｂを発現させた形質転換体系統番号6の細胞塊抽出物。レーン17：改良型頭部欠損HA-M1融合タンパク質Ｂを発現させた形質転換体系統番号7の細胞塊抽出物。レーン18：改良型頭部欠損HA-M1融合タンパク質Ｂを発現させた形質転換体系統番号8の細胞塊抽出物。レーン19：改良型頭部欠損HA-M1融合タンパク質Ｂを発現させた形質転換体系統番号9の細胞塊抽出物。レーン20：改良型頭部欠損HA-M1融合タンパク質Ｂを発現させた形質転換体系統番号10の細胞塊抽出物。 The extracted protein was subjected to SDS-PAGE and Western blotting. Detection was performed by anti-His antibody. The results of SDS-PAGE and Western blotting of 10 independent strains of rice transformant callus expressing the improved head-deficient HA-M1 fusion protein A and the improved head-deficient HA-M1 fusion protein B are shown. Shown in 4. In FIG. 4, the contents of each lane are as follows. Lane 1: Cell mass extract of transformant lineage No. 1 expressing the improved head-deficient HA-M1 fusion protein A. Lane 2: Cell mass extract of transformant lineage No. 2 expressing the improved head-deficient HA-M1 fusion protein A. Lane 3: Cell mass extract of transformant lineage No. 3 expressing the improved head-deficient HA-M1 fusion protein A. Lane 4: Cell mass extract of transformant lineage No. 4 expressing the improved head defect HA-M1 fusion protein A. Lane 5: Cell mass extract of transformant lineage No. 5 expressing the improved head-deficient HA-M1 fusion protein A. Lane 6: Cell mass extract of transformant lineage No. 6 expressing the improved head defect HA-M1 fusion protein A. Lane 7: Cell mass extract of transformant lineage number 7 expressing the improved head-deficient HA-M1 fusion protein A. Lane 8: Cell mass extract of transformant lineage No. 8 expressing the improved head defect HA-M1 fusion protein A. Lane 9: Cell mass extract of transformant lineage No. 9 expressing the improved head-deficient HA-M1 fusion protein A. Lane 10: Cell mass extract of transformant lineage No. 10 expressing the improved head defect HA-M1 fusion protein A. Lane 11: Cell mass extract of transformant lineage No. 1 expressing the improved head-deficient HA-M1 fusion protein B. Lane 12: Cell mass extract of transformant lineage No. 2 expressing the improved head-deficient HA-M1 fusion protein B. Lane 13: Cell mass extract of transformant lineage No. 3 expressing improved head defect HA-M1 fusion protein B. Lane 14: Cell mass extract of transformant lineage No. 4 expressing the improved head defect HA-M1 fusion protein B. Lane 15: Cell mass extract of transformant lineage No. 5 expressing improved head defect HA-M1 fusion protein B. Lane 16: Cell mass extract of transformant lineage No. 6 expressing the improved head defect HA-M1 fusion protein B. Lane 17: Cell mass extract of transformant lineage number 7 expressing the improved head defect HA-M1 fusion protein B. Lane 18: Cell mass extract of transformant lineage No. 8 expressing improved head defect HA-M1 fusion protein B. Lane 19: Cell mass extract of transformant lineage number 9 expressing the improved head defect HA-M1 fusion protein B. Lane 20: Cell mass extract of transformant lineage No. 10 expressing improved head defect HA-M1 fusion protein B.
　どちらの頭部欠損HA-M1融合タンパク質を発現させたイネ形質転換体カルスも、SDS-PAGEでははっきりとしたバンドを確認できなかったが、ウエスタンブロッティングでは、想定分子量の位置に目的タンパク質のバンドを確認できた。このことより、HAの頭部欠損領域を拡大することにより、イネでの頭部欠損HA-M1融合タンパク質の発現量が増加することが確認できた。 In the rice transformant callus expressing both head-deficient HA-M1 fusion proteins, no clear band could be confirmed by SDS-PAGE, but in Western blotting, the band of the target protein was found at the position of the assumed molecular weight. It could be confirmed. From this, it was confirmed that the expression level of the head-deficient HA-M1 fusion protein in rice was increased by expanding the head-deficient region of HA.
実施例３：再改良型頭部欠損HA-M1融合タンパク質のイネによる生産
１．再改良型頭部欠損HA-M1融合タンパク質のイネ発現用プラスミドの構築
ア）　再改良型頭部欠損HA-M1融合タンパク質の設計
　頭部欠損HA-M1融合タンパク質のイネでの発現量を増やすために、以下のように、さらなる改良を実施した。 Example 3: Production of re-improved head defect HA-M1 fusion protein by rice
1. 1. Construction of a plasmid for rice expression of the re-improved head-deficient HA-M1 fusion protein
A) Design of re-improved head-deficient HA-M1 fusion protein In order to increase the expression level of the head-deficient HA-M1 fusion protein in rice, further improvements were carried out as follows.
（i）再改良型頭部欠損融合タンパク質Ｂ＋KDEL（配列番号１中の第５１～３３５番残基および第５２９～５５４番残基に加え、第５５５～５６６番残基が欠損）
　National Center for Biotechnology Informationのデータベースより、イネのGlobulin-1遺伝子（Glb-1）の、分泌シグナルをコードするヌクレオチド配列を取得した（配列番号５０）。配列番号１４に示されたヌクレオチド配列にコードされた改良型頭部欠損融合タンパク質Ｂについて、イネα-アミラーゼ3Dの分泌シグナルをコードする、１０番目のアデニンから８４番目のチミンまでの７５ヌクレオチド配列を、Glb-1の分泌シグナルをコードするヌクレオチド配列と置換し、さらにC末端の６ｘHisタグをコードする配列の下流に、小胞体係留シグナルであるKDEL配列をコードするヌクレオチド配列（配列番号２２）を連結した、改良型頭部欠損融合タンパク質Ｂ+KDELをコードするヌクレオチド配列をデザインした（配列番号２３）。また改良型頭部欠損融合タンパク質Ｂ+KDELについて、HAのC末端に存在する、１２アミノ酸配列からなるサイトプラズミックテールをコードする、配列番号２３の７７５番目のチミンから８１０番目のチミンまでの３６塩基配列を除去し、そこにフレキシブルな構造を取るＧＳＡＧＳＡのリンカーをコードするヌクレオチド配列（配列番号２４）を挿入した、再改良型頭部欠損融合タンパク質Ｂ+KDELをコードするヌクレオチド配列をデザインした（配列番号２５）。なお、分泌シグナル、6xHisタグおよびKDELを連結しない再改良型頭部欠損融合タンパク質Ｂの配列は配列番号６５とした。 (I) Re-improved head defect fusion protein B + KDEL (in addition to residues 51 to 335 and residues 529 to 554 in SEQ ID NO: 1, residues 555 to 566 are deleted)
From the database of National Center for Biotechnology Information, the nucleotide sequence encoding the secretory signal of the Globulin-1 gene (Glb-1) of rice was obtained (SEQ ID NO: 50). For the improved head-deficient fusion protein B encoded by the nucleotide sequence shown in SEQ ID NO: 14, the 75 nucleotide sequence from the 10th adenine to the 84th timine encoding the secretory signal of rice α-amylase 3D was obtained. , Substituted with the nucleotide sequence encoding the Glb-1 secretion signal, and further linked the nucleotide sequence encoding the KDEL sequence, which is the vesicle mooring signal, downstream of the sequence encoding the 6xHis tag at the C-terminal (SEQ ID NO: 22). The nucleotide sequence encoding the improved head defect fusion protein B + KDEL was designed (SEQ ID NO: 23). For the improved head defect fusion protein B + KDEL, 36 from 775th timine to 810th timine of SEQ ID NO: 23, which encodes a cytoplasmic tail consisting of a 12-amino acid sequence present at the C-terminal of HA. A nucleotide sequence encoding the re-improved head-deficient fusion protein B + KDEL was designed by removing the base sequence and inserting a nucleotide sequence (SEQ ID NO: 24) encoding a linker of GSAGSA having a flexible structure therein (SEQ ID NO: 24). SEQ ID NO: 25). The sequence of the re-improved head defect fusion protein B not linked with the secretory signal, 6xHis tag and KDEL was designated as SEQ ID NO: 65.
（ii）再改良型頭部欠損HA-M1融合タンパク質Ｄ+KDEL（配列番号１中の第５９～３３５番残基および第５２９～５５４番残基に加え、第５５５～５６６番残基が欠損）
　また、配列番号７に示された頭部欠損HA-M1融合タンパク質遺伝子を元に、イネα-アミラーゼ3Dの分泌シグナルをコードする、１０番目のアデニンから８４番目のチミンまでの７５ヌクレオチド配列を、Glb-1の分泌シグナルをコードするヌクレオチド配列と置換し、また２０８番目のチミンから２１０番目のグアニンまでのコドンにコードされたシステインから、３３７番目のシトシンから３３９番目のグアニンまでのコドンにコードされたロイシンまでの４４アミノ酸よりなる配列を欠損させ、配列番号１３にコードされたリンカーにコードされたアミノ酸配列を挿入し（改良型頭部欠損HA-M1融合タンパク質D）、さらにC末端の６ｘHisタグをコードする配列の下流に、小胞体係留シグナルであるKDEL配列をコードするヌクレオチド配列（配列番号２２）を連結した、改良型頭部欠損HA-M1融合タンパク質Ｄ+KDELをコードするヌクレオチド（配列番号２６）をデザインした。改良型頭部欠損融合タンパク質Ｄについて、HAのC末端に存在する、１２アミノ酸配列からなるサイトプラズミックテールをコードする、配列番号２６の７９９番目のチミンから８３４番目のチミンまでの３６塩基配列を除去し、そこにフレキシブルな構造を取るＧＳＡＧＳＡのリンカーをコードするヌクレオチド配列（配列番号２４）を挿入した、再改良型頭部欠損融合タンパク質Ｄ+KDELをコードするヌクレオチド配列をデザインした（配列番号２７）。なお、分泌シグナル、6xHisタグおよびKDELを連結しない再改良型頭部欠損融合タンパク質Ｄの配列は配列番号６６とした。 (Ii) Re-improved head defect HA-M1 fusion protein D + KDEL (in addition to residues 59 to 335 and residues 529 to 554 in SEQ ID NO: 1, residues 555 to 566 are deleted. )
In addition, based on the head-deficient HA-M1 fusion protein gene shown in SEQ ID NO: 7, a 75-nucleotide sequence from the 10th adenine to the 84th timine encoding the secretory signal of rice α-amylase 3D was obtained. Substituted with the nucleotide sequence encoding the Glb-1 secretory signal, and also encoded from the cysteine encoded by the codon from the 208th timine to the 210th guanine to the codon from the 337th cytosine to the 339th guanine. The sequence consisting of 44 amino acids up to Leucine was deleted, the amino acid sequence encoded by the linker encoded by SEQ ID NO: 13 was inserted (improved head-deficient HA-M1 fusion protein D), and the C-terminal 6xHis tag was added. A nucleotide encoding the improved head-deficient HA-M1 fusion protein D + KDEL (SEQ ID NO: 22), in which a nucleotide sequence encoding the KDEL sequence, which is a vesicle mooring signal, is ligated downstream of the sequence encoding 26) was designed. For the improved head-deficient fusion protein D, the 36-base sequence from 799th timine to 834th timine of SEQ ID NO: 26, which encodes a cytoplasmic tail consisting of a 12-amino acid sequence, located at the C-terminal of HA. We designed a nucleotide sequence encoding the re-improved head-deficient fusion protein D + KDEL (SEQ ID NO: 27), which was removed and a nucleotide sequence encoding the linker of GSAGSA having a flexible structure was inserted therein (SEQ ID NO: 24). ). The sequence of the re-improved head defect fusion protein D not linked with the secretory signal, 6xHis tag and KDEL was designated as SEQ ID NO: 66.
イ）プラスミドの構築
　イネでの発現用ベクター構築の概略を、図５に示す。National Center for Biotechnology Informationのデータベースより、イネのGlobulin-1遺伝子（Glb-1）のプロモーター（配列番号２８）およびターミネーター（配列番号２９）のヌクレオチド配列を取得した。そして、図５に示す構造の、二種のRNA干渉を引き起こさせるための構築物であるグルテリン発現抑制カセット及びプロラミン発現抑制カセットをデザインした。具体的には、まず、イネの貯蔵タンパク質であるグルテリンＡ、Ｂ、Ｃの発現を抑制するために、イネのUbi1遺伝子の約2 kbのプロモーター（配列番号３０）と、pRI 101-AN（タカラバイオ）に由来するNOSのターミネーター（配列番号３１）の間に、rice aspartic proteaseの3rd intron（RAP 3rd intron、配列番号３２）を挟んで、グルテリンＡ、Ｂ、Ｃ遺伝子の一部の配列（配列番号３３）およびその配列と相補な関係にある配列（配列番号３４）を配置した構築物をデザインし、グルテリン発現抑制カセットと命名した。同様に、イネの貯蔵タンパク質である13 KDおよび16 KDのプロラミンの発現を抑制するために、イネのUbi1遺伝子の約2 kbのプロモーター（配列番号２８）と、pRI 101-AN（タカラバイオ）に由来するNOSのターミネーター（配列番号２９）の間に、rice aspartic proteaseの3rd intron（RAP 3rd intron、配列番号３２）を挟んで、13 KDおよび16 KDのプロラミン遺伝子の一部の配列（配列番号３５）およびその配列と相補な関係にある配列（配列番号３６）を配置した構築物をデザインし、プロラミン発現抑制カセットと命名した。これら二種の発現抑制カセットを、Ex-Taq（タカラバイオ）を用いたPCRで合成し、エントリーベクターであるpKS4-MCSおよびpKS221MCS（いずれも、Wakasaら、2006、Plant Biotechnol. J. 4: 499-510に詳細を記載）にサブクローニングした。一方で、改良型もしくは再改良型頭部欠損融合タンパク質ＢもしくはＤの発現カセットをEx-Taq（タカラバイオ）を用いたPCRで合成し、エントリーベクターpK
S2-3MCS（Wakasaら、2006、Plant Biotechnol. J. 4: 499-510に詳細を記載）にサブクローニングした。そして、上記三種のカセットを、得られた三種のエントリーベクターより、GatewayTMクローニング技術を用いてイネ形質転換用ベクターp35SHPTAg7-43GW（Wakasaら、2006、Plant Biotechnol. J. 4: 499-510）へ移し替えた。具体的には、pKS4-MCSにサブクローニングされたグルテリン発現抑制カセットを挟んで存在するattL4配列およびattR1配列、pKS221MCSにサブクローニングされたプロラミン発現抑制カセットを挟んで存在するattL1配列およびattL2配列、pKS2-3MCSにサブクローニングされた改良型もしくは再改良型頭部欠損融合タンパク質ＢもしくはＤの発現カセットを挟んで存在するattR2配列およびattL3配列、イネ形質転換用ベクターp35SHPTAg7-43GWに存在するattR4配列およびattR3配列について、attL1とattR1、attL2とattR2、attL3とattR3、attL4とattR4の各配列の間で、Gateway(R)LR クロナーゼTMII 酵素ミックス（インビトロジェン）により、その添付プロトコールに従って組換えを起こさせ、三種のカセットを、イネ形質転換用ベクターp35SHPTAg7-43GWにまとめて移し替えた。その結果、図５に示すとおり、Glbpro:HA52-335(-linker)、Glbpro:HA52-335(+linker)、Glbpro:HA59-335(-linker)、Glbpro:HA59-335(+linker)の、４種類のプラスミドが構築された。 B) Construction of plasmid Figure 5 shows an outline of the construction of a vector for expression in rice. From the database of National Center for Biotechnology Information, the nucleotide sequences of the promoter (SEQ ID NO: 28) and terminator (SEQ ID NO: 29) of the Globulin-1 gene (Glb-1) of rice were obtained. Then, a glutelin expression-suppressing cassette and a prolamin expression-suppressing cassette, which are structures for causing two kinds of RNA interference, having the structure shown in FIG. 5 were designed. Specifically, first, in order to suppress the expression of glutelin A, B, and C, which are storage proteins of rice, a promoter (SEQ ID NO: 30) of about 2 kb of the Ubi1 gene of rice and pRI 101-AN (Takara). A part of the glutelin A, B, and C genes (sequence) is sandwiched between the NOS terminator (SEQ ID NO: 31) derived from bio) and the 3rd intron (RAP 3rd intron, SEQ ID NO: 32) of the rice aspartic promoter. A construct in which a sequence complementary to the sequence (SEQ ID NO: 34) was arranged was designed and named as a glutelin expression-suppressing cassette. Similarly, in order to suppress the expression of prolamins of the rice storage proteins 13 KD and 16 KD, the promoter of about 2 kb of the Ubi1 gene of rice (SEQ ID NO: 28) and pRI 101-AN (Takarabio) The 3rd intron (RAP 3rd intron, SEQ ID NO: 32) of the rice aspartic protease is sandwiched between the terminators of the NOS from which it is derived (SEQ ID NO: 29), and a partial sequence of the prolamin gene of 13 KD and 16 KD (SEQ ID NO: 35) is sandwiched. ) And a sequence in which a sequence complementary to the sequence (SEQ ID NO: 36) was arranged was designed and named as a promoter expression inhibitory cassette. These two expression-suppressing cassettes were synthesized by PCR using Ex-Taq (Takara Bio), and the entry vectors pKS4-MCS and pKS221MCS (both Wakasa et al., 2006, Plant Biotechnol. J. 4: 499). Subcloned into -510). On the other hand, an expression cassette of the improved or re-improved head defect fusion protein B or D was synthesized by PCR using Ex-Taq (Takara Bio), and the entry vector pK
Subcloned into S2-3MCS (detailed in Wakasa et al., 2006, Plant Biotechnol. J. 4: 499-510). Then, the above three types of cassettes were transferred from the obtained three types of entry vectors to the rice transformation vector p35SHPTAg7-43GW (Wakasa et al., 2006, Plant Biotechnol. J. 4: 499-510) using Gateway TM cloning technology. Transferred. Specifically, the attL4 and attR1 sequences subcloned into pKS4-MCS sandwiching the gluterin expression-suppressing cassette, the attL1 and attL2 sequences subcloned into pKS221MCS, and the attL1 and attL2 sequences, pKS2-3MCS. Regarding the attR2 and attL3 sequences that are present across the expression cassette of the improved or re-improved head defect fusion protein B or D subcloned into, and the attR4 and attR3 sequences that are present in the rice transformation vector p35SHPTAg7-43GW. AttL1 and attR1, attL2 and attR2, attL3 and attR3, attL4 and attR4 sequences are recombined by Gateway (R) LR clonase TM II enzyme mix (invitrogen) according to the attached protocol, and three cassettes are used. Was collectively transferred to the rice transformation vector p35SHPTAg7-43GW. As a result, as shown in FIG. 5, of Glbpro: HA52-335 (-linker), Glbpro: HA52-335 (+ linker), Glbpro: HA59-335 (-linker), Glbpro: HA59-335 (+ linker), Four types of plasmids were constructed.
２．イネ形質転換体作成とタンパク生産能の評価
　完成したプラスミドにより、アグロバクテリウム法にてイネ（品種：キタアケ（Kitaake））の形質転換を実施し各プラスミドにつき20-25個体の独立したハイグロマイシンＢ（50 μg/mL）に耐性の形質転換体カルスを得た。イネ形質転換体カルスから、定法に従って植物体を再分化させ、閉鎖系温室にて短日条件下で栽培を行ない、種子を採種した。得られた種子からタンパク質を抽出し、SDS-PAGEおよびウエスタンブロッティングに供試した。すなわち、種子をマルチビーズショッカー（登録商標。安井器械）で粉砕後、1粒あたり　400μLの尿素-SDS バッファー（50 mM Tris-HCl pH 6.8, 8 M Urea, 4 % SDS, 5 % 2-mercaptoethanol, 20 % Glycerol）を加え、1時間、ボルテックスにて撹拌し、14,000rpmで10分遠心後、上清 (3-6　μL）をSDS-PAGEに供試した。SDS-PAGEには12%のポリアクリルアミドゲルを用いた。ウエスタンブロッティングの検出は、抗His抗体によって行なった。 2. 2. Preparation of rice transformants and evaluation of protein-producing ability The completed plasmids were transformed with rice (variety: Kitaake ) by the Agrobacterium method, and 20-25 independent hygromycin Bs were used for each plasmid. A transformant callus resistant to (50 μg / mL) was obtained. From the rice transformant callus, the plant was redifferentiated according to a conventional method, cultivated in a closed greenhouse under short-day conditions, and seeds were collected. Proteins were extracted from the obtained seeds and subjected to SDS-PAGE and Western blotting. That is, after crushing the seeds with a multi-bead shocker (registered trademark, Yasui Kikai), 400 μL of urea-SDS buffer (50 mM Tris-HCl pH 6.8, 8 M Urea, 4% SDS, 5% 2-mercaptoethanol,) per grain. 20% Glycerol) was added, stirred by vortex for 1 hour, centrifuged at 14,000 rpm for 10 minutes, and the supernatant (3-6 μL) was subjected to SDS-PAGE. A 12% polyacrylamide gel was used for SDS-PAGE. Western blotting was detected by anti-His antibody.
　結果を図６に示す。図６において、各レーンの内容は、左から順に次の通りである。図5のGlbpro:HA52-335-M1(-linker)の形質転換体の系統#02, #03, #06, #12, #18。図5のGlbpro:HA59-335-M1(-linker)の形質転換体の系統#02, #04, #16, #21, #23。図5のGlbpro:HA52-335-M1(+linker)の形質転換体の系統#03, #08, #14, #19, #25。図5のGlbpro:HA59-335-M1(+linker)の形質転換体の系統#01, #04, #05, #18。 The results are shown in Fig. 6. In FIG. 6, the contents of each lane are as follows in order from the left. Glbpro: HA52-335-M1 (-linker) transformant lineage # 02, # 03, # 06, # 12, # 18 in Fig. 5. Glbpro: HA59-335-M1 (-linker) transformant lineage # 02, # 04, # 16, # 21, # 23 in FIG. Glbpro: HA52-335-M1 (+ linker) transformant lineage # 03, # 08, # 14, # 19, # 25 in Fig. 5. Glbpro: HA59-335-M1 (+ linker) transformant lineage # 01, # 04, # 05, # 18 in Fig. 5.
　HAの頭部欠損領域の違いは、頭部欠損融合タンパク質の発現量に大きな違いをもたらさなかった。一方で、HAのC末端に存在する１２アミノ酸配列からなるサイトプラズミックテールの除去および／またはフレキシブルな構造を取るＧＳＡＧＳＡのリンカーの挿入は、頭部欠損融合タンパク質の発現量を増加させる効果が示された。 The difference in the head defect region of HA did not bring about a large difference in the expression level of the head defect fusion protein. On the other hand, removal of the cytoplasmic tail consisting of the 12 amino acid sequence present at the C-terminal of HA and / or insertion of a linker of GSAGSA having a flexible structure has been shown to have the effect of increasing the expression level of the head-deficient fusion protein. Was done.
３．再改良型HA-M1融合タンパク質の様々なイネカルス発現ベクター
ア）様々なイネカルス発現用ベクターの構築
　イネのカルスでの発現用ベクター構築の概略を、図７に示す。 3. 3. Various Inecalus Expression Vectors for Re-improved HA-M1 Fusion Protein
A) Construction of various rice callus expression vectors FIG. 7 shows an outline of the construction of rice callus expression vectors.
（i）ベクター甲
　ベクター構築のためのDNA断片はKOD-One（東洋紡）により増幅し、断片の連結はIn-Fusionクローニングキット（タカラバイオ）を用いて実施した。National Center for Biotechnology Informationのデータベースより、トウモロコシのUbiuitin遺伝子（Ubi）のプロモーター（配列番号５１）、3xFLAGタグをコードするヌクレオチド配列（配列番号５２）を取得した。配列番号２７に記載のヌクレオチド配列にコードされる再改良型HA-M1融合タンパク質Dの遺伝子発現用ベクターより、Glb-1プロモーターをUbiプロモーターに置換し、Glb-1の分泌シグナルをコードするヌクレオチド配列と、HAのN末端をコードするヌクレオチド配列の間に、配列番号５２の3xFLAGをコードするヌクレオチド配列をin frameで挿入し、配列番号５３に記載の遺伝子コード領域を持つベクター甲を構築した。 (I) Vector A DNA fragment for vector construction was amplified by KOD-One (Toyobo), and ligation of the fragment was carried out using an In-Fusion cloning kit (Takara Bio). From the database of National Center for Biotechnology Information, the promoter of the Ubiuitin gene (Ubi) of maize (SEQ ID NO: 51) and the nucleotide sequence encoding the 3xFLAG tag (SEQ ID NO: 52) were obtained. From the gene expression vector of the modified HA-M1 fusion protein D encoded by the nucleotide sequence set forth in SEQ ID NO: 27, the Glb-1 promoter is replaced with the Ubi promoter, and the nucleotide sequence encoding the Glb-1 secretion signal is obtained. And, the nucleotide sequence encoding 3xFLAG of SEQ ID NO: 52 was inserted in frame between the nucleotide sequences encoding the N-terminal of HA, and a vector instep having the gene coding region described in SEQ ID NO: 53 was constructed.
（ii）ベクター乙
　ベクター甲から、KDELをコードするヌクレオチド配列を除去し、配列番号５４に記載の遺伝子コード領域を持つベクター乙を構築した。 (Ii) Vector B The nucleotide sequence encoding KDEL was removed from Vector A, and Vector B having the gene coding region set forth in SEQ ID NO: 54 was constructed.
（iii）ベクター丙
　ベクター乙を元に、配列番号５４において、２９２番目のアデニンから１６４４番目のグアニンまでのヌクレオチド配列（配列番号５５）にコードされるアミノ酸配列について、GenScript社に委託し、同社プログラム "OptimumGene"にて、コドン使用頻度はイネに最適化したままで、配列番号５５とは異なるコドンからなるヌクレオチド配列（配列番号５６）をデザインし、合成した。 (Iii) Based on Vector B, the amino acid sequence encoded by the nucleotide sequence (SEQ ID NO: 55) from the 292nd adenine to the 1644th guanine in SEQ ID NO: 54 was outsourced to GenScript, and the company's program. In "OptimumGene", a nucleotide sequence (SEQ ID NO: 56) consisting of codons different from SEQ ID NO: 55 was designed and synthesized while keeping the codon usage frequency optimized for rice.
（iv）ベクター丁
　ベクター乙の、配列番号５５の領域を、配列番号５６で置換し、配列番号５７に記載の遺伝子コード領域を持つベクター丙を構築した。ベクター丙を元に、Glb-1の分泌シグナルをコードする領域を、開始コドンのみを残して削除し、配列番号５８に記載の遺伝子コード領域を持つベクター丁を構築した。 (Iv) Vector Ding The region of SEQ ID NO: 55 of Vector B was replaced with SEQ ID NO: 56 to construct a vector hei having the gene coding region set forth in SEQ ID NO: 57. Based on the vector 丙, the region encoding the secretory signal of Glb-1 was deleted leaving only the start codon, and a vector clove having the gene coding region shown in SEQ ID NO: 58 was constructed.
イ）形質転換体カルスの作成
　完成したプラスミドにより、アグロバクテリウム法にてイネ（品種：キタアケ(Kitaake)）の形質転換を実施し各プラスミドにつき20-25個体の独立したハイグロマイシンＢ（50 μg/mL）に耐性の形質転換体カルスを得た。イネ形質転換体カルスは、2回のハイグロマイシンＢによる選抜後、3%のスクロースを含むN6液体培地に置床し、28℃、暗黒下で2週間の振とう培養を行ない、所定時間の培養後、細胞（カルス）を回収した。回収したカルスは、液体窒素で凍結後、マルチビーズショッカー（登録商標。安井器械）で粉砕し、尿素-SDSバッファーでタンパク質を抽出した。抽出したタンパク質は、SDS-PAGEおよびウエスタンブロッティングに供試した。検出は抗FLAG抗体によって行なった。 B ) Preparation of transformant callus The completed plasmid was transformed into rice (variety: Kitaake ) by the Agrobacterium method, and 20-25 independent hygromycin B (50 μg) was used for each plasmid. A transformant callus resistant to / mL) was obtained. After selection with hygromycin B twice, the rice transformant callus was placed in an N6 liquid medium containing 3% sucrose, shake-cultured at 28 ° C. in the dark for 2 weeks, and after culturing for a predetermined time. , Cells (callus) were collected. The collected callus was frozen in liquid nitrogen, pulverized with a multi-bead shocker (registered trademark, Yasui Instrument), and the protein was extracted with a urea-SDS buffer. The extracted protein was subjected to SDS-PAGE and Western blotting. Detection was performed by anti-FLAG antibody.
ウ）タンパク質の分析
　ベクター甲による形質転換体の独立した6系統と、ベクター乙による形質転換体の独立した5系統の、SDS-PAGEおよびウエスタンブロッティングの結果を、図８に示す。図８において、各レーンの内容は次の通りである。レーン 1～6：ベクター甲を発現したイネのカルスのHA-M1融合タンパク質の発現（各レーンはそれぞれ独立したクローン）。レーン 7～11：ベクター乙を発現したイネのカルスのHA-M1融合タンパク質の発現（各レーンはそれぞれ独立したクローン）。系統によるばらつきはあるものの、KDELの付与されていない再改良型頭部欠損HA-M1融合タンパク質Ｄを発現するベクター乙による形質転換体の発現量は、KDELの付与されたベクター甲による形質転換体よりも高い傾向にあり、KDELの付与は、頭部欠損HA-M1融合タンパク質の発現を抑制する傾向があることが示された。 C) Protein analysis The results of SDS-PAGE and Western blotting of 6 independent strains of transformants by Vector A and 5 independent strains of transformants by Vector B are shown in FIG. In FIG. 8, the contents of each lane are as follows. Lanes 1-6: Expression of HA-M1 fusion protein of rice callus expressing vector instep (each lane is an independent clone). Lanes 7-11: Expression of HA-M1 fusion protein of rice callus expressing Vector B (each lane is an independent clone). Although there are variations depending on the strain, the expression level of the transformant by the vector B expressing the re-improved head defect HA-M1 fusion protein D to which KDEL was not given is the transformant by the vector A to which KDEL was given. It was shown that the addition of KDEL tended to suppress the expression of the head-deficient HA-M1 fusion protein.
　ベクター乙による形質転換体の独立した5系統と、ベクター丙による形質転換体の独立した4系統、ベクター丁による形質転換体の独立した5系統の、ウエスタンブロッティングの結果を、図９に示す。図９において、各レーンの内容は次の通りである。レーン 1～5：ベクター乙を発現したイネのカルスのHA-M1融合タンパク質の発現（各レーンはそれぞれ独立したクローン）。レーン 6～10：ベクター丙を発現したイネのカルスのHA-M1融合タンパク質の発現（各レーンはそれぞれ独立したクローン）。レーン 11～14：ベクター丁を発現したイネのカルスのHA-M1融合タンパク質の発現（各レーンはそれぞれ独立したクローン）。系統によるばらつきはあるものの、コドンを変えた再改良型頭部欠損融合タンパク質Ｄを発現するベクター丙による形質転換体の発現量は、ベクター乙による形質転換体の発現量と比較して顕著な差が認められず、コドン使用頻度を変えずに頭部欠損HA-M1融合タンパク質を発現させる遺伝子のコドンの変更は、発現量に大きな効果をもたらさないことが示された。一方で、頭部にGlb-1の分泌シグナルを持たない再改良型頭部欠損融合タンパク質Ｄを発現するベクター丁による形質転換体の発現量は、Glb-1の分泌シグナルを持つベクター丙による形質転換体よりも低い傾向にあり、Glb-1の分泌シグナルの付与は、頭部欠損HA-M1融合タンパク質の発現を促進する効果があることが示された。 FIG. 9 shows the results of Western blotting of 5 independent lines of the transformant by Vector B, 4 independent lines of the transformant by Vector Hei, and 5 independent lines of the transformant by Vector Ding. In FIG. 9, the contents of each lane are as follows. Lanes 1-5: Expression of HA-M1 fusion protein of rice callus expressing Vector B (each lane is an independent clone). Lanes 6-10: Expression of HA-M1 fusion protein of rice callus expressing Vector Hei (each lane is an independent clone). Lanes 11-14: Expression of the HA-M1 fusion protein of rice callus expressing Vector Ding (each lane is an independent clone). Although there are variations depending on the strain, the expression level of the transformant by the vector 丙 expressing the re-improved head defect fusion protein D in which the codon is changed is significantly different from the expression level of the transformant by the vector B. It was shown that the change of the codon of the gene expressing the head-deficient HA-M1 fusion protein without changing the frequency of codon use does not have a great effect on the expression level. On the other hand, the expression level of the transformant by the vector Ding expressing the re-improved head defect fusion protein D which does not have the Glb-1 secretory signal in the head is the trait by the vector 丙 which has the Glb-1 secretory signal. It tends to be lower than that of the transformant, and it was shown that the addition of the secretory signal of Glb-1 has the effect of promoting the expression of the head-deficient HA-M1 fusion protein.
実施例４：動物試験用抗原タンパク質の合成
１　頭部欠損 HA-M1融合タンパク質及び改良型頭部欠損 HA-M1融合タンパク質である、抗原タンパク質合成用プラスミドの構築
ア）頭部欠損 HA-M1融合タンパク質をコードするヌクレオチド配列を含むプラスミド
　配列番号８のヌクレオチド配列にコードされた頭部欠損HA-M1融合タンパク質について、イネのα-アミラーゼ3Dの分泌シグナル２５アミノ酸をコードする領域を除き、またM1のアミノ酸配列を（A/swine/IL/00685/2005(H1N1)）株のM1タンパク質のアミノ酸配列（登録番号：ACM17279.1、配列番号１６）に変更したものを、コドンをHA、M1それぞれ元のウイルスのコドンに戻し、すなわち、HAについては登録番号MK622940.1の配列、M1については登録番号FJ638301.1の配列のコドンに戻したヌクレオチド配列をデザインした（配列番号１７）。 Example 4: Synthesis of antigen protein for animal testing
1 Construction of a plasmid for antigen protein synthesis, which is a head-deficient HA-M1 fusion protein and an improved head-deficient HA-M1 fusion protein.
A) For the head-deficient HA-M1 fusion protein encoded by the nucleotide sequence of plasmid SEQ ID NO: 8, which contains the nucleotide sequence encoding the head-deficient HA-M1 fusion protein, 25 amino acids secreted from rice α-amylase 3D were used. Excluding the region encoding, the amino acid sequence of M1 was changed to the amino acid sequence of the M1 protein of the (A / swine / IL / 00685/2005 (H1N1)) strain (registration number: ACM17279.1, SEQ ID NO: 16). , A nucleotide sequence was designed in which the codons were returned to the codons of the original virus for HA and M1, that is, the sequence of registration number MK622940.1 for HA and the codon of registration number FJ638301.1 for M1 (sequence). Number 17).
　このヌクレオチド配列の、5'末端にEcoRV配列と開始コドン（ATG）を、3'末端の終始コドン（TGA）の後ろにNotI配列を付加した配列を人工遺伝子合成し、小麦無細胞タンパク合成系用プラスミドであるpEU-E01-MCS（セルフリーサイエンス）のEcoRVとNotIの間に挿入し、pKBac1199と命名した。挿入された配列に変異がないことをシーケンシングにより確認した。 An artificial gene of this nucleotide sequence with the EcoRV sequence and start codon (ATG) at the 5'end and the NotI sequence added after the stop codon (TGA) at the 3'end was synthesized for wheat cell-free protein synthesis. It was inserted between EcoRV and NotI of the plasmid pEU-E01-MCS (CellFree Science) and named pKBac1199. It was confirmed by sequencing that there was no mutation in the inserted sequence.
イ）改良型頭部欠損 HA-M1融合タパク質をコードするヌクレオチド配列を含むプラスミド
　改良型頭部欠損HA-M1融合タンパク質を小麦無細胞タンパク合成系で合成するために、pKBac1199を元に以下の改変を行なった。配列番号１７に記載のヌクレオチド配列について、１００番目のグアニンから３００番目のグアニンまでのヌクレオチド配列を削除し、そこに配列番号１１に記載の配列を挿入し、配列番号１８のヌクレオチド配列をデザインした。すなわち、pBac1199を鋳型として、KOD FX Neo（東洋紡）を用いたPCRにより、新版 植物のPCR実験プロトコール（島本功・佐々木卓治監修、秀潤社、1997、p95-p100）記載の合成遺伝子の作成方法により、配列番号１８のヌクレオチド配列の、5'末端にEcoRV配列と開始コドン（ATG）を、3'末端の終始コドン（TGA）の後ろにNotI配列を付加した断片を作製し、小麦無細胞タンパク合成系用プラスミドであるpEU-E01-MCS（セルフリーサイエンス）のEcoRVとNotIの間に挿入し、pKBac1201と命名した。挿入された配列に変異がないことをシーケンシングにより確認した。得られたプラスミドは、大腸菌DH5α（東洋紡社）にクローニングし、QIAGEN Plasmid Maxi Kit（キアゲン社）を用いて大量調製した。 B) A plasmid containing a nucleotide sequence encoding an improved head-deficient HA-M1 fusion protein In order to synthesize an improved head-deficient HA-M1 fusion protein in a wheat cell-free protein synthesis system, the following is based on pKBac1199. Modified. Regarding the nucleotide sequence set forth in SEQ ID NO: 17, the nucleotide sequence from the 100th guanine to the 300th guanine was deleted, and the sequence set forth in SEQ ID NO: 11 was inserted therein to design the nucleotide sequence of SEQ ID NO: 18. That is, the method for preparing a synthetic gene described in the new version of the plant PCR experimental plasmid (supervised by Ko Shimamoto and Takuji Sasaki, Shujunsha, 1997, p95-p100) by PCR using pBac1199 as a template and KOD FX Neo (Toyo Boseki). Created a fragment of the nucleotide sequence of SEQ ID NO: 18 with the EcoRV sequence and start codon (ATG) at the 5'end and the NotI sequence after the start codon (TGA) at the 3'end. It was inserted between EcoRV and NotI of pEU-E01-MCS (CellFree Science), which is a plasmid for synthetic systems, and named pKBac1201. It was confirmed by sequencing that there was no mutation in the inserted sequence. The obtained plasmid was cloned into Escherichia coli DH5α (Toyobo Co., Ltd.) and prepared in large quantities using the QIAGEN plasmid Maxi Kit (Qiagen Co., Ltd.).
２．抗原タンパク質の合成
　1 mg/mLの濃度に調製したプラスミドを鋳型に、頭部欠損HA-M1タンパク質および改良型頭部欠損HA-M1融合タンパク質Ｂの合成をした。合成の概要は以下のとおり。プラスミド上のSP6プロモーターを利用して37℃で6時間の転写反応を行ない、mRNAを合成した。合成したmRNAを用い、反応スケール6 mLの重層法で翻訳反応（15℃、20時間）を行なった。コムギ胚芽抽出液は、頭部欠損HA-M1タンパク質に関してはWEPRO7240H（セルフリーサイエンス）を、改良型頭部欠損HA-M1融合タンパク質に関してはWEPRO7240（セルフリーサイエンス）を、それぞれ使用した。合成されたタンパク質（総画分）を遠心（21,600xG、4℃、10分間）し、沈殿画分を、コムギ無細胞タンパク質合成系用翻訳バッファー（セルフリーサイエンス社）にて2回洗浄後、コムギ無細胞タンパク質合成系用翻訳バッファーに、約1 mg/mLとなるように懸濁した。頭部欠損HA-M1融合タンパク質の配列は配列番号１９、改良型頭部欠損HA-M1融合タンパク質の配列は配列番号２０とした。 2. 2. Synthesis of antigen protein Using a plasmid prepared at a concentration of 1 mg / mL as a template, head-deficient HA-M1 protein and improved head-deficient HA-M1 fusion protein B were synthesized. The outline of synthesis is as follows. Using the SP6 promoter on the plasmid, a transcription reaction was performed at 37 ° C for 6 hours to synthesize mRNA. Using the synthesized mRNA, a translation reaction (15 ° C., 20 hours) was carried out by a layered method with a reaction scale of 6 mL. For the wheat germ extract, WEPRO7240H (CellFree Science) was used for the head-deficient HA-M1 protein, and WEPRO7240 (CellFree Science) was used for the improved head-deficient HA-M1 fusion protein. Centrifuge the synthesized protein (total fraction) (21,600xG, 4 ° C, 10 minutes), wash the precipitated fraction twice with a translation buffer for wheat cell-free protein synthesis (CellFree Sciences, Inc.), and then wheat. It was suspended in a translation buffer for a cell-free protein synthesis system to a concentration of about 1 mg / mL. The sequence of the head-deficient HA-M1 fusion protein was SEQ ID NO: 19, and the sequence of the improved head-deficient HA-M1 fusion protein was SEQ ID NO: 20.
３．再改良型頭部欠損 HA-M1融合タンパク質である、抗原タンパク質合成用プラスミドの構築
ア）再改良型頭部欠損 HA-M1融合タンパク質をコードするヌクレオチド配列を含むプラスミド
　再改良型頭部欠損HA-M1融合タンパク質を小麦無細胞タンパク合成系で合成するためのベクター作製を、以下のように実施した。 3. 3. Construction of a plasmid for antigen protein synthesis, which is a re-improved head-deficient HA-M1 fusion protein
A) A plasmid containing a nucleotide sequence encoding the re-improved head-deficient HA-M1 fusion protein The following vector preparation for synthesizing the re-improved head-deficient HA-M1 fusion protein in a wheat cell-free protein synthesis system It was carried out as follows.
　改良型頭部欠損HA-M1融合タンパク質をコードするヌクレオチド配列を持つpKBac1201を元に、欠損させたいHAのC末端の１２アミノ酸配列をコードする領域から外側に向けた２本のプライマーを用いてPCRを行ない、この領域を欠損させた、開環化されたベクターの断片を作製した。一方で、この欠損させた領域を、ＧＳＡＧＳＡからなるアミノ酸配列をコードするヌクレオチドで連結するために、このアミノ酸配列をコードし、かつ両端に、開環化されたベクター配列の二つの末端と15 merの相同配列を持つ、お互いに相補鎖の関係となる２本のオリゴヌクレオチドを合成し、アニールさせた断片を作製した。これら2本の作製された断片を、In-Fusionクローニングキットを用いて連結し、プラスミドを完成させた。 Based on pKBac1201 which has a nucleotide sequence encoding the improved head-deficient HA-M1 fusion protein, PCR is performed using two primers pointing outward from the region encoding the 12 amino acid sequence of the C-terminal of the HA to be deleted. To prepare a fragment of the ring-opened vector lacking this region. On the other hand, in order to link this deleted region with a nucleotide encoding the amino acid sequence consisting of GSAGSA, this amino acid sequence is encoded, and at both ends, the two ends of the cyclized vector sequence and 15 mer. Two oligonucleotides having a homologous sequence of No. 2 and having a complementary strand relationship with each other were synthesized to prepare an annealed fragment. These two prepared fragments were ligated using an In-Fusion cloning kit to complete a plasmid.
　具体的には、４つのオリゴヌクレオチド、TKIWver3_1（配列番号３７）、TKIWver3_2（配列番号３８）、TKIWver3_3（配列番号３９）、TKIWver3_4（配列番号４０）を合成した。プラスミドpKBac1201を鋳型とし、TKIWver3_1とTKIWver3_2をプライマーセットとして、KOD One（東洋紡）を用いて、その添付プロトコールに従ってPCRを実施し、増幅された断片をアガロースゲル電気泳動後のゲルより、illustra GFX PCR DNA and Gel Band Purification Kit（GE ヘルスケア）で精製・回収した。また、TKIWver3_3およびTKIWver3_4（それぞれ、0.1 nmol/μLの濃度の水溶液として調製されている）を40 μLずつ混合し、鍋に張った水の上で1分間、煮沸した後に、そのまま鍋の水が室温に戻るまで放置し、アニーリングを促した。得られた断片を定法に従ってエタノール沈殿により濃縮した。このようにして得た二種類の断片を、In-Fusionクローニングキットを用いて環状化し、大腸菌DH5α（東洋紡）の形質転換を行なった。このようにして作製されたプラスミドが、目的のとおりの構築がなされていることは、シーケンシングにより確認し、pKBac1211と命名した。得られたプラスミドは、大腸菌DH5α（東洋紡）にクローニングし、QIAGEN Plasmid Maxi Kit（キアゲン）を用いて調製した。 Specifically, four oligonucleotides, TKIWver3_1 (SEQ ID NO: 37), TKIWver3_2 (SEQ ID NO: 38), TKIWver3_3 (SEQ ID NO: 39), and TKIWver3_4 (SEQ ID NO: 40) were synthesized. PCR was performed using the plasmid pKBac1201 as a template, TKIWver3_1 and TKIWver3_2 as primer sets, and KODOne (Toyo Spinning Co., Ltd.) according to the attached protocol. Purified and recovered with andGelBandPurificationKit (GE Healthcare). Also, mix 40 μL of TKIWver3_3 and TKIWver3_4 (each prepared as an aqueous solution with a concentration of 0.1 nmol / μL), boil for 1 minute on water in a pot, and then keep the water in the pot at room temperature. I left it until I returned to, and urged annealing. The obtained fragments were concentrated by ethanol precipitation according to a conventional method. The two types of fragments thus obtained were cyclized using an In-Fusion cloning kit and transformed with Escherichia coli DH5α (Toyobo). It was confirmed by sequencing that the plasmid thus prepared was constructed as intended, and the name was pKBac1211. The obtained plasmid was cloned into Escherichia coli DH5α (Toyobo) and prepared using QIAGEN Plasmamid MaxiKit (Qiagen).
４．抗原タンパク質の合成
　1 mg/mLの濃度に調製したプラスミドpKBac1211を鋳型に、再改良型頭部欠損HA-M1融合タンパク質を合成した。合成の概要は以下のとおり。プラスミド上のSP6プロモーターを利用して37℃で6時間の転写反応を行ない、mRNAを合成した。合成したmRNAを用い、反応スケール6 mLの重層法で翻訳反応（15℃、20時間）を行なった。コムギ胚芽抽出液はWEPRO7240を使用した。合成されたタンパク質（総画分）を遠心（21,600xG、4℃、10分間）し、沈殿画分を、コムギ無細胞タンパク質合成系用翻訳バッファー（セルフリーサイエンス社）にて2回洗浄後、コムギ無細胞タンパク質合成系用翻訳バッファーに、約1 mg/mLとなるように懸濁した。このように調製したタンパク質を、抗原タンパク質Ｃ（再改良型頭部欠損HA-M1融合タンパク質、配列番号４１）とした。 4. Synthesis of antigen protein Using the plasmid pKBac1211 prepared at a concentration of 1 mg / mL as a template, a re-improved head-deficient HA-M1 fusion protein was synthesized. The outline of synthesis is as follows. Using the SP6 promoter on the plasmid, a transcription reaction was performed at 37 ° C for 6 hours to synthesize mRNA. Using the synthesized mRNA, a translation reaction (15 ° C., 20 hours) was carried out by a layered method with a reaction scale of 6 mL. WEPRO7240 was used as the wheat germ extract. Centrifuge the synthesized protein (total fraction) (21,600xG, 4 ° C, 10 minutes), wash the precipitated fraction twice with a translation buffer for wheat cell-free protein synthesis (CellFree Sciences, Inc.), and then wheat. It was suspended in a translation buffer for a cell-free protein synthesis system to a concentration of about 1 mg / mL. The protein thus prepared was designated as antigen protein C (re-improved head-deficient HA-M1 fusion protein, SEQ ID NO: 41).
実施例５：頭部欠損HA-M1融合タンパク質および改良型頭部欠損HA-M1融合タンパク質Ｂの酵母での発現
　頭部欠損HA-M1融合タンパク質および改良型頭部欠損HA-M1融合タンパク質Ｂ（上記イネの実施例において作成したＢ）について、出芽酵母Saccharomyces cerevisiaeによる発現を試みた。 Example 5: Expression of head-deficient HA-M1 fusion protein and improved head-deficient HA-M1 fusion protein B in yeast Head-deficient HA-M1 fusion protein and improved head-deficient HA-M1 fusion protein B ( For B) prepared in the above rice example, expression was attempted by the budding yeast Saccharomyces cerevisiae .
１．酵母発現用プラスミドの構築
　酵母で発現させるためのプロモーターに関しては、Saccharomyces Genome Database（https://www.yeastgenome.org/）より、出芽酵母Saccharomyces cerevisiaeの0.7 kbのTDH3遺伝子プロモーターのヌクレオチド配列を取得した（配列番号２１）。この配列の5'側にSpeIサイト、3'側にEcoRVを付加するプライマーを作製し、BY4741（フナコシ）の染色体DNAを鋳型として、KOD FX Neoにより、その添付プロトコールに従ってPCRを行なった。その後、SpeIとEcoRVで消化し、illustra GFX PCR DNA and Gel Band Purification Kit（GE ヘルスケア）で精製し、TDH3プロモーター断片とした。また小麦無細胞タンパク合成系用ベクター、pKBac1199およびpKbac1201より、それぞれ頭部欠損HA-M1融合タンパク質および改良型頭部欠損HA-M1融合タンパク質Ｂをコードするヌクレオチド配列を含む断片について、EcoRV-NotI断片として、アガロースゲル電気泳動後のゲルより、illustra GFX PCR DNA and Gel Band Purification Kitで精製・回収した。また、骨格となるベクター、pYES2（インビトロジェン）についてSpeI（New England Biolabs）とNotI（New England Biolabs）で消化後、1% アガロースゲルで電気泳動を行ない、臭化エチジウムで染色後、ベクターのバンドをUV照射下で切り出した。 1. 1. Construction of plasmid for yeast expression Regarding the promoter for expression in yeast, the nucleotide sequence of the 0.7 kb TDH3 gene promoter of Saccharomyces cerevisiae in Saccharomyces cerevisiae was obtained from the Saccharomyces Genome Database (https://www.yeastgenome.org/). (SEQ ID NO: 21). Primers to add SpeI site on the 5'side and EcoRV on the 3'side of this sequence were prepared, and PCR was performed by KOD FX Neo using the chromosomal DNA of BY4741 (Funakoshi) as a template according to the attached protocol. It was then digested with SpeI and EcoRV and purified with the illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare) to give the TDH3 promoter fragment. EcoRV-NotI fragments from pKBac1199 and pKbac1201 vectors for wheat cell-free protein synthesis, regarding fragments containing nucleotide sequences encoding head-deficient HA-M1 fusion protein and improved head-deficient HA-M1 fusion protein B, respectively. From the gel after agarose gel electrophoresis, it was purified and recovered with the illustra GFX PCR DNA and Gel Band Purification Kit. In addition, the skeletal vector, pYES2 (Invitrogen), was digested with SpeI (New England Biolabs) and NotI (New England Biolabs), electrophoresed on a 1% agarose gel, stained with ethidium bromide, and then the band of the vector was formed. It was cut out under UV irradiation.
　図１０に示すように、酵母用プラスミドpYES2のSpeI部位とNotI部位の間に、5'側からTDH3プロモーター、頭部欠損HA-M1融合タンパク質もしくは改良型頭部欠損HA-M1融合タンパク質Bをコードするヌクレオチドを、この順番で、Ligation High ver.2（東洋紡）を用いて連結した。連結によって製成したプラスミドが、目的の形および配列となっていることを、シーケンシングによって確認した。 As shown in FIG. 10, the TDH3 promoter, head-deficient HA-M1 fusion protein or improved head-deficient HA-M1 fusion protein B is encoded between the SpeI and NotI sites of the yeast plasmid pYES2 from the 5'side. The nucleotides to be subjected to were ligated in this order using Ligation High ver.2 (Toyobo). Sequencing confirmed that the plasmid produced by ligation had the desired shape and sequence.
　このようにして、頭部欠損HA-M1融合タンパク質および改良型頭部欠損HA-M1融合タンパク質Ｂを酵母で発現させるためのプラスミドを構築し、それぞれpKBac1207およびpKBac1208と命名した。これらのプラスミドは、多コピーで、酵母の核外で自律複製するタイプのものであり、選択マーカーとして宿主のウラシル要求性を相補するURA3遺伝子を持っている。 In this way, plasmids for expressing the head-deficient HA-M1 fusion protein and the improved head-deficient HA-M1 fusion protein B in yeast were constructed and named pKBac1207 and pKBac1208, respectively. These plasmids are multicopy, of the type that autonomously replicates outside the nucleus of yeast, and carry the URA3 gene, which complements the host's uracil requirement as a selectable marker.
２．酵母形質転換体作製と頭部欠損HA-M1融合タンパク質および改良型頭部欠損 HA-M1融合タンパク質Ｂの生産能評価
ア）酵母形質転換体による生産
　酵母を用いた実験は、他に記載がない限り、Current Protocol in Molecular Biology（John Wiley & Sons）に記載の方法を用いた。完成したプラスミドpKBac1207およびpKBac1208を、リチウム法にて出芽酵母Saccharomyces cerevisiae（菌株：BY4741、BY4742、フナコシ）に導入した。得られた形質転換体を、10 mLのSD培地（ウラシルを含まない）を入れた100 mLの三角フラスコにて、25℃、120 rpmで一晩、振とう培養を行なった。 2. 2. Yeast transformant preparation and evaluation of productivity of head-deficient HA-M1 fusion protein and improved head-deficient HA-M1 fusion protein B
A) Experiments using yeast produced by yeast transformants used the method described in Current Protocol in Molecular Biology (John Wiley & Sons) unless otherwise stated. The completed plasmids pKBac1207 and pKBac1208 were introduced into Saccharomyces cerevisiae (strains: BY4741, BY4742, Funakoshi) by the lithium method. The obtained transformants were shake-cultured overnight at 25 ° C. and 120 rpm in a 100 mL Erlenmeyer flask containing 10 mL of SD medium (without uracil).
イ）タンパク質の分析
　培養後、遠心分離にて酵母を回収し、0.1 mLの抽出バッファー（50 mM Tris-HCl pH 6.8, 8 M Urea, 4 % SDS, 50 mM DTT, 20 % Glycerol）の存在下でガラスビーズ（直径：425-600 μm、シグマ）と一緒に激しく撹拌することにより、細胞を破砕した。細胞抽出液を遠心分離により回収し、アトー社のシステムを用いてSDS-PAGEおよびウエスタンブロッティングに供試した。検出は抗His抗体によって行なった。 B) Protein analysis After culturing, yeast is collected by centrifugation and in the presence of 0.1 mL extraction buffer (50 mM Tris-HCl pH 6.8, 8 M Urea, 4% SDS, 50 mM DTT, 20% Glycerol). Cells were disrupted by vigorous stirring with glass beads (diameter: 425-600 μm, sigma). Cell extracts were collected by centrifugation and tested on SDS-PAGE and Western blotting using Atto's system. Detection was performed by anti-His antibody.
　結果を図１１に示す。図１１において、各レーンの内容は次の通りである。1: 分子量マーカー。2,3: オリジナル型/BY4741。4,5: 改良型B/BY4741。6: BY4741(ネガティブコントロール）。7,8: オリジナル型/BY4742。9,10: 改良型/BY4742。11: BY4742(ネガティブコントロール)。オリジナル型：頭部欠損HA-M1融合タンパク質。改良型：改良型頭部欠損HA-M1融合タンパク質B。 The results are shown in FIG. In FIG. 11, the contents of each lane are as follows. 1: Molecular weight marker. 2,3: Original type / BY4741. 4,5: Improved type B / BY4741. 6: BY4741 (negative control). 7,8: Original type / BY4742. 9,10: Improved type / BY4742. 11: BY4742 (negative control). Original type: Head defect HA-M1 fusion protein. Improved: Improved head defect HA-M1 fusion protein B.
　宿主にBY4741、BY4742のいずれを用いた場合にも、改良型頭部欠損HA-M1融合タンパク質Ｂの発現量は、頭部欠損HA-M1融合タンパク質の発現量よりも多かった。このことから、イネを宿主とした場合のみならず酵母を宿主とした場合でも、HAの頭部欠損領域を拡大することにより、頭部欠損HA-M1融合タンパク質の発現量が増加することが確認できた。　 When either BY4741 or BY4742 was used as the host, the expression level of the improved head-deficient HA-M1 fusion protein B was higher than the expression level of the head-deficient HA-M1 fusion protein. From this, it was confirmed that the expression level of the head-deficient HA-M1 fusion protein is increased by expanding the head-deficient region of HA not only when rice is used as the host but also when yeast is used as the host. did it. It was
　植物界被子植物門イネ目イネ科のイネと、菌界子嚢菌門サッカロミケス目サッカロミケス科の出芽酵母で、HAの頭部欠損領域の拡大が、頭部欠損HA-M1融合タンパク質の発現量増加に効果があったことから、植物界および菌界で共通して、HAの頭部欠損領域の拡大が、頭部欠損HA-M1融合タンパク質の発現量増加に効果があることが示された。 In Saccharomycetale, Saccharomycetale, Saccharomycetale, Saccharomycetale, Saccharomycetale, Saccharomycetale, Saccharomycetale, Saccharomycetale, Saccharomycetale, Saccharomycetale, Saccharomycetale, H. Since there was an effect, it was shown that the expansion of the head-deficient region of HA is effective in increasing the expression level of the head-deficient HA-M1 fusion protein, which is common in both the plant and fungal fields.
実施例６：酵母による再改良型頭部欠損HA-M1融合タンパク質の発現
１．融合タンパク質の設計
　頭部欠損HA-M1融合タンパク質のHA部分の改変が、酵母における発現量に与える影響を調べるため、図１２に概略を示すように、新たな発現ベクターのシリーズを構築した。すなわち、頭部欠損HA-M1融合タンパク質の発現に用いたベクターを元に、酵母での選択マーカー遺伝子をURA3からPGK1プロモーターとPGK1ターミネーターに挟まれたG418耐性遺伝子からなる、G418耐性カセットと置換した。また、HAの頭部は、頭部欠損HA-M1融合タンパク質と同じ領域を欠損させたものと、改良型頭部欠損HA-M1融合タンパク質と同じ領域を欠損させたものの２種類、HAのC末端は、再改良型頭部欠損HA-M1融合タンパク質と同じC末端の１２アミノ酸配列をコードする領域を欠損させたものと欠損させていないものの２種類を組合せ、合計４種類の遺伝子合成を行なった。またこれらの遺伝子には、各融合タンパク質をコードするヌクレオチド配列の5'側に、FLAGタグコードするヌクレオチド配列（配列番号４２）をタンデムに３コピー連結させたヌクレオチド配列と、さらにその5'側に、MFA1遺伝子の分泌シグナルをコードするヌクレオチド配列（配列番号４３）と、さらにその5'側に、HindIII配列を付加した。また、HAとM1の間には、ＧＳＡＧＳＡからなるアミノ酸配列をコードするヌクレオチド配列（配列番号４４）を挿入した。このようにして、４種類の融合タンパク質のデザインを行なった（配列番号４５～４８）。 Example 6: Expression of re-improved head-deficient HA-M1 fusion protein by yeast
1. 1. Design of fusion protein In order to investigate the effect of modification of the HA portion of the head-deficient HA-M1 fusion protein on the expression level in yeast, a new series of expression vectors was constructed as outlined in FIG. That is, based on the vector used for the expression of the head-deficient HA-M1 fusion protein, the selectable marker gene in yeast was replaced with a G418 resistance cassette consisting of a G418 resistance gene sandwiched between the PGK1 promoter and PGK1 terminator from URA3. .. In addition, there are two types of HA heads, one in which the same region as the head-deficient HA-M1 fusion protein is deleted and the other in which the same region as the improved head-deficient HA-M1 fusion protein is deleted. A total of 4 types of genes are synthesized by combining two types of terminals, one with the region encoding the 12-amino acid sequence at the C-terminal, which is the same as the re-improved head-deficient HA-M1 fusion protein, and the other without the deletion. rice field. In addition, these genes have a nucleotide sequence in which 3 copies of the FLAG tag-encoding nucleotide sequence (SEQ ID NO: 42) are tandemly linked to the 5'side of the nucleotide sequence encoding each fusion protein, and further to the 5'side. , The nucleotide sequence encoding the secretory signal of the MFA1 gene (SEQ ID NO: 43) and the Hind III sequence were added to the 5'side thereof. In addition, a nucleotide sequence (SEQ ID NO: 44) encoding an amino acid sequence consisting of GSAGSA was inserted between HA and M1. In this way, four types of fusion proteins were designed (SEQ ID NOs: 45-48).
２．ベクターの構築
　pKBac1207の選択マーカーであるURA3遺伝子を、Yamanoらの論文（J. Biotechnol., 32: 173-178, 1994）に記載のプラスミドpZNEOを鋳型としてPCRで取得したG418耐性カセットと、In-Fusionクローニングにより置換し、pKBac1207NEOを得た。MFA1遺伝子の分泌シグナルをコードするDNA断片は、BY4741（フナコシ）の染色体DNAを鋳型としてPCRにより取得した。FLAGタグをコードするヌクレオチド配列をタンデムに3コピー連結させたDNA断片は、人工合成によって取得した。融合タンパク質をコードするDNA断片は、pKBac1199、pKBac1201、pKBac1211を鋳型としたPCRを組み合わせて、KOD One（東洋紡）により断片を増幅し、最終的にはすべての断片をIn-Fusionクローニングによって、pKBac1207NEOのTDH3プロモーターとCYC1ターミネーターの間に図１２の形に連結させ、pYHAM1-1、pYHAM1-2、pYHAM1-15、pYHAM1-16の４つのプラスミドを得た。発現させる融合タンパク質をコードするヌクレオチド配列が正しいことはシーケンシングによって確認した。 2. 2. Construction of vector The URA3 gene, which is a selectable marker for pKBac1207, was obtained by PCR using the plasmid pZNEO described in the paper by Yamano et al. (J. Biotechnol., 32: 173-178, 1994) as a template, and an In- Substituting by Fusion cloning gave pKBac1207NEO. The DNA fragment encoding the secretory signal of the MFA1 gene was obtained by PCR using the chromosomal DNA of BY4741 (Funakoshi) as a template. A DNA fragment in which 3 copies of the nucleotide sequence encoding the FLAG tag were tandemly linked was obtained by artificial synthesis. The DNA fragment encoding the fusion protein is amplified by KOD One (Toyobo) in combination with PCR using pKBac1199, pKBac1201 and pKBac1211 as templates, and finally all the fragments are cloned into pKBac1207NEO. By linking between the TDH3 promoter and the CYC1 terminator in the form shown in FIG. 12, four plasmids, pYHAM1-1, pYHAM1-2, pYHAM1-15, and pYHAM1-16, were obtained. Sequencing confirmed that the nucleotide sequence encoding the fusion protein to be expressed was correct.
３．酵母形質転換体作製と各種融合タンパク質の生産能評価
　酵母を用いた実験は、他に記載がない限り、Current Protocol in Molecular Biology（John Wiley & Sons）に記載の方法を用いた。完成したプラスミドpYHAM1-1、pYHAM1-2、pYHAM1-15、pYHAM1-16を、リチウム法にて出芽酵母Saccharomyces cerevisiae（菌株：Wyeast3724、Wyeast社）に導入した。得られた形質転換体を、10 mLのYPD培地（200 μg/LのG418を含む）を入れた100 mLの三角フラスコにて、30℃、150 rpmで一晩、振とう培養を行なった。 3. 3. Preparation of yeast transformants and evaluation of productivity of various fusion proteins Experiments using yeast used the methods described in Current Protocol in Molecular Biology (John Wiley & Sons) unless otherwise stated. The completed plasmids pYHAM1-1, pYHAM1-2, pYHAM1-15 and pYHAM1-16 were introduced into Saccharomyces cerevisiae (strain: Wyeast3724, Wyeast) by the lithium method. The obtained transformants were shake-cultured overnight at 30 ° C. and 150 rpm in a 100 mL Erlenmeyer flask containing 10 mL of YPD medium (containing 200 μg / L of G418).
　培養後、遠心分離にて酵母を回収し、0.1 mLの抽出バッファー（50 mM Tris-HCl pH 6.8, 8 M Urea, 4 % SDS, 50 mM DTT, 20 % Glycerol）の存在下でガラスビーズ（直径：425-600 μm、シグマ）と一緒に激しく撹拌することにより、細胞を破砕した。細胞抽出液を遠心分離により回収し、アトー社のシステムを用いてSDS-PAGEおよびウエスタンブロッティングに供試した。検出は抗His抗体によって行なった。 After culturing, yeast is collected by centrifugation, and glass beads (diameter) are present in the presence of 0.1 mL extraction buffer (50 mM Tris-HCl pH 6.8, 8 M Urea, 4% SDS, 50 mM DTT, 20% Glycerol). : 425-600 μm, Sigma) was vigorously stirred to disrupt the cells. Cell extracts were collected by centrifugation and tested on SDS-PAGE and Western blotting using Atto's system. Detection was performed by anti-His antibody.
　結果を図１３に示す。図１３において、各レーンの内容は次の通りである。1: pYHAM1-15, 2: pYHAM1-16, 3: pYHAM1-1, 4: pYHAM1-2。pYHAM1-15、pYHAM1-16を導入した株とpYHAM1-1、pYHAM1-2を導入した株の比較から、HAの頭部欠損領域を拡大することにより、頭部欠損HA-M1融合タンパク質の発現量が増加することが確認できた。一方、pYHAM1-1を導入した株とpYHAM1-2を導入した株の比較から、HAのC末端の１２アミノ酸配列を欠損させることにより、すなわち改良型頭部欠損融合タンパク質から再改良型頭部欠損融合タンパク質に改良することにより、頭部欠損HA-M1融合タンパク質の発現量がさらに増加することが示された。植物界被子植物門イネ目イネ科のイネと、菌界子嚢菌門サッカロミケス目サッカロミケス科の出芽酵母で、HAのC末端の１２アミノ酸配列を欠損が、頭部欠損HA-M1融合タンパク質の発現量増加に効果があったことから、植物界および菌界で共通して、HAのC末端の１２アミノ酸配列の欠損が、頭部欠損HA-M1融合タンパク質の発現量増加に効果があることが示された。 The results are shown in FIG. In FIG. 13, the contents of each lane are as follows. 1: pYHAM1-15, 2: pYHAM1-16, 3: pYHAM1-1, 4: pYHAM1-2. From the comparison between the strains introduced with pYHAM1-15 and pYHAM1-16 and the strains introduced with pYHAM1-1 and pYHAM1-2, the expression level of the head-deficient HA-M1 fusion protein was expanded by expanding the head-deficient region of HA. Was confirmed to increase. On the other hand, from the comparison between the strain into which pYHAM1-1 was introduced and the strain into which pYHAM1-2 was introduced, by deleting the 12 amino acid sequence at the C-terminal of HA, that is, the improved head defect from the improved head defect fusion protein was re-improved head defect. It was shown that the expression level of the head-deficient HA-M1 fusion protein was further increased by improving the fusion protein. The expression level of the head-deficient HA-M1 fusion protein is deleted in the sprouting yeast of the family Saccharomycetale, Saccharomycetale, and the phylum Saccharomycetale of the family Saccharomycetale. Since it was effective in increasing the amount, it was shown that the deletion of the C-terminal 12 amino acid sequence of HA is effective in increasing the expression level of the head-deficient HA-M1 fusion protein, which is common in both the plant and fungal fields. Was done.
実施例７：インフルエンザ感染マウスによる薬効試験（改良型。皮下・経鼻）
１．試験の概要
　被験物質のインフルエンザウイルスに対する抑制効果を確認する目的で、マウスに、抗原タンパク質（被験物質）を予め投与（皮下投与又は経鼻投与）し、インフルエンザウイルスに対する反応を検討した。概要を図１４に示す。試験群は12群（表１）とした。 Example 7: Drug efficacy test using influenza-infected mice (improved type. Subcutaneous / nasal)
1. 1. Outline of the test In order to confirm the inhibitory effect of the test substance on influenza virus, an antigen protein (test substance) was administered to mice in advance (subcutaneous administration or nasal administration), and the reaction to influenza virus was examined. The outline is shown in FIG. The test group was 12 groups (Table 1).
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
２．試験方法
ア）被験物質
　実施例６により調製した改良型頭部欠損HA-M1融合タンパク質を抗原タンパク質とした。
改良型; HA51-335融合タンパク質Ｂ（配列番号１中の第５１～３３５残基および第５２９～５５４残基が欠損） 2. 2. Test method
A) Test substance The improved head-deficient HA-M1 fusion protein prepared in Example 6 was used as an antigen protein.
Improved version; HA51-335 fusion protein B (deficient in residues 51-335 and 529-554 in SEQ ID NO: 1)
イ）動物
　動物は、マウスBALB/c、メス、4週令（試験開始時）（日本チャールス・リバーより購入し馴化して使用）を用いた。 B) Animals For animals, mice BALB / c, females, and 4-week-old animals (at the start of the test) (purchased from Charles River Japan and used after acclimatization) were used.
　動物は5頭/ケージで、室温24±3℃、湿度50±20%、換気10-25回/時間、照明12時間の環境下で飼育した。飼料はMF（オリエンタル酵母工業）を自由摂取させることによって給餌した。馴化終了時（試験開始時）の体重を基に群分けした。動物は１群10頭とした。 The animals were bred in an environment of 5 animals / cage, room temperature 24 ± 3 ° C, humidity 50 ± 20%, ventilation 10-25 times / hour, and lighting 12 hours. The feed was fed by free intake of MF (Oriental Yeast Co., Ltd.). Groups were grouped based on body weight at the end of acclimation (at the start of the test). The number of animals was 10 per group.
ウ）インフルエンザウイルス
　インフルエンザウイルスはH1N1（株名：A/PR/8/34、ATCC. No.：VR-1469、BSL：2、ウイルス力価：1.6×108 TCID50/mL）及びH3N2（株名：A/Port Chalmers/1/73、ATCC. No.：VR-810、BSL：2、ウイルス力価：1.3×107 TCID50/mL）の2亜型を用い、両ウイルスとも既報（中野ら,2015年,第62回日本試験動物学会）に従いマウス高感受性に変異させ、凍結保存したウイルス液を解凍し、PBS（Life Technologies Corporation）を用いて6 × 104 TCID50/mL（3 × 103 TCID50/50 μL）に調製したもの（以下、ウイルス接種液ともいう）を用いた。 C) Influenza virus Influenza virus is H1N1 (strain name: A / PR / 8/34, ATCC. No .: VR-1469, BSL: 2, virus titer: 1.6 × 10 8 TCID 50 / mL) and H3N2 (strain). Name: A / Port Chalmers / 1/73, ATCC. No .: VR-810, BSL: 2, virus titer: 1.3 × 10 7 TCID 50 / mL), both viruses have been reported (Nakano). Et al., 2015, 62nd Annual Meeting of the Japanese Society of Test Animal Science), mutated to high sensitivity in mice, thawed the cryopreserved virus solution, and used PBS (Life Technologies Corporation) for 6 × 10 4 TCID 50 / mL (3 ×). 10 3 TCID 50/50 μL) was used (hereinafter, also referred to as virus inoculum).
エ）抗原タンパク質を含む投与液の調製と投与方法
　抗原タンパク質は皮下、経鼻それぞれで投与した。投与方法は以下のとおりである。 D) Preparation and administration method of administration solution containing antigen protein The antigen protein was administered subcutaneously and nasally. The administration method is as follows.
（i）皮下投与
　タンパク質改良型溶液（タンパク質濃度250μg/mL）に同体積のアジュバント（InjectTM Alum (Thermo scientific)）を加え混合し、これを高用量群投与液とした（タンパク質濃度125μg/mL）。この高用量群投与液をアジュバントで10倍希釈した溶液を低用量群投与液とした（タンパク質濃度12.5μg/mL）。また、ベヒクル投与液は、タンパク質を含まないベヒクル液（前記タンパク質溶液用のベヒクル液にタンパク質を溶解させないもの）とアジュバントを1/１（体積）混合して調製した。 (I) Subcutaneous administration A protein-improved solution (protein concentration 250 μg / mL) was mixed with the same volume of adjuvant (Inject TM Alum (Thermo scientific)) to prepare a high-dose group administration solution (protein concentration 125 μg / mL). ). This high-dose group administration solution was diluted 10-fold with an adjuvant and used as the low-dose group administration solution (protein concentration 12.5 μg / mL). The vehicle administration solution was prepared by mixing 1/1 (volume) of a protein-free vehicle solution (one in which the protein is not dissolved in the vehicle solution for the protein solution) and an adjuvant.
　上記投与液は、マウス1頭1回あたり0.2mL、7日の間隔をあけて2回、計0.4mLを皮下投与した（高用量群のマウスには1回あたり25μg/head、計50μg/headのタンパク質を、低用量群のマウスには1回あたり2.5μg/head、計5μg/headのタンパク質を投与した）。 The above-mentioned administration solution was subcutaneously administered at 0.2 mL per mouse, twice at 7-day intervals, for a total of 0.4 mL (25 μg / head for mice in the high-dose group, for a total of 50 μg / head). The low-dose group of mice received 2.5 μg / head of protein at a time, for a total of 5 μg / head).
(ii)経鼻投与
　生理食塩液にて250μg/mLに調製したタンパク質改良型懸濁液を高用量群投与液とし、生理食塩液にてさらに10倍希釈したものを低用量群投与液とした。また、生理食塩液をベヒクル投与液とした。 (ii) Nasal administration A protein-improved suspension prepared to 250 μg / mL with physiological saline was used as the high-dose group administration solution, and a 10-fold diluted protein with physiological saline was used as the low-dose group administration solution. .. In addition, the physiological saline solution was used as the vehicle administration solution.
　上記投与液は、マウス1頭あたり50μLを、7日の間隔をあけて2回、計100μLを経鼻投与した（高用量群のマウスには1回あたり12.5μg/head、計25μg/headのタンパク質を、低用量群のマウスには1回あたり1.25μg/head、計2.5μg/headのタンパク質を投与した）。　 The above administration solution was nasally administered at 50 μL per mouse, twice at 7-day intervals, for a total of 100 μL (12.5 μg / head for mice in the high-dose group, for a total of 25 μg / head). Mice in the low-dose group received 1.25 μg / head of protein at a time, for a total of 2.5 μg / head). It was
オ）インフルエンザウイルスの接種
　インフルエンザウイルス接種は、2回目の抗原タンパク質投与後7日目（day 0）に、イソフルラン麻酔下で、ウイルス接種液を50μL経鼻接種した。 E) Influenza virus inoculation On the 7th day (day 0) after the second administration of the antigen protein, 50 μL of the virus inoculum was nasally inoculated under isoflurane anesthesia.
カ）評価
　マウスの状態把握のため、ウイルス接種の14日前(Day-14)、7日前(Day-7)、ウイルス接種日(Day0)、ウイルス接種日から3日後(Day3)、7日後(Day7)、10日後(Day10)、14日後(Day14)に体重測定を行った。またウイルス接種日の14日前からウイルス接種日14日後までの期間、マウスの生死、一般状態（活動性の低下及び被毛の粗剛）についても評価した。試験データとして得られた試験群ごとの計量データは平均値±標準偏差で記載した。 F) 14 days (Day-14), 7 days (Day-7), virus inoculation day (Day 0), 3 days (Day 3), 7 days (Day 7) after virus inoculation to grasp the condition of the evaluated mice. ), 10 days later (Day 10), and 14 days later (Day 14). In addition, the life and death of mice and the general condition (decreased activity and coarse coat) were also evaluated during the period from 14 days before the virus inoculation date to 14 days after the virus inoculation date. The measurement data for each test group obtained as test data are described as mean ± standard deviation.
３．結果
　結果を図１５(生存率)に示す。 3. 3. Results The results are shown in FIG. 15 (survival rate).
ア）H1N1接種に対する、抗原タンパク質皮下投与の効果
　改良型 25 μg/body投与群（1群）および2.5 μg/body投与群（2群）、ならびにベヒクル投与群（3群）のday14時点の生存率（平均生存日数）はそれぞれ100%（14.0日）、90%（13.2日）および50%（11.0日）であり、改良型投与群はベヒクル群に比して生存率と平均生存日数の改善が認められた。生存率の改善については、3群に対し1群で有意差が認められた。 A) Effect of subcutaneous administration of antigen protein on H1N1 inoculation Survival rate of 25 μg / body administration group (1 group), 2.5 μg / body administration group (2 group), and vehicle administration group (3 group) at day 14 (Average survival days) were 100% (14.0 days), 90% (13.2 days) and 50% (11.0 days), respectively, and the improved-type group had improved survival rates and average survival days compared to the vehicle group. Admitted. Regarding the improvement of survival rate, a significant difference was observed in 1 group compared to 3 groups.
　一般状態において、1群および2群（改良型投与群）では被毛の粗剛が認められ、その他一部の個体で活動性の低下が散見されたが、その後回復し、観察終了時においては、ほとんどの個体で異常は認められなかった。一方、3群（ベヒクル群）ではほとんどすべての個体において被毛の粗剛および活動性の低下が認められ、観察終了時まで継続した。すなわち、改良型投与群は少なくとも被毛の粗剛化が少ない又は回復が早く、活動性低下が少ない又は回復が早いことが認められた。 In the general condition, coarse stiffness of the coat was observed in the 1st and 2nd groups (improved administration group), and the activity decreased in some other individuals, but it recovered after that, and at the end of the observation, it recovered. , No abnormalities were observed in most of the individuals. On the other hand, in almost all the individuals in the 3 groups (vehicle group), the coarseness of the coat and the decrease in activity were observed, and it continued until the end of the observation. That is, it was confirmed that in the improved administration group, at least the coarsening of the coat was less or the recovery was quicker, and the decrease in activity was less or the recovery was faster.
　また、接種後10日目に3群（ベヒクル群）の体重減少がピーク（14.5 ± 0.9 g）であり、この時点における1群および2群の体重は、それぞれ17.4 ± 2.3 gおよび20.4 ± 1.2 gであった。体重減少の抑制は、3群（ベヒクル群）に比して、1群では接種後3、7および14日目、2群では接種後3～14日目において有意差が認められた。すなわち、ベヒクル群に比して、改良型投与群では体重減少の抑制が認められた。 On the 10th day after inoculation, the weight loss of the 3rd group (Vehicle group) peaked (14.5 ± 0.9g), and the body weights of the 1st and 2nd groups at this time were 17.4 ± 2.3g and 20.4 ± 1.2g, respectively. Met. The suppression of weight loss was significantly different in the 1st group on the 3rd, 7th and 14th days after the inoculation, and in the 2nd group on the 3rd to 14th days after the inoculation, compared with the 3rd group (Vehicle group). That is, the suppression of weight loss was observed in the improved administration group as compared with the vehicle group.
イ）H1N1接種に対する、抗原タンパク質経鼻投与の効果
　改良型 12.5 μg/head投与群（4群）および1.25 μg/head投与群（5群）、ならびにベヒクル液投与群（6群）の14日時点の群生存率（平均生存日数）はそれぞれ100%（14.0日）、100%（14.0日）および70%（13.0日）であった。改良型投与群はベヒクル群に比して生存率と平均生存日数の改善が認められた。 B ) Effect of nasal administration of antigen protein on H1N1 inoculation 14 days of improved 12.5 μg / head administration group (4 groups), 1.25 μg / head administration group (5 groups), and vehicle solution administration group (6 groups) The group survival rates (mean survival days) were 100% (14.0 days), 100% (14.0 days) and 70% (13.0 days), respectively. The improved administration group showed improvement in survival rate and average survival time compared to the vehicle group.
　また、接種後10日目に6群（ベヒクル群）の体重減少がピーク（14.9 ± 1.2 g）であり、この時点における4群および5群ではそれぞれ20.4 ± 1.3 gおよび21.1 ± 1.4 gであった。体重減少の抑制は、6群（ベヒクル群）に比して、4群、5群では接種後３～14日目において有意差が認められた。すなわち、ベヒクル群に比して、改良型投与群では体重減少の抑制が認められた。 On the 10th day after inoculation, the weight loss of the 6th group (vehicle group) peaked (14.9 ± 1.2g), and at this time, it was 20.4 ± 1.3g and 21.1 ± 1.4g in the 4th and 5th groups, respectively. .. There was a significant difference in the suppression of weight loss between the 4th and 5th groups compared to the 6th group (vehicle group) on the 3rd to 14th days after inoculation. That is, the suppression of weight loss was observed in the improved administration group as compared with the vehicle group.
ウ）H3N2接種に対する抗原タンパク質皮下投与の効果
　改良型25 μg/head投与群（7群）および2.5 μg/head投与群（8群）、ならびにベヒクル液投与群（9群）の14日時点の生存率（平均生存日数）はそれぞれ90%（13.7日）、100%（14.0日）および100%（14.0日）であった。 C) Effect of subcutaneous administration of antigen protein on H3N2 inoculation Survival of improved 25 μg / head administration group (7 groups), 2.5 μg / head administration group (8 groups), and vehicle solution administration group (9 groups) at 14 days The rates (mean survival days) were 90% (13.7 days), 100% (14.0 days) and 100% (14.0 days), respectively.
　7群および8群において多くの個体で被毛の粗剛が認められたが、死亡個体を除いてその後回復し、7群では接種後11日目、8群では9日目から回復した。9群では全例において被毛の粗剛が認められ、観察終了日（接種14日目）にほとんどの個体で回復した。すなわち、改良型投与群は少なくとも被毛の粗剛化が少ない又は回復が早いことが認められた。 Coarse coat was observed in many individuals in groups 7 and 8, but recovered afterwards except for dead individuals, and recovered from the 11th day after inoculation in the 7th group and from the 9th day in the 8th group. Coarse coat was observed in all cases in group 9, and most of the individuals recovered on the day of the end of observation (14th day of inoculation). That is, it was confirmed that the improved administration group had at least less coarsening of the coat or faster recovery.
　接種後7日目に9群（ベヒクル群）の体重減少がピーク（17.2 ± 1.0 g）であり、この時点における7群および8群ではそれぞれ17.9 ± 1.3 gおよび18.9 ± 1.3 gであった。体重減少の抑制は、9群（ベヒクル群）に比して、8群（改良型2.5μg/head群）では接種後７および10日目において有意差が認められた。すなわち、ベヒクル群に比して、改良型投与群では体重減少の抑制が認められた。 On the 7th day after inoculation, the weight loss of the 9th group (vehicle group) peaked (17.2 ± 1.0g), and at this time, it was 17.9 ± 1.3g and 18.9 ± 1.3g in the 7th and 8th groups, respectively. There was a significant difference in the suppression of weight loss in the 8 groups (improved 2.5 μg / head group) compared to the 9 groups (vehicle group) on the 7th and 10th days after inoculation. That is, the suppression of weight loss was observed in the improved administration group as compared with the vehicle group.
エ）H3N2接種に対する、抗原タンパク質経鼻投与の効果
　改良型 12.5 μg/head投与群（10群）および1.25 μg/head投与群（11群）、ならびにベヒクル液投与群（12群）の14日時点の生存率（平均生存日数）はそれぞれ90%（13.7日）、100%（14.0日）および70%（13.0日）であり、改良型投与群はベヒクル群に比して生存率と平均生存日数の改善が認められた。 D) Effect of nasal administration of antigen protein on H3N2 inoculation 14 days of improved 12.5 μg / head administration group (10 groups), 1.25 μg / head administration group (11 groups), and vehicle solution administration group (12 groups) The survival rate (average survival time) was 90% (13.7 days), 100% (14.0 days) and 70% (13.0 days), respectively, and the improved administration group had a survival rate and an average survival time compared to the vehicle group. Improvement was observed.
　10群および11群の半数程度の個体において被毛の粗剛が認められたが、死亡個体を除きほとんどの個体は9～11日目に回復した。12群（ベヒクル群）では全例で被毛の粗剛が認められ、接種後13日目にほとんどの個体が回復した。すなわち、改良型投与群は少なくとも被毛の粗剛化が少ない又は回復が早いことが認められた。 Coarse coat was observed in about half of the individuals in the 10th and 11th groups, but most of the individuals recovered on the 9th to 11th days except the dead individuals. In the 12th group (vehicle group), coarse stiffness of the coat was observed in all cases, and most of the individuals recovered on the 13th day after inoculation. That is, it was confirmed that the improved administration group had at least less coarsening of the coat or faster recovery.
　また、接種後10日目に12群（ベヒクル群）の体重減少がピーク（17.0 ± 2.7 g）であり、この時点における10群および11群ではそれぞれ19.2 ± 2.4 gおよび20.6 ± 1.2 gであった。体重減少の抑制は、12群（ベヒクル群）に比して、11群（改良型1.25μg/head投与群）では接種後10および14日目に有意差が認められた。すなわち、改良型投与群はベヒクル群に比して体重減少の抑制が認められた。 On the 10th day after inoculation, the weight loss of the 12th group (vehicle group) peaked (17.0 ± 2.7g), and at this time, it was 19.2 ± 2.4g and 20.6 ± 1.2g in the 10th and 11th groups, respectively. .. The suppression of weight loss was significantly different between the 12 groups (vehicle group) and the 11 groups (improved 1.25 μg / head administration group) on the 10th and 14th days after inoculation. That is, the improved administration group showed suppression of weight loss as compared with the vehicle group.
オ）結論
　インフルエンザウイルスH1N1を接種した個体において、生存率、一般状態および体重推移の結果から改良型はH1N1のインフルエンザに対し、薬効を有することが示された。 E) Conclusion In individuals inoculated with influenza virus H1N1, the improved survival rate, general condition, and body weight transition showed that the improved version had a medicinal effect on H1N1 influenza.
　H3N2を接種した個体においては、改良型投与群とベヒクル投与群の間における生存率の差が有意でなかった。しかし、一般状態や体重推移で改善傾向を認め、とくに体重減少の抑制においては有意差が認められたため、H3N2のインフルエンザウイルスに対する有効性が示されたと言える。 In the individuals inoculated with H3N2, the difference in survival rate between the improved administration group and the vehicle administration group was not significant. However, it can be said that the effectiveness of H3N2 against influenza virus was shown because an improvement tendency was observed in the general condition and body weight transition, and a significant difference was observed especially in the suppression of body weight loss.
　さらに、上述のように、改良型抗原タンパク質を投与したマウスは、該抗原タンパク質を投与されていないベヒクル群と比較して、インフルエンザウイルス(H1N1型及びH3N2型)に対する抵抗性を獲得していることから、抗原タンパク質改良型はA型インフルエンザウイルス亜種間で交差免疫を付与し得るワクチンとして有用であることが示された。 Furthermore, as described above, the mice treated with the improved antigen protein have acquired resistance to influenza virus (H1N1 type and H3N2 type) as compared with the vehicle group not treated with the antigen protein. Therefore, it was shown that the improved antigenic protein is useful as a vaccine capable of imparting cross-immunity between influenza A virus subspecies.
実施例８：インフルエンザウイルス感染マウスによる薬効試験(改良・再改良。経鼻)
１．試験の概要
　被験物質のインフルエンザウイルスに対する抑制効果を確認する目的で、マウスに、抗原タンパク質（被験物質）を予め経鼻投与し、インフルエンザウイルスに対する反応を検討した。概要を図１６に示す。試験群は14群（表２）とした。 Example 8: Drug efficacy test using influenza virus-infected mice (improved / re-improved. Nasal)
1. 1. Outline of the test In order to confirm the inhibitory effect of the test substance on influenza virus, an antigen protein (test substance) was administered nasally to mice in advance, and the reaction to influenza virus was examined. The outline is shown in FIG. The test group was 14 groups (Table 2).
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
２．試験方法
ア）被験物質
　実施例４により調製した下記頭部欠損HA-M1融合タンパク質３種を抗原タンパク質とした。
オリジナル型; オリジナル融合タンパク質（配列番号１中の第７６～３０８残基および第５２９～５５４残基が欠損）
改良型; HA51-335融合タンパク質Ｂ（配列番号１中の第５１～３３５残基および第５２９～５５４残基が欠損）
再改良型; HA51-335, 555-566融合タンパク質Ｂ（配列番号１中の第５１～３３５残基、第５２９～５５４残基および第５５５～５６６残基が欠損） 2. 2. Test method
A) Test substance The following three head-deficient HA-M1 fusion proteins prepared in Example 4 were used as antigen proteins.
Original type; Original fusion protein (deficient in residues 76-308 and 529-554 in SEQ ID NO: 1)
Improved version; HA51-335 fusion protein B (deficient in residues 51-335 and 529-554 in SEQ ID NO: 1)
Re-improved; HA51-335, 555-566 Fusion Protein B (deficient in residues 51-335, 529-554 and 555-566 in SEQ ID NO: 1)
イ）動物
　動物は、マウスBALB/c、メス、６週令（試験開始時）（日本チャールス・リバーより購入し馴化して使用）を用いた。 B) Animals For animals, mice BALB / c, females, and 6-week-old animals (at the start of the test) (purchased from Charles River Japan and used after acclimatization) were used.
　動物は5頭/ケージで、室温24±3℃、湿度50±20%、換気10-25回/時間、照明12時間の環境下で飼育した。飼料はMF（オリエンタル酵母工業）を自由摂取させることによって給餌した。馴化終了時（試験開始時）の体重を基に群分けした。動物は１群10頭とした。 The animals were bred in an environment of 5 animals / cage, room temperature 24 ± 3 ° C, humidity 50 ± 20%, ventilation 10-25 times / hour, and lighting 12 hours. The feed was fed by free intake of MF (Oriental Yeast Co., Ltd.). Groups were grouped based on body weight at the end of acclimation (at the start of the test). The number of animals was 10 per group.
ウ）インフルエンザウイルス
　インフルエンザウイルスはH1N1（株名：A/PR/8/34、ATCC. No.：VR-1469、BSL：2、ウイルス力価：1.6×108 TCID50/mL）及びH3N2（株名：A/Port Chalmers/1/73、ATCC. No.：VR-810、BSL：2、ウイルス力価：1.3×107 TCID50/mL）の2亜型を用い、両ウイルスとも既報（中野ら,2015年,第62回日本試験動物学会総会）にしたがいマウス高感受性に変異させ、凍結保存したウイルス液を解凍し、PBS（Life Technologies Corporation）を用いて2 × 105 TCID50/mL（1 × 104 TCID50/50 μL）に調製したもの（以下、ウイルス接種液ともいう）を用いた。 C) Influenza virus Influenza virus is H1N1 (strain name: A / PR / 8/34, ATCC. No .: VR-1469, BSL: 2, virus titer: 1.6 × 10 8 TCID 50 / mL) and H3N2 (strain). Name: A / Port Chalmers / 1/73, ATCC. No .: VR-810, BSL: 2, virus titer: 1.3 × 10 7 TCID 50 / mL), both viruses have been reported (Nakano). Et al., 2015, 62nd Annual Meeting of the Japanese Society of Test Animal Science), mutated to highly sensitive mice, thawed the cryopreserved virus solution, and used PBS (Life Technologies Corporation) for 2 × 10 5 TCID 50 / mL ( 1 × 10 4 TCID 50/50 μL) was used (hereinafter, also referred to as virus inoculum).
エ）抗原タンパク質を含む投与液の調製と投与方法
　抗原タンパク質の投与方法は以下のとおりである。すなわち、生理食塩液にて250μg/mLに調製したオリジナル型懸濁液、改良型懸濁液又は再改良型懸濁液を高用量群投与液とし、生理食塩液にてさらに10倍希釈したものを低用量群投与液とした。また、生理食塩液をベヒクル投与液とした。 D) Preparation and administration method of administration solution containing antigen protein The administration method of antigen protein is as follows. That is, the original suspension, the improved suspension, or the re-improved suspension prepared to 250 μg / mL with physiological saline was used as a high-dose group administration solution, and further diluted 10-fold with physiological saline. Was used as a low-dose group administration solution. In addition, the physiological saline solution was used as the vehicle administration solution.
　上記投与液は、マウス1頭あたり50μLを、7日の間隔をあけて2回、計100μLを経鼻投与した（高用量群のマウスには1回あたり12.5μg/head、計25μg/headのタンパク質を、低用量群のマウスには1回あたり1.25μg/head、計2.5μg/headのタンパク質を投与した）。 The above administration solution was nasally administered at 50 μL per mouse, twice at 7-day intervals, for a total of 100 μL (12.5 μg / head for mice in the high-dose group, for a total of 25 μg / head). Mice in the low-dose group received 1.25 μg / head of protein at a time, for a total of 2.5 μg / head).
オ）インフルエンザウイルスの接種
　インフルエンザウイルス接種は、2回目の抗原タンパク質投与後7日目（day 0）に、イソフルラン麻酔下で、ウイルス接種液を50μL経鼻接種した。 E) Influenza virus inoculation On the 7th day (day 0) after the second administration of the antigen protein, 50 μL of the virus inoculum was nasally inoculated under isoflurane anesthesia.
カ）評価
　マウスの状態把握のため、ウイルス接種の14日前(Day-14)、7日前(Day-7)、ウイルス接種日(Day0)、ウイルス接種日から3日後(Day3)、7日後(Day7)、10日後(Day10)、14日後(Day14)に体重測定を行った。またウイルス接種日の14日前からウイルス接種日14日後までの期間、マウスの生死、一般状態（活動性の低下及び被毛の粗剛）についても評価した。試験データとして得られた試験群ごとの計量データは平均値±標準偏差で記載した。 F) 14 days (Day-14), 7 days (Day-7), virus inoculation day (Day 0), 3 days (Day 3), 7 days (Day 7) after virus inoculation to grasp the condition of the evaluated mice. ), 10 days later (Day 10), and 14 days later (Day 14). In addition, the life and death of mice and the general condition (decreased activity and coarse coat) were also evaluated during the period from 14 days before the virus inoculation date to 14 days after the virus inoculation date. The measurement data for each test group obtained as test data are described as mean ± standard deviation.
３．結果
　結果を図１７(生存率)に示す。 3. 3. Results The results are shown in FIG. 17 (survival rate).
ア）H1N1に対する抗原タンパク質経鼻投与の効果
　１群（オリジナル型懸濁液 12.5 μg/ head）、２群（オリジナル型懸濁液 1.25 μg/ head）、３群（改良型懸濁液 12.5 μg/ head）、４群（改良型懸濁液 1.25 μg/50 μL）、５群（再改良型懸濁液 12.5 μg/ head）、６群（再改良型懸濁液 1.25 μg/ head）および７群（生理食塩液 50 μL）の平均生存日数（ウイルス接種14日目の生存率）はそれぞれ13.0日（70%）、10.3日（20%）、12.8日（80%）、10.9日（40%）、12.0日（60%）、11.0日（30%）および9.0日（0%）であった。すなわち、オリジナル型（１群、２群）、改良型（３群、４群）、再改良型（５群、６群）のいずれの群も、ベヒクル（生食）群に比して平均生存日数と接種14日目の生存率に改善が認められた。生存率の改善について、１群、３群、４群、５群および６群は７群と比較して有意なものであった。 A) Effect of nasal administration of antigenic protein on H1N1 Group 1 (original suspension 12.5 μg / head), group 2 (original suspension 1.25 μg / head), group 3 (improved suspension 12.5 μg / head) Head), 4 groups (improved suspension 1.25 μg / 50 μL), 5 groups (re-improved suspension 12.5 μg / head), 6 groups (re-improved suspension 1.25 μg / head) and 7 groups The average survival days (survival rate on the 14th day of virus inoculation) of (physiological saline 50 μL) were 13.0 days (70%), 10.3 days (20%), 12.8 days (80%), and 10.9 days (40%), respectively. , 12.0 days (60%), 11.0 days (30%) and 9.0 days (0%). That is, the average survival days of all of the original type (1 group, 2 groups), the improved type (3 groups, 4 groups), and the re-improved type (5 groups, 6 groups) are compared with the vehicle (raw food) group. The survival rate on the 14th day of inoculation was improved. Regarding the improvement of survival rate, the 1st group, 3rd group, 4th group, 5th group and 6th group were significant as compared with the 7th group.
イ）H3N2 に対する抗原タンパク質経鼻投与の効果
　８群（オリジナル型懸濁液 12.5 μg/ head）、９群（オリジナル型懸濁液 1.25 μg/ head）、10群（改良型懸濁液 12.5 μg/ head）、11群（改良型懸濁液 1.25 μg/ head）、12群（再改良型懸濁液 12.5 μg/ head）、13群（再改良型懸濁液 1.25 μg/ head）および14群（生理食塩液50 μL）の平均生存日数（ウイルス接種14日目の生存率）はそれぞれ13.0日（80%）、13.5日（80%）、12.9日（70%）、12.4日（50%）、13.6日（90%）、12.7日（60%）および11.8日（50%）であった。すなわち、オリジナル型（８群、９群）、改良型（10群、11群）、再改良型（12群、13群）のいずれの群も、ベヒクル（生食）群に比して平均生存日数と接種14日目の生存率の両方または一方に改善が認められた。生存率の改善について、12群は14群と比較して有意なものであった。 B ) Effect of nasal administration of antigenic protein on H3N2 Group 8 (original suspension 12.5 μg / head), group 9 (original suspension 1.25 μg / head), group 10 (improved suspension 12.5 μg / head) Head), 11 groups (improved suspension 1.25 μg / head), 12 groups (re-improved suspension 12.5 μg / head), 13 groups (re-improved suspension 1.25 μg / head) and 14 groups (re-improved suspension 1.25 μg / head) The average survival time (survival rate on the 14th day of virus inoculation) of 50 μL of physiological saline was 13.0 days (80%), 13.5 days (80%), 12.9 days (70%), 12.4 days (50%), respectively. They were 13.6 days (90%), 12.7 days (60%) and 11.8 days (50%). That is, the average survival time of all of the original type (8 groups, 9 groups), the improved type (10 groups, 11 groups), and the re-improved type (12 groups, 13 groups) compared with the vehicle (raw food) group. And on the 14th day of inoculation, improvement was observed in both or one of the survival rates. The improvement in survival rate was significant in the 12 group compared to the 14 group.
　また、８群（Day 10およびDay 14）および12群（Day 10）は14群と比較して有意に体重減少を抑制した。 In addition, the 8th group (Day 10 and Day 14) and the 12th group (Day 10) significantly suppressed the weight loss as compared with the 14th group.
ウ）結論
　上述のように、本発明の抗原タンパク質を経鼻投与したマウスは該抗原タンパク質を投与されていない群と比較して、インフルエンザウイルス(H1N1型及びH3N2型)に対する抵抗性を獲得しており、従って、本発明の抗原タンパク質はA型インフルエンザウイルス亜種間で交差免疫を付与し得るワクチンとして有用であることが示された。 C) Conclusion As described above, mice nasally administered with the antigenic protein of the present invention acquired resistance to influenza virus (H1N1 type and H3N2 type) as compared with the group not administered with the antigenic protein. Therefore, it has been shown that the antigenic protein of the present invention is useful as a vaccine capable of imparting cross-immunity between influenza A virus subspecies.
実施例９：被験物質投与マウスによるインフルエンザウイルス中和抗体試験
１．試験の概要
　被験物質を投与したマウスの血中に、インフルエンザウイルスを中和する活性を持った抗体が生産されていることを確認するための試験を実施した。マウスに、抗原タンパク質（被験物質）を予め投与（経鼻投与）し、インフルエンザウイルスに対する反応を検討した。試験群は９群（以下の表３を参照）とした。 Example 9: Influenza virus neutralizing antibody test using test substance-administered mice
1. 1. Outline of the test A test was conducted to confirm that an antibody having an activity of neutralizing influenza virus was produced in the blood of mice to which the test substance was administered. Antigen protein (test substance) was administered to mice in advance (nasal administration), and the reaction to influenza virus was examined. The test group was 9 groups (see Table 3 below).
２．試験方法
ア）被験物質
（i）試験物質
　実施例４により調製した以下の抗原タンパク質を使用した。すなわち、コムギ無細胞タンパク質合成系を用いて合成した各タンパク質は、15,000 rpm、４℃で10分間の遠心分離により沈殿として回収後、500 μg/mLとなるように生理食塩液（大塚薬品工場）に懸濁し、実験に用いるまで-80℃の超低温槽にて保管したものを用いた。
改良型：改良型頭部欠損HA-M1キメラタンパク質Ｂ（配列番号２０）；および
再改良型：再改良型頭部欠損HA-M1キメラタンパク質（配列番号４１）。 2. 2. Test method
A) Test substance
(I) Test substance The following antigen proteins prepared according to Example 4 were used. That is, each protein synthesized using the wheat cell-free protein synthesis system is recovered as a precipitate by centrifugation at 15,000 rpm for 10 minutes at 4 ° C, and then the physiological saline solution (Otsuka Pharmaceutical Factory) is adjusted to 500 μg / mL. It was suspended in the water and stored in an ultra-low temperature bath at -80 ° C until it was used for the experiment.
Improved: improved head-deficient HA-M1 chimeric protein B (SEQ ID NO: 20); and re-improved: re-improved head-deficient HA-M1 chimeric protein (SEQ ID NO: 41).
　改良型頭部欠損HA-M1キメラタンパク質Ｂおよび再改良型頭部欠損HA-M1キメラタンパク質は、解凍後に緩やかに転倒混和し、超音波処理でタンパク質を懸濁液中に拡散させた後（タンパク原液）、一匹1回あたりの投与量が、タンパク原液40 μL + Adj A 5 μL + 生理食塩液5 μL（+ Adj A群）、 タンパク原液40 μL + Adj B 10 μL（+ Adj B群）、またはタンパク原液40 μL + 生理食塩液10 μL（Adj なし群）になるように調製した（表３）。 The improved head-deficient HA-M1 chimeric protein B and the re-improved head-deficient HA-M1 chimeric protein are gently inverted and mixed after thawing, and the protein is diffused into the suspension by ultrasonic treatment (protein). Undiluted solution), the dose per animal is protein undiluted solution 40 μL + Adj A 5 μL + physiological saline solution 5 μL (+ Adj A group), protein undiluted solution 40 μL + Adj B 10 μL (+ Adj B group) , Or protein stock solution 40 μL + physiological saline solution 10 μL (Adj-free group) was prepared (Table 3).
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
（ii）対照物質およびアジュバント
　生理食塩液は、大塚製薬工場社から入手した。表３に記載のAdj A（2',3'-cGAMP (InvivoGen)）は、生理食塩液を加えてピペッティングで緩やかに混和し、1 μg/μLとなるように調製した。Adj B（Poly(I:C) (InvivoGen)）は、生理食塩液を加えてピペッティングで緩やかに混和し、1 μg/μLとなるように調製し、アニーリングを促すため、使用直前に65～70℃の範囲で10分間保温した後、室温で一時間静置してから使用した。Adj AおよびAdj Bは上記のように処理したものを、上記（i）のようにタンパク原液と共に、またはアジュバントのみを生理食塩液に混和した被験液として、投与に供した。なお、生理食塩液のみを投与する対照試験群も設けた。 (Ii) The control substance and the adjuvant physiological saline solution were obtained from Otsuka Pharmaceutical Factory. The Adj A (2', 3'-cGAMP (InvivoGen)) shown in Table 3 was prepared by adding physiological saline and gently mixing by pipetting to a concentration of 1 μg / μL. Adj B (Poly (I: C) (InvivoGen)) is prepared by adding physiological saline and gently mixing by pipetting to a concentration of 1 μg / μL to promote annealing. After keeping warm in the range of 70 ° C. for 10 minutes, it was allowed to stand at room temperature for 1 hour before use. Adj A and Adj B were treated as described above and subjected to administration together with the protein stock solution as described in (i) above or as a test solution in which only the adjuvant was mixed with a physiological saline solution. A control test group in which only physiological saline was administered was also provided.
イ）動物
　マウスは、5週齢（入荷時）、雌のBALB/c系統を、日本チャールス・リバーより入手し馴化して使用した。 B) Animal mice were 5 weeks old (at the time of arrival), and female BALB / c strains were obtained from Japan Charles River and used after being acclimatized.
（i）飼育
　マウスの飼育は、オートクレーブ滅菌した耐熱性ポリサルフォン製ケージ（207W×365D×140H mm, TECNIPLAST Ltd）を使用し、ケージ当たり五匹で飼育した。室温24 ± 3 ℃、湿度50 ± 20 %、換気（10～25回/1時間）、照明12時間（8:00～20:00）の飼育条件で飼育した。飼料はMF（オリエンタル酵母工業株式会社）を自由摂取とした。また飲料水はオートクレーブ滅菌した市水を自由摂取とした。 (I) Breeding mice were bred using autoclave-sterilized heat-resistant polysulfon cages (207W x 365D x 140H mm, TECNIPLAST Ltd) with five mice per cage. The animals were bred under the conditions of room temperature 24 ± 3 ° C, humidity 50 ± 20%, ventilation (10 to 25 times / hour), and lighting for 12 hours (8:00 to 20:00). The feed was MF (Oriental Yeast Co., Ltd.) as a free intake. As drinking water, autoclave-sterilized city water was used as free intake.
（ii）群分け
　入荷日ならびに検疫および馴化終了日に体重を測定し、検疫馴化終了時の体重を用いて、１群５匹となるように体重層別化無作為抽出法により各群にマウスを振り分けた。 (Ii) Grouping Weigh the mice on the arrival date and the end date of quarantine and acclimatization, and use the weight at the end of quarantine acclimatization to make 5 animals per group by weight stratification random sampling method. Was sorted.
ウ）インフルエンザウイルス
　インフルエンザウイルスはH1N1（株名：A/PR/8/34、ATCC. No.：VR-1469、BSL：2、ウイルス力価：1.6×108 TCID50/mL）及びH3N2（株名：A/Port Chalmers/1/73、ATCC. No.：VR-810、BSL：2、ウイルス力価：1.3×107 TCID50/mL）の2亜型をATCCから購入し、両ウイルスとも既報（中野ら, 2015年, 第62回日本試験動物学会）に従ってマウス高感受性に変異させ、凍結保存したウイルス液を解凍し、PBS（Life Technologies Corporation）を用いて6×104 TCID50/mL（3×103 TCID50/50 μL）に調製したもの（以下「ウイルス液」ともいう）を用いた。 C) Influenza virus Influenza virus is H1N1 (strain name: A / PR / 8/34, ATCC. No .: VR-1469, BSL: 2, virus titer: 1.6 × 10 8 TCID50 / mL) and H3N2 (strain name). : A / Port Chalmers / 1/73, ATCC. No .: VR-810, BSL: 2, virus titer: 1.3 × 10 7 TCID50 / mL) 2 subtypes were purchased from ATCC, and both viruses have already been reported ( Nakano et al., 2015, 62nd Annual Meeting of the Japanese Society of Test Animal Science), mutated to high sensitivity in mice, thawed the cryopreserved virus solution, and used PBS (Life Technologies Corporation) for 6 × 10 4 TCID50 / mL (3 ×). 10 3 TCID 50/50 μL) was used (hereinafter also referred to as “virus solution”).
エ）被験物質の投与
　被験物質の投与は、1週間間隔で2回（day0、7）、イソフルラン吸入麻酔下でピペットを用いて50 μL/bodyで経鼻投与とした。 D) Administration of test substance The test substance was administered nasally at 50 μL / body using a pipette under isoflurane inhalation anesthesia twice at weekly intervals (day 0, 7).
オ）中和抗体価の測定
　採血は、観察終了日（Day 14）にイソフルラン吸入麻酔下で腹大静脈もしくは心臓より採取可能全量の血液を採取した。採種した血液は、室温で30分程度放置後に6000 rpm、15分間、4℃で遠心分離を行ない、血清採取後に2本に分注して凍結保存した。 E) Measurement of neutralizing antibody titer On the end of observation (Day 14), the total amount of blood that could be collected from the abdominal vena cava or the heart was collected under isoflurane inhalation anesthesia. The collected blood was left at room temperature for about 30 minutes, then centrifuged at 6000 rpm for 15 minutes at 4 ° C, and after serum collection, it was dispensed into two and stored frozen.
　採取した血清は使用する前に56℃で30分間静置し、その後、96穴プレート内で測定用DMEM培地（BSA 0.2%（w/v）、ペニシリン100単位/mL、ストレプトマイシン100 μg/mL、ファンギゾン0.5 μg/mL、トリプシン1.0 μg/mLを含む）を用いて10, 20, 40, 80, 160, 320, 640, 1280, 2560, 5120, 10240, 20480倍まで希釈（50μL/穴）し、そのそれぞれの希釈液について、測定用DMEM培地で調製したウイルス液（102 TCID50/50μL/穴）を加え、37℃で30分間静置した（血清/ウイルス混合液）。 The collected serum is allowed to stand at 56 ° C for 30 minutes before use, and then in a 96-well plate, DMEM medium for measurement (BSA 0.2% (w / v), penicillin 100 units / mL, streptomycin 100 μg / mL, Dilute to 10, 20, 40, 80, 160, 320, 640, 1280, 2560, 5120, 10240, 20480-fold (50 μL / hole) with fungizone 0.5 μg / mL, trypsin 1.0 μg / mL). A virus solution (10 2 TCID 50/50 μL / hole) prepared in DMEM medium for measurement was added to each diluted solution, and the mixture was allowed to stand at 37 ° C. for 30 minutes (serum / virus mixed solution).
　培養用DMEM培地（10% FBS, ペニシリン100単位/mL、ストレプトマイシン100 μg/mLを含む）を用いて、あらかじめMadin-Darby canine kidney細胞（MDCK細胞）を培養しておいた24穴プレートに1 mLずつPBSを加えて洗浄し、その後、血清/ウイルス混合液0.1 mLを24穴プレートに播種した。また培地0.1 mL（細胞対照）およびウイルス液/培地等量混合液0.1 mL（ウイルス対照）をそれぞれ6穴分ずつ播種した。34℃で1時間静置した後に、培地0.5 mLを加え、2～3日間程度培養しウイルス増殖の有無を判定した。 Using DMEM medium for culture (containing 10% FBS, penicillin 100 units / mL, streptomycin 100 μg / mL), 1 mL on a 24-well plate in which Madin-Darby canine kidney cells (MDCK cells) have been cultured in advance. PBS was added one by one and washed, and then 0.1 mL of serum / virus mixture was inoculated on a 24-well plate. In addition, 0.1 mL of medium (cell control) and 0.1 mL of virus / medium equivalent mixture (virus control) were inoculated for 6 holes each. After allowing to stand at 34 ° C for 1 hour, 0.5 mL of the medium was added, and the cells were cultured for about 2 to 3 days to determine the presence or absence of viral replication.
　ウイルス増殖を完全に抑制する血清の最大希釈倍率の逆数を中和抗体価とした。 The reciprocal of the maximum dilution ratio of serum that completely suppresses viral growth was defined as the neutralizing antibody titer.
　試験データとして得られた計量データは幾何平均および95% 信頼区間を算出した。また有意差の検定は、JMP（SAS Institute Japan）を用い、Dunnettの検定法によってp値を算出することによって行なった。p値の計算の際、中和抗体価が10未満の場合は、10と読み換えて計算を行なった。 For the measurement data obtained as test data, the geometric mean and 95% confidence interval were calculated. The significance test was performed by using JMP (SAS Institute Japan) and calculating the p-value by Dunnett's test method. When the p-value was calculated, if the neutralizing antibody titer was less than 10, it was read as 10 and calculated.
３．結果
　被験物質を投与したマウス血清中における、H1N1のA型インフルエンザウイルスに対する中和抗体価を表４に示す。 3. 3. Results Table 4 shows the neutralizing antibody titers of H1N1 against influenza A virus in the serum of mice to which the test substance was administered.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　生理食塩液（9群）を投与物質とした場合は、10倍希釈でも、H1N1の亜型のウイルスの増殖を抑制できなかった。また、アジュバントのみを投与物質とした場合（7群、8群）は、20倍以上の希釈率から（20倍以上薄くすると）、H1N1の亜型のウイルスの増殖を抑制できなかった。 When physiological saline (group 9) was used as the administration substance, the growth of H1N1 subtype virus could not be suppressed even with 10-fold dilution. In addition, when only the adjuvant was used as the administered substance (groups 7 and 8), the growth of the H1N1 subtype virus could not be suppressed from the dilution ratio of 20 times or more (when diluted 20 times or more).
　一方、改良型頭部欠損HA-M1キメラタンパク質B（1群、2群、3群）や、再改良型頭部欠損HA-M1キメラタンパク質（4群、5群、6群）を投与物質とした場合は、アジュバントの有無に関わらず、40倍から160倍の希釈率で、なおウイルスの増殖を抑制した。 On the other hand, the improved head-deficient HA-M1 chimeric protein B ( groups 1, 2, and 3) and the re-improved head-deficient HA-M1 chimeric protein ( groups 4, 5, and 6) were used as administration substances. In this case, the growth of the virus was still suppressed at a dilution ratio of 40 to 160 times with or without an adjuvant.
　改良型頭部欠損HA-M1キメラタンパク質B単独投与群（3群）および、再改良型頭部欠損HA-M1キメラタンパク質単独投与群（6群）については、Dunnettの検定法により、生理食塩液投与群（9群）を対照としての比較を行なったところ、どちらのタンパク質に関しても、生理食塩液投与群に対して、統計学的に有意な中和抗体価の差が認められた。 For the improved head-deficient HA-M1 chimeric protein B alone-administered group (group 3) and the re-improved head-deficient HA-M1 chimeric protein alone-administered group (group 6), a physiological saline solution was used according to Dunnett's test method. When comparison was performed using the administration group (9 groups) as a control, a statistically significant difference in neutralizing antibody titer was observed between the administration group and the physiological saline administration group.
　以上の結果から、改良型頭部欠損HA-M1キメラタンパク質Bおよび再改良型頭部欠損HA-M1キメラタンパク質は、H1N1の亜型のウイルスを中和する抗体を産生することにより、投与マウスに、このウイルスに対する抵抗性を付与していることが示された。 From the above results, the improved head-deficient HA-M1 chimeric protein B and the re-improved head-deficient HA-M1 chimeric protein were administered to treated mice by producing an antibody that neutralizes the H1N1 subtype virus. , Showed to confer resistance to this virus.
　次に、被験物質を投与したマウス血清中における、H3N2のA型インフルエンザウイルスに対する中和抗体価を表５に示す。 Next, Table 5 shows the neutralizing antibody titers of H3N2 against influenza A virus in the serum of mice to which the test substance was administered.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　生理食塩液（9群）を投与物質とした場合は、10倍希釈でも、H3N2の亜型のウイルスの増殖を抑制できなかった。また、アジュバントのみを投与物質とした場合（7群、8群）は、10倍もしくは20倍を以上の希釈率から、H1N1の亜型のウイルスの増殖を抑制できなくなった。 When physiological saline (group 9) was used as the administration substance, the growth of H3N2 subtype virus could not be suppressed even with 10-fold dilution. In addition, when only the adjuvant was used as the administration substance (groups 7 and 8), the growth of the H1N1 subtype virus could not be suppressed from the dilution ratio of 10 times or 20 times or more.
　一方、改良型頭部欠損HA-M1キメラタンパク質B（1群、2群、3群）や、再改良型頭部欠損HA-M1キメラタンパク質（4群、5群、6群）を投与物質とした場合は、アジュバントの有無に関わらず、40倍から160倍の希釈率で、なおウイルスの増殖を抑制した。 On the other hand, the improved head-deficient HA-M1 chimeric protein B ( groups 1, 2, and 3) and the re-improved head-deficient HA-M1 chimeric protein ( groups 4, 5, and 6) were used as administration substances. In this case, the growth of the virus was still suppressed at a dilution ratio of 40 to 160 times with or without an adjuvant.
　改良型頭部欠損HA-M1キメラタンパク質B単独投与群（3群）および、再改良型頭部欠損HA-M1キメラタンパク質単独投与群（6群）については、Dunnettの検定法により、生理食塩液投与群（9群）を対照としての比較を行なったところ、どちらのタンパク質に関しても、生理食塩液投与群に対して、統計学的に有意な中和抗体価の差が認められた。 For the improved head-deficient HA-M1 chimeric protein B alone-administered group (group 3) and the re-improved head-deficient HA-M1 chimeric protein alone-administered group (group 6), a physiological saline solution was used according to Dunnett's test method. When comparison was performed using the administration group (9 groups) as a control, a statistically significant difference in neutralizing antibody titer was observed between the administration group and the physiological saline administration group.
　以上の結果から、改良型頭部欠損HA-M1キメラタンパク質Bおよび再改良型頭部欠損HA-M1キメラタンパク質は、H3N2の亜型のウイルスを中和する抗体を産生することにより、投与マウスに、このウイルスに対する抵抗性を付与していることが示された。 From the above results, the improved head-deficient HA-M1 chimeric protein B and the re-improved head-deficient HA-M1 chimeric protein were administered to treated mice by producing an antibody that neutralizes the H3N2 subtype virus. , Showed to confer resistance to this virus.
４．結論
　改良型頭部欠損HA-M1キメラタンパク質Ｂ、再改良型頭部欠損HA-M1キメラタンパク質はいずれも、H1N1、H3N2のいずれの亜型のA型インフルエンザウイルスに対しても中和活性を有する抗体をマウス血清中に産生させることが示され、ワクチンとして使用できることが確認された。 4. Conclusion Both the improved head-deficient HA-M1 chimeric protein B and the re-improved head-deficient HA-M1 chimeric protein have neutralizing activity against both H1N1 and H3N2 subtypes of influenza A virus. It was shown that the antibody was produced in mouse serum, confirming that it could be used as a vaccine.

Claims (22)

  1. 　インフルエンザウイルスに由来するＨＡタンパク質とＭ１タンパク質との融合タンパク質において、特定の領域のアミノ酸残基が欠損してなる部分欠損ＨＡ－Ｍ１融合タンパク質であって、
    　前記ＨＡタンパク質が、配列番号１中の第１８～５６６アミノ酸残基で表されるアミノ酸配列を有するタンパク質、または該アミノ酸配列に対して少なくとも９０％の配列同一性を有するアミノ酸配列を有し、かつ、インフルエンザウイルス由来のＨＡタンパク質として機能するタンパク質であり、
    　前記Ｍ１タンパク質が、配列番号５中の第１～２５２アミノ酸残基で表されるアミノ酸配列を有するタンパク質、または該アミノ酸配列に対して少なくとも９０％の配列同一性を有するアミノ酸配列を有し、かつ、インフルエンザウイルス由来のＭ１タンパク質として機能するタンパク質であり、
    　配列番号１中の第５９～３３５アミノ酸残基および第５２９～５５４アミノ酸残基に相当する領域のアミノ酸残基が少なくとも欠損しており、かつ、
    　配列番号１中の第１８～５０アミノ酸残基、第３４０～５２８アミノ酸残基および配列番号５中の第１～２５２アミノ酸残基に相当する領域のアミノ酸残基が少なくとも保持されている、部分欠損ＨＡ－Ｍ１融合タンパク質。     A partially deficient HA-M1 fusion protein in which an amino acid residue in a specific region is deleted in a fusion protein of an HA protein derived from influenza virus and an M1 protein.
    The HA protein has a protein having an amino acid sequence represented by the 18th to 566 amino acid residues in SEQ ID NO: 1, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence, and has an amino acid sequence. , A protein that functions as an HA protein derived from influenza virus,
    The M1 protein has a protein having an amino acid sequence represented by the 1st to 252nd amino acid residues in SEQ ID NO: 5, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence, and , A protein that functions as an M1 protein derived from influenza virus.
    At least the amino acid residues in the regions corresponding to the 59th to 335th amino acid residues and the 529th to 554th amino acid residues in SEQ ID NO: 1 are deleted, and the amino acid residues are deleted.
    A partial defect in which at least the amino acid residues in the regions corresponding to the 18th to 50th amino acid residues, the 340th to 528th amino acid residues in SEQ ID NO: 1 and the 1st to 252nd amino acid residues in SEQ ID NO: 5 are retained. HA-M1 fusion protein.
  2. 　配列番号１中の第５９～３３５アミノ酸残基、第５１～３３５アミノ酸残基、第５９～３３９アミノ酸残基、または第５１～３３９アミノ酸残基に相当する領域のアミノ酸残基が欠損している、請求項１に記載の部分欠損ＨＡ－Ｍ１融合タンパク質。 The amino acid residues in the region corresponding to the 59th to 335th amino acid residues, the 51st to 335th amino acid residues, the 59th to 339th amino acid residues, or the 51st to 339th amino acid residues in SEQ ID NO: 1 are deleted. , The partially deficient HA-M1 fusion protein according to claim 1.
  3. 　配列番号１中の第５５５～５６６アミノ酸残基に相当する領域のアミノ酸残基が欠損している、請求項１または２に記載の部分欠損ＨＡ－Ｍ１融合タンパク質。 The partially deficient HA-M1 fusion protein according to claim 1 or 2, wherein the amino acid residue in the region corresponding to the 555 to 566 amino acid residues in SEQ ID NO: 1 is deficient.
  4. 　前記ＨＡタンパク質と前記Ｍ１タンパク質とを、Ｎ末端からＣ末端に向けてこの順で含んでなる、請求項１～３のいずれか一項に記載の部分欠損ＨＡ－Ｍ１融合タンパク質。 The partially deficient HA-M1 fusion protein according to any one of claims 1 to 3, wherein the HA protein and the M1 protein are contained in this order from the N-terminal to the C-terminal.
  5. 　欠損した領域および／または部分欠損ＨＡタンパク質とＭ１タンパク質の融合部位にリンカーが挿入されている、請求項１～４のいずれか一項に記載の部分欠損ＨＡ－Ｍ１融合タンパク質。 The partially deficient HA-M1 fusion protein according to any one of claims 1 to 4, wherein the linker is inserted into the deficient region and / or the fusion site of the partially deficient HA protein and the M1 protein.
  6. 　前記リンカーが、ＧＳＧリンカー、ＧＳＧＳＧリンカー、ＧＳＧＳＧＳＧＳリンカー、ＧＳＡＧＳＡリンカー、またはＧＧＧＧＳＧＧＧＧＳＧＧＧＧＳリンカーである、請求項５に記載の部分欠損ＨＡ－Ｍ１融合タンパク質。 The partially deficient HA-M1 fusion protein according to claim 5, wherein the linker is a GSG linker, a GSGSG linker, a GSGSGSGS linker, a GSAGSA linker, or a GGGGGSGGGGGSGGGS linker.
  7. 　請求項１～６のいずれか一項に記載の部分欠損ＨＡ－Ｍ１融合タンパク質をコードする、核酸分子。 A nucleic acid molecule encoding the partially deficient HA-M1 fusion protein according to any one of claims 1 to 6.
  8. 　請求項７に記載の核酸分子を含んでなる、発現ベクター。 An expression vector comprising the nucleic acid molecule according to claim 7.
  9. 　請求項７に記載の核酸分子または請求項８に記載の発現ベクターを含んでなる、形質転換体。 A transformant comprising the nucleic acid molecule according to claim 7 or the expression vector according to claim 8.
  10. 　前記形質転換体がイネまたは酵母である、請求項９に記載の形質転換体。 The transformant according to claim 9, wherein the transformant is rice or yeast.
  11. 　請求項９または１０に記載の形質転換体を培養または育成することを含んでなる、部分欠損ＨＡ－Ｍ１融合タンパク質を製造する方法。 A method for producing a partially deficient HA-M1 fusion protein, which comprises culturing or growing the transformant according to claim 9 or 10.
  12. 　請求項１～６のいずれか一項に記載の部分欠損ＨＡ－Ｍ１融合タンパク質、請求項７に記載の核酸分子または請求項８に記載の発現ベクターを含んでなる、医薬組成物。 A pharmaceutical composition comprising the partially defective HA-M1 fusion protein according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, or the expression vector according to claim 8.
  13. 　インフルエンザウイルスの感染症を予防または治療するための、請求項１２に記載の医薬組成物。 The pharmaceutical composition according to claim 12, for preventing or treating an influenza virus infection.
  14. 　インフルエンザウイルスに対するワクチンとして用いるための、請求項１２に記載の医薬組成物。 The pharmaceutical composition according to claim 12, which is used as a vaccine against influenza virus.
  15. 　前記インフルエンザウイルスがインフルエンザウイルスＡ型である、請求項１３または１４に記載の医薬組成物。 The pharmaceutical composition according to claim 13 or 14, wherein the influenza virus is influenza virus type A.
  16. 　療法に用いるための、請求項１～６のいずれか一項に記載の部分欠損ＨＡ－Ｍ１融合タンパク質、請求項７に記載の核酸分子または請求項８に記載の発現ベクター。 The partially defective HA-M1 fusion protein according to any one of claims 1 to 6, the nucleic acid molecule according to claim 7, or the expression vector according to claim 8 for use in therapy.
  17. 　インフルエンザウイルスの感染症を予防または治療するための、請求項１６に記載の部分欠損ＨＡ－Ｍ１融合タンパク質、核酸分子または発現ベクター。 The partially deficient HA-M1 fusion protein, nucleic acid molecule or expression vector according to claim 16, for preventing or treating an influenza virus infection.
  18. 　インフルエンザウイルスに対するワクチンとして用いるための、請求項１６に記載の部分欠損ＨＡ－Ｍ１融合タンパク質、核酸分子または発現ベクター。 The partially deficient HA-M1 fusion protein, nucleic acid molecule or expression vector according to claim 16, for use as a vaccine against influenza virus.
  19. 　前記インフルエンザウイルスがインフルエンザウイルスＡ型である、請求項１７または１８に記載の部分欠損ＨＡ－Ｍ１融合タンパク質、核酸分子または発現ベクター。 The partially defective HA-M1 fusion protein, nucleic acid molecule or expression vector according to claim 17 or 18, wherein the influenza virus is influenza virus type A.
  20. 　請求項１～６のいずれか一項に記載の部分欠損ＨＡ－Ｍ１融合タンパク質、請求項７に記載の核酸分子または請求項８に記載の発現ベクターを被験体に投与することを含んでなる、該被験体におけるインフルエンザウイルスの感染症を予防または治療する方法。 The present invention comprises administering to a subject a partially defective HA-M1 fusion protein according to any one of claims 1 to 6, a nucleic acid molecule according to claim 7, or an expression vector according to claim 8. A method for preventing or treating an influenza virus infection in the subject.
  21. 　請求項１～６のいずれか一項に記載の部分欠損ＨＡ－Ｍ１融合タンパク質、請求項７に記載の核酸分子または請求項８に記載の発現ベクターを被験体に投与することを含んでなる、該被験体においてインフルエンザウイルスに対する防御免疫反応を誘導する方法。 The present invention comprises administering to a subject a partially defective HA-M1 fusion protein according to any one of claims 1 to 6, a nucleic acid molecule according to claim 7, or an expression vector according to claim 8. A method of inducing a protective immune response against influenza virus in the subject.
  22. 　前記インフルエンザウイルスがインフルエンザウイルスＡ型である、請求項２０または２１に記載の方法。 The method according to claim 20 or 21, wherein the influenza virus is influenza virus type A.
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