WO2015186721A1 - Antibody or antibody fragment including variable region thereof, antigen polypeptide, and uses thereof - Google Patents

Antibody or antibody fragment including variable region thereof, antigen polypeptide, and uses thereof Download PDF

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WO2015186721A1
WO2015186721A1 PCT/JP2015/065986 JP2015065986W WO2015186721A1 WO 2015186721 A1 WO2015186721 A1 WO 2015186721A1 JP 2015065986 W JP2015065986 W JP 2015065986W WO 2015186721 A1 WO2015186721 A1 WO 2015186721A1
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antibody
seq
variable region
amino acid
acid sequence
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PCT/JP2015/065986
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French (fr)
Japanese (ja)
Inventor
裕子 内田
岳彦 西藤
鈴木　康司
雄二 庄屋
雅義 豊浦
有希子 石田
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国立研究開発法人農業・食品産業技術総合研究機構
株式会社ファーマフーズ
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Priority to JP2016525195A priority Critical patent/JP6525214B2/en
Publication of WO2015186721A1 publication Critical patent/WO2015186721A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • 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
    • 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
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses

Definitions

  • the present invention relates to antibody fragments comprising an antibody or its variable region, antigenic polypeptides, and uses thereof.
  • H5 subtype highly pathogenic avian influenza virus including H5N1 subtype highly pathogenic avian influenza virus (HPAIV) causes outbreak of avian influenza in poultry from 2003 onwards, aspect of localization mainly in Asia It is When such highly pathogenic avian influenza virus infects birds and the infection spreads, it causes great damage to the livestock industry and the like, and there is also a fear of human infection. Since HPAIV's epidemic prevention in Japan is based on a cover-up investigation, early detection at the field level is important. At present, as a test system that is simply used in the field, one that detects influenza A virus is used, but it is not one that rapidly determines H5N1 subtype highly pathogenic avian influenza.
  • a diagnostic kit using influenza nucleoprotein as a target antigen is mentioned, but there is a problem that it has no H5N1 avian influenza virus specificity and also responds to other influenza viruses.
  • Other examples include a diagnostic kit using recombinant H5 hemagglutinin (HA, virus hemagglutinin) protein of H5N1 subtype inactivated virus as a target antigen, but because an antibody against an immunodominant antigen can be produced, the virus If there is a mutation in the gene, there is a problem that the reactivity may be reduced or undetectable. Therefore, development of a diagnostic reagent for H5 subtype avian influenza virus infection is desired. In order to develop an effective diagnostic reagent, it is necessary to produce an antibody that broadly recognizes the H5 subtype avian influenza virus.
  • Patent Document 1 describes a monoclonal antibody that specifically binds to HA envelope glycoprotein of H5 subtype avian influenza virus or neuraminidase glycoprotein of N1 subtype.
  • Patent Document 2 describes a monoclonal antibody and the like which can recognize and bind an epitope in the HA2 subunit of HA, and have a neutralizing activity against the H5 subtype avian influenza virus.
  • Patent Document 3 describes an isolated monoclonal antibody that binds to an epitope of the stem region of the avian influenza virus HA protein and neutralizes influenza A virus including H5N1 subtype.
  • evaluation of antibody activity is carried out by virus neutralization test. For example, it is carried out by testing the presence or absence of HA protein activity by a hemagglutination inhibition test or the like. That is, when the antibody in the prior art infects a human etc., it aims at neutralizing the H5N1 subtype avian influenza virus using the said antibody. Therefore, even if these antibodies are used as detection reagents, if a mutation occurs in the virus, there is a high possibility that the reactivity of the antibody is lost and the virus can not be detected.
  • hemagglutinin hemagglutinin, HA
  • HA hemagglutinin
  • the present invention includes any one of the following aspects.
  • ⁇ 1> An antibody or a variable region thereof characterized by specifically binding to a hemagglutinin HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 in the hemagglutinin HA1 region of H5 subtype avian influenza virus Antibody fragment.
  • a method for producing an antibody that specifically binds to a hemagglutinin HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 of the hemagglutinin HA1 region of H5 subtype avian influenza virus which comprises the following (a A) a method comprising the steps of (c): (a) immunizing the avian hemagglutinin HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 with a bird; (b) the above step (a) Obtaining a phage antibody library comprising a phage antibody which specifically binds to the above-mentioned hemagglutinin HA1 antigen polypeptide from the birds immunized with the above; and (c) the above-mentioned hemagglutinin HA1 among the phage antibodies obtained in the above step (b) Concentrating and selecting antibodies that specifically bind to the antigenic polypeptide.
  • the antibody according to the present invention and the antibody fragment containing the variable region exhibit an excellent effect of specifically binding to H5 subtype avian influenza virus and not binding to other subtypes of avian influenza virus other than H5. . Therefore, H5 subtype avian influenza virus can be detected accurately, rapidly and simply by using the antibody according to the present invention.
  • Figure 7 shows the steps of immunization of a chicken and preparation of a scFv phage antibody library from immunized chicken spleen. It is a figure which shows 1 process in the preparation methods of the antibody based on Example 1 of this invention. The process of panning selection of scFv phage library is shown. It is a figure which shows 1 process in the preparation methods of the antibody based on Example 1 of this invention. The steps of screening an scFv phage library using ELISA are shown. It is a figure which shows the variable region of the amino acid sequence of the obtained wild type single chain antibody based on Example 1 of this invention.
  • FIG. 6 shows the results of CBB staining of a wild-type bivalent antibody protein according to Example 1 of the present invention after electrophoresis. It is a figure which shows the antibody titer of the wild type bivalent antibody based on Example 1 of this invention. It is a figure which shows the antibody binding property with respect to the variant of H5N1 subtype avian influenza virus of the wild type bivalent antibody, and two or more types of subtype avian influenza viruses other than H5 based on Example 1 of this invention.
  • polynucleotide may also be referred to as “nucleic acid” or “nucleic acid molecule” and is intended to be a polymer of nucleotides.
  • Base sequence can also be referred to as “nucleic acid sequence” or “nucleotide sequence”, and unless otherwise stated, is intended the sequence of deoxyribonucleotides or the sequence of ribonucleotides.
  • the polynucleotide may be single-stranded or double-stranded, and in the case of single-stranded polynucleotide, it may be a sense strand or an antisense strand.
  • polypeptide can also be referred to as “protein” or “protein fragment”.
  • H5 subtype avian influenza virus and “H5N1 subtype avian influenza virus” refer to a type of avian influenza virus subtype.
  • Avian influenza viruses are classified according to their nuclear and matrix protein antigen specificity. Avian influenza virus is mainly classified into A, B and C serotypes. Among these, type A avian influenza virus has eight RNA segments and encodes ten viral proteins.
  • Hemagglutinin refers to the envelope glycoprotein of influenza virus. HA enables adsorption and entry of influenza virus into host cells.
  • Neuroaminidase (hereinafter referred to as "NA”) has a function of cleaving sialic acid when virus particles leave the cell surface at the late stage of infection, and plays a role in acquiring infectivity.
  • influenza A viruses are derived from birds, and influenza viruses of type A are further classified into subtypes by the antigenic nature of HA and NA.
  • HA has 16 subtypes of H1 to H16, and NA has 9 subtypes of N1 to N9.
  • H5 and H7 subtypes of influenza viruses are not highly pathogenic to the natural host waterfowl, but exhibit high virulence that causes death when infected with poultry such as chickens It is known that it is called highly pathogenic avian influenza virus (HPAIV). Furthermore, in recent years, cases of human infection have been successively reported from the outbreak of influenza caused by H5N1 subtype virus infection in poultry. From such a situation, there is concern that H5N1 subtype HPAIV is mutated so as to be able to efficiently infect human to human, and it becomes a novel influenza virus.
  • HPAIV highly pathogenic avian influenza virus
  • the H5N1 avian influenza virus which has been localized to poultry in Asia in recent years, can be further classified into multiple clades based on the homology of the base sequences of the HA gene. Accession number (Accession) in the GenBank database (web page: http://www.ncbi.nlm.nih.gov/genbank/) provided by the National Center for Biotechnology Information (NCBI).
  • NCBI National Center for Biotechnology Information
  • HA gene of H5N1 subtype HPAIV registered by No. is classified into clades 0 to 9 and further sub-clades thereof by WHO / OIE / FAO H5N1 Evolution Working Group.
  • the “HA1 region” and the “HA2 region” respectively indicate two regions flanking the proteolytic cleavage site of the hemagglutinin protein (HA) of influenza virus and the fusion peptide region linked thereto. Moreover, proteolytic cleavage at the HA1-HA2 junction of the HA protein is associated with viral proliferative properties, and the continuous presence of hydrophobic amino acids around this cleavage site is characteristic of HPAIV . And the HA1 and HA2 regions are responsible for the high virulence in chickens.
  • HA hemagglutinin protein
  • sequence identity across the HA1 region between all clades in the H5N1 subtypes described above is 91-99 for all different clade combinations, using those listed above for each clade strain. % Sequence identity.
  • sequence identity across the HA1 region among all the H1 to H16 subtypes is in the range of 7 to 51% for any combination of different subtypes.
  • sequence identity between the entire HA1 region of each of the H1 to H4 subtypes and the H6 to H16 subtypes and the entire HA1 region of the H5 type is very low, all in the range of 8% to 21%.
  • sequence identity across the HA2 region between all clades in the above mentioned H5N1 subtypes is 94 to 100% in all clade combinations, using the ones listed above for each clade strain Have the sequence identity of
  • sequence identity across the HA1 region between all H1 to H16 subtypes is in the range of 42 to 81% for any combination of different subtypes.
  • sequence identity between the entire HA2 region of each of the H1 to H4 type and the H6 to H16 subtype and the entire HA2 region of the H5 subtype is 45 to 75%.
  • bivalent antibody means an antibody having two antigen binding sites per molecule, that is, an antibody having a bivalent binding to an antigen.
  • the above “bivalent antibody” is composed of two light chains (a light chain variable region and a light chain constant region) and two heavy chains (a heavy chain variable region and a heavy chain constant region) which are respectively homologous to each other and which are disulfide linked (S An antibody having a structure bound by -S bond).
  • the antibody molecule be a full-length antibody molecule, and a “bivalent antibody” as long as it has a structure in which two heavy chains can be linked by S—S bond, ie, a structure having at least F (ab ′) 2 fragment It is included in the category of
  • single-chain variable fragment refers to a light chain (also referred to as L chain) variable region and a heavy chain (Heavy chain, H) It refers to an antibody in which one variable region is linked to a variable region (also referred to as a chain) and two variable regions are close to form one antigen binding site.
  • the above “single-chain variable region fragment” is an antibody having one antigen binding site per molecule. For example, it refers to an antibody obtained by an antibody production method using phage display. Also described herein as “single chain antibodies”.
  • a and / or B is a concept including both A and B and A or B, and can be reworded as “at least one of A and B”.
  • HA1 antigen polypeptide according to the present invention may be a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10.
  • the HA1 antigen polypeptide according to the present invention may be a polypeptide consisting of 15 or more and 16 or less consecutive amino acids in a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10.
  • a “linker peptide to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10” May be added, other amino acids or proteins may be linked (for example, a tagged protein or a fusion protein).
  • the linker polypeptide linked to the HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 includes a polypeptide consisting of 10 to 25 amino acids, etc.
  • the tag includes His tag, Myc tag and Flag tag etc. are mentioned.
  • the method for producing the HA1 antigen polypeptide according to the present invention may be chemical synthesis or may use an expression vector.
  • the HA1 antigen polypeptide of the present invention can be produced by known polypeptide synthesis methods.
  • polypeptide synthesis methods include, but are not limited to, chemical synthesis methods such as liquid phase polypeptide synthesis methods and solid phase peptide synthesis methods.
  • the polypeptide may be produced from a transformant into which the expression vector has been introduced, or may be produced using an in vitro translation system.
  • a target polypeptide can be produced in a host cell into which an expression vector into which a polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 10 has been inserted has been introduced.
  • an expression vector into which a polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 10 has been inserted has been introduced.
  • the method described in the item of recombinant vector] is preferably used.
  • the transformant for example, the following [5.
  • the method described in item [transformant] is used for example.
  • the HA1 antigen polypeptide according to the present invention is preferably stably expressed in the host cell, but may be transiently expressed.
  • the polypeptide thus produced can be purified according to known methods.
  • the method for purifying the polypeptide is not particularly limited, and examples thereof include gel filtration chromatography, ion exchange chromatography, affinity chromatography and the like.
  • the HA1 antigen polypeptide according to the present invention is very highly retained among strains having different H5N1 subtypes among the amino acid sequences of the HA1 region of the H5N1 avian influenza virus virus, as shown in Examples and FIG. And it is the amino acid sequence of the site
  • the sequence identity of the amino acid sequence corresponding to this amino acid sequence is very low. That is, the HA1 antigen polypeptide according to the present invention is resistant to mutation even in the host, and is structurally very excellent in its ability to bind to an antibody. Therefore, the HA1 antigen polypeptide according to the present invention can be suitably used for the production of an antibody very specific for the H5N1 avian influenza virus.
  • Antibody according to the present invention and antibody fragment containing the variable region thereof specifically bind to a hemagglutinin HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 in the HA1 region of H5 subtype avian influenza virus. It features.
  • HA1 antigen polypeptide specifically binds to HA1 antigen polypeptide means to recognize and bind to the sequence of the HA1 antigen polypeptide, and includes, for example, the sequence of the HA1 antigen polypeptide, It also binds to polypeptides having longer sequences.
  • antibody means immunoglobulin (IgA, IgD, IgE, IgY, IgG, IgM and Fab fragments thereof, F (ab ') 2 fragment, Fc fragment) thereof, for example, a monoclonal antibody
  • bivalent antibodies such as polyclonal antibodies, anti-idiotype antibodies, chimerized antibodies, humanized antibodies and single chain variable region fragments and peptides including the complementarity determining regions (CDRs) of the antibodies, etc. It is not limited to these.
  • an antibody fragment of the present invention which comprises the variable region of the above antibody, is also included in the scope of the present invention.
  • the antibody fragment is not particularly limited as long as it is an antibody fragment containing the variable region of the above-mentioned antibody and recognizes the H5 subtype avian influenza virus.
  • Such an antibody fragment may be a fragment of any of the full-length H chain, full-length L chain, variable region of H chain, and variable region of L chain of the antibody of the present invention.
  • Such antibody fragments can be suitably used, for example, in flow cytometry or ELISA.
  • antibody fragments can be suitably used to produce chimeric antibodies by transplantation into antibodies of human or other animals.
  • one embodiment of the antibody according to the present invention and an antibody fragment containing the variable region thereof is specific for consecutive 15 or more and 16 or less amino acids in the HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 Bond to
  • specific binding of the antibody fragment containing the above-mentioned antibody or its variable region to the HA1 antigen polypeptide may occur in one specific region in the HA1 antigen polypeptide, or at two or more places. It may occur simultaneously in different areas.
  • One aspect of the antibody according to the present invention is a bivalent antibody in which the H chain consists of the amino acid sequence shown in SEQ ID NO: 26 and the L chain consists of the amino acid sequence shown in SEQ ID NO: 28.
  • another example of the antibody according to the present invention is a bivalent antibody in which the H chain consists of the amino acid sequence shown in SEQ ID NO: 52 and the L chain consists of the amino acid sequence shown in SEQ ID NO: 28.
  • the amino acid sequence shown in SEQ ID NO: 2 is the amino acid sequence of the H chain variable region of the antibody according to one aspect of the present invention
  • the amino acid sequence shown in SEQ ID NO: 3 is the L chain of the antibody according to one aspect of the present invention It is an amino acid sequence of a variable region.
  • FIG. 4 shows the amino acid sequence of the H chain variable region and the amino acid sequence of the L chain variable region of the antibody according to one aspect of the present invention.
  • the H chain variable region of the antibody according to one embodiment of the present invention has CDR1, CDR2 and CDR3 which are regions of CDRs shown by squares.
  • the heavy chain variable region of the antibody according to one aspect of the present invention consists of the amino acid sequence of 1st to 130th amino acid sequences shown in SEQ ID NO: 1 and CDR1 consisting of the 31st to 35th amino acid sequences;
  • the CDR2 consists of the 50th to 66th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 1 and the CDR3 consists of the 99th to 119th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 1.
  • the light chain variable region of the antibody according to one aspect of the present invention has CDR1, CDR2 and CDR3 which are indicated by a box.
  • the L chain variable region of the antibody according to one aspect of the present invention consists of the amino acid sequence 148 to 270 of the amino acid sequence shown in SEQ ID NO: 1, and the amino acid 168 to 176 in the amino acid sequence shown in SEQ ID NO: 1 It has CDR1 consisting of the sequence, CDR2 consisting of the amino acid sequence 193 to 203 of the amino acid sequence shown in SEQ ID NO: 1, and CDR3 consisting of the amino acid sequence 232 to 240 of the amino acid sequence shown in SEQ ID NO: 1.
  • the antibody of one embodiment of the present invention is an antibody consisting of the amino acid sequence shown in SEQ ID NO: 1.
  • the antibody of another aspect of the present invention comprises the amino acid sequence of which the heavy chain variable region is shown in SEQ ID NO: 45, and the amino acid sequence of which the light chain variable region is shown in SEQ ID NO: 3. Furthermore, an antibody of another aspect of the present invention is an antibody consisting of the amino acid sequence shown in SEQ ID NO: 44.
  • amino acid sequences of CDR1, CDR2 and CDR3 of the H chain variable region of the antibody according to one aspect of the present invention are shown in SEQ ID NOs: 4 to 6, respectively, and amino acids of CDR1, CDR2 and CDR3 of the L chain variable region of the antibody according to the present invention The sequences are shown in SEQ ID NOs: 7-9, respectively.
  • amino acid sequence and nucleotide sequence of CDR3 of the H chain variable region of the antibody according to another aspect of the present invention are shown in SEQ ID NOs: 46 and 47, respectively.
  • another embodiment of the antibody according to the present invention comprises all of CDR1 shown in SEQ ID NO: 4, CDR2 shown in SEQ ID NO: 5 and CDR3 shown in SEQ ID NO: 6 in the H chain variable region, And an antibody fragment comprising an antibody or its variable region, having all of CDR1 shown in SEQ ID NO: 7, CDR2 shown in SEQ ID NO: 8 and CDR3 shown in SEQ ID NO: 9 in the L chain variable region. is there.
  • still another embodiment of the antibody according to the present invention comprises all of CDR1 shown in SEQ ID NO: 4, CDR2 shown in SEQ ID NO: 5 and CDR3 shown in SEQ ID NO: 46 in the H chain variable region. And an antibody fragment comprising an antibody or its variable region, having all of CDR1 shown in SEQ ID NO: 7, CDR2 shown in SEQ ID NO: 8 and CDR3 shown in SEQ ID NO: 9 in the L chain variable region It is.
  • still another embodiment of the antibody according to the present invention is the CDR1 represented by SEQ ID NO: 4, the CDR2 represented by SEQ ID NO: 5 and the CDR3 represented by SEQ ID NO: 6 or SEQ ID NO: 46 in the H chain variable region. And at least one of CDR1 shown in SEQ ID NO: 7, CDR2 shown in SEQ ID NO: 8 and CDR3 shown in SEQ ID NO: 9 in the L chain variable region An antibody or an antibody fragment containing the variable region thereof.
  • still another embodiment of the antibody according to the present invention is the CDR1 represented by SEQ ID NO: 4, the CDR2 represented by SEQ ID NO: 5 and the CDR3 represented by SEQ ID NO: 6 or SEQ ID NO: 46 in the H chain variable region. Or at least one of CDR1 shown in SEQ ID NO: 7, CDR2 shown in SEQ ID NO: 8 and CDR3 shown in SEQ ID NO: 9 in the L chain variable region An antibody or an antibody fragment containing the variable region thereof.
  • the antibody according to the present invention or an antibody fragment containing the variable region thereof also includes those having the following characteristics.
  • the H chain variable region is 1 to 13, preferably 1 to 6, more preferably 1 to 2 or 3 More preferably, one amino acid consists of a substituted, deleted, inserted and / or added amino acid sequence, and the light chain variable region is shown in the amino acid sequence shown in SEQ ID NO: 3 or in SEQ ID NO: 3 Amino acid in which 1 to 10, preferably 1 to 5, more preferably 1 to 2 or 3 and even more preferably 1 amino acid is substituted, deleted, inserted and / or added in the amino acid sequence An antibody or an antibody fragment comprising a variable region thereof consisting of a sequence.
  • an antibody according to the present invention and an antibody fragment containing the variable region thereof is an antibody or antibody wherein the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 2 and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 3 Mention may be made of antibody fragments comprising the variable region.
  • Another example of the antibody according to the present invention and an antibody fragment containing the variable region thereof, wherein the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 45 and the light chain variable region is the amino acid shown in SEQ ID NO: 3 Mention may be made of an antibody consisting of a sequence or an antibody fragment comprising the variable region thereof.
  • the amino acid sequence shown in SEQ ID NO: 45 the 105th amino acid Y of the amino acid sequence shown in SEQ ID NO: 2 is substituted with R, the 106th amino acid S with R, and the 107th amino acid Y with V Sequence.
  • An antibody having such a sequence has higher reactivity to the HA1 antigen while maintaining its specificity for the H5 subtype avian influenza virus, which is more preferable.
  • an antibody fragment according to the present invention or an antibody fragment containing the variable region thereof specifically binds to a hemagglutinin HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 in the hemagglutinin HA1 region of H5 subtype avian influenza virus Or an antibody fragment containing the variable region thereof, which is shown in any of the following (1) to (4): (1) An antibody consisting of the amino acid sequence shown in SEQ ID NO: 1 or 44 or an antibody fragment containing the variable region thereof (2) An antibody fragment comprising an amino acid sequence in which 1 to 35 amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 1 or 44, or an antibody fragment comprising the variable region thereof (3) An antibody fragment having a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO: 1 or 44, or an antibody fragment comprising the variable region thereof (4) A polynucleotide that hybridizes under stringent conditions to a polynucleo
  • one aspect of the antibody according to the present invention is obtained by the production method including the steps of (a) to (c) shown below.
  • the antibody according to the present invention and the antibody fragment containing the variable region thereof are a single chain variable region fragment or a bivalent antibody, preferably a bivalent antibody.
  • Examples of single chain antibodies encompassed by the present invention include antibodies consisting of the amino acid sequence shown in SEQ ID NO: 44.
  • amino acids 252 to 351 of the amino acid sequence shown in SEQ ID NO: 1 to amino acids 252 to 359 in the amino acid sequence shown in SEQ ID NO: 44 The antibody which consists of the substituted amino acid sequence is mentioned.
  • amino acids 252 to 359 of the amino acid sequence shown in SEQ ID NO: 44 are the amino acids 252 to 351 of the amino acid sequence shown in SEQ ID NO: 1
  • an example of the bivalent antibody according to the present invention is a bivalent mouse chimeric anti-HA1 antibody having the following characteristics: a variable region derived from a chicken and a constant region derived from a mouse IgG1, which is an immunoglobulin The subtype is IgG1 and the molecular weight is about 150 kDa.
  • a further specific example of the bivalent antibody according to the present invention is that in which the H chain consists of the amino acid sequence shown in SEQ ID NO: 26 and the L chain consists of the amino acid sequence shown in SEQ ID NO: 28. Furthermore, as another example of a more preferable bivalent antibody according to the present invention, one in which the H chain consists of the amino acid sequence shown in SEQ ID NO: 52 and the L chain consists of the amino acid sequence shown in SEQ ID NO: 28 Be
  • the antibody according to the present invention and the antibody fragment containing the variable region also encompass an antibody labeled with a labeling agent.
  • the labeled antibody has the following [7. Kit according to the present invention] and [8. Method for detecting H5 subtype avian influenza virus].
  • Examples of the above-mentioned labeling agent include enzymes, enzyme substrates, radioactive isotopes, luminescent substances, fluorescent substances, biotin, coloring substances and the like.
  • Examples of enzymes include peroxidase, ⁇ -galactosidase, alkaline phosphatase, glucose oxidase, acetylcholinesterase and glucose-6-phosphate dehydrogenase. Coupling between these enzymes and antibodies can be carried out by known methods using crosslinking agents such as maleimide compounds and N-hydroxysuccinimide ester compounds.
  • As the enzyme substrate known substances can be used depending on the enzyme used.
  • OPD orthophenylenediamine
  • TMB tetramethylbenzidine
  • nitro blue tetrazolium (NBT) and 5-bromo- A mixed substrate of 4-chloro-3-indolylphosphatase p-toluidinyl salt (BCIP) or the like can be used.
  • radioactive isotopes those used in conventional radioimmunoassays such as 125 I, 3 H or 35 S can be used.
  • Radiolabeling to the antibody according to the present invention can be performed using a known method.
  • the fluorescent dye those used for ordinary fluorescent antibody methods such as fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate and phycoerythrin can be used.
  • fluorescent silica nanoparticles may be used as the fluorescent material.
  • the light emitting substance isoluminol, acridine ester, lucigenin or the like can be used.
  • a known method can be used as a method of labeling.
  • a coloring latex particle, a gold colloid, etc. can be mentioned, for example.
  • the antibody according to the present invention and an antibody fragment containing the variable region thereof may be immobilized on a solid support.
  • Solid supports that can be used include polystyrene, polycarbonate, polypropylene or polyvinyl microtiter plates, test tubes, capillaries, beads (such as latex particles and metal compounds), membranes (such as liposomes) and filters.
  • polystyrene is particularly preferred.
  • the antibody thus immobilized is, for example, described later [8. [Method for detecting H5 subtype avian influenza virus]
  • the antibody according to the present invention and the antibody fragment containing the variable region thereof are highly retained among strains differing in H5 subtype among the amino acid sequences of HA1 region of H5 subtype avian influenza virus It binds to the site exposed to the surface of the H5 subtype avian influenza virus protein in a three-dimensional structure. Furthermore, the binding to subtypes other than H5 is significantly lower. Therefore, the antibody according to the present invention and the antibody fragment containing the variable region thereof can detect H5 subtype avian influenza virus specifically, simply and at high sensitivity. Therefore, the antibody according to the present invention can be suitably used in a kit for detecting H5 subtype avian influenza virus, a detection method, and the like.
  • H5 subtype avian influenza virus includes subtypes of N1 to N9, and examples of H5 subtype avian influenza virus used as a target of detection in the present invention include H5N1 subtype, H5N2 subtype and H5N8. There are subtypes.
  • the polynucleotide according to the present invention encodes an antibody fragment comprising the above-mentioned antibody and its variable region.
  • Specific examples of this polynucleotide include the polynucleotides described in any of the following (1) to (4). (1) specifically binding to an HA1 antigen polypeptide having the amino acid sequence shown in SEQ ID NO: 10, having the amino acid sequence shown in SEQ ID NO: 1 or 44 and within the HA1 region of H5 subtype avian influenza virus A polynucleotide encoding an antibody characterized by or an antibody fragment comprising the variable region thereof.
  • (2) has an amino acid sequence in which 1 to 35 amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 1 or 44, and the HA1 region of H5 subtype avian influenza virus Among them, a polynucleotide encoding an antibody or antibody fragment comprising a variable region thereof, which is characterized by specifically binding to an HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10.
  • the number of amino acids substituted, deleted, inserted, and / or added is preferably 1 to 17, preferably 1 to 13, and more preferably 1 to 10. , 1 to 8 is more preferable, 1 to 5 is more preferable, and 1 to 2 or 3 is particularly preferable.
  • HA1 consisting of the amino acid sequence shown in SEQ ID NO: 10, having a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO: 1 or 44 and of the HA1 region of H5 subtype avian influenza virus
  • the sequence identity of the amino acid sequence is preferably 95% or more, more preferably 96% or more, and particularly preferably 97% or more, 98% or more, or 99% or more.
  • stringent conditions for example, conditions described in Reference: "Molecular cloning-a Laboratory manual 2nd edition (Sambrook et al., 1989)" can be mentioned. More specifically, under stringent conditions, for example, 6 ⁇ SSC (composition of 1 ⁇ SSC: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5 A condition in which a solution containing ⁇ Denhardt and 100 mg / mL herring sperm DNA is incubated with a probe at 65 ° C.
  • this polynucleotide has 90% or more of sequence identity with the base sequence of the polynucleotide described in the above (1), 95% or more, 96% or more, 97% or more, 98% It is more preferable to have sequence identity of 99% or more.
  • the polynucleotide of the present invention may exist in the form of RNA (eg, mRNA) or in the form of DNA (eg, cDNA or genomic DNA).
  • the DNA may be double stranded or single stranded.
  • An example of the polynucleotide according to the present invention is DNA encoding the polypeptide shown in SEQ ID NO: 1 and SEQ ID NO: 44.
  • An example of the sequence of a polynucleotide encoding the polypeptide shown in SEQ ID NO: 1 is shown in SEQ ID NO: 54
  • an example of the sequence of a polynucleotide encoding the polypeptide shown in SEQ ID NO: 44 is shown in SEQ ID NO: 55.
  • the polynucleotide according to the present invention may contain additional sequences such as untranslated region (UTR) sequences, signal sequences and introns.
  • UTR untranslated region
  • the method for obtaining (isolating) the polynucleotide according to the present invention is not particularly limited.
  • a probe that specifically hybridizes with a part of the base sequence of the above polynucleotide is prepared, and a genome is prepared.
  • the DNA library or cDNA library may be screened.
  • the polynucleotide according to the present invention may be synthesized according to a nucleic acid synthesis method such as the phosphoroamidite method.
  • a method for obtaining the polynucleotide according to the present invention a method using amplification means such as PCR can be mentioned.
  • primers are prepared from the 5 'and 3' sequences (or their complementary sequences) of the cDNA of the polynucleotide, and genomic DNA (or cDNA) or the like is used as a template using these primers.
  • genomic DNA or cDNA or the like is used as a template using these primers.
  • a large amount of DNA fragment containing the polynucleotide according to the present invention can be obtained by performing PCR etc. and amplifying the DNA region sandwiched between both primers.
  • Examples of the polynucleotide according to the present invention further include DNAs encoding the bivalent antibody of the present invention (SEQ ID NOS: 27 and 29 and 53).
  • the polynucleotide (eg, DNA) according to the present invention can also be used as a recombinant vector inserted into a suitable vector.
  • the type of the vector may be, for example, a vector that replicates autonomously (such as a plasmid), or is integrated into the host cell's genome when introduced into a host cell and replicated together with the integrated chromosome. It may be
  • the vector is preferably an expression vector.
  • the polynucleotide according to the present invention is functionally linked to elements necessary for transcription (for example, a promoter and the like).
  • the promoter is a DNA sequence that exhibits transcriptional activity in a host cell, and can be appropriately selected depending on the type of host.
  • Promoters operable in bacterial cells include E. coli lac, trp and tac promoters and the like.
  • promoters operable in insect cells include polh promoter, p10 promoter, and pB1 promoter.
  • promoters operable in yeast cells include promoters derived from yeast glycolytic genes, alcohol dehydrogenase gene promoters, and phosphoglycerate kinase promoters.
  • promoters operable in filamentous fungal cells include the ADH3 promoter, and the tpiA promoter.
  • promoters operable in mammalian cells include SV40 promoter, bovine papilloma virus (BPV) promoter and human cytomegalovirus (CMV) promoter.
  • SV40 promoter bovine papilloma virus (BPV) promoter
  • CMV human cytomegalovirus
  • polynucleotide according to the present invention may optionally be operatively linked to a suitable terminator, such as, for example, a human growth hormone terminator or, for fungal hosts, a TPI1 terminator or an ADH3 terminator.
  • a suitable terminator such as, for example, a human growth hormone terminator or, for fungal hosts, a TPI1 terminator or an ADH3 terminator.
  • the recombinant vector according to the present invention may further have elements such as polyadenylation signal, transcription enhancer sequence and translation enhancer sequence.
  • the recombinant vector according to the present invention may further comprise a DNA sequence enabling the vector to replicate in the host cell, for example the SV40 origin of replication (when the host cell is a mammalian cell) Can be mentioned.
  • the recombinant vector according to the present invention may further contain a selectable marker.
  • a selectable marker for example, drug resistance genes such as ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin or hygromycin can be mentioned.
  • a transformant can be produced by introducing the polynucleotide according to the present invention or the recombinant vector according to the present invention (generally referred to as the nucleic acid construct of the present invention) into a suitable host cell.
  • Host cells include, for example, bacterial cells, yeast cells, fungal cells and higher eukaryotic cells.
  • bacterial cells examples include gram positive bacteria such as Bacillus and Streptomyces or gram negative bacteria such as E. coli. Transformation of these bacterial cells may be performed by, for example, a protoplast method or a method using competent cells.
  • yeast cells include cells of organisms belonging to Saccharomyces or Schizosaccharomyces, such as Saccharomyces cerevisiae and Saccharomyces kluyveri.
  • electroporation, spheroplast method, lithium acetate method and the like can be mentioned.
  • fungal cells other than yeast cells are cells of filamentous fungi, eg, organisms belonging to Aspergillus, Neurospora, Fusarium, or Trichoderma.
  • filamentous fungi eg, organisms belonging to Aspergillus, Neurospora, Fusarium, or Trichoderma.
  • transformation can be performed by integrating the nucleic acid construct of the present invention into a host chromosome to obtain a recombinant host cell. Integration of the nucleic acid construct into the host chromosome can be performed, for example, by homologous recombination or heterologous recombination.
  • Examples of higher eukaryotic cells include plant cells, animal cells and insect cells, and the like, and animal cells further include mammalian cells and avian cells.
  • insect cells examples include Sf9 cells and Sf21 cells.
  • a recombinant gene transfer vector and a baculovirus are co-introduced into the insect cell to obtain a recombinant virus in the insect cell culture supernatant, and the insect cell is further infected with the recombinant virus , Protein can be expressed.
  • a co-introduction method for example, a calcium phosphate method or a lipofection method can be mentioned.
  • plant cells examples include T87 cells.
  • electroporation calcium phosphate method, lipofection method, liposome method, DEAE dextran method, microinjection method and the like can be used for transformation of plant cells.
  • mammalian cells examples include HEK 293 cells, HeLa cells, COS cells, BHK cells, CHL cells, CHO cells, SP2 / 0 cells and NSO cells.
  • mammalian cells When mammalian cells are used as host cells, for example, electroporation, calcium phosphate method, lipofection method, liposome method, DEAE dextran method, microinjection method and the like can be used for transformation of mammalian cells.
  • transformants described above are cultured in an appropriate culture medium under conditions that allow expression of the introduced nucleic acid construct. Then, if necessary, an antibody fragment according to the present invention or an antibody fragment containing the variable region thereof is isolated and purified from a culture of transformants.
  • the transformant is not limited to cells. That is, the transformant may be, for example, a tissue, an organ, and an individual transformed with the nucleic acid construct of the present invention.
  • the non-cell transformant is of non-human origin, and in particular, the individual is preferably of non-human origin.
  • One example of a method for producing an antibody according to the present invention and an antibody fragment containing the variable region thereof is specific to a hemagglutinin HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 in the hemagglutinin HA1 region of H5 subtype avian influenza virus It is a method of producing an antibody which binds to the antibody, which comprises the following steps (a) to (c): (a) hemagglutinin consisting of the amino acid sequence shown in SEQ ID NO: 10 Immunizing the bird with an HA1 antigen polypeptide; (b) obtaining a phage antibody library containing a phage antibody that specifically binds to the hemagglutinin HA1 antigen polypeptide from the bird immunized in the step (a); c) Among the phage antibodies obtained in the above step (
  • Step (a) is a step of immunizing birds with an HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10.
  • the method for administering the antigen to birds is not particularly limited.
  • the antigenic polypeptide may be administered intraperitoneally, or the antigenic polypeptide may be administered intravenously.
  • the primary immunization be administered by mixing an equal amount of the antigenic polypeptide and the immunostimulatory agent.
  • immunostimulatory agent for example, those commonly used in the field such as complete Freund's adjuvant, incomplete Freund's adjuvant, or aluminum hydroxide gel adjuvant can be used.
  • a boost (secondary immunization) can be performed 4 weeks after the primary immunization and a boost (third immunization) can be performed 3 to 4 weeks later as an administration interval of the antigenic polypeptide. If serum elevation is not observed, boosting can be performed after 2 to 3 weeks.
  • Birds that immunize the HA1 antigen polypeptide include chicken, ostrich, quail, turkey and the like, with chicken being most preferable in terms of easiness of breeding and securing of feed.
  • Step (b) is a step of obtaining a phage antibody library containing phage antibodies that specifically bind to the antigenic polypeptide from the birds immunized in step (a) above.
  • the method for obtaining an antibody that specifically binds to an antigenic polypeptide is not particularly limited. For example, a method of producing a monoclonal antibody by producing a hybridoma (Reference document: J. Vet. Med. Sci 58 1053 1996) or a method of producing avian phage antibody by phage display (Japanese Patent Application Publication "Patent No. 3908257), etc.). In Examples described later, phage display is used because antibodies can be easily and efficiently produced.
  • a method of obtaining a phage antibody that specifically binds to an HA1 antigen polypeptide using a phage display method is described.
  • the spleen is isolated from the birds immunized in the above step (a), and RNA is extracted from the spleen.
  • RT-PCR is performed using the obtained RNA as a template, and cDNAs containing various heavy chain variable region and light chain variable region genes of the avian antibody are recovered.
  • a plurality of combinations of the obtained heavy chain variable region and light chain variable region genes are linked via a gene encoding a linker ((GGGGS) ⁇ 3) and a gene encoding a spacer (Asp-Val) , Construct a scFv gene.
  • the resulting scFv gene is introduced into a plasmid incorporating avian C ⁇ and the g3p gene to prepare a phagemid vector.
  • the prepared phagemid vector is transformed into a host (eg, E. coli etc.).
  • a host eg, E. coli etc.
  • E. coli transformed with a phagemid vector is further infected with a helper phage.
  • phages expressing scFv can be obtained.
  • panning selection can be performed as a method of concentrating an antibody that specifically binds to the HA1 antigen polypeptide from among a plurality of single-chain phage antibodies (phage antibody library).
  • the method of “panning selection” is not particularly limited.
  • the antigen polypeptide is immobilized on an immuno module plate, and the immobilized antibody polypeptide is reacted with a phage antibody group, and phage not bound is removed by washing. Then, there is a method of repeating several times the operation of eluting only the phages bound to the antigenic polypeptide, infecting E. coli and propagating. By performing panning selection, phages that specifically bind to only the antigenic polypeptide can be enriched.
  • the panning selection operation is preferably repeated once to six times, and more preferably six times.
  • Step (c) is a step of selecting an antibody that specifically binds to the above HA1 antigen polypeptide among the antibodies that specifically bind to the antigen polypeptide obtained in the above step (b).
  • the selection method is not particularly limited, and for example, ELISA method, Western blot method, flow cytometry method, intermolecular interaction analysis and the like can be performed.
  • the above-mentioned solid phase ELISA is performed on the phage antibody concentrated by the above panning selection using the HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 as the solid phase antigen.
  • Antibodies that specifically bind to the HA1 antigen polypeptide can be selected.
  • a step of producing a chicken-type divalent antibody based on the obtained phage antibody producing a chicken-type divalent antibody
  • a step of producing a chimerized antibody having a variable region of a natural chicken antibody for the method of producing a chimerized antibody, see Japanese Patent Laid-Open Publication
  • Japanese Patent Laid-Open Publication No. 2006-282521 and Japanese Patent Laid-Open Publication No. 2005-245337 or a step of preparing a humanized antibody having an antigen-binding region of a chicken natural antibody
  • the method for producing a humanized antibody may include the Japanese Patent Application Publication (JP 2006-241026A).
  • Recombinant vector preferably using the expression vector for H chain and the expression vector for L chain, and introducing the variable region of chicken antibody into them, respectively.
  • a method of expressing in a host mammalian cell can be mentioned.
  • a method including amplification of a target region by PCR using a DNA encoding the obtained phage antibody as a template can be mentioned.
  • random mutagenesis using Inverse PCR or the like, or known mutagenesis, using the obtained phage antibody or a DNA encoding a bivalent or chimeric antibody as a template The method may include the step of mutating the amino acid sequence of the target site by performing point mutation introduction and the like using a method.
  • kits according to the present invention are kits for detecting H5 subtype avian influenza virus contained in a sample, and comprises at least an antibody according to the present invention or an antibody fragment containing a variable region thereof.
  • the antibody according to the present invention and an antibody fragment containing the variable region thereof [1. Antibody according to the present invention].
  • the kit according to the present invention further comprises at least one antibody that recognizes H5 subtype avian influenza virus or an antibody fragment containing the variable region thereof other than the antibody according to the present invention or the antibody fragment containing the variable region thereof.
  • an antibody that specifically binds to an HA2 antigen polypeptide having an amino acid sequence contained in the HA2 region of H5 subtype avian influenza virus and an antibody fragment containing its variable region can be mentioned.
  • an antibody that specifically binds to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 17 in the HA2 region of H5 subtype avian influenza virus and an antibody fragment containing its variable region can be mentioned.
  • a bivalent anti-HA2 antibody consisting of the amino acid sequence in which the H chain is shown in SEQ ID NO: 18 and an L chain consisting of the amino acid sequence shown in SEQ ID NO: 20 and the H chain in SEQ ID NO: 22
  • the bivalent anti-HA2 antibody which consists of an amino acid sequence shown, and whose L chain consists of an amino acid sequence shown by sequence number 24 is mentioned.
  • a DNA encoding the H chain consisting of the amino acid sequence shown in the above SEQ ID NO: 18 consists of the base sequence shown in SEQ ID NO: 19 and shown in the above SEQ ID NO: 20
  • the DNA encoding the L chain consisting of the amino acid sequence is an anti-HA2 antibody consisting of the base sequence shown in SEQ ID NO: 21
  • the DNA encoding the H chain consisting of the amino acid sequence is an anti-HA2 antibody which comprises a DNA sequence consisting of the nucleotide sequence shown in SEQ ID NO: 23 and encoding an L chain consisting of the amino acid sequence shown above in SEQ ID NO: 24 comprises the nucleotide sequence shown in SEQ ID NO: 25 .
  • the kit according to the present invention includes, in addition to the antibody according to the present invention, a member (secondary antibody, a coloring reagent, a blocking reagent, etc.) necessary for performing an immune reaction such as immunochromatography, ELISA and Western blotting. Plates (96-well plate etc.) and tubes etc. may be included.
  • the membrane, gel for electrophoresis, electrophoresis apparatus, blotting apparatus, reagents for blotting and the like necessary for performing Western blotting may be included.
  • a secondary antibody for detecting the antibody according to the present invention and a substrate of a labeled enzyme bound to the secondary antibody may be provided.
  • the secondary antibody examples include alkaline phosphatase-labeled anti-IgG antibody and horseradish peroxidase (HRP) -labeled anti-IgG antibody.
  • HRP horseradish peroxidase
  • the origin of the anti-IgG antibody to be used is not specifically limited, According to the objective, it selects suitably.
  • a substrate for HRP detection OPD, TMB and ECL (Electro-generated chemiluminescence) etc. can be mentioned, for example.
  • a substrate of alkaline phosphatase for example, a mixed substrate of nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphatase p-toluidinyl salt (BCIP) can be mentioned.
  • NBT nitroblue tetrazolium
  • BCIP 5-bromo-4-chloro-3-indolylphosphatase p-toluidinyl salt
  • kits eg, vials, tubes, ampoules, bottles, etc.
  • instructions for use in the above kit may be further included.
  • the detection method will be described later [8. Methods for detecting H5 subtype avian influenza virus] can be mentioned.
  • the kit according to the present invention includes, for example, an immunochromatographic kit comprising the antibody according to the present invention, an antibody other than the antibody according to the present invention, and members necessary for enhancing the detection sensitivity.
  • the method for detecting H5 subtype avian influenza virus according to the present invention comprises the step of reacting an antibody according to the present invention or an antibody fragment containing the variable region thereof with a sample prepared from a living body.
  • an antibody according to the present invention [2. Antibody according to the present invention and an antibody fragment containing the variable region thereof].
  • the step of reacting the above-mentioned antibody according to the present invention or the antibody fragment containing the variable region thereof with a sample prepared from the living body is prepared from the living body into a composition containing the antibody according to the present invention or the antibody fragment containing the variable region
  • the method may include the steps of contacting the sample, and detecting a reaction occurring between the sample and a composition containing the antibody or an antibody fragment containing the variable region thereof.
  • Methods used for detection of the above reaction include ELISA, radioimmunoassay, fluorescent immunoassay, western blot, immunochromatography, affinity chromatography, immunoprecipitation, immunodiffusion, hemagglutination inhibition test, etc. Can be mentioned.
  • Examples of methods for detecting the above-mentioned reaction include a sandwich method, a competitive method, a direct adsorption method and the like using an antibody labeled with a labeling agent. By these methods, the amount of the target antigen present in the sample can be measured.
  • the sample used in the detection method according to the present invention is not particularly limited as long as it can contain at least the protein of interest.
  • biological samples include cell samples, tissue samples and wiping fluid samples, and from the viewpoint of the ease of sampling method, wiping fluid samples are preferred.
  • the wiping fluid samples include tracheal wiping fluid, laryngopharyngeal wiping fluid, oral cavity wiping fluid, and wiping solution of the general excretory space of birds.
  • Organisms from which a biological sample is derived include, for example, mammals such as birds, humans and pigs. Among these, preferably, it is a bird, and among the birds, more preferably a fowl chicken.
  • the collected biological sample may be subjected to a test after extracting the protein as necessary or after removing an unnecessary component.
  • the obtained biological sample may be stored by a method suitable for the type of biological sample, such as cryopreservation, if necessary.
  • the detection method according to the present invention is a reaction between a sample prepared from a living body and an antibody fragment containing at least one type of antibody that recognizes H5 subtype avian influenza virus or its variable region other than the antibody according to the present invention
  • the method may further comprise the step of With respect to antibodies other than the antibody according to the present invention and the antibody fragment containing the variable region thereof and the antibody fragment containing the variable region [7. Kit according to the present invention].
  • the test method according to the present invention further determines the presence or absence of a virus in the biological sample, or the amount of H5 subtype avian influenza virus in the biological sample, more specifically, per unit amount of biological sample.
  • the method may include the step of measuring the amount of H5 subtype avian influenza virus included.
  • the amount of H5 subtype avian influenza virus is, for example, the amount of H5 subtype avian influenza virus protein.
  • the concept of measuring the amount of H5 subtype avian influenza virus contained per unit amount of biological sample includes both quantitative measurement and qualitative measurement, and in addition to concentration measurement, a format comparable to a control It includes presenting the amount of H5 subtype avian influenza virus. More specifically, for example, data comparison before acquisition of concentration conversion using a calibration curve or the like, or in the form of whether the amount of H5 subtype avian influenza virus exceeds a certain threshold or not The presentation of the results of
  • the method for detecting H5 subtype avian influenza virus according to the present invention may include the step of determining the possibility of infection of H5 subtype avian influenza virus in a subject individual.
  • determining the possibility of infection with the H5 subtype avian influenza virus means that the target individual is infected with the influenza virus regardless of whether or not the influenza has developed. Indicates to determine whether or not.
  • avian influenza in chickens include symptoms such as depression, flesh crown, pits and legs cyanosis, facial edema, neurological symptoms, feathering, and diarrhea.
  • a step of determining whether or not the H5 subtype avian influenza virus is excreted is mentioned as the step of the determination.
  • the method of judging by visual observation of the data of color development and numerical value etc. which were obtained in the method used in the process of detecting H5 subtype avian influenza virus mentioned above is mentioned about the virus-derived substance which has been developed .
  • the test individual when the amount of the virus in the biological sample of the test individual exceeds a predetermined threshold value as compared to a healthy control, the test individual is excreting the virus by H5 subtype avian influenza virus infection It is determined that Incidentally, the fact that the amount of virus exceeds a predetermined threshold value may be a result of quantitative measurement or a result of qualitative measurement, and it is needless to say that the comparison of specific numerical values is a relative amount of It is a concept that includes comparison (it is not necessary to actually calculate the amount, and it is determined whether it is higher or lower than a certain standard).
  • the above test of the control sample may be performed simultaneously with the test of the sample of the test individual, or may be performed separately. That is, the numerical value of the control sample to be compared with the numerical value of the test individual may be the value obtained in the test performed when the sample of the test individual is different from when it is tested.
  • the test of the control sample does not have to be performed by the individual who tests the test individual, and for example, the test value of the control sample already acquired and accumulated in a database or the like can be used as a threshold.
  • the numerical value of the sample of the healthy individual may be used directly, or the average value obtained when the numerical value of the sample of the healthy individual of a certain number is used as the population It is also good.
  • a cutoff value may be set in advance, and the numerical value of the test individual may be compared with this cutoff value.
  • the H5 subtype avian influenza virus contained in a sample is specifically, accurately, rapidly and highly sensitively detected than the existing detection method. Can.
  • the present invention includes any one of the following aspects.
  • the heavy chain variable region includes all of CDR1 shown in SEQ ID NO: 4, CDR2 shown in SEQ ID NO: 5 and CDR3 shown in SEQ ID NO: 6 or SEQ ID NO: 46, and a light chain variable region
  • the heavy chain variable region is substituted, deleted, inserted, and / or added with 1 to 13 amino acids in the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 2 1 to 10 amino acids are substituted, deleted, inserted, and / or made up of the amino acid sequence, and the light chain variable region is the amino acid sequence shown in SEQ ID NO: 3 or the amino acid sequence shown in SEQ ID NO: 3
  • An antibody fragment comprising the antibody according to ⁇ 1> or ⁇ 2>, or a variable region thereof, characterized in that it comprises an added amino acid sequence.
  • ⁇ 4> The antibody according to ⁇ 3>, wherein the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 45, and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 3 Or an antibody fragment comprising the variable region thereof.
  • ⁇ 5> Of the hemagglutinin HA1 region of the H5 subtype avian influenza virus shown in any of the following (1) to (4), specifically to the hemagglutinin HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10
  • An antibody fragment or antibody fragment according to any one of ⁇ 1> to ⁇ 4> characterized in that it binds: (1) an antibody consisting of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 44 or An antibody fragment comprising the variable region, (2) an antibody comprising an amino acid sequence in which 1 to 35 amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 44
  • An antibody fragment containing the variable region (3) 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 44
  • the present invention is directed to a polynucleotide comprising a sequence complementary to the polynucleot
  • An antibody consisting of an amino acid sequence encoded by a polynucleotide hybridizing under various conditions or an antibody fragment comprising the variable region thereof.
  • ⁇ 7> An antibody fragment comprising the antibody according to any one of ⁇ 1> to ⁇ 6> or a variable region thereof, which is a single-chain variable region fragment or a bivalent antibody.
  • ⁇ 8> The bivalent antibody according to ⁇ 1>, wherein the heavy chain consists of the amino acid sequence shown in SEQ ID NO: 26 or 52, and the light chain consists of the amino acid sequence shown in SEQ ID NO: 28; An antibody fragment comprising the antibody or the variable region thereof described in any of ⁇ 7>.
  • ⁇ 9> A polynucleotide encoding the antibody according to any one of ⁇ 1> to ⁇ 8> or an antibody fragment containing the variable region thereof.
  • ⁇ 10> A recombinant vector comprising the polynucleotide according to ⁇ 9>.
  • ⁇ 11> A transformant, wherein the polynucleotide according to ⁇ 9> or the recombinant vector according to ⁇ 10> is introduced.
  • a method for producing an antibody that specifically binds to a hemagglutinin HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 of the hemagglutinin HA1 region of H5 subtype avian influenza virus which comprises the following (a A) a method comprising the steps of (c): (a) immunizing the avian hemagglutinin HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 with a bird; (b) the above step (a) Obtaining a phage antibody library comprising a phage antibody which specifically binds to the above-mentioned hemagglutinin HA1 antigen polypeptide from the birds immunized with the above; and (c) the above-mentioned hemagglutinin HA1 among the phage antibodies obtained in the above step (b) Concentrating and selecting antibodies that specifically bind to the antigenic polypeptide.
  • ⁇ 13> The method according to ⁇ 12>, wherein the antibody is a single chain variable region fragment or a bivalent antibody.
  • ⁇ 14> A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 10.
  • ⁇ 15> A kit for detecting H5 subtype avian influenza virus contained in a sample, comprising an antibody according to any one of ⁇ 1> to ⁇ 8> or an antibody fragment containing a variable region thereof
  • a kit that is characterized by ⁇ 16> A method for detecting H5 subtype avian influenza virus contained in a sample, comprising: the antibody according to any one of ⁇ 1> to ⁇ 8> or an antibody fragment containing the variable region thereof Reacting the sample with the sample.
  • ⁇ 17> The method according to ⁇ 16>, wherein the sample is a swab of trachea, laryngopharynx, oral cavity or common excretory.
  • ⁇ 20> The antibody fragment according to ⁇ 18> or ⁇ 19>, or a variable region thereof, wherein the H5 subtype avian influenza virus is an H5N1 avian influenza virus.
  • ⁇ 21> Any one of ⁇ 18> to ⁇ 20>, which specifically binds to the region represented by the amino acid sequence shown in SEQ ID NO: 10 in the hemagglutinin HA1 region of H5 subtype avian influenza virus An antibody fragment comprising the described antibody or its variable region.
  • FIG. 1 is a diagram showing one step in the method for producing an antibody, and shows the steps for producing a scFv phage antibody library from the above-described chicken immunity and immune chicken spleen.
  • ⁇ Panning selection> The scFv phage antibody library was used to perform panning on a plate on which a synthetic peptide was immobilized. Panning was performed according to the method described in the reference: "Nakamura et al., J Vet Med Sci. 2004 Ju; 66 (7): 807-814". After five rounds of panning, the reactivity of the library was confirmed by ELISA using a plate on which a synthetic peptide was immobilized, and phages were screened from the library whose reactivity began to increase. The screening method is as follows. The phage was infected into E.
  • FIG. 2 is a diagram showing one step in the method for producing an antibody, and shows a step of panning selection of scFv phage library.
  • the screening was performed using the ELISA method shown in the item of ⁇ ELISA> below.
  • ⁇ ELISA> For screening, PBS containing 5 ⁇ g / ml of synthetic peptide was placed at 50 ⁇ l / well in a 96-well plate (442404, Thermo), and the antigen was immobilized overnight at 4 ° C. After immobilization, the wells were blocked with PBS containing 25% Block Ace (UK-B80, DS Pharma Biomedical), and the culture supernatant containing the scFv phage antibody was reacted.
  • PBS containing 5 ⁇ g / ml of synthetic peptide was placed at 50 ⁇ l / well in a 96-well plate (442404, Thermo), and the antigen was immobilized overnight at 4 ° C. After immobilization, the wells were blocked with PBS containing 25% Block Ace (UK-B80, DS Pharma Biomedical), and the culture supernatant containing the scFv phage antibody was reacted.
  • FIG. 3 is a diagram showing one step in the method for producing an antibody according to an example of the present invention, and shows a step of screening a scFv phage library using ELISA.
  • FIG. 4 is a diagram showing the variable region of the amino acid sequence of the obtained antibody.
  • Example 1 the amino acid sequence of full length L chain is shown in SEQ ID NO: 28 and the base sequence of full length L chain is shown in SEQ ID NO: 29
  • SEQ ID NO: 28 the sequence of No. 6-2-5 obtained in Example 1 is referred to as wild type, and a sequence obtained by modifying the same in Example 2 below is described as a variant type sequence.
  • FIG. 5 is a diagram showing one step in the method for producing an antibody, and shows steps for producing a bivalent antibody by recombination into a bivalent antibody expression vector.
  • the resulting purified antibody was subjected to SDS-PAGE electrophoresis using 5-20% c-Page (C520L, ATTO), and the degree of purification was confirmed by CBB staining (178-00551, Wako).
  • FIG. 6 shows the results of CBB staining of wild-type bivalent antibody protein after electrophoresis.
  • the antibody titer was measured as follows. Similarly, PBS containing 5 ⁇ g / ml of recombinant hemagglutinin (rHA) (derived from H5N1 virus, A / Vietnam / 1203/2004, CT6450, Protein Sciences Corp.) or bovine serum albumin (BSA (bovine serum albumin)) was similarly immobilized After blocking, purified mouse / chicken chimeric antibody (No. 6-2-5) was reacted at a concentration of 0 to 2 ⁇ g / ml.
  • rHA recombinant hemagglutinin
  • BSA bovine serum albumin
  • FIG. 7 shows the antibody titer of a wild-type bivalent antibody.
  • FIG. 8 shows antibody binding to wild-type bivalent antibody H5N1 avian influenza virus variants and to a plurality of H5 avian influenza virus subtypes other than H5.
  • the purified wild-type bivalent antibody (No. 6-2-5) obtained in this example has almost no binding ability to avian influenza virus except subtype H5, It also showed high binding ability and specificity to the H5 subtype avian influenza virus.
  • Example 2 Preparation of Mutant Antibody and Evaluation of Reactivity (2-1. Preparation of antibody) ⁇ Preparation of mutant library> Using the H chain of the above-mentioned clone No. 6-2-5 as a template, random mutations in which 9 base random mutations were introduced into CDR3 of the H chain of No. 6-2-5 using Inverse PCR method were used. Seven types were prepared (SMK-101, TOYOBO). The primer sets used are shown in SEQ ID NOS: 30-43.
  • variable regions of the H chain and the variable region of the L chain of the seven random variants obtained were amplified by PCR, respectively, and joined with a linker to prepare a scFv phage library.
  • the details of the method of preparing the scFv phage library were performed according to the method described in the reference: "Nakamura et al., J Vet Med Sci. 2004 Ju; 66 (7): 807-814".
  • ⁇ Panning selection> The scFv phage antibody library was used to perform panning against rHA (11700-V08H, Sino Biological Inc.). Panning was performed according to the method described in the reference: "Nakamura et al., J Vet Med Sci. 2004 Ju; 66 (7): 807-814". After 4 rounds of panning, the increase in reactivity was confirmed by ELISA. Screening was performed from the second round of panning library whose reactivity has been increased. The screening was performed using the same method as that described in (1-1. Preparation of antibody) in ⁇ Example 1> ⁇ Panningselection>.
  • Sequence confirmation was performed by outsourcing (Eurofin Genomics). As a result, acquisition of an antibody of a novel sequence was confirmed.
  • the amino acid sequence of full length single chain antibody of newly obtained clone No. 6-2-5-21 is shown in SEQ ID NO: 44.
  • the sequence of No. 6-2-5-21, which is a newly obtained clone, had the same sequence as that of No. 6-2-5 except for the sequence of CDR3 of the H chain.
  • the amino acid sequence of the H chain variable region of No. 6-2-521 is shown in SEQ ID NO: 45, the amino acid sequence of CDR3 in SEQ ID NO: 46, and the base sequence of CDR3 in SEQ ID NO: 47.
  • FIG. 9 is a diagram showing an alignment of the amino acid sequence of the CDR3 of the H chain of wild type antibody No. 6-2-5 with the amino acid sequence of the CDR3 of the H chain of mutant antibody No. 6-2-5-21. is there.
  • mouse / chicken chimeric antibody (IgG1) expression vector H chain expression vector: restriction vector treated with SacII (R0157S, BioLabs) and NheI (R0131S, BioLabs) expression vector: pcDNA4 / myc-His, L chain expression vector: Recombination into pcDNA3 / myc-His, Invitrogen) using GeneArt® Seamless Cloning and Assembly (A14606, lifetechnologies).
  • the mouse chimeric expression vector used was the vector described in Tateishi et al., J Vet Med Sci. 2008 Apr; 70 (4): 397-400.
  • the sequences of the variable region amplification primers are shown in SEQ ID NOs: 48 to 51.
  • FIG. 10 is a diagram showing the antibody titer of a mutated bivalent antibody.
  • Biacore T200 Biacore T200, GE Healthcare
  • Biacore T200 Biacore T200, GE Healthcare
  • the affinity was measured using Mouse Antibody Capture Kit (BR-1008-38, GE Healthcare) .
  • a rabbit anti-mouse polyclonal antibody was immobilized on the surface of the CM5 chip by amine coupling using free carboxyl groups on the surface of the CM5 chip using NHS / EDC according to a standard protocol provided by the manufacturer.
  • Nos. 6-2-5 and 6-2-5-21 were captured on rabbit anti-mouse polyclonal antibodies.
  • Different concentrations of rHA were applied to Biacore T200 to generate kinetic sensorgrams and to calculate binding constants. The results are shown in Table 1 below.
  • FIG. 11 is a view showing antibody binding of each of the mutant bivalent antibodies to each of the avian influenza viruses.
  • the three-dimensional structure analysis software Pymol (web page: http://www.pymol.org/) was used, and the H5 subtype registered in the protein data bank (web page: http://pdbj.org/)
  • the three-dimensional structure of HA protein (PDB ID: 2IBX) was compared with the sequence of the amino acid of H5 subtype.
  • FIG. 12 shows the sequence (right) of a synthetic polypeptide used as an antigen of H5N1 avian influenza virus, and the three-dimensional structure (left) of an HA protein.
  • the amino acid sequence consisting of 16 amino acids shown as SEQ ID NO: 10 is a clade 0-9 different in H5N1 subtype in the HA1 region. It was saved among the stocks of In addition, this amino acid sequence was conserved in 99.4% of H5 subtypes of 2812 strains isolated after 1997 registered in NCBI.
  • amino acid sequence consisting of 16 amino acids was highly retained between strains different in H5 subtype, and appeared on the surface of the protein in terms of conformation.
  • H1 to H16 subtype avian influenza viruses 8781 for H1 subtypes from NCBI influenza virus resources (web page: http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html) H2 subtype 231, H3 subtype 4536, H4 subtype 608, H5 subtype 2812, H6 subtype 687, H7 subtype 787, H8 subtype 65, 739 for H9 subtype, 190 for H10 subtype, 187 for H11 subtype, 84 for H12 subtype, 53 for H13 subtype, 4 for H14 subtype, 12 for H15 subtype, H16 The amino acid sequence of the full-length HA gene of 19 avian influenza viruses was obtained for the subtype.
  • FIG. 13 is a diagram showing sequence identity among all subtypes of H1-H16 subtype avian influenza virus and among all clades present in H5N1 subtype avian influenza virus.
  • A shows sequence identity among all subtypes in H1-H16 subtype avian influenza virus
  • b shows sequence identity among all clades present in H5N1 subtype avian influenza virus Show the sex.

Abstract

　In order to provide an antibody or antibody fragment including the variable region of said antibody for the H5 subtypes of the avian influenza virus, an antigen polypeptide, and uses thereof, the present invention provides: an antibody or antibody fragment including the variable region of said antibody, which specifically binds to the HA1 antigen polypeptide of the HA1 region of the H5 subtypes of the avian influenza virus, said antigen polypeptide comprising the amino acid sequence represented by SEQ ID NO: 10; the HA1 antigen polypeptide comprising the amino acid sequence represented by SEQ ID NO: 10; and uses of the antibody and antibody fragment including the variable region of the antibody.

Description

抗体またはその可変領域を含む抗体断片、抗原ポリペプチド、およびその利用Antibody or antibody fragment containing the variable region thereof, antigenic polypeptide, and use thereof
　本発明は、抗体またはその可変領域を含む抗体断片、抗原ポリペプチド、およびその利用に関する。 The present invention relates to antibody fragments comprising an antibody or its variable region, antigenic polypeptides, and uses thereof.
　Ｈ５Ｎ１亜型高病原性鳥インフルエンザウイルス（ＨＰＡＩＶ）をはじめとするＨ５亜型高病原性鳥インフルエンザウイルスは、２００３年以降各地で家禽における鳥インフルエンザの発生を引き起こし、アジアを中心に常在化の様相を呈している。このような高病原性鳥インフルエンザウイルスが鳥類に感染し、その感染症が蔓延すると、畜産業等に甚大な損害を及ぼすと共に、ヒトへの感染も危惧される。日本国内におけるＨＰＡＩＶの防疫は摘発淘汰を基本としているため、現場レベルにおける早期検出が重要である。現在現場で簡易的に用いられる検査系として、Ａ型インフルエンザウイルスを検出するものが用いられているが、Ｈ５Ｎ１亜型高病原性鳥インフルエンザを即座に判定するものではない。既存の診断キットとして、例えば、インフルエンザ核タンパク質を標的抗原とした診断キットが挙げられるが、Ｈ５Ｎ１亜型鳥インフルエンザウイルス特異性がなく、他のインフルエンザウイルスにも反応するという問題がある。また、その他の例としては、Ｈ５Ｎ１亜型不活化ウイルスのリコンビナントＨ５ヘマグルチニン（ＨＡ、ウイルス赤血球凝集素）タンパク質を標的抗原とした診断キットが挙げられるが、イムノドミナントな抗原に対する抗体ができるため、ウイルスの遺伝子に変異があった場合、反応性が低下または検出できないという可能性があるという問題がある。そのため、Ｈ５亜型鳥インフルエンザウイルス感染の診断試薬の開発が望まれている。効果的な診断試薬の開発のためには、広くＨ５亜型鳥インフルエンザウイルスを認識する抗体作製が必要である。 H5 subtype highly pathogenic avian influenza virus including H5N1 subtype highly pathogenic avian influenza virus (HPAIV) causes outbreak of avian influenza in poultry from 2003 onwards, aspect of localization mainly in Asia It is When such highly pathogenic avian influenza virus infects birds and the infection spreads, it causes great damage to the livestock industry and the like, and there is also a fear of human infection. Since HPAIV's epidemic prevention in Japan is based on a cover-up investigation, early detection at the field level is important. At present, as a test system that is simply used in the field, one that detects influenza A virus is used, but it is not one that rapidly determines H5N1 subtype highly pathogenic avian influenza. As an existing diagnostic kit, for example, a diagnostic kit using influenza nucleoprotein as a target antigen is mentioned, but there is a problem that it has no H5N1 avian influenza virus specificity and also responds to other influenza viruses. Other examples include a diagnostic kit using recombinant H5 hemagglutinin (HA, virus hemagglutinin) protein of H5N1 subtype inactivated virus as a target antigen, but because an antibody against an immunodominant antigen can be produced, the virus If there is a mutation in the gene, there is a problem that the reactivity may be reduced or undetectable. Therefore, development of a diagnostic reagent for H5 subtype avian influenza virus infection is desired. In order to develop an effective diagnostic reagent, it is necessary to produce an antibody that broadly recognizes the H5 subtype avian influenza virus.
　例えば、特許文献１には、Ｈ５亜型鳥インフルエンザウイルスのＨＡエンベロープ糖タンパク質またはＮ１亜型のノイラミニダーゼ糖タンパク質に特異的に結合するモノクローナル抗体が記載されている。また、特許文献２には、ＨＡのＨＡ２サブユニットにおけるエピトープを認識および結合することができ、Ｈ５亜型の鳥インフルエンザウイルスに対して中和活性を有するモノクローナル抗体等が記載されている。 For example, Patent Document 1 describes a monoclonal antibody that specifically binds to HA envelope glycoprotein of H5 subtype avian influenza virus or neuraminidase glycoprotein of N1 subtype. In addition, Patent Document 2 describes a monoclonal antibody and the like which can recognize and bind an epitope in the HA2 subunit of HA, and have a neutralizing activity against the H5 subtype avian influenza virus.
　また、特許文献３には、鳥インフルエンザウイルスのＨＡタンパク質のステム領域のエピトープに結合し、Ｈ５Ｎ１亜型を包含するＡ型インフルエンザウイルスを中和する、単離されたモノクローナル抗体が記載されている。 In addition, Patent Document 3 describes an isolated monoclonal antibody that binds to an epitope of the stem region of the avian influenza virus HA protein and neutralizes influenza A virus including H5N1 subtype.
日本国公開特許公報「特表２０１０－５３９１６２号公報（２０１０年１２月１６日公表）」Japanese Published Patent Publication "Tokukai 2010-539162 (published on December 16, 2010)" 日本国公開特許公報「特表２０１０－５０２２０７号公報（２０１０年１月２８日公表）」Japanese Published Patent Publication "Tokukai 2010-502207" (published on January 28, 2010) 日本国公開特許公報「特表２０１１－５０６３４４号公報（２０１１年３月３日公表）」Japanese published patent publication "Japanese Patent Application Publication No. 2011-506344 (released on March 3, 2011)"
　上述の特許文献１～３における抗体のスクリーニングに際し、抗体の活性の評価は、ウイルスの中和試験によって行われている。例えば血球凝集阻害試験などにより、ＨＡタンパク質活性の有無を試験することによって行われている。すなわち、従来技術における抗体は、ヒト等に感染した場合、当該抗体を用いてＨ５Ｎ１亜型鳥インフルエンザウイルスを中和することを目的としている。その為、それらの抗体を検出試薬に供したとしても、ウイルスの変異が起こってしまった場合、抗体の反応性が失われ、ウイルスを検出できなくなる可能性が高い。 In screening of antibodies in the above-mentioned Patent Documents 1 to 3, evaluation of antibody activity is carried out by virus neutralization test. For example, it is carried out by testing the presence or absence of HA protein activity by a hemagglutination inhibition test or the like. That is, when the antibody in the prior art infects a human etc., it aims at neutralizing the H5N1 subtype avian influenza virus using the said antibody. Therefore, even if these antibodies are used as detection reagents, if a mutation occurs in the virus, there is a high possibility that the reactivity of the antibody is lost and the virus can not be detected.
　しかし、亜型ウイルス特異的な検出の標的となるウイルス赤血球凝集素（ヘマグルチニン、ＨＡ）タンパク質においてアミノ酸置換が頻繁に起こっており、広くＨ５亜型ＨＡを認識する抗体の作製が困難である。そのため、抗原検出によるＨ５亜型鳥インフルエンザウイルス感染の診断試薬の普及が遅れている。 However, amino acid substitution frequently occurs in virus hemagglutinin (hemagglutinin, HA) protein, which is a target of subtype-specific virus detection, and it is difficult to produce an antibody that widely recognizes H5 subtype HA. Therefore, the spread of diagnostic reagents for H5 subtype avian influenza virus infection by antigen detection has been delayed.
　上記の課題を解決するために、本願発明者らは鋭意検討をし、広くＨ５亜型ＨＡを認識し得る抗体の作出に成功した。 In order to solve the above problems, the present inventors diligently studied and succeeded in producing an antibody capable of broadly recognizing H5 subtype HA.
　本発明は以下の何れかの一態様を包含する。
＜１＞　Ｈ５亜型鳥インフルエンザウイルスのヘマグルチニンＨＡ１領域のうち、配列番号１０に示されるアミノ酸配列からなるヘマグルチニンＨＡ１抗原ポリペプチドに特異的に結合することを特徴とする、抗体またはその可変領域を含む抗体断片。
＜２＞　Ｈ５亜型鳥インフルエンザウイルスのヘマグルチニンＨＡ１領域のうち、配列番号１０に示されるアミノ酸配列からなるヘマグルチニンＨＡ１抗原ポリペプチドに特異的に結合する抗体を製造する方法であって、下記の（ａ）～（ｃ）の工程を含むことを特徴とする、方法：（ａ）配列番号１０に示されるアミノ酸配列からなるヘマグルチニンＨＡ１抗原ポリペプチドをトリに免疫する工程；（ｂ）上記工程（ａ）で免疫したトリから上記ヘマグルチニンＨＡ１抗原ポリペプチドに特異的に結合するファージ抗体を含むファージ抗体ライブラリーを得る工程；および（ｃ）上記工程（ｂ）で得られたファージ抗体のうち、上記ヘマグルチニンＨＡ１抗原ポリペプチドに特異的に結合する抗体を濃縮および選抜する工程。
＜３＞　配列番号１０に示されるアミノ酸配列からなることを特徴とする、ポリペプチド。 The present invention includes any one of the following aspects.
<1> An antibody or a variable region thereof characterized by specifically binding to a hemagglutinin HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 in the hemagglutinin HA1 region of H5 subtype avian influenza virus Antibody fragment.
<2> A method for producing an antibody that specifically binds to a hemagglutinin HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 of the hemagglutinin HA1 region of H5 subtype avian influenza virus, which comprises the following (a A) a method comprising the steps of (c): (a) immunizing the avian hemagglutinin HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 with a bird; (b) the above step (a) Obtaining a phage antibody library comprising a phage antibody which specifically binds to the above-mentioned hemagglutinin HA1 antigen polypeptide from the birds immunized with the above; and (c) the above-mentioned hemagglutinin HA1 among the phage antibodies obtained in the above step (b) Concentrating and selecting antibodies that specifically bind to the antigenic polypeptide.
<3> A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 10.
　本発明に係る抗体およびその可変領域を含む抗体断片は、Ｈ５亜型鳥インフルエンザウイルスに特異的に結合し、且つＨ５以外の他の亜型の鳥インフルエンザウイルスには結合しないという優れた効果を奏する。したがって、本発明に係る抗体を用いれば、Ｈ５亜型鳥インフルエンザウイルスを正確、迅速且つ簡便に検出することができる。 The antibody according to the present invention and the antibody fragment containing the variable region exhibit an excellent effect of specifically binding to H5 subtype avian influenza virus and not binding to other subtypes of avian influenza virus other than H5. . Therefore, H5 subtype avian influenza virus can be detected accurately, rapidly and simply by using the antibody according to the present invention.
本発明の実施例１に係る、抗体の作製方法における一工程を示す図である。ニワトリの免疫および免疫ニワトリ脾臓からのｓｃＦｖファージ抗体ライブラリーの作製の工程を示している。It is a figure which shows 1 process in the preparation methods of the antibody based on Example 1 of this invention. Figure 7 shows the steps of immunization of a chicken and preparation of a scFv phage antibody library from immunized chicken spleen. 本発明の実施例１に係る、抗体の作製方法における一工程を示す図である。ｓｃＦｖファージライブラリーの、パニング選択の工程を示している。It is a figure which shows 1 process in the preparation methods of the antibody based on Example 1 of this invention. The process of panning selection of scFv phage library is shown. 本発明の実施例１に係る、抗体の作製方法における一工程を示す図である。ｓｃＦｖファージライブラリーの、ＥＬＩＳＡを用いたスクリーニングの工程を示している。It is a figure which shows 1 process in the preparation methods of the antibody based on Example 1 of this invention. The steps of screening an scFv phage library using ELISA are shown. 本発明の実施例１に係る、得られた野生型の単鎖抗体のアミノ酸配列の可変領域を示す図である。It is a figure which shows the variable region of the amino acid sequence of the obtained wild type single chain antibody based on Example 1 of this invention. 本発明の実施例１に係る、抗体の作製方法における一工程を示す図である。二価抗体発現ベクターへの組換えによる二価抗体の作製の工程を示している。It is a figure which shows 1 process in the preparation methods of the antibody based on Example 1 of this invention. The process of production of bivalent antibody by recombination to bivalent antibody expression vector is shown. 本発明の実施例１に係る、野生型の二価抗体タンパク質を電気泳動後にＣＢＢ染色した結果を示す図である。FIG. 6 shows the results of CBB staining of a wild-type bivalent antibody protein according to Example 1 of the present invention after electrophoresis. 本発明の実施例１に係る、野生型の二価抗体の抗体価を示す図である。It is a figure which shows the antibody titer of the wild type bivalent antibody based on Example 1 of this invention. 本発明の実施例１に係る、野生型の二価抗体のＨ５Ｎ１亜型鳥インフルエンザウイルスの変異体および複数種類のＨ５以外の亜型鳥インフルエンザウイルスに対する抗体結合性を示す図である。It is a figure which shows the antibody binding property with respect to the variant of H5N1 subtype avian influenza virus of the wild type bivalent antibody, and two or more types of subtype avian influenza viruses other than H5 based on Example 1 of this invention. 本発明の実施例２に係る、野生型の単鎖抗体のＨ鎖のＣＤＲ３のアミノ酸配列と変異型抗体のＨ鎖のＣＤＲ３のアミノ酸配列とのアライメントを示す図である。It is a figure which shows alignment with the amino acid sequence of CDR3 of the heavy chain of wild type single chain antibody based on Example 2 of this invention, and the amino acid sequence of CDR3 of the heavy chain of variant antibody. 本発明の実施例２に係る、変異型の二価抗体の抗体価を示す図である。It is a figure which shows the antibody titer of a mutated bivalent antibody based on Example 2 of this invention. 本発明の実施例２に係る、変異型の二価抗体の、各亜型鳥インフルエンザウイルスに対する抗体結合性を示す図である。It is a figure which shows the antibody binding property with respect to each subtype avian influenza virus of the mutated bivalent antibody based on Example 2 of this invention. 本発明の実施例に係る、Ｈ５亜型鳥インフルエンザウイルスの抗原として用いた合成ペプチドの配列（右）およびＨＡタンパク質の立体構造（左）を示す図である。It is a figure which shows the sequence (right) of the synthetic peptide used as an antigen of H5 subtype avian influenza virus based on the Example of this invention, and the three-dimensional structure (left) of HA protein. 本発明の参考例に係る、Ｈ１～Ｈ１６亜型鳥インフルエンザウイルスにおけるすべての亜型間（ａ）およびＨ５Ｎ１亜型鳥インフルエンザウイルスに存在しているすべてのクレード間（ｂ）の配列同一性を示す図である。Show the sequence identity among all subtypes (a) in H1 to H16 subtype avian influenza virus and among all clades (b) present in H5N1 subtype avian influenza virus according to the reference example of the present invention FIG.
　本発明の実施の形態について説明すれば、以下の通りである。なお、本発明は、これに限定されるものではない。 It will be as follows if embodiment of this invention is described. The present invention is not limited to this.
　〔用語等の定義〕
　本明細書において、「ポリヌクレオチド」は、「核酸」または「核酸分子」とも換言でき、ヌクレオチドの重合体を意図している。また、「塩基配列」は、「核酸配列」または「ヌクレオチド配列」とも換言でき、特に言及のない限り、デオキシリボヌクレオチドの配列またはリボヌクレオチドの配列を意図している。また、ポリヌクレオチドは、一本鎖であっても二本鎖であってもよく、一本鎖の場合はセンス鎖であってもアンチセンス鎖であってもよい。 [Definition of terms, etc.]
As used herein, "polynucleotide" may also be referred to as "nucleic acid" or "nucleic acid molecule" and is intended to be a polymer of nucleotides. "Base sequence" can also be referred to as "nucleic acid sequence" or "nucleotide sequence", and unless otherwise stated, is intended the sequence of deoxyribonucleotides or the sequence of ribonucleotides. The polynucleotide may be single-stranded or double-stranded, and in the case of single-stranded polynucleotide, it may be a sense strand or an antisense strand.
　本明細書において、「ポリペプチド」は、「タンパク質」または「タンパク質断片」とも換言できる。 In the present specification, "polypeptide" can also be referred to as "protein" or "protein fragment".
　本明細書において、「Ｈ５亜型鳥インフルエンザウイルス」および「Ｈ５Ｎ１亜型鳥インフルエンザウイルス」は、鳥インフルエンザウイルスのサブタイプの一種を指している。 As used herein, "H5 subtype avian influenza virus" and "H5N1 subtype avian influenza virus" refer to a type of avian influenza virus subtype.
　「鳥インフルエンザウイルス」は、それらの核タンパク質およびマトリックスタンパク質の抗原特異性に従って分類される。鳥インフルエンザウイルスは主にＡ、ＢおよびＣ血清型に類別される。このうち、Ａ型鳥インフルエンザウイルスは、８個のＲＮＡ分節を有し、１０個のウイルスタンパク質をコードしている。 "Avian influenza viruses" are classified according to their nuclear and matrix protein antigen specificity. Avian influenza virus is mainly classified into A, B and C serotypes. Among these, type A avian influenza virus has eight RNA segments and encodes ten viral proteins.
　「ヘマグルチニン」（以下、“ＨＡ”と称する）は、インフルエンザウイルスのエンベロープ糖タンパク質を意味する。ＨＡは、インフルエンザウイルスの宿主細胞への吸着および侵入を可能にする。「ノイラミニダーゼ」（以下、“ＮＡ”と称する）は、ウイルス粒子が感染後期に細胞表面から離れる際にシアル酸を切断する働きを有しており、感染性を獲得する役割を担っている。 "Hemagglutinin" (hereinafter referred to as "HA") refers to the envelope glycoprotein of influenza virus. HA enables adsorption and entry of influenza virus into host cells. "Neuraminidase" (hereinafter referred to as "NA") has a function of cleaving sialic acid when virus particles leave the cell surface at the late stage of infection, and plays a role in acquiring infectivity.
　全ての既知のＡ型インフルエンザウイルスは鳥類に由来し、Ａ型のインフルエンザウイルスは、ＨＡおよびＮＡの抗原性の性質によって亜型にさらに分類される。ＨＡには、Ｈ１～Ｈ１６の１６個のサブタイプがあり、ＮＡには、Ｎ１～Ｎ９の９個のサブタイプがある。これらのＨＡとＮＡとの種々の組合せを有する、種々のＡ型インフルエンザウイルスのサブタイプが存在している。これらの組み合わせのうち、鳥由来インフルエンザウイルスのＨ５Ｎ１、Ｈ６Ｎ１、Ｈ７Ｎ３、Ｈ７Ｎ７、Ｈ７Ｎ９、Ｈ９Ｎ２およびＨ１０Ｎ８の亜型は、ヒトへの感染を引き起こすことが知られている。Ｈ５およびＨ７亜型のインフルエンザウイルスのうち、一部のウイルスは本来の宿主である水禽類に対する病原性はそれほど高くないが、鶏等の家禽に感染した場合死に至らしめるほどの高い病原性を示すことが知られ、高病原性鳥インフルエンザウイルス（ＨＰＡＩＶ）と呼ばれている。さらに、近年では、家禽におけるＨ５Ｎ１亜型ウイルス感染によるインフルエンザの発生から、ヒトへの感染事例が相次いで報告されている。このような状況から、Ｈ５Ｎ１亜型ＨＰＡＩＶが、ヒトからヒトへ効率的に感染できるように変異し、新型インフルエンザウイルスとなることが懸念されている。 All known influenza A viruses are derived from birds, and influenza viruses of type A are further classified into subtypes by the antigenic nature of HA and NA. HA has 16 subtypes of H1 to H16, and NA has 9 subtypes of N1 to N9. There are different subtypes of influenza A virus with different combinations of these HA and NA. Among these combinations, the H5N1, H6N1, H7N3, H7N7, H7N9, H9N2 and H10N8 subtypes of avian influenza viruses are known to cause human infection. Of the H5 and H7 subtypes of influenza viruses, some viruses are not highly pathogenic to the natural host waterfowl, but exhibit high virulence that causes death when infected with poultry such as chickens It is known that it is called highly pathogenic avian influenza virus (HPAIV). Furthermore, in recent years, cases of human infection have been successively reported from the outbreak of influenza caused by H5N1 subtype virus infection in poultry. From such a situation, there is concern that H5N1 subtype HPAIV is mutated so as to be able to efficiently infect human to human, and it becomes a novel influenza virus.
　近年アジアを中心に家禽に常在化しているＨ５Ｎ１亜型鳥インフルエンザウイルスは、ＨＡ遺伝子の塩基配列の相同性に基づいて、さらに複数種のクレードに分類することができる。米国生物工学情報センター（NCBI; National Center for Biotechnology Information）により提供されるＧｅｎＢａｎｋデータベース（ウェブページ：http://www.ncbi.nlm.nih.gov/genbank/）において、所定のアクセッション番号（Accession No.）により登録されているＨ５Ｎ1亜型ＨＰＡＩＶのＨＡ遺伝子は、WHO/OIE/FAO H5N1 Evolution Working Groupによって、クレード０から９まで、およびさらにそのサブクレードに分類されている。例えば、クレード０としてA/Hong Kong/156/1997、クレード１としてA/duck/Vietnam/2/2007、クレード２．１．１としてA/chicken/Indonesia/7/2003、クレード２．１．２としてA/Indonesia/CDC596/2006、クレード２．１．３としてA/Indonesia/CDC940/2006、クレード２．２としてA/Egypt/1394-NAMRU3/2007、クレード２．３．１としてA/duck/Hunan/127/2005、クレード２．３．２としてA/whooper swan/Akita/1/2008、クレード２．３．３としてA/chicken/Guiyang/3055/2005、クレード２．３．４としてA/Viet Nam/HN31242/2007、クレード２．４としてA/Chicken/Yunnan/493/2005、クレード２．５としてA/chicken/Yamaguchi/7/2004、クレード３としてA/Pheasant/HongKong/FY155/2001、クレード４としてA/goose/Guiyang/337/2006、クレード５としてA/goose/Guangxi/914/2004、クレード６としてA/duck/Hubei/wg/2002、クレード７としてA/Beijing/01/2003、クレード８としてA/chicken/Hong Kong/61.9/2002、およびクレード９としてA/Goose/Shantou/1621/2005が、それぞれのクレードの代表株として挙げられる。 The H5N1 avian influenza virus, which has been localized to poultry in Asia in recent years, can be further classified into multiple clades based on the homology of the base sequences of the HA gene. Accession number (Accession) in the GenBank database (web page: http://www.ncbi.nlm.nih.gov/genbank/) provided by the National Center for Biotechnology Information (NCBI). HA gene of H5N1 subtype HPAIV registered by No.) is classified into clades 0 to 9 and further sub-clades thereof by WHO / OIE / FAO H5N1 Evolution Working Group. For example, A / Hong Kong / 156/1997 as clade 0, A / duck / Vietnam / 2/2007 as clade 1, A / chicken / Indonesia / 7/2003 as clade 2.1.1, clade 2.1.2 As A / Indonesia / CDC 596/2006, Clade 2.1.3 as A / Indonesia / CDC 940/2006, Clade 2.2 as A / Egypt / 1394-NAMRU 3/2007, Clade 2.3.1 as A / duck / Hunan / 127/2005, A / whooper swan / Akita / 1/2008 as clade 2.3.2, A / chicken / Guiyang / 3055/2005 as clade 2.3.3, A / whol as clade 2.3.4 Viet Nam / HN 31242/2007, A / Chicken / Yunnan / 493/2005 as clade 2.4, A / chicken / Yamaguchi / 7/2004 as clade 2.5, A / Pheasant / Hong Kong / FY 155/2001, as clade 3 A / goose / Guiyang / 337/2006 as clade A / goose / Guangxi / 914/2004 as clade 5, A / duck / Hubei / wg / 2002 as clade 6, A / Beijing / 01/2003 as clade 7 Craded 8 A / chicken / Hong Kong / 61.9 / 2002, and A / Goose / Shantou / 1621/2005 as clade 9 it may be mentioned as representative strains of each clade.
　また、本明細書において、「ＨＡ１領域」および「ＨＡ２領域」とは、それぞれ、インフルエンザウイルスのヘマグルチニンタンパク質（ＨＡ）のタンパク質分解酵素切断部位およびそれに連なる融合ペプチド領域を挟む２つの領域を指している。また、ＨＡタンパク質のＨＡ１－ＨＡ２接合部におけるタンパク質分解による切断はウイルスの増殖性と関連しており、この切断部位周辺に疎水性アミノ酸が連続して存在していることがＨＰＡＩＶに特徴的である。そして、ＨＡ１領域およびＨＡ２領域は、鶏における高病原性に関与する。 Furthermore, in the present specification, the “HA1 region” and the “HA2 region” respectively indicate two regions flanking the proteolytic cleavage site of the hemagglutinin protein (HA) of influenza virus and the fusion peptide region linked thereto. . Moreover, proteolytic cleavage at the HA1-HA2 junction of the HA protein is associated with viral proliferative properties, and the continuous presence of hydrophobic amino acids around this cleavage site is characteristic of HPAIV . And the HA1 and HA2 regions are responsible for the high virulence in chickens.
　また、上述のＨ５Ｎ１亜型におけるすべてのクレード間のＨＡ１領域全域の配列同一性は、各クレードの株について、上に挙げたものを用いた場合、いずれの異なるクレードの組み合わせにおいても、９１～９９％の配列同一性を有している。一方で、Ｈ１～Ｈ１６すべての亜型間のＨＡ１領域全域の配列同一性は、いずれの異なる亜型の組み合わせにおいても、７～５１％の範囲である。中でも、Ｈ１～Ｈ４亜型およびＨ６～Ｈ１６亜型それぞれのＨＡ１領域全域と、Ｈ５型のＨＡ１領域全域との配列同一性は、非常に低く、すべて８％～２１％の範囲である。 Also, the sequence identity across the HA1 region between all clades in the H5N1 subtypes described above is 91-99 for all different clade combinations, using those listed above for each clade strain. % Sequence identity. On the other hand, the sequence identity across the HA1 region among all the H1 to H16 subtypes is in the range of 7 to 51% for any combination of different subtypes. Above all, the sequence identity between the entire HA1 region of each of the H1 to H4 subtypes and the H6 to H16 subtypes and the entire HA1 region of the H5 type is very low, all in the range of 8% to 21%.
　また、上述のＨ５Ｎ１亜型におけるすべてのクレード間のＨＡ２領域全域の配列同一性は、各クレードの株について、上に挙げたものを用いた場合、いずれのクレードの組み合わせにおいても、９４～１００％の配列同一性を有している。一方で、Ｈ１～Ｈ１６すべての亜型間のＨＡ１領域全域の配列同一性は、いずれの異なる亜型の組み合わせにおいても、４２～８１％の範囲である。中でも、Ｈ１～Ｈ４型およびＨ６～Ｈ１６亜型それぞれのＨＡ２領域全域と、Ｈ５亜型のＨＡ２領域全域との配列同一性は、４５～７５％である。 Also, the sequence identity across the HA2 region between all clades in the above mentioned H5N1 subtypes is 94 to 100% in all clade combinations, using the ones listed above for each clade strain Have the sequence identity of On the other hand, the sequence identity across the HA1 region between all H1 to H16 subtypes is in the range of 42 to 81% for any combination of different subtypes. Above all, the sequence identity between the entire HA2 region of each of the H1 to H4 type and the H6 to H16 subtype and the entire HA2 region of the H5 subtype is 45 to 75%.
　また、本明細書において、「二価抗体」とは、１分子あたり抗原結合部位を２つ有する抗体、つまり、抗原との結合価が二価の抗体を意味する。上記「二価抗体」は、それぞれ相同な２本の軽鎖（軽鎖可変領域および軽鎖定常領域）と２本の重鎖（重鎖可変領域および重鎖定常領域）とがジスルフィド結合（Ｓ－Ｓ結合）により結合した構造を有する抗体である。但し、完全長の抗体分子である必要はなく、２本の重鎖がＳ－Ｓ結合により結合することができる構造、すなわち少なくともＦ（ａｂ’）２フラグメントを有する構造であれば「二価抗体」の範疇に含まれる。 Moreover, in the present specification, the term "bivalent antibody" means an antibody having two antigen binding sites per molecule, that is, an antibody having a bivalent binding to an antigen. The above "bivalent antibody" is composed of two light chains (a light chain variable region and a light chain constant region) and two heavy chains (a heavy chain variable region and a heavy chain constant region) which are respectively homologous to each other and which are disulfide linked (S An antibody having a structure bound by -S bond). However, it is not necessary that the antibody molecule be a full-length antibody molecule, and a “bivalent antibody” as long as it has a structure in which two heavy chains can be linked by S—S bond, ie, a structure having at least F (ab ′) 2 fragment It is included in the category of
　また、本明細書において、「一本鎖可変領域断片（single-chain variable fragment、（ｓｃＦｖ））」とは、軽鎖（Light chain、Ｌ鎖とも称する）可変領域と重鎖（Heavy chain、Ｈ鎖とも称する）可変領域とがリンカーによってつながれ、２種類の可変領域が接近することよって、１つの抗原結合部位を形成した抗体を意味する。上記「一本鎖可変領域断片」は、１分子あたり１つの抗原結合部位を有する抗体である。例えば、ファージディスプレイ法を用いた抗体作成方法によって得られる抗体を指す。本明細書においては「単鎖抗体」とも記載する。 Moreover, in the present specification, “single-chain variable fragment (scFv)” refers to a light chain (also referred to as L chain) variable region and a heavy chain (Heavy chain, H) It refers to an antibody in which one variable region is linked to a variable region (also referred to as a chain) and two variable regions are close to form one antigen binding site. The above "single-chain variable region fragment" is an antibody having one antigen binding site per molecule. For example, it refers to an antibody obtained by an antibody production method using phage display. Also described herein as "single chain antibodies".
　本明細書において、「Ａおよび／またはＢ」は、ＡおよびＢとＡまたはＢとの双方を含む概念であり、「ＡおよびＢの少なくとも一方」とも換言できる。 In the present specification, “A and / or B” is a concept including both A and B and A or B, and can be reworded as “at least one of A and B”.
　〔１．本発明に係るＨＡ１抗原ポリペプチド〕
　本発明に係るＨＡ１抗原ポリペプチドは、配列番号１０に示されるアミノ酸配列からなるポリペプチドであり得る。 [1. HA1 antigen polypeptide according to the present invention]
The HA1 antigen polypeptide according to the present invention may be a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10.
　また、本発明に係るＨＡ１抗原ポリペプチドは、配列番号１０に示されるアミノ酸配列からなるポリペプチド中の、連続する１５個以上１６個以下のアミノ酸からなるポリペプチドであり得る。 Furthermore, the HA1 antigen polypeptide according to the present invention may be a polypeptide consisting of 15 or more and 16 or less consecutive amino acids in a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10.
　本明細書中において使用される場合、「配列番号１０に示されるアミノ酸配列からなるＨＡ１抗原ポリペプチド」の機能が阻害されない限り、「配列番号１０に示されるアミノ酸配列からなるポリペプチド」にリンカーペプチドが付加されたもの、他のアミノ酸またはタンパク質が連結されたもの（例えば、タグ化されたタンパク質または融合タンパク質）であってもよい。 As used herein, as long as the function of the “HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10” is not inhibited, a “linker peptide to the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10” May be added, other amino acids or proteins may be linked (for example, a tagged protein or a fusion protein).
　配列番号１０に示されるアミノ酸配列からなるＨＡ１抗原ポリペプチドに連結される、リンカーポリペプチドとしては、１０～２５個のアミノ酸からなるポリペプチド等が挙げられ、タグとしては、Ｈｉｓタグ、ＭｙｃタグおよびＦｌａｇタグ等が挙げられる。 The linker polypeptide linked to the HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 includes a polypeptide consisting of 10 to 25 amino acids, etc. The tag includes His tag, Myc tag and Flag tag etc. are mentioned.
　本発明に係るＨＡ１抗原ポリペプチドの作製方法は、化学合成であっても発現ベクターを用いるものであってもよい。化学合成による場合は、本発明に係るＨＡ１抗原ポリペプチドは、公知のポリペプチド合成法によって製造することができる。ポリペプチド合成法としては、液相ポリペプチド合成法、固相ペプチド合成法等の化学合成法が挙げられるが、これらに限定されない。発現ベクターを用いる場合は、発現ベクターを導入した形質転換体からポリペプチドを生成しても、インビトロ翻訳系を用いてポリペプチドを生成してもよい。例えば、配列番号１０に示されるアミノ酸配列をコードするポリヌクレオチドを挿入した発現ベクターを導入した宿主細胞中にて目的のポリペプチドを生成することができる。例えば、以下の〔４．組換えベクター〕の項目に記載の方法が好適に用いられる。また、形質転換体についても、例えば、以下の〔５．形質転換体〕の項目に記載の方法が用いられる。 The method for producing the HA1 antigen polypeptide according to the present invention may be chemical synthesis or may use an expression vector. In the case of chemical synthesis, the HA1 antigen polypeptide of the present invention can be produced by known polypeptide synthesis methods. Examples of polypeptide synthesis methods include, but are not limited to, chemical synthesis methods such as liquid phase polypeptide synthesis methods and solid phase peptide synthesis methods. When an expression vector is used, the polypeptide may be produced from a transformant into which the expression vector has been introduced, or may be produced using an in vitro translation system. For example, a target polypeptide can be produced in a host cell into which an expression vector into which a polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 10 has been inserted has been introduced. For example, the following [4. The method described in the item of recombinant vector] is preferably used. Moreover, for the transformant, for example, the following [5. The method described in item [transformant] is used.
　なお、本発明に係るＨＡ１抗原ポリペプチドは、宿主細胞中において安定的に発現していることが好ましいが、一過性に発現していてもよい。このようにして生成されたポリペプチドを、公知の方法に従って精製することができる。ポリペプチドの精製方法としては、特に限定されないが、例えば、ゲル濾過クロマトグラフィー、イオン交換クロマトグラフィー、およびアフィニティクロマトグラフィー等が挙げられる。 The HA1 antigen polypeptide according to the present invention is preferably stably expressed in the host cell, but may be transiently expressed. The polypeptide thus produced can be purified according to known methods. The method for purifying the polypeptide is not particularly limited, and examples thereof include gel filtration chromatography, ion exchange chromatography, affinity chromatography and the like.
　本発明に係るＨＡ１抗原ポリペプチドは、実施例および図１２にも示す通り、Ｈ５Ｎ１亜型鳥インフルエンザウイルスのＨＡ１領域のアミノ酸配列のうち、Ｈ５Ｎ１亜型の異なる株間において非常に高く保持されており、且つ立体構造上、Ｈ５Ｎ１亜型鳥インフルエンザウイルスタンパク質の表面に出ている部位のアミノ酸配列である。一方で、Ｈ１～Ｈ４型およびＨ６～Ｈ１６型において、このアミノ酸配列に対応するアミノ酸配列の配列同一性は非常に低い。すなわち本発明に係るＨＡ１抗原ポリペプチドは、宿主内においても変異が起こりにくく且つ、構造上、抗体との結合性能が非常に優れている。したがって、本発明に係るＨＡ１抗原ポリペプチドであれば、Ｈ５Ｎ１亜型鳥インフルエンザウイルスに非常に特異的な抗体の作製に好適に利用することができる。 The HA1 antigen polypeptide according to the present invention is very highly retained among strains having different H5N1 subtypes among the amino acid sequences of the HA1 region of the H5N1 avian influenza virus virus, as shown in Examples and FIG. And it is the amino acid sequence of the site | part which has come out on the surface of H5N1 subtype avian influenza virus protein by three-dimensional structure. On the other hand, in H1-H4 and H6-H16, the sequence identity of the amino acid sequence corresponding to this amino acid sequence is very low. That is, the HA1 antigen polypeptide according to the present invention is resistant to mutation even in the host, and is structurally very excellent in its ability to bind to an antibody. Therefore, the HA1 antigen polypeptide according to the present invention can be suitably used for the production of an antibody very specific for the H5N1 avian influenza virus.
　〔２．本発明に係る抗体およびその可変領域を含む抗体断片〕
　本発明に係る抗体およびその可変領域を含む抗体断片は、Ｈ５亜型鳥インフルエンザウイルスのＨＡ１領域のうち、配列番号１０に示されるアミノ酸配列からなるヘマグルチニンＨＡ１抗原ポリペプチドに特異的に結合することを特徴とする。 [2. Antibody according to the present invention and antibody fragment containing the variable region thereof
The antibody according to the present invention and an antibody fragment containing the variable region thereof specifically bind to a hemagglutinin HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 in the HA1 region of H5 subtype avian influenza virus. It features.
　ここで、「ＨＡ１抗原ポリペプチドに特異的に結合する」とは、当該ＨＡ１抗原ポリペプチドの配列を認識して結合することを意味しており、例えば、当該ＨＡ１抗原ポリペプチドの配列を含む、より長い配列を有するポリペプチド等にも結合する。 Here, "specifically binds to HA1 antigen polypeptide" means to recognize and bind to the sequence of the HA1 antigen polypeptide, and includes, for example, the sequence of the HA1 antigen polypeptide, It also binds to polypeptides having longer sequences.
　本明細書において、上記「抗体」は、免疫グロブリン（ＩｇＡ、ＩｇＤ、ＩｇＥ、ＩｇＹ、ＩｇＧ、ＩｇＭおよびこれらのＦａｂフラグメント、Ｆ（ａｂ’）２フラグメント、Ｆｃフラグメント）を意味し、例えば、モノクローナル抗体、ポリクローナル抗体、抗イディオタイプ抗体、キメラ化抗体、ヒト化抗体等の二価抗体および一本鎖可変領域断片および抗体の相補性決定領域（ＣＤＲ）を含むペプチド等が挙げられるが、本発明はこれらに限定されない。 In the present specification, the above "antibody" means immunoglobulin (IgA, IgD, IgE, IgY, IgG, IgM and Fab fragments thereof, F (ab ') 2 fragment, Fc fragment) thereof, for example, a monoclonal antibody And bivalent antibodies such as polyclonal antibodies, anti-idiotype antibodies, chimerized antibodies, humanized antibodies and single chain variable region fragments and peptides including the complementarity determining regions (CDRs) of the antibodies, etc. It is not limited to these.
　また、本発明の抗体の断片であって、上記抗体の可変領域を含む抗体断片も本発明の範囲に含まれる。かかる抗体断片としては、上記抗体の可変領域を含む抗体断片であってＨ５亜型鳥インフルエンザウイルスを認識する抗体断片であれば特に限定されるものではない。かかる抗体断片は、本発明の抗体のＨ鎖全長、Ｌ鎖全長、Ｈ鎖の可変領域、およびＬ鎖の可変領域のいずれかの領域の断片であってもよい。このような抗体断片であれば、例えば、フローサイトメトリー法またはＥＬＩＳＡ法に好適に用いることができる。また、抗体断片は、ヒトその他の動物の抗体に移植することによって、キメラ抗体を作製するために好適に用いることができる。 In addition, an antibody fragment of the present invention, which comprises the variable region of the above antibody, is also included in the scope of the present invention. The antibody fragment is not particularly limited as long as it is an antibody fragment containing the variable region of the above-mentioned antibody and recognizes the H5 subtype avian influenza virus. Such an antibody fragment may be a fragment of any of the full-length H chain, full-length L chain, variable region of H chain, and variable region of L chain of the antibody of the present invention. Such antibody fragments can be suitably used, for example, in flow cytometry or ELISA. Also, antibody fragments can be suitably used to produce chimeric antibodies by transplantation into antibodies of human or other animals.
　また、本発明に係る抗体およびその可変領域を含む抗体断片の一態様は、配列番号１０に示されるアミノ酸配列からなるＨＡ１抗原ポリペプチド中の、連続する１５個以上１６個以下のアミノ酸に特異的に結合する。 In addition, one embodiment of the antibody according to the present invention and an antibody fragment containing the variable region thereof is specific for consecutive 15 or more and 16 or less amino acids in the HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 Bond to
　また、上述の抗体またはその可変領域を含む抗体断片と、ＨＡ１抗原ポリペプチドとの特異的な結合は、ＨＡ１抗原ポリペプチド中の１箇所の特定の領域に生じているか、または２か所以上の異なる領域に同時に生じていてもよい。 In addition, specific binding of the antibody fragment containing the above-mentioned antibody or its variable region to the HA1 antigen polypeptide may occur in one specific region in the HA1 antigen polypeptide, or at two or more places. It may occur simultaneously in different areas.
　本発明に係る抗体の一態様は、Ｈ鎖が配列番号２６に示されるアミノ酸配列からなり、且つ、Ｌ鎖が配列番号２８に示されるアミノ酸配列からなる二価抗体である。また、本発明に係る抗体の別の例は、Ｈ鎖が配列番号５２に示されるアミノ酸配列からなり、且つ、Ｌ鎖が配列番号２８に示されるアミノ酸配列からなる二価抗体である。 One aspect of the antibody according to the present invention is a bivalent antibody in which the H chain consists of the amino acid sequence shown in SEQ ID NO: 26 and the L chain consists of the amino acid sequence shown in SEQ ID NO: 28. In addition, another example of the antibody according to the present invention is a bivalent antibody in which the H chain consists of the amino acid sequence shown in SEQ ID NO: 52 and the L chain consists of the amino acid sequence shown in SEQ ID NO: 28.
　また、配列番号２に示すアミノ酸配列は、本発明の一態様に係る抗体のＨ鎖可変領域のアミノ酸配列であり、配列番号３に示すアミノ酸配列は、本発明の一態様に係る抗体のＬ鎖可変領域のアミノ酸配列である。図４に本発明の一態様に係る抗体のＨ鎖可変領域のアミノ酸配列およびＬ鎖可変領域のアミノ酸配列を示す。図４に示すように、本発明の一態様に係る抗体のＨ鎖可変領域は、四角で囲んで示しているＣＤＲの領域である、ＣＤＲ１、ＣＤＲ２およびＣＤＲ３を有している。すなわち本発明の一態様に係る抗体のＨ鎖可変領域は、配列番号１に示すアミノ酸配列の１番目～１３０番目のアミノ酸配列からなり、３１番目～３５番目のアミノ酸配列からなるＣＤＲ１、配列番号１に示すアミノ酸配列の５０番目～６６番目のアミノ酸配列からなるＣＤＲ２および配列番号１に示すアミノ酸配列の９９番目～１１９番目のアミノ酸配列からなるＣＤＲ３を有する。また、図４に示すように、本発明の一態様に係る抗体のＬ鎖可変領域は、四角で囲んで示しているＣＤＲ１、ＣＤＲ２およびＣＤＲ３を有している。すなわち本発明の一態様に係る抗体のＬ鎖可変領域は、配列番号１に示すアミノ酸配列の１４８番目～２７０番目のアミノ酸配列からなり、配列番号１に示すアミノ酸配列の１６８番目～１７６番目のアミノ酸配列からなるＣＤＲ１、配列番号１に示すアミノ酸配列の１９３番目～２０３番目のアミノ酸配列からなるＣＤＲ２、および配列番号１に示すアミノ酸配列の２３２番目～２４０番目のアミノ酸配列からなるＣＤＲ３を有する。また、本発明の一態様の抗体は、配列番号１に示すアミノ酸配列からなる抗体である。また、本発明の別の一態様の抗体はＨ鎖可変領域が配列番号４５に示すアミノ酸配列からなり、且つ、Ｌ鎖可変領域が配列番号３に示すアミノ酸配列からなる。さらに、本発明の別の一態様の抗体は配列番号４４に示すアミノ酸配列からなる抗体である。 The amino acid sequence shown in SEQ ID NO: 2 is the amino acid sequence of the H chain variable region of the antibody according to one aspect of the present invention, and the amino acid sequence shown in SEQ ID NO: 3 is the L chain of the antibody according to one aspect of the present invention It is an amino acid sequence of a variable region. FIG. 4 shows the amino acid sequence of the H chain variable region and the amino acid sequence of the L chain variable region of the antibody according to one aspect of the present invention. As shown in FIG. 4, the H chain variable region of the antibody according to one embodiment of the present invention has CDR1, CDR2 and CDR3 which are regions of CDRs shown by squares. That is, the heavy chain variable region of the antibody according to one aspect of the present invention consists of the amino acid sequence of 1st to 130th amino acid sequences shown in SEQ ID NO: 1 and CDR1 consisting of the 31st to 35th amino acid sequences; The CDR2 consists of the 50th to 66th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 1 and the CDR3 consists of the 99th to 119th amino acid sequence of the amino acid sequence shown in SEQ ID NO: 1. In addition, as shown in FIG. 4, the light chain variable region of the antibody according to one aspect of the present invention has CDR1, CDR2 and CDR3 which are indicated by a box. That is, the L chain variable region of the antibody according to one aspect of the present invention consists of the amino acid sequence 148 to 270 of the amino acid sequence shown in SEQ ID NO: 1, and the amino acid 168 to 176 in the amino acid sequence shown in SEQ ID NO: 1 It has CDR1 consisting of the sequence, CDR2 consisting of the amino acid sequence 193 to 203 of the amino acid sequence shown in SEQ ID NO: 1, and CDR3 consisting of the amino acid sequence 232 to 240 of the amino acid sequence shown in SEQ ID NO: 1. In addition, the antibody of one embodiment of the present invention is an antibody consisting of the amino acid sequence shown in SEQ ID NO: 1. Furthermore, the antibody of another aspect of the present invention comprises the amino acid sequence of which the heavy chain variable region is shown in SEQ ID NO: 45, and the amino acid sequence of which the light chain variable region is shown in SEQ ID NO: 3. Furthermore, an antibody of another aspect of the present invention is an antibody consisting of the amino acid sequence shown in SEQ ID NO: 44.
　本発明の一態様に係る抗体のＨ鎖可変領域のＣＤＲ１、ＣＤＲ２およびＣＤＲ３のアミノ酸配列をそれぞれ配列番号４～６に示し、本発明に係る抗体のＬ鎖可変領域のＣＤＲ１、ＣＤＲ２およびＣＤＲ３のアミノ酸配列をそれぞれ配列番号７～９に示す。本発明の別の一態様に係る抗体のＨ鎖可変領域のＣＤＲ３のアミノ酸配列および塩基配列をそれぞれ配列番号４６および４７に示す。 The amino acid sequences of CDR1, CDR2 and CDR3 of the H chain variable region of the antibody according to one aspect of the present invention are shown in SEQ ID NOs: 4 to 6, respectively, and amino acids of CDR1, CDR2 and CDR3 of the L chain variable region of the antibody according to the present invention The sequences are shown in SEQ ID NOs: 7-9, respectively. The amino acid sequence and nucleotide sequence of CDR3 of the H chain variable region of the antibody according to another aspect of the present invention are shown in SEQ ID NOs: 46 and 47, respectively.
　また、本発明に係る抗体の他の態様は、Ｈ鎖可変領域中に、配列番号４に示されるＣＤＲ１、配列番号５に示されるＣＤＲ２および配列番号６に示されるＣＤＲ３のすべてを含んでおり、且つ、Ｌ鎖可変領域中に、配列番号７に示されるＣＤＲ１、配列番号８に示されるＣＤＲ２および配列番号９に示されるＣＤＲ３のすべてを有している、抗体またはその可変領域を含む抗体断片である。 In addition, another embodiment of the antibody according to the present invention comprises all of CDR1 shown in SEQ ID NO: 4, CDR2 shown in SEQ ID NO: 5 and CDR3 shown in SEQ ID NO: 6 in the H chain variable region, And an antibody fragment comprising an antibody or its variable region, having all of CDR1 shown in SEQ ID NO: 7, CDR2 shown in SEQ ID NO: 8 and CDR3 shown in SEQ ID NO: 9 in the L chain variable region. is there.
　また、本発明に係る抗体のさらに他の態様は、Ｈ鎖可変領域中に、配列番号４に示されるＣＤＲ１、配列番号５に示されるＣＤＲ２および配列番号４６に示されるＣＤＲ３のすべてを含んでおり、且つ、Ｌ鎖可変領域中に、配列番号７に示されるＣＤＲ１、配列番号８に示されるＣＤＲ２および配列番号９に示されるＣＤＲ３のすべてを有している、抗体またはその可変領域を含む抗体断片である。 Furthermore, still another embodiment of the antibody according to the present invention comprises all of CDR1 shown in SEQ ID NO: 4, CDR2 shown in SEQ ID NO: 5 and CDR3 shown in SEQ ID NO: 46 in the H chain variable region. And an antibody fragment comprising an antibody or its variable region, having all of CDR1 shown in SEQ ID NO: 7, CDR2 shown in SEQ ID NO: 8 and CDR3 shown in SEQ ID NO: 9 in the L chain variable region It is.
　また、本発明に係る抗体のさらに他の態様は、Ｈ鎖可変領域中に、配列番号４に示されるＣＤＲ１、配列番号５に示されるＣＤＲ２および配列番号６または配列番号４６に示されるＣＤＲ３のうち少なくとも１つを含んでおり、且つ、Ｌ鎖可変領域中に、配列番号７に示されるＣＤＲ１、配列番号８に示されるＣＤＲ２および配列番号９に示されるＣＤＲ３のうちの少なくとも１つを有している、抗体またはその可変領域を含む抗体断片である。 Furthermore, still another embodiment of the antibody according to the present invention is the CDR1 represented by SEQ ID NO: 4, the CDR2 represented by SEQ ID NO: 5 and the CDR3 represented by SEQ ID NO: 6 or SEQ ID NO: 46 in the H chain variable region. And at least one of CDR1 shown in SEQ ID NO: 7, CDR2 shown in SEQ ID NO: 8 and CDR3 shown in SEQ ID NO: 9 in the L chain variable region An antibody or an antibody fragment containing the variable region thereof.
　また、本発明に係る抗体のさらに他の態様は、Ｈ鎖可変領域中に、配列番号４に示されるＣＤＲ１、配列番号５に示されるＣＤＲ２および配列番号６または配列番号４６に示されるＣＤＲ３のうち少なくとも１つを有しているか、またはＬ鎖可変領域中に、配列番号７に示されるＣＤＲ１、配列番号８に示されるＣＤＲ２および配列番号９に示されるＣＤＲ３のうちの少なくとも１つを有している、抗体またはその可変領域を含む抗体断片である。 Furthermore, still another embodiment of the antibody according to the present invention is the CDR1 represented by SEQ ID NO: 4, the CDR2 represented by SEQ ID NO: 5 and the CDR3 represented by SEQ ID NO: 6 or SEQ ID NO: 46 in the H chain variable region. Or at least one of CDR1 shown in SEQ ID NO: 7, CDR2 shown in SEQ ID NO: 8 and CDR3 shown in SEQ ID NO: 9 in the L chain variable region An antibody or an antibody fragment containing the variable region thereof.
　また、本発明に係る抗体、またはその可変領域を含む抗体断片は、以下の特徴を有するものも包含している。 Furthermore, the antibody according to the present invention, or an antibody fragment containing the variable region thereof also includes those having the following characteristics.
　Ｈ鎖可変領域が、配列番号２に示されるアミノ酸配列、または、配列番号２に示されるアミノ酸配列において、１～１３個、好ましくは１～６個、より好ましくは１～２個または３個、さらに好ましくは１個のアミノ酸が置換、欠失、挿入、および／もしくは付加されたアミノ酸配列からなり、且つ
　Ｌ鎖可変領域が、配列番号３に示されるアミノ酸配列、または、配列番号３に示されるアミノ酸配列において、１～１０個、好ましくは１～５個、より好ましくは１～２個または３個、さらにより好ましくは１個のアミノ酸が置換、欠失、挿入、および／もしくは付加されたアミノ酸配列からなる、抗体またはその可変領域を含む抗体断片。 In the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 2, the H chain variable region is 1 to 13, preferably 1 to 6, more preferably 1 to 2 or 3 More preferably, one amino acid consists of a substituted, deleted, inserted and / or added amino acid sequence, and the light chain variable region is shown in the amino acid sequence shown in SEQ ID NO: 3 or in SEQ ID NO: 3 Amino acid in which 1 to 10, preferably 1 to 5, more preferably 1 to 2 or 3 and even more preferably 1 amino acid is substituted, deleted, inserted and / or added in the amino acid sequence An antibody or an antibody fragment comprising a variable region thereof consisting of a sequence.
　一例として、本発明に係る抗体およびその可変領域を含む抗体断片は、Ｈ鎖可変領域が配列番号２に示すアミノ酸配列からなり、且つＬ鎖可変領域が配列番号３に示すアミノ酸配列からなる抗体またはその可変領域を含む抗体断片を挙げることができる。 As one example, an antibody according to the present invention and an antibody fragment containing the variable region thereof is an antibody or antibody wherein the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 2 and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 3 Mention may be made of antibody fragments comprising the variable region.
　本発明に係る抗体およびその可変領域を含む抗体断片の別の例は、Ｈ鎖可変領域が、配列番号４５に示されるアミノ酸配列からなり、且つＬ鎖可変領域が、配列番号３に示されるアミノ酸配列からなる抗体またはその可変領域を含む抗体断片を挙げることができる。ここで、配列番号４５に示されるアミノ酸配列は、配列番号２に示すアミノ酸配列の１０５番目のアミノ酸ＹをＲに、１０６番目のアミノ酸ＳをＲに、１０７番目のアミノ酸ＹをＶに、それぞれ置換した配列である。このような配列を有する抗体は、Ｈ５亜型鳥インフルエンザウイルスへの特異性を維持しながら、ＨＡ１抗原に対するより高い反応性を有しており、より好ましい。 Another example of the antibody according to the present invention and an antibody fragment containing the variable region thereof, wherein the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 45 and the light chain variable region is the amino acid shown in SEQ ID NO: 3 Mention may be made of an antibody consisting of a sequence or an antibody fragment comprising the variable region thereof. Here, in the amino acid sequence shown in SEQ ID NO: 45, the 105th amino acid Y of the amino acid sequence shown in SEQ ID NO: 2 is substituted with R, the 106th amino acid S with R, and the 107th amino acid Y with V Sequence. An antibody having such a sequence has higher reactivity to the HA1 antigen while maintaining its specificity for the H5 subtype avian influenza virus, which is more preferable.
　さらに、本発明に係る抗体またはその可変領域を含む抗体断片は、Ｈ５亜型鳥インフルエンザウイルスのヘマグルチニンＨＡ１領域のうち、配列番号１０に示されるアミノ酸配列からなるヘマグルチニンＨＡ１抗原ポリペプチドに特異的に結合する抗体またはその可変領域を含む抗体断片であって、以下の（１）～（４）の何れかに示すものを包含する。
（１）配列番号１または４４に示されるアミノ酸配列からなる抗体またはその可変領域を含む抗体断片、
（２）配列番号１または４４に示されるアミノ酸配列において１～３５個のアミノ酸が置換、欠失、挿入、および/または付加されたアミノ酸配列からなる抗体またはその可変領域を含む抗体断片、
（３）配列番号１または４４に示されるアミノ酸配列に対して９０％以上の配列同一性を有する抗体またはその可変領域を含む抗体断片、
（４）上記（１）に記載の抗体またはその可変領域を含む抗体断片をコードするポリヌクレオチドと相補的な配列からなるポリヌクレオチドに対して、ストリンジェントな条件下においてハイブリダイズするポリヌクレオチドによってコードされるアミノ酸配列からなる抗体またはその可変領域を含む抗体断片。なお、ストリンジェントな条件については、本発明に係るポリヌクレオチドの欄で後述する。 Furthermore, an antibody fragment according to the present invention or an antibody fragment containing the variable region thereof specifically binds to a hemagglutinin HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 in the hemagglutinin HA1 region of H5 subtype avian influenza virus Or an antibody fragment containing the variable region thereof, which is shown in any of the following (1) to (4):
(1) An antibody consisting of the amino acid sequence shown in SEQ ID NO: 1 or 44 or an antibody fragment containing the variable region thereof
(2) An antibody fragment comprising an amino acid sequence in which 1 to 35 amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 1 or 44, or an antibody fragment comprising the variable region thereof
(3) An antibody fragment having a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO: 1 or 44, or an antibody fragment comprising the variable region thereof
(4) A polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of a sequence complementary to a polynucleotide encoding an antibody described in (1) above or an antibody fragment containing the variable region thereof Or an antibody fragment comprising the variable region thereof. The stringent conditions will be described later in the section of the polynucleotide according to the present invention.
　また、本発明に係る抗体の一態様は、下記に示す、（ａ）～（ｃ）の工程を含む製造方法によって得られたものである。 In addition, one aspect of the antibody according to the present invention is obtained by the production method including the steps of (a) to (c) shown below.
　（ａ）配列番号１０に示されるアミノ酸配列からなるヘマグルチニンＨＡ１抗原ポリペプチドをトリに免疫する工程；（ｂ）上記工程（ａ）で免疫したトリから上記ヘマグルチニンＨＡ１抗原ポリペプチドに特異的に結合するファージ抗体を含むファージ抗体ライブラリーを得る工程；および（ｃ）上記工程（ｂ）で得られたファージ抗体のうち、上記ヘマグルチニンＨＡ１抗原ポリペプチドに特異的に結合する抗体を濃縮および選抜する工程。 (A) immunizing the bird with a hemagglutinin HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10; (b) specifically binding to the hemagglutinin HA1 antigen polypeptide from the birds immunized in the step (a) A step of obtaining a phage antibody library containing a phage antibody; and (c) a step of concentrating and selecting an antibody which specifically binds to the hemagglutinin HA1 antigen polypeptide among the phage antibodies obtained in the step (b).
　上記工程を含む製造方法については、以下の〔６．本発明に係る抗体の製造方法〕において詳細に記載する。 The following [6. The method for producing an antibody according to the present invention] will be described in detail.
　また、本発明に係る抗体およびその可変領域を含む抗体断片は、一本鎖可変領域断片または二価抗体であり、好ましくは二価抗体である。 Furthermore, the antibody according to the present invention and the antibody fragment containing the variable region thereof are a single chain variable region fragment or a bivalent antibody, preferably a bivalent antibody.
　本発明に包含される単鎖抗体の例としては、配列番号４４に示されるアミノ酸配列からなる抗体が挙げられる。 Examples of single chain antibodies encompassed by the present invention include antibodies consisting of the amino acid sequence shown in SEQ ID NO: 44.
　本発明に包含される単鎖抗体の他の例としては、配列番号１に示されるアミノ酸配列の２５２番目～３５１番目のアミノ酸を配列番号４４に示されるアミノ酸配列の２５２番目～３５９番目のアミノ酸に置換したアミノ酸配列からなる抗体が挙げられる。 As another example of the single-chain antibody included in the present invention, amino acids 252 to 351 of the amino acid sequence shown in SEQ ID NO: 1 to amino acids 252 to 359 in the amino acid sequence shown in SEQ ID NO: 44 The antibody which consists of the substituted amino acid sequence is mentioned.
　本発明に包含される単鎖抗体のさらに他の例としては、配列番号４４に示されるアミノ酸配列の２５２番目～３５９番目のアミノ酸を配列番号１に示されるアミノ酸配列の２５２番目～３５１番目のアミノ酸に置換したアミノ酸配列からなる抗体が挙げられる。 As yet another example of the single-chain antibody included in the present invention, the amino acids 252 to 359 of the amino acid sequence shown in SEQ ID NO: 44 are the amino acids 252 to 351 of the amino acid sequence shown in SEQ ID NO: 1 And antibodies consisting of the amino acid sequence substituted for
　また、本発明に係る二価抗体の一例は、以下の特性：可変領域がニワトリ由来であり、定常領域がマウスＩｇＧ１由来である、二価のマウスキメラ型抗ＨＡ１抗体であって、免疫グロブリンのサブタイプはＩｇＧ１であり、分子量は約１５０ｋＤａである；を有するものである。 In addition, an example of the bivalent antibody according to the present invention is a bivalent mouse chimeric anti-HA1 antibody having the following characteristics: a variable region derived from a chicken and a constant region derived from a mouse IgG1, which is an immunoglobulin The subtype is IgG1 and the molecular weight is about 150 kDa.
　本発明に係る二価抗体のさらに具体的な一例としては、Ｈ鎖が配列番号２６に示されるアミノ酸配列からなり、且つ、Ｌ鎖が配列番号２８に示されるアミノ酸配列からなるものが挙げられる。さらに、本発明に係るより好ましい二価抗体の他の例としては、Ｈ鎖が配列番号５２に示されるアミノ酸配列からなり、且つ、Ｌ鎖が配列番号２８に示されるアミノ酸配列からなるものが挙げられる。 A further specific example of the bivalent antibody according to the present invention is that in which the H chain consists of the amino acid sequence shown in SEQ ID NO: 26 and the L chain consists of the amino acid sequence shown in SEQ ID NO: 28. Furthermore, as another example of a more preferable bivalent antibody according to the present invention, one in which the H chain consists of the amino acid sequence shown in SEQ ID NO: 52 and the L chain consists of the amino acid sequence shown in SEQ ID NO: 28 Be
　本発明に係る抗体およびその可変領域を含む抗体断片は、標識剤によって、標識化された抗体も包含している。標識化された抗体は、以下の〔７．本発明に係るキット〕および〔８．Ｈ５亜型鳥インフルエンザウイルスを検出する方法〕において用いることができる。 The antibody according to the present invention and the antibody fragment containing the variable region also encompass an antibody labeled with a labeling agent. The labeled antibody has the following [7. Kit according to the present invention] and [8. Method for detecting H5 subtype avian influenza virus].
　上述の標識剤としては、酵素、酵素基質、放射性同位元素、発光物質、蛍光物質、ビオチンおよび着色物質等が挙げられる。酵素の例としては、ペルオキシダーゼ、β－ガラクトシダーゼ、アルカリホスファターゼ、グルコースオキシダーゼ、アセチルコリンエステラーゼおよびグルコース－６－リン酸脱水素酵素を挙げることができる。これら酵素と抗体との結合は、マレイミド化合物およびＮ－ヒドロキシスクシンイミドエステル化合物等の架橋剤を用いる公知の方法により行うことができる。酵素基質としては、使用する酵素に応じて公知の物質を使用することができる。例えば、酵素としてペルオキシダーゼを使用する場合には、ＯＰＤ（オルトフェニレンジアミン）およびＴＭＢ（テトラメチルベンジジン）等を、また酵素としてアルカリホスファターゼを用いる場合には、ニトロブルーテトラゾリウム（ＮＢＴ）および５－ブロモ－４－クロロ－３－インドリルホスファターゼｐ－トルイジニル塩（ＢＣＩＰ）の混合基質等を用いることができる。 Examples of the above-mentioned labeling agent include enzymes, enzyme substrates, radioactive isotopes, luminescent substances, fluorescent substances, biotin, coloring substances and the like. Examples of enzymes include peroxidase, β-galactosidase, alkaline phosphatase, glucose oxidase, acetylcholinesterase and glucose-6-phosphate dehydrogenase. Coupling between these enzymes and antibodies can be carried out by known methods using crosslinking agents such as maleimide compounds and N-hydroxysuccinimide ester compounds. As the enzyme substrate, known substances can be used depending on the enzyme used. For example, when using peroxidase as an enzyme, OPD (orthophenylenediamine) and TMB (tetramethylbenzidine) etc., and when using alkaline phosphatase as an enzyme, nitro blue tetrazolium (NBT) and 5-bromo- A mixed substrate of 4-chloro-3-indolylphosphatase p-toluidinyl salt (BCIP) or the like can be used.
　放射性同位元素としては、１２５Ｉ、３Ｈまたは３５Ｓ等の通常のラジオイムノアッセイで用いられているものを使用することができる。本発明に係る抗体への放射標識は公知の方法を用いて行うことができる。上記蛍光色素としては、フルオレセインイソチオシアネート、テトラメチルローダミンイソチオシアネートおよびフィコエリスリン等の通常の蛍光抗体法に用いられるものを使用することができる。また、蛍光物質として、蛍光シリカナノ粒子を使用してもよい。また、上記発光物質としては、イソルミノール、アクリジンエステルおよびルシゲニン等を用いることができる。この際、標識の方法は、公知の方法を用いることができる。また、上記着色物質としては、例えば、着色ラテックス粒子および金コロイド等を挙げることができる。 As radioactive isotopes, those used in conventional radioimmunoassays such as 125 I, 3 H or 35 S can be used. Radiolabeling to the antibody according to the present invention can be performed using a known method. As the fluorescent dye, those used for ordinary fluorescent antibody methods such as fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate and phycoerythrin can be used. Alternatively, fluorescent silica nanoparticles may be used as the fluorescent material. In addition, as the light emitting substance, isoluminol, acridine ester, lucigenin or the like can be used. At this time, a known method can be used as a method of labeling. Moreover, as said coloring substance, a coloring latex particle, a gold colloid, etc. can be mentioned, for example.
　また、本発明に係る抗体およびその可変領域を含む抗体断片は、固体支持体に固相化されていてもよい。使用できる固体支持体としては、ポリスチレン、ポリカーボネート、ポリプロピレンまたはポリビニール性のマイクロタイタープレート、試験管、キャピラリー、ビーズ（ラテックス粒子および金属化合物など）、膜（リポソームなど）およびフィルターなどが挙げられる。これらのうち、特にポリスチレンが好ましい。このように固相化された抗体は、例えば、後述する〔８．Ｈ５亜型鳥インフルエンザウイルスを検出する方法〕における方法において好適に用いられる。 In addition, the antibody according to the present invention and an antibody fragment containing the variable region thereof may be immobilized on a solid support. Solid supports that can be used include polystyrene, polycarbonate, polypropylene or polyvinyl microtiter plates, test tubes, capillaries, beads (such as latex particles and metal compounds), membranes (such as liposomes) and filters. Among these, polystyrene is particularly preferred. The antibody thus immobilized is, for example, described later [8. [Method for detecting H5 subtype avian influenza virus]
　後述する実施例においても示す通り、本発明に係る抗体およびその可変領域を含む抗体断片は、Ｈ５亜型鳥インフルエンザウイルスのＨＡ１領域のアミノ酸配列のうち、Ｈ５亜型の異なる株間において高く保持されており、且つ立体構造上、Ｈ５亜型鳥インフルエンザウイルスタンパク質の表面に出ている部位に結合する。さらに、Ｈ５以外の亜型に対する結合性は著しく低い。そのため、本発明に係る抗体およびその可変領域を含む抗体断片は、Ｈ５亜型鳥インフルエンザウイルスを特異的に、簡便、且つ高感度に検出することができる。したがって、本発明に係る抗体は、Ｈ５亜型鳥インフルエンザウイルスの検出するキットおよび検出方法等に、好適に利用することができる。 As also shown in the examples described later, the antibody according to the present invention and the antibody fragment containing the variable region thereof are highly retained among strains differing in H5 subtype among the amino acid sequences of HA1 region of H5 subtype avian influenza virus It binds to the site exposed to the surface of the H5 subtype avian influenza virus protein in a three-dimensional structure. Furthermore, the binding to subtypes other than H5 is significantly lower. Therefore, the antibody according to the present invention and the antibody fragment containing the variable region thereof can detect H5 subtype avian influenza virus specifically, simply and at high sensitivity. Therefore, the antibody according to the present invention can be suitably used in a kit for detecting H5 subtype avian influenza virus, a detection method, and the like.
　また、Ｈ５亜型鳥インフルエンザウイルスは、Ｎ１～Ｎ９のサブタイプを含んでおり、本発明において検出の対象として用いられるＨ５亜型鳥インフルエンザウイルスとしては、例えば、Ｈ５Ｎ１亜型、Ｈ５Ｎ２亜型およびＨ５Ｎ８亜型が挙げられる。 Furthermore, H5 subtype avian influenza virus includes subtypes of N1 to N9, and examples of H5 subtype avian influenza virus used as a target of detection in the present invention include H5N1 subtype, H5N2 subtype and H5N8. There are subtypes.
　〔３．本発明に係る抗体およびその可変領域を含む抗体断片をコードするポリヌクレオチド〕
　本発明に係るポリヌクレオチドは、上記抗体およびその可変領域を含む抗体断片をコードするものである。このポリヌクレオチドは、具体的には、例えば、以下の（１）～（４）の何れかに記載のポリヌクレオチドが挙げられる。
（１）配列番号１または４４に示されるアミノ酸配列を有し、Ｈ５亜型鳥インフルエンザウイルスのＨＡ１領域のうち、配列番号１０に示されるアミノ酸配列からなるＨＡ１抗原ポリペプチドに特異的に結合することを特徴とする抗体またはその可変領域を含む抗体断片をコードするポリヌクレオチド。
（２）配列番号１または４４に示されるアミノ酸配列において１～３５個のアミノ酸が置換、欠失、挿入、および/または付加されたアミノ酸配列を有し、Ｈ５亜型鳥インフルエンザウイルスのＨＡ１領域のうち、配列番号１０に示されるアミノ酸配列からなるＨＡ１抗原ポリペプチドに特異的に結合することを特徴とする抗体またはその可変領域を含む抗体断片をコードするポリヌクレオチド。なお、置換、欠失、挿入、および／または付加されたアミノ酸の個数は、１～１７個であることが好ましく、１～１３個であることが好ましく、１～１０個であることがより好ましく、１～８個であることがさらに好ましく、１～５個であることがさらに好ましく、１～２個または３個であることが特に好ましい。
（３）配列番号１または４４に示されるアミノ酸配列に対して９０％以上の配列同一性を有し、Ｈ５亜型鳥インフルエンザウイルスのＨＡ１領域のうち、配列番号１０に示されるアミノ酸配列からなるＨＡ１抗原ポリペプチドに特異的に結合することを特徴とする抗体またはその可変領域を含む抗体断片をコードするポリヌクレオチド。なお、アミノ酸配列の配列同一性は、９５％以上であることが好ましく、９６％以上であることがさらに好ましく、９７％以上、９８％以上、或いは９９％以上であることが特に好ましい。
（４）上記（１）に記載のポリヌクレオチドと相補的な配列からなるポリヌクレオチドに対して、ストリンジェントな条件下においてハイブリダイズするポリヌクレオチド。なお、ストリンジェントな条件下とは、例えば、参考文献：“Molecular cloning-a Laboratory manual 2nd edition（Sambrookら、1989）”に記載の条件などが挙げられる。ストリンジェントな条件下とは、より具体的には例えば、６×ＳＳＣ（１×ＳＳＣの組成：０．１５Ｍ塩化ナトリウム、０．０１５Ｍクエン酸ナトリウム、ｐＨ７．０）、０．５％ＳＤＳ、５×デンハートおよび１００ｍｇ／ｍＬニシン***ＤＮＡを含む溶液にプローブとともに６５℃で８～１６時間恒温し、ハイブリダイズさせる条件が挙げられる。なお、このポリヌクレオチドは、上記（１）に記載のポリヌクレオチドの塩基配列に対して９０％以上の配列同一性を有することがさらに好ましく、９５％以上、９６％以上、９７％以上、９８％以上、或いは９９％以上の配列同一性を有することがさらに好ましい。 [3. Antibody according to the present invention and polynucleotide encoding an antibody fragment containing the variable region thereof]
The polynucleotide according to the present invention encodes an antibody fragment comprising the above-mentioned antibody and its variable region. Specific examples of this polynucleotide include the polynucleotides described in any of the following (1) to (4).
(1) specifically binding to an HA1 antigen polypeptide having the amino acid sequence shown in SEQ ID NO: 10, having the amino acid sequence shown in SEQ ID NO: 1 or 44 and within the HA1 region of H5 subtype avian influenza virus A polynucleotide encoding an antibody characterized by or an antibody fragment comprising the variable region thereof.
(2) has an amino acid sequence in which 1 to 35 amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 1 or 44, and the HA1 region of H5 subtype avian influenza virus Among them, a polynucleotide encoding an antibody or antibody fragment comprising a variable region thereof, which is characterized by specifically binding to an HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10. The number of amino acids substituted, deleted, inserted, and / or added is preferably 1 to 17, preferably 1 to 13, and more preferably 1 to 10. , 1 to 8 is more preferable, 1 to 5 is more preferable, and 1 to 2 or 3 is particularly preferable.
(3) HA1 consisting of the amino acid sequence shown in SEQ ID NO: 10, having a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO: 1 or 44 and of the HA1 region of H5 subtype avian influenza virus A polynucleotide encoding an antibody or an antibody fragment containing a variable region thereof, which is characterized by specifically binding to an antigenic polypeptide. The sequence identity of the amino acid sequence is preferably 95% or more, more preferably 96% or more, and particularly preferably 97% or more, 98% or more, or 99% or more.
(4) A polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of a sequence complementary to the polynucleotide described in (1) above. In addition, under stringent conditions, for example, conditions described in Reference: "Molecular cloning-a Laboratory manual 2nd edition (Sambrook et al., 1989)" can be mentioned. More specifically, under stringent conditions, for example, 6 × SSC (composition of 1 × SSC: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, 5 A condition in which a solution containing × Denhardt and 100 mg / mL herring sperm DNA is incubated with a probe at 65 ° C. for 8 to 16 hours and hybridized is mentioned. In addition, it is more preferable that this polynucleotide has 90% or more of sequence identity with the base sequence of the polynucleotide described in the above (1), 95% or more, 96% or more, 97% or more, 98% It is more preferable to have sequence identity of 99% or more.
　本発明にかかるポリヌクレオチドは、ＲＮＡ（例えば、ｍＲＮＡ）の形態、またはＤＮＡの形態（例えば、ｃＤＮＡまたはゲノムＤＮＡ）で存在し得る。ＤＮＡは、二本鎖であっても、一本鎖であってもよい。本発明にかかるポリヌクレオチドの一例としては、配列番号１、および配列番号４４に示すポリペプチドをコードするＤＮＡである。配列番号１に示すポリペプチドをコードするポリヌクレオチドの配列の一例を配列番号５４に示し、配列番号４４に示すポリペプチドをコードするポリヌクレオチドの配列の一例を配列番号５５に示す。本発明に係るポリヌクレオチドは、非翻訳領域（ＵＴＲ）の配列、シグナル配列およびイントロンなどの付加的な配列を含むものであってもよい。 The polynucleotide of the present invention may exist in the form of RNA (eg, mRNA) or in the form of DNA (eg, cDNA or genomic DNA). The DNA may be double stranded or single stranded. An example of the polynucleotide according to the present invention is DNA encoding the polypeptide shown in SEQ ID NO: 1 and SEQ ID NO: 44. An example of the sequence of a polynucleotide encoding the polypeptide shown in SEQ ID NO: 1 is shown in SEQ ID NO: 54, and an example of the sequence of a polynucleotide encoding the polypeptide shown in SEQ ID NO: 44 is shown in SEQ ID NO: 55. The polynucleotide according to the present invention may contain additional sequences such as untranslated region (UTR) sequences, signal sequences and introns.
　本発明に係るポリヌクレオチドを取得する（単離する）方法は、特に限定されるものではないが、例えば、上記ポリヌクレオチドの塩基配列の一部と特異的にハイブリダイズするプローブを調製し、ゲノムＤＮＡライブラリーまたはｃＤＮＡライブラリーをスクリーニングすればよい。或いは、本発明に係るポリヌクレオチドを、ホスホロアミダイト法などの核酸合成法に従って合成してもよい。 The method for obtaining (isolating) the polynucleotide according to the present invention is not particularly limited. For example, a probe that specifically hybridizes with a part of the base sequence of the above polynucleotide is prepared, and a genome is prepared. The DNA library or cDNA library may be screened. Alternatively, the polynucleotide according to the present invention may be synthesized according to a nucleic acid synthesis method such as the phosphoroamidite method.
　また、本発明にかかるポリヌクレオチドを取得する方法として、ＰＣＲなどの増幅手段を用いる方法を挙げることができる。例えば、当該ポリヌクレオチドのｃＤＮＡのうち、５’側および３’側の配列（またはその相補配列）の中からそれぞれプライマーを調製し、これらプライマーを用いてゲノムＤＮＡ（またはｃＤＮＡ）などを鋳型にしてＰＣＲなどを行い、両プライマー間に挟まれるＤＮＡ領域を増幅することで、本発明にかかるポリヌクレオチドを含むＤＮＡ断片を大量に取得できる。 Further, as a method for obtaining the polynucleotide according to the present invention, a method using amplification means such as PCR can be mentioned. For example, primers are prepared from the 5 'and 3' sequences (or their complementary sequences) of the cDNA of the polynucleotide, and genomic DNA (or cDNA) or the like is used as a template using these primers. A large amount of DNA fragment containing the polynucleotide according to the present invention can be obtained by performing PCR etc. and amplifying the DNA region sandwiched between both primers.
　本発明に係るポリヌクレオチドとしては、さらに、本発明の二価抗体をコードしているＤＮＡ（配列番号２７および２９および５３）などを挙げることができる。 Examples of the polynucleotide according to the present invention further include DNAs encoding the bivalent antibody of the present invention (SEQ ID NOS: 27 and 29 and 53).
　〔４．組換えベクター〕
　本発明に係るポリヌクレオチド（例えばＤＮＡ）は、適当なベクター中に挿入された組換えベクターとして利用に供することもできる。当該ベクターの種類は、例えば、自立的に複製するベクター（例えばプラスミドなど）でもよいし、或いは、宿主細胞に導入された際に宿主細胞のゲノムに組み込まれ、組み込まれた染色体と共に複製されるものであってもよい。 [4. Recombinant vector]
The polynucleotide (eg, DNA) according to the present invention can also be used as a recombinant vector inserted into a suitable vector. The type of the vector may be, for example, a vector that replicates autonomously (such as a plasmid), or is integrated into the host cell's genome when introduced into a host cell and replicated together with the integrated chromosome. It may be
　上記ベクターは、好ましくは発現ベクターである。発現ベクターにおいて本発明に係るポリヌクレオチドは、転写に必要な要素（例えば、プロモーターなど）が機能的に連結されている。プロモータは宿主細胞において転写活性を示すＤＮＡ配列であり、宿主の種類に応じて適宜することができる。 The vector is preferably an expression vector. In the expression vector, the polynucleotide according to the present invention is functionally linked to elements necessary for transcription (for example, a promoter and the like). The promoter is a DNA sequence that exhibits transcriptional activity in a host cell, and can be appropriately selected depending on the type of host.
　細菌細胞で作動可能なプロモータとしては、大腸菌のlac、trpおよびtacプロモータなどが挙げられる。 Promoters operable in bacterial cells include E. coli lac, trp and tac promoters and the like.
　昆虫細胞で作動可能なプロモータの例としては、ｐｏｌｈプロモータ、ｐ１０プロモータ、およびｐＢ１プロモータなどが挙げられる。酵母細胞で作動可能なプロモータの例としては、酵母解糖系遺伝子由来のプロモータ、アルコールデヒドロゲナーゼ遺伝子プロモータ、およびホスホグリセリン酸キナーゼプロモータなどが挙げられる。糸状菌細胞で作動可能なプロモータの例としては、ＡＤＨ３プロモータ、およびｔｐｉＡプロモータなどが挙げられる。 Examples of promoters operable in insect cells include polh promoter, p10 promoter, and pB1 promoter. Examples of promoters operable in yeast cells include promoters derived from yeast glycolytic genes, alcohol dehydrogenase gene promoters, and phosphoglycerate kinase promoters. Examples of promoters operable in filamentous fungal cells include the ADH3 promoter, and the tpiA promoter.
　哺乳動物細胞で作動可能なプロモータの例としては、ＳＶ４０プロモータ、ウシ・パピローマ・ウイルス（ＢＰＶ）プロモータおよびヒトサイトメガロウイルス（ＣＭＶ）プロモータなどが挙げられる。 Examples of promoters operable in mammalian cells include SV40 promoter, bovine papilloma virus (BPV) promoter and human cytomegalovirus (CMV) promoter.
　また、本発明に係るポリヌクレオチドは必要に応じて、例えばヒト成長ホルモンターミネータまたは真菌宿主についてはＴＰＩ１ターミネータもしくはＡＤＨ３ターミネータのような適切なターミネータに機能的に結合されてもよい。本発明に係る組換えベクターは更に、ポリアデニレーションシグナル、転写エンハンサ配列および翻訳エンハンサ配列のような要素を有していてもよい。 Also, the polynucleotide according to the present invention may optionally be operatively linked to a suitable terminator, such as, for example, a human growth hormone terminator or, for fungal hosts, a TPI1 terminator or an ADH3 terminator. The recombinant vector according to the present invention may further have elements such as polyadenylation signal, transcription enhancer sequence and translation enhancer sequence.
　本発明に係る組換えベクターは、さらに、該ベクターが宿主細胞内で複製することを可能にするＤＮＡ配列を具備してもよく、その一例としてはＳＶ４０複製起点（宿主細胞が哺乳類細胞のとき）が挙げられる。 The recombinant vector according to the present invention may further comprise a DNA sequence enabling the vector to replicate in the host cell, for example the SV40 origin of replication (when the host cell is a mammalian cell) Can be mentioned.
　本発明に係る組換えベクターはさらに選択マーカーを含有してもよい。選択マーカーとしては、例えば、アンピシリン、カナマイシン、テトラサイクリン、クロラムフェニコール、ネオマイシンまたはヒグロマイシンのような薬剤耐性遺伝子を挙げることができる。 The recombinant vector according to the present invention may further contain a selectable marker. As a selection marker, for example, drug resistance genes such as ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin or hygromycin can be mentioned.
　〔５．形質転換体〕
　本発明に係るポリヌクレオチド、または、本発明に係る組換えベクター（本発明の核酸構築物と総称する）を適当な宿主細胞に導入することによって形質転換体を作製することができる。 [5. Transformant]
A transformant can be produced by introducing the polynucleotide according to the present invention or the recombinant vector according to the present invention (generally referred to as the nucleic acid construct of the present invention) into a suitable host cell.
　宿主細胞としては、例えば、細菌細胞、酵母細胞、真菌細胞および高等真核細胞などが挙げられる。 Host cells include, for example, bacterial cells, yeast cells, fungal cells and higher eukaryotic cells.
　細菌細胞の例としては、バチルスおよびストレプトマイセスなどのグラム陽性菌または大腸菌などのグラム陰性菌が挙げられる。これら細菌細胞の形質転換は、例えば、プロトプラスト法、またはコンピテント細胞を用いる方法などによって行えばよい。 Examples of bacterial cells include gram positive bacteria such as Bacillus and Streptomyces or gram negative bacteria such as E. coli. Transformation of these bacterial cells may be performed by, for example, a protoplast method or a method using competent cells.
　酵母細胞の例としては、サッカロマイセスまたはシゾサッカロマイセスに属する生物の細胞が挙げられ、例えば、サッカロマイセス・セレビシエ（Saccharomyces cerevisiae）およびサッカロマイセス・クルイベリ（Saccharomyces kluyveri）などが挙げられる。本発明の核酸構築物の酵母宿主への導入方法としては、例えば、エレクトロポレーション法、スフェロブラスト法および酢酸リチウム法などを挙げることができる。 Examples of yeast cells include cells of organisms belonging to Saccharomyces or Schizosaccharomyces, such as Saccharomyces cerevisiae and Saccharomyces kluyveri. As a method for introducing the nucleic acid construct of the present invention into a yeast host, for example, electroporation, spheroplast method, lithium acetate method and the like can be mentioned.
　酵母細胞以外の真菌細胞の例は、糸状菌、例えば、アスペルギルス、ニューロスポラ、フザリウム、またはトリコデルマに属する生物の細胞である。宿主細胞として糸状菌を用いる場合、本発明の核酸構築物を宿主染色体に組み込んで組換え宿主細胞を得ることによって形質転換を行うことができる。核酸構築物の宿主染色体への組み込みは、例えば、相同組換えまたは異種組換えによって行うことができる。 Examples of fungal cells other than yeast cells are cells of filamentous fungi, eg, organisms belonging to Aspergillus, Neurospora, Fusarium, or Trichoderma. When a filamentous fungus is used as a host cell, transformation can be performed by integrating the nucleic acid construct of the present invention into a host chromosome to obtain a recombinant host cell. Integration of the nucleic acid construct into the host chromosome can be performed, for example, by homologous recombination or heterologous recombination.
　高等真核細胞の例としては、植物細胞、動物細胞および昆虫細胞等が挙げられ、動物細胞としては、哺乳動物細胞および鳥類細胞がさらに挙げられる。 Examples of higher eukaryotic cells include plant cells, animal cells and insect cells, and the like, and animal cells further include mammalian cells and avian cells.
　昆虫細胞の例としては、Ｓｆ９細胞およびＳｆ２１細胞などが挙げられる。宿主細胞として昆虫細胞を用いる場合、組換え遺伝子導入ベクターおよびバキュロウイルスを昆虫細胞に共導入して昆虫細胞培養上清中に組換えウイルスを得た後、さらに組換えウイルスを昆虫細胞に感染させ、タンパク質を発現させることができる。共導入方法としては、例えば、リン酸カルシウム法またはリポフェクション法などを挙げることができる。 Examples of insect cells include Sf9 cells and Sf21 cells. When an insect cell is used as a host cell, a recombinant gene transfer vector and a baculovirus are co-introduced into the insect cell to obtain a recombinant virus in the insect cell culture supernatant, and the insect cell is further infected with the recombinant virus , Protein can be expressed. As a co-introduction method, for example, a calcium phosphate method or a lipofection method can be mentioned.
　植物細胞の例としては、Ｔ８７細胞などが挙げられる。宿主細胞として植物細胞を用いる場合、植物細胞の形質転換には、例えば、エレクトロポレーション法、リン酸カルシウム法、リポフェクション法、リポソーム法、ＤＥＡＥデキストラン法およびマイクロインジェクション法などを用いることができる。 Examples of plant cells include T87 cells. When plant cells are used as host cells, for example, electroporation, calcium phosphate method, lipofection method, liposome method, DEAE dextran method, microinjection method and the like can be used for transformation of plant cells.
　哺乳動物細胞の例としては、ＨＥＫ２９３細胞、ＨｅＬａ細胞、ＣＯＳ細胞、ＢＨＫ細胞、ＣＨＬ細胞、ＣＨＯ細胞、ＳＰ２／０細胞およびＮＳＯ細胞などが挙げられる。宿主細胞として哺乳動物細胞を用いる場合、哺乳動物細胞の形質転換には、例えば、エレクトロポレーション法、リン酸カルシウム法、リポフェクション法、リポソーム法、ＤＥＡＥデキストラン法およびマイクロインジェクション法などを用いることができる。 Examples of mammalian cells include HEK 293 cells, HeLa cells, COS cells, BHK cells, CHL cells, CHO cells, SP2 / 0 cells and NSO cells. When mammalian cells are used as host cells, for example, electroporation, calcium phosphate method, lipofection method, liposome method, DEAE dextran method, microinjection method and the like can be used for transformation of mammalian cells.
　上記の形質転換体は、導入された核酸構築物の発現を可能にする条件下で、適切な培養培地中で培養する。次いで、必要に応じて、形質転換体の培養物から、本発明に係る抗体またはその可変領域を含む抗体断片を単離精製する。 The transformants described above are cultured in an appropriate culture medium under conditions that allow expression of the introduced nucleic acid construct. Then, if necessary, an antibody fragment according to the present invention or an antibody fragment containing the variable region thereof is isolated and purified from a culture of transformants.
　なお、形質転換体は、細胞に限定されない。すなわち、形質転換体は、例えば、本発明に係る核酸構築物で形質転換された組織、器官、および個体であってもよい。 The transformant is not limited to cells. That is, the transformant may be, for example, a tissue, an organ, and an individual transformed with the nucleic acid construct of the present invention.
　ただし、細胞以外の形質転換体は、非ヒト由来のものであることが好ましい場合があり、特に個体は非ヒト由来のものであることが好ましい。 However, it may be preferable that the non-cell transformant is of non-human origin, and in particular, the individual is preferably of non-human origin.
　〔６．本発明に係る抗体およびその可変領域を含む抗体断片の製造方法〕
　本発明に係る抗体およびその可変領域を含む抗体断片の製造方法の一例は、Ｈ５亜型鳥インフルエンザウイルスのヘマグルチニンＨＡ１領域のうち、配列番号１０に示されるアミノ酸配列からなるヘマグルチニンＨＡ１抗原ポリペプチドに特異的に結合する抗体を製造する方法であって、下記の（ａ）～（ｃ）の工程を含むことを特徴とする、方法である：（ａ）配列番号１０に示されるアミノ酸配列からなるヘマグルチニンＨＡ１抗原ポリペプチドをトリに免疫する工程；（ｂ）上記工程（ａ）で免疫したトリから上記ヘマグルチニンＨＡ１抗原ポリペプチドに特異的に結合するファージ抗体を含むファージ抗体ライブラリーを得る工程；および（ｃ）上記工程（ｂ）で得られたファージ抗体のうち、上記ヘマグルチニンＨＡ１抗原ポリペプチドに特異的に結合する抗体を濃縮および選抜する工程。 [6. Method of Producing Antibody According to the Present Invention and Antibody Fragment Containing Variable Region Thereof]
One example of a method for producing an antibody according to the present invention and an antibody fragment containing the variable region thereof is specific to a hemagglutinin HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 in the hemagglutinin HA1 region of H5 subtype avian influenza virus It is a method of producing an antibody which binds to the antibody, which comprises the following steps (a) to (c): (a) hemagglutinin consisting of the amino acid sequence shown in SEQ ID NO: 10 Immunizing the bird with an HA1 antigen polypeptide; (b) obtaining a phage antibody library containing a phage antibody that specifically binds to the hemagglutinin HA1 antigen polypeptide from the bird immunized in the step (a); c) Among the phage antibodies obtained in the above step (b), the above hemagglutinin HA1 antigen polypeptide Step of concentrating and selection of antibodies that specifically bind to tides.
　以下に、それぞれの工程について説明する。 Below, each process is demonstrated.
　（工程（ａ））
　工程（ａ）は、配列番号１０に示されるアミノ酸配列からなるＨＡ１抗原ポリペプチドをトリに免疫する工程である。抗原をトリに投与する方法は特に限定されない。例えば、抗原ポリペプチドを腹腔内に投与してもよいし、抗原ポリペプチドを静脈内に投与してもよい。また、抗原の免疫原性を高める観点から、初回免疫は、抗原ポリペプチドと免疫賦活剤とを等量混合して投与することが好ましい。上記「免疫賦活剤」としては、例えば、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、または水酸化アルミニウムゲルアジュバント等のこの分野で通常用いられるものを利用することができる。 (Step (a))
Step (a) is a step of immunizing birds with an HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10. The method for administering the antigen to birds is not particularly limited. For example, the antigenic polypeptide may be administered intraperitoneally, or the antigenic polypeptide may be administered intravenously. Also, from the viewpoint of enhancing the immunogenicity of the antigen, it is preferable that the primary immunization be administered by mixing an equal amount of the antigenic polypeptide and the immunostimulatory agent. As the above-mentioned "immunostimulatory agent", for example, those commonly used in the field such as complete Freund's adjuvant, incomplete Freund's adjuvant, or aluminum hydroxide gel adjuvant can be used.
　抗原ポリペプチドを投与する間隔としては、例えば、初回免疫後４週間後に追加免疫（２次免疫）を行ない、さらに３～４週間後に追加免疫（３次免疫）を行なうことができる。血清の上昇が認められない場合は、さらに２～３週間後に追加免疫を行なうことができる。 For example, a boost (secondary immunization) can be performed 4 weeks after the primary immunization and a boost (third immunization) can be performed 3 to 4 weeks later as an administration interval of the antigenic polypeptide. If serum elevation is not observed, boosting can be performed after 2 to 3 weeks.
　ＨＡ１抗原ポリペプチドを免疫するトリとしては、ニワトリ、ダチョウ、ウズラおよび七面鳥などが挙げられ、飼育の容易さおよび飼料の確保の観点からニワトリが最も好ましい。 Birds that immunize the HA1 antigen polypeptide include chicken, ostrich, quail, turkey and the like, with chicken being most preferable in terms of easiness of breeding and securing of feed.
　（工程（ｂ））
　工程（ｂ）は、上記工程（ａ）で免疫したトリから抗原ポリペプチドに特異的に結合するファージ抗体を含むファージ抗体ライブラリーを得る工程である。抗原ポリペプチドに特異的に結合する抗体を得る方法は特に限定されない。例えば、ハイブリドーマを作製することによってモノクローナル抗体を作製する方法（参照文献：J.Vet. Med. Sci 58 1053 1996）またはファージディスプレイ法によりトリ型ファージ抗体を作製する方法（日本国公開特許公報「特許３９０８２５７号公報」の記載を参照）等によって製造することができる。後述する実施例では、簡便且つ効率よく抗体を製造できることから、ファージディスプレイ法を用いている。 (Step (b))
Step (b) is a step of obtaining a phage antibody library containing phage antibodies that specifically bind to the antigenic polypeptide from the birds immunized in step (a) above. The method for obtaining an antibody that specifically binds to an antigenic polypeptide is not particularly limited. For example, a method of producing a monoclonal antibody by producing a hybridoma (Reference document: J. Vet. Med. Sci 58 1053 1996) or a method of producing avian phage antibody by phage display (Japanese Patent Application Publication "Patent No. 3908257), etc.). In Examples described later, phage display is used because antibodies can be easily and efficiently produced.
　ここで、一例として、ファージディスプレイ法を用いてＨＡ１抗原ポリペプチドに特異的に結合するファージ抗体を得る方法について説明する。 Here, as an example, a method of obtaining a phage antibody that specifically binds to an HA1 antigen polypeptide using a phage display method is described.
　ファージディスプレイ法では、まず、上記工程（ａ）で免疫したトリから脾臓を摘出し、当該脾臓からＲＮＡを抽出する。得られたＲＮＡを鋳型としてＲＴ－ＰＣＲを行ない、トリ抗体の、様々なＨ鎖可変領域とＬ鎖可変領域の遺伝子を含むｃＤＮＡを回収する。 In the phage display method, first, the spleen is isolated from the birds immunized in the above step (a), and RNA is extracted from the spleen. RT-PCR is performed using the obtained RNA as a template, and cDNAs containing various heavy chain variable region and light chain variable region genes of the avian antibody are recovered.
　次いで、得られたＨ鎖可変領域とＬ鎖可変領域の遺伝子の複数の組合せを、リンカー（（ＧＧＧＧＳ）×３）をコードする遺伝子およびスペーサー（Ａｓｐ－Ｖａｌ）をコードする遺伝子を介して結合させ、ｓｃＦｖ遺伝子を構築する。 Then, a plurality of combinations of the obtained heavy chain variable region and light chain variable region genes are linked via a gene encoding a linker ((GGGGS) × 3) and a gene encoding a spacer (Asp-Val) , Construct a scFv gene.
　次いで、トリのＣλと、ｇ３ｐ遺伝子とを組み込んだプラスミドに、得られたｓｃＦｖ遺伝子を導入し、ファージミドベクターを作製する。 Then, the resulting scFv gene is introduced into a plasmid incorporating avian Cλ and the g3p gene to prepare a phagemid vector.
　次いで、作製したファージミドベクターを宿主（例えば、大腸菌等）に形質転換する。 Then, the prepared phagemid vector is transformed into a host (eg, E. coli etc.).
　次いで、ファージミドベクターを形質転換した大腸菌にさらにヘルパーファージを感染させる。このようにして得られた大腸菌を培養すればｓｃＦｖを発現するファージを得ることができる。 Then, E. coli transformed with a phagemid vector is further infected with a helper phage. By culturing the thus obtained E. coli, phages expressing scFv can be obtained.
　これらの複数の単鎖のファージ抗体（ファージ抗体ライブラリー）の中からＨＡ１抗原ポリペプチドに特異的に結合する抗体を濃縮する方法として、例えば、パニング選択を行なうことができる。 For example, panning selection can be performed as a method of concentrating an antibody that specifically binds to the HA1 antigen polypeptide from among a plurality of single-chain phage antibodies (phage antibody library).
　上記「パニング選択」の方法としては特に限定されないが、例えば、抗原ポリペプチドをイムノモジュールプレートに固定化し、固定化した抗原ポリペプチドにファージ抗体群を反応させ、結合しなかったファージを洗浄により除去し、抗原ポリペプチドに結合したファージだけを溶出して大腸菌に感染させて増殖させるという操作を数回繰り返す方法がある。パニング選択を行なうことによって、抗原ポリペプチドのみに特異的に結合するファージを濃縮することができる。 The method of “panning selection” is not particularly limited. For example, the antigen polypeptide is immobilized on an immuno module plate, and the immobilized antibody polypeptide is reacted with a phage antibody group, and phage not bound is removed by washing. Then, there is a method of repeating several times the operation of eluting only the phages bound to the antigenic polypeptide, infecting E. coli and propagating. By performing panning selection, phages that specifically bind to only the antigenic polypeptide can be enriched.
　なお、このパニング選択の操作は、１回～６回繰り返すことが好ましく、６回繰り返すことがより好ましい。 The panning selection operation is preferably repeated once to six times, and more preferably six times.
　（工程（ｃ））
　工程（ｃ）は、上記工程（ｂ）で得られた抗原ポリペプチドに特異的に結合する抗体のうち、上記ＨＡ１抗原ポリペプチドに特異的に結合する抗体を選抜する工程である。 (Step (c))
Step (c) is a step of selecting an antibody that specifically binds to the above HA1 antigen polypeptide among the antibodies that specifically bind to the antigen polypeptide obtained in the above step (b).
　選抜する方法としては、特に限定されるものではないが、例えば、ＥＬＩＳＡ法、ウエスタンブロット法、フローサイトメトリー法および分子間相互作用解析等を行なうことができる。 The selection method is not particularly limited, and for example, ELISA method, Western blot method, flow cytometry method, intermolecular interaction analysis and the like can be performed.
　例えば、ＥＬＩＳＡ法を行なう場合は、配列番号１０に示されるアミノ酸配列からなるＨＡ１抗原ポリペプチドを固相抗原として用いた、上記パニング選択によって濃縮されたファージ抗体について固相ＥＬＩＳＡを行なうことによって、上記ＨＡ１抗原ポリペプチドに特異的に結合する抗体を、選抜することができる。 For example, when performing the ELISA method, the above-mentioned solid phase ELISA is performed on the phage antibody concentrated by the above panning selection using the HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 as the solid phase antigen. Antibodies that specifically bind to the HA1 antigen polypeptide can be selected.
　本発明に係る抗体の製造方法では、上記工程（ａ）～（ｃ）に加えて、得られたファージ抗体をもとにニワトリ型二価抗体を作製する工程（ニワトリ型二価抗体を作製する方法については、国際公開第２００６／０９３０８０号パンフレットを参照のこと）、またはニワトリの天然抗体の可変領域を有するキメラ化抗体を作製する工程（キメラ化抗体を作製する方法については、日本国公開特許公報「特開２００６－２８２５２１号公報」および日本国公開特許公報「特開２００５－２４５３３７号公報」を参照のこと）、またはニワトリの天然抗体の抗原結合領域を有するヒト化抗体を作製する工程（ヒト化抗体を作製する方法については、日本国公開特許公報「特開２００６-２４１０２６号公報」を参照のこと）を含んでいてもよい。 In the method of producing an antibody according to the present invention, in addition to the above steps (a) to (c), a step of producing a chicken-type divalent antibody based on the obtained phage antibody (producing a chicken-type divalent antibody For the method, see WO 2006/093080) or the step of producing a chimerized antibody having a variable region of a natural chicken antibody (for the method of producing a chimerized antibody, see Japanese Patent Laid-Open Publication) Japanese Patent Laid-Open Publication No. 2006-282521 and Japanese Patent Laid-Open Publication No. 2005-245337, or a step of preparing a humanized antibody having an antigen-binding region of a chicken natural antibody The method for producing a humanized antibody may include the Japanese Patent Application Publication (JP 2006-241026A).
　このようなニワトリ型二価抗体またはキメラ化抗体の作製方法として、具体的には、例えば、〔４．組換えベクター〕において記載した発現用ベクターを用いる方法が挙げられ、好ましくは、Ｈ鎖用発現用ベクターとＬ鎖用発現用ベクターとを用い、これらにニワトリ抗体の可変域領域をそれぞれ導入して宿主となる哺乳動物細胞で発現させる方法を挙げることができる。 As a method for producing such a chicken-type bivalent antibody or a chimerized antibody, specifically, for example, [4. Recombinant vector], preferably using the expression vector for H chain and the expression vector for L chain, and introducing the variable region of chicken antibody into them, respectively. A method of expressing in a host mammalian cell can be mentioned.
　また、本発明に係る抗体の可変領域を含む抗体断片を作製する場合は、得られたファージ抗体をコードしているＤＮＡを鋳型としたＰＣＲによる目的の領域の増幅等を含む方法が挙げられる。例えば、本発明に係る抗体の作製方法は、得られたファージ抗体または二価抗体もしくはキメラ抗体をコードしているＤＮＡを鋳型として、Ｉｎｖｅｒｓｅ　ＰＣＲ法等を用いたランダム変異導入、または公知の変異導入法を用いた点変異導入等を行うことによって、目的の部位のアミノ酸配列を変異させる工程を包含していてもよい。 In the case of producing an antibody fragment containing the variable region of the antibody according to the present invention, a method including amplification of a target region by PCR using a DNA encoding the obtained phage antibody as a template can be mentioned. For example, in the method for producing an antibody according to the present invention, random mutagenesis using Inverse PCR or the like, or known mutagenesis, using the obtained phage antibody or a DNA encoding a bivalent or chimeric antibody as a template The method may include the step of mutating the amino acid sequence of the target site by performing point mutation introduction and the like using a method.
　例えば、ニワトリ型二価抗体であれば、ニワトリ由来の試料における非特異的反応が低いと考えられるため、養鶏場などの現場におけるＨ５亜型鳥インフルエンザウイルスの検出等に好適且つより効果的に用いることができる。 For example, if it is a chicken-type bivalent antibody, it is considered that nonspecific reaction in a sample derived from chicken is considered to be low, so it is suitably and more effectively used for detection of H5 subtype avian influenza virus in the field such as a poultry farm be able to.
　なお、上記工程（ａ）～（ｃ）を含むものは本発明に係る製造方法の一例であり、本発明に係る他の一例としては、上述の〔５．形質転換体〕に記載された形質転換体を導入された核酸構築物の発現を可能にする条件下で、宿主細胞に適した適切な培養培地中で培養し、次いで、必要に応じて、形質転換体の培養物から、本発明に係る抗体またはその可変領域を含む抗体断片を単離精製する方法が挙げられる。 In addition, what contains the said process (a)-(c) is an example of the manufacturing method based on this invention, As other examples based on this invention, above-mentioned [5. The transformant described in [1] is cultured in a suitable culture medium suitable for the host cell under conditions which allow expression of the introduced nucleic acid construct, and then, if necessary, transformation. A method of isolating and purifying an antibody according to the present invention or an antibody fragment containing the variable region thereof from the culture of the body can be mentioned.
　また、ヒト化抗体であれば、ヒト由来の試料における非特異的反応が低いと考えられるため、ヒトに対するＨ５亜型鳥インフルエンザウイルスの検出方法等に好適に用いることができる。 Moreover, if it is a humanized antibody, since it is thought that the nonspecific reaction in the sample derived from human is low, it can be used suitably for the detection method etc. of H5 subtype avian influenza virus with respect to a human.
　〔７．本発明に係るキット〕
　本発明に係るキットは、試料中に含まれるＨ５亜型鳥インフルエンザウイルスを検出するためのキットであって、少なくとも本発明に係る抗体またはその可変領域を含む抗体断片を備えている。本発明に係る抗体およびその可変領域を含む抗体断片については、〔１．本発明に係る抗体〕で説明した通りである。 [7. Kit according to the present invention]
A kit according to the present invention is a kit for detecting H5 subtype avian influenza virus contained in a sample, and comprises at least an antibody according to the present invention or an antibody fragment containing a variable region thereof. The antibody according to the present invention and an antibody fragment containing the variable region thereof [1. Antibody according to the present invention].
　また、本発明に係るキットは、本発明に係る抗体またはその可変領域を含む抗体断片以外の、Ｈ５亜型鳥インフルエンザウイルスを認識する少なくとも一種類の抗体またはその可変領域を含む抗体断片をさらに備えていてもよい。例えば、Ｈ５亜型鳥インフルエンザウイルスのＨＡ２領域に含まれるアミノ酸配列を有するＨＡ２抗原ポリペプチドに特異的に結合する抗体およびその可変領域を含む抗体断片が挙げられる。より具体的には、Ｈ５亜型鳥インフルエンザウイルスのＨＡ２領域のうち、配列番号１７のアミノ酸配列からなるポリペプチドに特異的に結合する抗体およびその可変領域を含む抗体断片が挙げられる。このような抗体としては、Ｈ鎖が配列番号１８に示されるアミノ酸配列からなり、且つ、Ｌ鎖が配列番号２０に示されるアミノ酸配列からなる二価の抗ＨＡ２抗体およびＨ鎖が配列番号２２に示されるアミノ酸配列からなり、且つ、Ｌ鎖が配列番号２４に示されるアミノ酸配列からなる二価の抗ＨＡ２抗体が挙げられる。また、これらの抗ＨＡ２抗体の例としては、上記配列番号１８に示されるアミノ酸配列からなるＨ鎖をコードしているＤＮＡが配列番号１９に示される塩基配列からなり、且つ上記配列番号２０に示されるアミノ酸配列からなるＬ鎖をコードしているＤＮＡが、配列番号２１に示される塩基配列からなる抗ＨＡ２抗体、および上記配列番号２２に示されるアミノ酸配列からなるＨ鎖をコードしているＤＮＡが配列番号２３に示される塩基配列からなり、且つ上記配列番号２４に示されるアミノ酸配列からなるＬ鎖をコードしているＤＮＡが、配列番号２５に示される塩基配列からなる抗ＨＡ２抗体がさらに挙げられる。 In addition, the kit according to the present invention further comprises at least one antibody that recognizes H5 subtype avian influenza virus or an antibody fragment containing the variable region thereof other than the antibody according to the present invention or the antibody fragment containing the variable region thereof. It may be For example, an antibody that specifically binds to an HA2 antigen polypeptide having an amino acid sequence contained in the HA2 region of H5 subtype avian influenza virus and an antibody fragment containing its variable region can be mentioned. More specifically, an antibody that specifically binds to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 17 in the HA2 region of H5 subtype avian influenza virus and an antibody fragment containing its variable region can be mentioned. As such an antibody, a bivalent anti-HA2 antibody consisting of the amino acid sequence in which the H chain is shown in SEQ ID NO: 18 and an L chain consisting of the amino acid sequence shown in SEQ ID NO: 20 and the H chain in SEQ ID NO: 22 The bivalent anti-HA2 antibody which consists of an amino acid sequence shown, and whose L chain consists of an amino acid sequence shown by sequence number 24 is mentioned. Moreover, as an example of these anti-HA2 antibodies, a DNA encoding the H chain consisting of the amino acid sequence shown in the above SEQ ID NO: 18 consists of the base sequence shown in SEQ ID NO: 19 and shown in the above SEQ ID NO: 20 Wherein the DNA encoding the L chain consisting of the amino acid sequence is an anti-HA2 antibody consisting of the base sequence shown in SEQ ID NO: 21 and the DNA encoding the H chain consisting of the amino acid sequence Further included is the anti-HA2 antibody which comprises a DNA sequence consisting of the nucleotide sequence shown in SEQ ID NO: 23 and encoding an L chain consisting of the amino acid sequence shown above in SEQ ID NO: 24 comprises the nucleotide sequence shown in SEQ ID NO: 25 .
　本発明に係るキットは、本発明に係る抗体以外にも、イムノクロマトグラフィー法、ＥＬＩＳＡ法およびウエスタンブロット法等の免疫反応を行うために必要な部材（二次抗体、発色試薬、ブロッキング試薬等）、プレート（９６ウェルプレート等）およびチューブ等が含まれていてもよい。またウエスタンブロット法を行うために必要なメンブレン、電気泳動用ゲル、電気泳動装置、ブロッティング装置およびブロッティング用試薬等が含まれていてもよい。例えば、本発明に係る抗体を検出するための二次抗体および二次抗体に結合させた標識酵素の基質等を備えていてもよい。 The kit according to the present invention includes, in addition to the antibody according to the present invention, a member (secondary antibody, a coloring reagent, a blocking reagent, etc.) necessary for performing an immune reaction such as immunochromatography, ELISA and Western blotting. Plates (96-well plate etc.) and tubes etc. may be included. In addition, the membrane, gel for electrophoresis, electrophoresis apparatus, blotting apparatus, reagents for blotting and the like necessary for performing Western blotting may be included. For example, a secondary antibody for detecting the antibody according to the present invention and a substrate of a labeled enzyme bound to the secondary antibody may be provided.
　上記二次抗体としては、例えば、アルカリホスファターゼ標識抗ＩｇＧ抗体および西洋ワサビペルオキシダーゼ（ＨＲＰ）標識抗ＩｇＧ抗体等を挙げることができる。なお、用いる抗ＩｇＧ抗体の由来は特に限定されず、目的に応じて適宜選択される。 Examples of the secondary antibody include alkaline phosphatase-labeled anti-IgG antibody and horseradish peroxidase (HRP) -labeled anti-IgG antibody. In addition, the origin of the anti-IgG antibody to be used is not specifically limited, According to the objective, it selects suitably.
　また、ＨＲＰ検出用の基質としては、例えば、ＯＰＤ、ＴＭＢおよびＥＣＬ（Electro-generated chemiluminesence）等を挙げることができる。また、アルカリホスファターゼの基質としては、例えば、ニトロブルーテトラゾリウム（ＮＢＴ）および５－ブロモ－４－クロロ－３－インドリルホスファターゼｐ－トルイジニル塩（ＢＣＩＰ）の混合基質等を挙げることができる。 Moreover, as a substrate for HRP detection, OPD, TMB and ECL (Electro-generated chemiluminescence) etc. can be mentioned, for example. Moreover, as a substrate of alkaline phosphatase, for example, a mixed substrate of nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphatase p-toluidinyl salt (BCIP) can be mentioned.
　また上記キットを構成する成分を格納するための１つ以上の容器（例えば、バイアル、管、アンプルおよびビンなど）が含まれていてもよい。また、上記キットにおいて使用する検出方法について詳細が記載された使用説明書がさらに含まれていてもよい。当該検出方法としては、後述する〔８．Ｈ５亜型鳥インフルエンザウイルスを検出する方法〕で説明される検出方法が挙げられる。 It may also include one or more containers (eg, vials, tubes, ampoules, bottles, etc.) for storing the components that make up the kit. In addition, instructions for use in the above kit may be further included. The detection method will be described later [8. Methods for detecting H5 subtype avian influenza virus] can be mentioned.
　本発明に係るキットとしては、例えば、本発明に係る抗体、本発明に係る抗体以外の抗体、および検出感度を高めるために必要な部材を備えた、イムノクロマトグラフィーを利用したキットが挙げられる。 The kit according to the present invention includes, for example, an immunochromatographic kit comprising the antibody according to the present invention, an antibody other than the antibody according to the present invention, and members necessary for enhancing the detection sensitivity.
　〔８．Ｈ５亜型鳥インフルエンザウイルスを検出する方法〕
　本発明に係るＨ５亜型鳥インフルエンザウイルスを検出する方法は、本発明に係る抗体またはその可変領域を含む抗体断片と、生体から調製した試料とを反応させる工程を含む。ここで、本発明に係る抗体については、〔２．本発明に係る抗体およびその可変領域を含む抗体断片〕に記載した通りである。 [8. Method for detecting H5 subtype avian influenza virus]
The method for detecting H5 subtype avian influenza virus according to the present invention comprises the step of reacting an antibody according to the present invention or an antibody fragment containing the variable region thereof with a sample prepared from a living body. Here, for the antibody according to the present invention, [2. Antibody according to the present invention and an antibody fragment containing the variable region thereof].
　上述の本発明に係る抗体またはその可変領域を含む抗体断片と、生体から調製した試料とを反応させる工程は、本発明に係る抗体またはその可変領域を含む抗体断片を含む組成物に生体から調製した試料を接触させる工程と、上記抗体またはその可変領域を含む抗体断片を含む組成物と上記試料との間に生じる反応を検出する工程とを含んでいてもよい。 The step of reacting the above-mentioned antibody according to the present invention or the antibody fragment containing the variable region thereof with a sample prepared from the living body is prepared from the living body into a composition containing the antibody according to the present invention or the antibody fragment containing the variable region The method may include the steps of contacting the sample, and detecting a reaction occurring between the sample and a composition containing the antibody or an antibody fragment containing the variable region thereof.
　上記反応を検出に用いられる手法としては、ＥＬＩＳＡ法、放射線免疫測定法、蛍光免疫測定法、ウエスタンブロット法、イムノクロマトグラフィー法、アフィニティクロマトグラフィー法、免疫沈降法、免疫拡散法および赤血球凝集抑制試験等が挙げられる。 Methods used for detection of the above reaction include ELISA, radioimmunoassay, fluorescent immunoassay, western blot, immunochromatography, affinity chromatography, immunoprecipitation, immunodiffusion, hemagglutination inhibition test, etc. Can be mentioned.
　上述の反応を検出する方法は、例えば、標識剤で標識された抗体を用いた、サンドイッチ法、競合法および直接吸着法等が挙げられる。これらの方法により、試料中に存在する、目的とする抗原の量を測定することができる。 Examples of methods for detecting the above-mentioned reaction include a sandwich method, a competitive method, a direct adsorption method and the like using an antibody labeled with a labeling agent. By these methods, the amount of the target antigen present in the sample can be measured.
　本発明に係る検出方法に用いる試料としては、特に限定されるものではないが、対象のタンパク質を少なくとも含み得るものであればよい。生体試料としては、例えば、細胞試料、組織試料および拭い液試料が挙げられ、中でも試料採取方法の容易性の観点から、拭い液試料が好ましい。拭い液試料としては、気管の拭い液、咽喉頭の拭い液、口腔の拭い液、および鳥類の総***腔の拭い液等が挙げられる。また、最適なウイルス増殖部位がウイルス毎に異なるため、一個体から採取した、気管の拭い液、咽喉頭の拭い液、口腔の拭い液および総***腔（鳥類の場合）の拭い液試料を全て検査に用いることが好ましい。生体試料の由来となる生物は、例えば、鳥類、ヒトおよびブタ等の哺乳動物が挙げられる。このうち、好ましくは、鳥類であり、鳥類のうち、より好ましくは家禽のニワトリである。 The sample used in the detection method according to the present invention is not particularly limited as long as it can contain at least the protein of interest. Examples of biological samples include cell samples, tissue samples and wiping fluid samples, and from the viewpoint of the ease of sampling method, wiping fluid samples are preferred. The wiping fluid samples include tracheal wiping fluid, laryngopharyngeal wiping fluid, oral cavity wiping fluid, and wiping solution of the general excretory space of birds. In addition, since the optimal virus growth site differs from virus to virus, all swabs of trachea, throat and throat, swabs of the oral cavity, and swabs of the entire cavity (for birds) collected from one individual It is preferred to use for inspection. Organisms from which a biological sample is derived include, for example, mammals such as birds, humans and pigs. Among these, preferably, it is a bird, and among the birds, more preferably a fowl chicken.
　採取された生体試料は、必要に応じてタンパク質を抽出する操作を行ってからか、または、不要な成分を除去する操作を行ってから、検査に供してもよい。 The collected biological sample may be subjected to a test after extracting the protein as necessary or after removing an unnecessary component.
　また、得られた生体試料は、必要に応じて凍結保存等の、生体試料の種類に適した方法で保存してもよい。 In addition, the obtained biological sample may be stored by a method suitable for the type of biological sample, such as cryopreservation, if necessary.
　また、本発明に係る検出方法は、本発明に係る抗体以外の、Ｈ５亜型鳥インフルエンザウイルスを認識する少なくとも一種類の抗体またはその可変領域を含む抗体断片と、生体から調製した試料とを反応させる工程をさらに含んでいてもよい。本発明に係る抗体およびその可変領域を含む抗体断片以外の抗体およびその可変領域を含む抗体断片については、〔７．本発明に係るキット〕に記載した通りである。 In addition, the detection method according to the present invention is a reaction between a sample prepared from a living body and an antibody fragment containing at least one type of antibody that recognizes H5 subtype avian influenza virus or its variable region other than the antibody according to the present invention The method may further comprise the step of With respect to antibodies other than the antibody according to the present invention and the antibody fragment containing the variable region thereof and the antibody fragment containing the variable region [7. Kit according to the present invention].
　本発明に係る検査方法は、さらに、上記生体試料におけるウイルスの存在の有無を判定するか、上記生体試料におけるＨ５亜型鳥インフルエンザウイルスの量、より具体的には、生体試料の単位量あたりに含まれるＨ５亜型鳥インフルエンザウイルスの量を測定する工程を含んでいてもよい。ここでＨ５亜型鳥インフルエンザウイルスの量とは、例えばＨ５亜型鳥インフルエンザウイルスタンパク質の量である。 The test method according to the present invention further determines the presence or absence of a virus in the biological sample, or the amount of H5 subtype avian influenza virus in the biological sample, more specifically, per unit amount of biological sample. The method may include the step of measuring the amount of H5 subtype avian influenza virus included. Here, the amount of H5 subtype avian influenza virus is, for example, the amount of H5 subtype avian influenza virus protein.
　但し、生体試料の単位量あたりに含まれるＨ５亜型鳥インフルエンザウイルスの量を測定するという概念には定量的測定および定性的測定の両方が含まれ、濃度測定の他、対照と比較可能な形式でＨ５亜型鳥インフルエンザウイルスの量を提示することが含まれる。より具体的には、例えば、検量線等を用いて濃度換算する以前の取得した時点でのデータ比較、またはＨ５亜型鳥インフルエンザウイルスの量がある一定の閾値を超えているか否かという形式での結果の提示等も含まれる。 However, the concept of measuring the amount of H5 subtype avian influenza virus contained per unit amount of biological sample includes both quantitative measurement and qualitative measurement, and in addition to concentration measurement, a format comparable to a control It includes presenting the amount of H5 subtype avian influenza virus. More specifically, for example, data comparison before acquisition of concentration conversion using a calibration curve or the like, or in the form of whether the amount of H5 subtype avian influenza virus exceeds a certain threshold or not The presentation of the results of
　また、本発明に係るＨ５亜型鳥インフルエンザウイルスを検出する方法は、対象となる個体における、Ｈ５亜型鳥インフルエンザウイルスの感染の可能性を判定する工程を包含していてもよい。 In addition, the method for detecting H5 subtype avian influenza virus according to the present invention may include the step of determining the possibility of infection of H5 subtype avian influenza virus in a subject individual.
　ここで、「Ｈ５亜型鳥インフルエンザウイルスの感染の可能性を判定する」とは、対象となる生物個体について、当該インフルエンザを発症しているか否かにかかわらず、当該インフルエンザウイルスに感染しているか否かを判定することを指す。 Here, “determining the possibility of infection with the H5 subtype avian influenza virus” means that the target individual is infected with the influenza virus regardless of whether or not the influenza has developed. Indicates to determine whether or not.
　また、ここで、Ｈ５亜型鳥インフルエンザウイルスによって引き起こされる鳥インフルエンザの発症とは、対象となる生物に、Ｈ５亜型鳥インフルエンザウイルスが感染することによって、当該感染した生物に鳥インフルエンザの症状が生じることを指している。鳥インフルエンザのニワトリにおける症状としては、沈うつ、肉冠、肉垂および脚部のチアノーゼ、顔面浮腫、神経症状、羽毛削ごう、ならびに下痢などの症状が挙げられる。 In addition, here, with the onset of avian influenza caused by the H5 subtype avian influenza virus, infection of the target organism with the H5 subtype avian influenza virus causes a symptom of avian influenza to the infected organism. Point to that. Symptoms of avian influenza in chickens include symptoms such as depression, flesh crown, pits and legs cyanosis, facial edema, neurological symptoms, feathering, and diarrhea.
　当該判定する工程として、具体的には、例えば、対象となる生物がニワトリの場合、Ｈ５亜型鳥インフルエンザウイルスを***しているか否かを判定する工程が挙げられる。 Specifically, for example, when the target organism is a chicken, a step of determining whether or not the H5 subtype avian influenza virus is excreted is mentioned as the step of the determination.
　当該判定方法としては、顕性化したウイルス由来物質について、上述したＨ５亜型鳥インフルエンザウイルスを検出する工程において用いられる手法において得られた発色および数値等のデータの目視により判定する方法が挙げられる。 As the said determination method, the method of judging by visual observation of the data of color development and numerical value etc. which were obtained in the method used in the process of detecting H5 subtype avian influenza virus mentioned above is mentioned about the virus-derived substance which has been developed .
　当該工程を行うことにより、Ｈ５亜型鳥インフルエンザウイルスによって引き起こされる鳥インフルエンザを発症する前に、感染しているか否かを知ることができる。 By performing this step, it is possible to know whether or not an infection has occurred before the onset of avian influenza caused by the H5 subtype avian influenza virus.
　判定の一例では、健常な対照と比較して被験個体の生体試料における当該ウイルスの量があらかじめ規定した閾値を超えた場合、被験個体は、Ｈ５亜型鳥インフルエンザウイルス感染によってウイルスを***していると判定される。なお、ウイルスの量があらかじめ規定した閾値を超えることとは、定量的測定による結果であっても定性的測定による結果であってもよく、具体的な数値の比較はもちろん、相対的な量の比較（実際に量を算出する必要は無く、ある基準より高いか低いかを判断する）も含む概念である。 In one example of the determination, when the amount of the virus in the biological sample of the test individual exceeds a predetermined threshold value as compared to a healthy control, the test individual is excreting the virus by H5 subtype avian influenza virus infection It is determined that Incidentally, the fact that the amount of virus exceeds a predetermined threshold value may be a result of quantitative measurement or a result of qualitative measurement, and it is needless to say that the comparison of specific numerical values is a relative amount of It is a concept that includes comparison (it is not necessary to actually calculate the amount, and it is determined whether it is higher or lower than a certain standard).
　対照試料の上記検査は、被験個体の試料の検査と同時に行われてもよく、また別々に行われてもよい。すなわち、被験個体の数値と比較される対照試料の数値は、被験個体の試料が検査されるときとは異なるときに行われた検査で得られた値であってもよい。また、対照試料の検査は、被験個体の検査を行う個体自身が行う必要は無く、例えば、既に取得されデータベース等に蓄積されている対照試料の検査値を閾値として用いることもできる。 The above test of the control sample may be performed simultaneously with the test of the sample of the test individual, or may be performed separately. That is, the numerical value of the control sample to be compared with the numerical value of the test individual may be the value obtained in the test performed when the sample of the test individual is different from when it is tested. In addition, the test of the control sample does not have to be performed by the individual who tests the test individual, and for example, the test value of the control sample already acquired and accumulated in a database or the like can be used as a threshold.
　判定に用いられる対照試料の数値については、健常個体の試料の数値を直接利用してもよく、一定の人数の健常個体の試料の数値を母集団としたときに得られる平均値を利用してもよい。また、カットオフ値をあらかじめ設定しておき、被験個体の数値とこのカットオフ値とを比較してもよい。 Regarding the numerical value of the control sample used for the determination, the numerical value of the sample of the healthy individual may be used directly, or the average value obtained when the numerical value of the sample of the healthy individual of a certain number is used as the population It is also good. In addition, a cutoff value may be set in advance, and the numerical value of the test individual may be compared with this cutoff value.
　このような方法により、Ｈ５亜型鳥インフルエンザウイルスに感染の可能性を早期に判定できるため、Ｈ５亜型鳥インフルエンザウイルス感染個体を有している農場内および当該農場から他の家禽農場への感染拡大を最小限に食い止める為の早期初動対応をとることができる。 By such a method, since the possibility of infection with H5 subtype avian influenza virus can be determined early, infection from farms having H5 subtype avian influenza virus-infected individuals to other poultry farms from the said farm Early initial response can be taken to minimize expansion.
　本発明に係るＨ５亜型鳥インフルエンザウイルスを検出する方法によれば、既存の検出方法よりも試料中に含まれるＨ５亜型鳥インフルエンザウイルスを特異的に、正確、迅速且つ高感度に検出することができる。 According to the method for detecting H5 subtype avian influenza virus according to the present invention, the H5 subtype avian influenza virus contained in a sample is specifically, accurately, rapidly and highly sensitively detected than the existing detection method. Can.
　〔９．まとめ〕
　本発明は以下の何れかの一態様を包含する。
＜１＞　Ｈ５亜型鳥インフルエンザウイルスのヘマグルチニンＨＡ１領域のうち、配列番号１０に示されるアミノ酸配列からなるヘマグルチニンＨＡ１抗原ポリペプチドに特異的に結合することを特徴とする、抗体またはその可変領域を含む抗体断片。
＜２＞　重鎖可変領域中に、配列番号４に示されるＣＤＲ１、配列番号５に示されるＣＤＲ２および配列番号６または配列番号４６に示されるＣＤＲ３のすべてを含んでおり、且つ、軽鎖可変領域中に、配列番号７に示されるＣＤＲ１、配列番号８に示されるＣＤＲ２および配列番号９に示されるＣＤＲ３のすべてを含んでいることを特徴とする、＜１＞に記載の抗体またはその可変領域を含む抗体断片。
＜３＞　重鎖可変領域が、配列番号２に示されるアミノ酸配列、または、配列番号２に示されるアミノ酸配列において、１～１３個のアミノ酸が置換、欠失、挿入、および／もしくは付加されたアミノ酸配列からなり、且つ軽鎖可変領域が、配列番号３に示されるアミノ酸配列、または、配列番号３に示されるアミノ酸配列において、１～１０個のアミノ酸が置換、欠失、挿入、および／もしくは付加されたアミノ酸配列からなることを特徴とする、＜１＞または＜２＞に記載の抗体またはその可変領域を含む抗体断片。
＜４＞　重鎖可変領域が、配列番号４５に示されるアミノ酸配列からなり、且つ
　軽鎖可変領域が、配列番号３に示されるアミノ酸配列からなることを特徴とする、＜３＞に記載の抗体またはその可変領域を含む抗体断片。
＜５＞　以下の（１）～（４）の何れかに示す、Ｈ５亜型鳥インフルエンザウイルスのヘマグルチニンＨＡ１領域のうち、配列番号１０に示されるアミノ酸配列からなるヘマグルチニンＨＡ１抗原ポリペプチドに特異的に結合することを特徴とする、＜１＞～＜４＞のいずれかに記載の抗体またはその可変領域を含む抗体断片：（１）配列番号１または配列番号４４に示されるアミノ酸配列からなる抗体またはその可変領域を含む抗体断片、（２）配列番号１または配列番号４４に示されるアミノ酸配列において１～３５個のアミノ酸が置換、欠失、挿入、および/または付加されたアミノ酸配列からなる抗体またはその可変領域を含む抗体断片、（３）配列番号１または配列番号４４に示されるアミノ酸配列に対して９０％以上の配列同一性を有する抗体またはその可変領域を含む抗体断片、（４）上記（１）に記載の抗体またはその可変領域を含む抗体断片をコードするポリヌクレオチドと相補的な配列からなるポリヌクレオチドに対して、ストリンジェントな条件下においてハイブリダイズするポリヌクレオチドによってコードされるアミノ酸配列からなる抗体またはその可変領域を含む抗体断片。
＜６＞　下記の（ａ）～（ｃ）の工程を含む製造方法によって得られたことを特徴とする、＜１＞～＜５＞のいずれかに記載の抗体またはその可変領域を含む抗体断片：
　（ａ）配列番号１０に示されるアミノ酸配列からなるヘマグルチニンＨＡ１抗原ポリペプチドをトリに免疫する工程；（ｂ）上記工程（ａ）で免疫したトリから上記ヘマグルチニンＨＡ１抗原ポリペプチドに特異的に結合するファージ抗体を含むファージ抗体ライブラリーを得る工程；および（ｃ）上記工程（ｂ）で得られたファージ抗体のうち、上記ヘマグルチニンＨＡ１抗原ポリペプチドに特異的に結合する抗体を濃縮および選抜する工程。
＜７＞　一本鎖可変領域断片または二価抗体であることを特徴とする、＜１＞～＜６＞のいずれかに記載の抗体またはその可変領域を含む抗体断片。
＜８＞　重鎖が配列番号２６または配列番号５２に示されるアミノ酸配列からなり、且つ、軽鎖が配列番号２８に示されるアミノ酸配列からなる二価抗体であることを特徴とする、＜１＞～＜７＞のいずれに記載の抗体またはその可変領域を含む抗体断片。
＜９＞　＜１＞～＜８＞のいずれかに記載の抗体またはその可変領域を含む抗体断片をコードすることを特徴とする、ポリヌクレオチド。
＜１０＞　＜９＞に記載のポリヌクレオチドを有することを特徴とする、組換えベクター。
＜１１＞　＜９＞に記載のポリヌクレオチドまたは＜１０＞に記載の組換えベクターが導入されていることを特徴とする、形質転換体。
＜１２＞　Ｈ５亜型鳥インフルエンザウイルスのヘマグルチニンＨＡ１領域のうち、配列番号１０に示されるアミノ酸配列からなるヘマグルチニンＨＡ１抗原ポリペプチドに特異的に結合する抗体を製造する方法であって、下記の（ａ）～（ｃ）の工程を含むことを特徴とする、方法：（ａ）配列番号１０に示されるアミノ酸配列からなるヘマグルチニンＨＡ１抗原ポリペプチドをトリに免疫する工程；（ｂ）上記工程（ａ）で免疫したトリから上記ヘマグルチニンＨＡ１抗原ポリペプチドに特異的に結合するファージ抗体を含むファージ抗体ライブラリーを得る工程；および（ｃ）上記工程（ｂ）で得られたファージ抗体のうち、上記ヘマグルチニンＨＡ１抗原ポリペプチドに特異的に結合する抗体を濃縮および選抜する工程。
＜１３＞　上記抗体は、一本鎖可変領域断片または二価抗体であることを特徴とする、＜１２＞に記載の方法。
＜１４＞　配列番号１０に示されるアミノ酸配列からなることを特徴とする、ポリペプチド。
＜１５＞　試料中に含まれるＨ５亜型鳥インフルエンザウイルスを検出するためのキットであって、＜１＞～＜８＞のいずれかに記載の抗体またはその可変領域を含む抗体断片を備えていることを特徴とする、キット。
＜１６＞　試料中に含まれるＨ５亜型鳥インフルエンザウイルスを検出する方法であって、＜１＞～＜８＞のいずれかに記載の抗体またはその可変領域を含む抗体断片と、生体から調製した試料とを反応させる工程を含むことを特徴とする、方法。
＜１７＞　上記試料は、気管、咽喉頭、口腔または総***腔の、拭い液であることを特徴とする＜１６＞に記載の方法。
＜１８＞　鳥インフルエンザウイルスのうちＨ５亜型鳥インフルエンザウイルスのみに特異的に結合することを特徴とする、抗体またはその可変領域を含む抗体断片。
＜１９＞　Ｈ５亜型鳥インフルエンザウイルスに特異的に結合し、かつＨ１～Ｈ４およびＨ６～Ｈ１６亜型鳥インフルエンザウイルスに特異的に結合しないことを特徴とする、抗体またはその可変領域を含む抗体断片。
＜２０＞　上記Ｈ５亜型鳥インフルエンザウイルスはＨ５Ｎ１亜型鳥インフルエンザウイルスであることを特徴とする、＜１８＞または＜１９＞に記載の抗体またはその可変領域を含む抗体断片。
＜２１＞　Ｈ５亜型鳥インフルエンザウイルスのヘマグルチニンＨＡ１領域における、配列番号１０に示されるアミノ酸配列で示される領域に特異的に結合することを特徴とする、＜１８＞～＜２０＞のいずれかに記載の抗体またはその可変領域を含む抗体断片。 [9. Summary]
The present invention includes any one of the following aspects.
<1> An antibody or a variable region thereof characterized by specifically binding to a hemagglutinin HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 in the hemagglutinin HA1 region of H5 subtype avian influenza virus Antibody fragment.
<2> The heavy chain variable region includes all of CDR1 shown in SEQ ID NO: 4, CDR2 shown in SEQ ID NO: 5 and CDR3 shown in SEQ ID NO: 6 or SEQ ID NO: 46, and a light chain variable region The antibody according to <1> or the variable region thereof, wherein the antibody comprises all of CDR1 shown in SEQ ID NO: 7, CDR2 shown in SEQ ID NO: 8 and CDR3 shown in SEQ ID NO: 9 Antibody fragments.
<3> The heavy chain variable region is substituted, deleted, inserted, and / or added with 1 to 13 amino acids in the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 2 1 to 10 amino acids are substituted, deleted, inserted, and / or made up of the amino acid sequence, and the light chain variable region is the amino acid sequence shown in SEQ ID NO: 3 or the amino acid sequence shown in SEQ ID NO: 3 An antibody fragment comprising the antibody according to <1> or <2>, or a variable region thereof, characterized in that it comprises an added amino acid sequence.
<4> The antibody according to <3>, wherein the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 45, and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 3 Or an antibody fragment comprising the variable region thereof.
<5> Of the hemagglutinin HA1 region of the H5 subtype avian influenza virus shown in any of the following (1) to (4), specifically to the hemagglutinin HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 An antibody fragment or antibody fragment according to any one of <1> to <4>, characterized in that it binds: (1) an antibody consisting of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 44 or An antibody fragment comprising the variable region, (2) an antibody comprising an amino acid sequence in which 1 to 35 amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 44 An antibody fragment containing the variable region, (3) 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 44 The present invention is directed to a polynucleotide comprising a sequence complementary to the polynucleotide encoding the antibody fragment or antibody fragment comprising the variable region thereof, (4) the antibody fragment described in (1) above or the antibody fragment comprising the variable region thereof. An antibody consisting of an amino acid sequence encoded by a polynucleotide hybridizing under various conditions or an antibody fragment comprising the variable region thereof.
<6> An antibody fragment according to any one of <1> to <5> or an antibody fragment containing a variable region thereof, characterized by being obtained by a production method comprising the following steps (a) to (c): :
(A) immunizing the bird with a hemagglutinin HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10; (b) specifically binding to the hemagglutinin HA1 antigen polypeptide from the birds immunized in the step (a) A step of obtaining a phage antibody library containing a phage antibody; and (c) a step of concentrating and selecting an antibody which specifically binds to the hemagglutinin HA1 antigen polypeptide among the phage antibodies obtained in the step (b).
<7> An antibody fragment comprising the antibody according to any one of <1> to <6> or a variable region thereof, which is a single-chain variable region fragment or a bivalent antibody.
<8> The bivalent antibody according to <1>, wherein the heavy chain consists of the amino acid sequence shown in SEQ ID NO: 26 or 52, and the light chain consists of the amino acid sequence shown in SEQ ID NO: 28; An antibody fragment comprising the antibody or the variable region thereof described in any of <7>.
<9> A polynucleotide encoding the antibody according to any one of <1> to <8> or an antibody fragment containing the variable region thereof.
<10> A recombinant vector comprising the polynucleotide according to <9>.
<11> A transformant, wherein the polynucleotide according to <9> or the recombinant vector according to <10> is introduced.
<12> A method for producing an antibody that specifically binds to a hemagglutinin HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 of the hemagglutinin HA1 region of H5 subtype avian influenza virus, which comprises the following (a A) a method comprising the steps of (c): (a) immunizing the avian hemagglutinin HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 with a bird; (b) the above step (a) Obtaining a phage antibody library comprising a phage antibody which specifically binds to the above-mentioned hemagglutinin HA1 antigen polypeptide from the birds immunized with the above; and (c) the above-mentioned hemagglutinin HA1 among the phage antibodies obtained in the above step (b) Concentrating and selecting antibodies that specifically bind to the antigenic polypeptide.
<13> The method according to <12>, wherein the antibody is a single chain variable region fragment or a bivalent antibody.
<14> A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 10.
<15> A kit for detecting H5 subtype avian influenza virus contained in a sample, comprising an antibody according to any one of <1> to <8> or an antibody fragment containing a variable region thereof A kit that is characterized by
<16> A method for detecting H5 subtype avian influenza virus contained in a sample, comprising: the antibody according to any one of <1> to <8> or an antibody fragment containing the variable region thereof Reacting the sample with the sample.
<17> The method according to <16>, wherein the sample is a swab of trachea, laryngopharynx, oral cavity or common excretory.
<18> An antibody or an antibody fragment containing a variable region thereof, which is characterized by specifically binding to only H5 subtype avian influenza virus among avian influenza viruses.
<19> An antibody or an antibody fragment comprising a variable region thereof, characterized in that it specifically binds to H5 subtype avian influenza virus and does not specifically bind to H1 to H4 and H6 to H16 subtype avian influenza virus .
<20> The antibody fragment according to <18> or <19>, or a variable region thereof, wherein the H5 subtype avian influenza virus is an H5N1 avian influenza virus.
<21> Any one of <18> to <20>, which specifically binds to the region represented by the amino acid sequence shown in SEQ ID NO: 10 in the hemagglutinin HA1 region of H5 subtype avian influenza virus An antibody fragment comprising the described antibody or its variable region.
　以下に実施例を示し、本発明の実施の形態についてさらに詳しく説明する。もちろん、本発明は以下の実施例に限定されるものではなく、細部については様々な態様が可能であることはいうまでもない。さらに、本発明は上述した実施形態に限定されるものではなく、請求項に示した範囲で種々の変更が可能であり、それぞれ開示された技術的手段を適宜組み合わせて得られる実施形態についても本発明の技術的範囲に含まれる。また、本明細書中に記載された文献の全てが参考として援用される。 Examples will be shown below, and the embodiment of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it is needless to say that various aspects are possible as to details. Furthermore, the present invention is not limited to the embodiments described above, and various modifications can be made within the scope of the claims, and embodiments obtained by appropriately combining the disclosed technical means are also included. It is included in the technical scope of the invention. Also, all of the documents described in the present specification are incorporated by reference.
　〔実施例１：野生型抗体の作製および反応性の評価〕
　（１－１．抗体の作製）
　＜ニワトリの免疫および免疫ニワトリ脾臓からのｓｃＦｖファージ抗体ライブラリーの作製＞
　６０日齡のメスのニワトリに、Ｈ５亜型インフルエンザウイルス由来ヘマグルチニンの合成ポリペプチド：ＨＡ１抗原ポリペプチド（“ペプチド番号：１１１１０５９Ｎ－１”）（配列番号１０）を免疫した（０．３ｍｇ／羽）。一次免疫には完全フロイントアジュバンド（014-09541、Wako）、二次および三次免疫には不完全フロイントアジュバンド（011-09551、Wako）を用いて抗原を免疫した。４回目の免疫はＰＢＳ（phosphate bufferedsaline）に希釈した抗原を静脈注射した。４回目の免疫から３日後にニワトリの脾臓を
回収し、Ficoll paque PLUS（17-1440-03、GE Healthcare）を用いた密度勾配遠心によりリンパ球を単離し、得られたリンパ球からTRIzole Reagent（15596026、Life Technologies）を用いてＲＮＡを抽出した。抽出したＲＮＡからPrimeScript II 1st Strand cDNA Synthesis Kit（6210A、TAKARA）を用いたＲＴ－ＰＣＲによりｃＤＮＡの合成を行い、ｓｃＦｖファージライブラリーを作製した。ｓｃＦｖファージライブラリーの作製は、参考文献：“Nakamura et al., J Vet Med Sci. 2004 Ju;66 (7): 807-814”に記載の方法に従って行った。 [Example 1: Production of wild-type antibody and evaluation of reactivity]
(1-1. Preparation of antibody)
Preparation of scFv Phage Antibody Library from Chicken Immunization and Immune Chicken Spleen
A 60-day-old female chicken was immunized with a synthetic polypeptide of hemagglutinin derived from H5 subtype influenza virus: HA1 antigen polypeptide ("peptide number: 1111059N-1") (SEQ ID NO: 10) (0.3 mg / feather) . Antigens were immunized with complete Freund's adjuvant (014-09541, Wako) for primary immunization and incomplete Freund's adjuvant (011-09551, Wako) for secondary and tertiary immunization. In the fourth immunization, antigen diluted in PBS (phosphate buffered saline) was intravenously injected. Three days after the fourth immunization, chicken spleen is collected, lymphocytes are isolated by density gradient centrifugation using Ficoll paque PLUS (17-1440-03, GE Healthcare), and TRIzole Reagent (from the obtained lymphocytes) RNA was extracted using 1559 026, Life Technologies). The cDNA was synthesized from the extracted RNA by RT-PCR using PrimeScript II 1 st Strand cDNA Synthesis Kit (6210A, TAKARA) to prepare a scFv phage library. Preparation of scFv phage library was performed according to the method described in the reference: "Nakamura et al., J Vet Med Sci. 2004 Ju; 66 (7): 807-814".
　上述の工程の模式図を図１に示す。図１は、抗体の作製方法における一工程を示す図であり、上述のニワトリの免疫および免疫ニワトリ脾臓からのｓｃＦｖファージ抗体ライブラリーの作製の工程を示している。 A schematic view of the above-mentioned process is shown in FIG. FIG. 1 is a diagram showing one step in the method for producing an antibody, and shows the steps for producing a scFv phage antibody library from the above-described chicken immunity and immune chicken spleen.
　＜パニング選択＞
　ｓｃＦｖファージ抗体ライブラリーを用いて、合成ペプチドを固相化したプレートによるパニングを行った。パニングは参考文献：“Nakamura et al., J Vet Med Sci. 2004 Ju;66 (7): 807-814”に記載の方法に従って行った。５回パニングを行った後、ライブラリーの反応性を、合成ペプチドを固相化したプレートを用いたＥＬＩＳＡによって確認し、反応性が上昇し始めたライブラリーからファージのスクリーニングを行った。スクリーニングの手法は以下の通りである。ファージを大腸菌に感染させてアンピシリン（５０μｇ／ｍｌ、nacalai）を含む２×ＹＴAgar plateにプレーティングし、得られたコロニーをアンピシリン含有２×ＹＴ液体培地中で培養した。ヘルパーファージに感染させた後、アンピシリン（５０μg／ｍｌ）、カナマイシン（２５μｇ／ｍｌ、明治製菓株式会社）、ＩＰＴＧ（１００μｇ／ｍｌ、nacalai）含有２×ＹＴ液体培地中でファージの誘導を行った。得られた培養上清中のｓｃＦｖファージ抗体の反応性を、抗原ＨＡ１固相化プレートを用いたＥＬＩＳＡによって確認した。 <Panning selection>
The scFv phage antibody library was used to perform panning on a plate on which a synthetic peptide was immobilized. Panning was performed according to the method described in the reference: "Nakamura et al., J Vet Med Sci. 2004 Ju; 66 (7): 807-814". After five rounds of panning, the reactivity of the library was confirmed by ELISA using a plate on which a synthetic peptide was immobilized, and phages were screened from the library whose reactivity began to increase. The screening method is as follows. The phage was infected into E. coli and plated on 2 × YTAgar plate containing ampicillin (50 μg / ml, nacalai), and the obtained colonies were cultured in 2 × YT liquid medium containing ampicillin. After infection with helper phage, induction of phage was carried out in 2 × YT liquid medium containing ampicillin (50 μg / ml), kanamycin (25 μg / ml, Meiji Seika Kaisha, Ltd.), IPTG (100 μg / ml, nacalai). The reactivity of the scFv phage antibody in the obtained culture supernatant was confirmed by ELISA using an antigen HA1 immobilized plate.
　上述の工程の模式図を図２に示す。図２は、抗体の作製方法における一工程を示す図であり、ｓｃＦｖファージライブラリーの、パニング選択の工程を示している。 A schematic view of the above-described process is shown in FIG. FIG. 2 is a diagram showing one step in the method for producing an antibody, and shows a step of panning selection of scFv phage library.
　続いて、上述のライブラリーのスクリーニングを行った。スクリーニングは以下の＜ＥＬＩＳＡ＞の項目に示す、ＥＬＩＳＡ法を用いて行った。 Subsequently, the above library was screened. The screening was performed using the ELISA method shown in the item of <ELISA> below.
　＜ＥＬＩＳＡ＞
　スクリーニングでは、５μg／ｍｌの合成ペプチドを含むＰＢＳを５０μｌ／ウェルで９６ウェルプレート（442404、Thermo）に入れ、一晩、４℃で抗原を固相化した。固相化の後、２５％Block Ace（UK-B80、DSファーマバイオメディカル）を含むＰＢＳでウェルをブロッキングし、ｓｃＦｖファージ抗体を含む培養上清を反応させた。二次抗体として、ＨＲＰ標識Goat anti-chickenIgG (H+L)（14-24-06 、KPL）を１０％Block Aceに１０００倍希釈した溶液を加え、基質として用いたＯＰＤの発色をプレートリーダー（Model 680、BIO-RAD社）で４９０ｎｍおよび６３０ｎｍの吸光度を測定した。 <ELISA>
For screening, PBS containing 5 μg / ml of synthetic peptide was placed at 50 μl / well in a 96-well plate (442404, Thermo), and the antigen was immobilized overnight at 4 ° C. After immobilization, the wells were blocked with PBS containing 25% Block Ace (UK-B80, DS Pharma Biomedical), and the culture supernatant containing the scFv phage antibody was reacted. As a secondary antibody, a solution of HRP-labeled Goat anti-chicken IgG (H + L) (14-24-06, KPL) diluted 10-fold in 10% Block Ace was added, and the color of the OPD used as a substrate was added to the plate reader ( The absorbance at 490 nm and 630 nm was measured using Model 680 (manufactured by BIO-RAD).
　上述の工程の模式図を図３に示す。図３は、本発明の実施例に係る、抗体の作製方法における一工程を示す図であり、ｓｃＦｖファージライブラリーの、ＥＬＩＳＡを用いたスクリーニングの工程を示している。 A schematic view of the above process is shown in FIG. FIG. 3 is a diagram showing one step in the method for producing an antibody according to an example of the present invention, and shows a step of screening a scFv phage library using ELISA.
　さらに、上述の工程で得られた陽性クローンを選択し、ＤＮＡシークエンサー（ABI PRISM 3100－Genetic Analyzer、Applied Biosystems）を用いてシークエンスを行うことによって、選択された陽性クローンのうち、陽性クローン番号No. 6-2-5のアミノ酸の配列を決定した。シークエンスに用いたプライマーを配列番号１１および配列番号１２として示し、No. 6-2-5のアミノ酸配列を配列番号１に示す。また、No. 6-2-5のＨ鎖およびＬ鎖におけるＣＤＲ１、２および３の配列を図４に示す。図４は、得られた抗体のアミノ酸配列の可変領域を示す図である。 Furthermore, positive clones obtained in the above-mentioned step are selected, and sequencing is performed using a DNA sequencer (ABI PRISM 3100-Genetic Analyzer, Applied Biosystems). The sequence of amino acids 6-2-5 was determined. The primers used for the sequence are shown as SEQ ID NO: 11 and SEQ ID NO: 12, and the amino acid sequence of No. 6-2-5 is shown in SEQ ID NO: 1. Further, the sequences of CDR1, 2 and 3 in the H chain and L chain of No. 6-2-5 are shown in FIG. FIG. 4 is a diagram showing the variable region of the amino acid sequence of the obtained antibody.
　＜組換えマウス／ニワトリキメラ（ＩｇＧ１）抗体発現ベクターへの組換え＞
　上記で選択したクローンのｓｃＦｖ抗体をコードするＤＮＡ鎖を鋳型にして、ニワトリ由来抗体遺伝子Ｈ鎖可変領域およびＬ鎖可変領域のＰＣＲ増幅を行った後、ＰＣＲ産物をＳａｃIIおよびＮｈｅＩ制限酵素処理（R0157S、R0131S、BioLabs）し、Ｈ鎖可変領域お
よびＬ鎖可変領域のそれぞれについて、同じように制限酵素処理したマウス／ニワトリキメラ抗体（ＩｇＧ１）発現ベクター（Ｈ鎖用発現ベクター：pcDNA4/myc-His、Ｌ鎖用発現ベクター：pcDNA3/myc-His、Invitrogen）に組換えた。作製したＨ鎖およびＬ鎖のコンストラクトをほ乳類培養細胞にトランスフェクトした後、発現した抗体（抗ＨＡ１マウス／ニワトリキメラモノクローナル抗体）の精製をProteinG Sepharose 4 Fast Flow（17-018-02、GE healthcare）を用いて行った。以上により、本発明に係る二価抗体のクローンを得た（No. 6-2-5）。得られたNo. 6-2-5の二価抗体のＨ鎖全長のアミノ酸配列を配列番号２６に、Ｈ鎖全長の塩基配列を配列番号２７にそれぞれ示す。また、Ｌ鎖全長のアミノ酸配列を配列番号２８に、Ｌ鎖全長の塩基配列を配列番号２９にそれぞれ示す。なお、以降、実施例１で得られたNo. 6-2-5の配列を野生型とし、以下の実施例２においてこれを改変した配列を変異型配列として記載する。 <Recombination to a recombinant mouse / chicken chimeric (IgG1) antibody expression vector>
Using the DNA chain encoding the scFv antibody of the clone selected above as a template, PCR amplification of the chicken-derived antibody gene H chain variable region and L chain variable region is performed, and then the PCR product is treated with SacII and NheI restriction enzymes (R0157S , R0131S, BioLabs) A mouse / chicken chimeric antibody (IgG1) expression vector (H chain expression vector: pcDNA4 / myc-His, similarly treated with restriction enzymes for each of the H chain variable region and L chain variable region) Expression vector for L chain: pcDNA3 / myc-His, Invitrogen) was recombined. After transfecting the prepared H-chain and L-chain constructs into cultured mammalian cells, purification of the expressed antibody (anti-HA1 mouse / chicken chimeric monoclonal antibody) was carried out using ProteinG Sepharose 4 Fast Flow (17-018-02, GE healthcare) Using the Thus, a clone of the bivalent antibody according to the present invention was obtained (No. 6-2-5). The amino acid sequence of the H chain full length of the obtained bivalent antibody No. 6-2-5 is shown in SEQ ID NO: 26 and the base sequence of the H chain full length in SEQ ID NO: 27, respectively. In addition, the amino acid sequence of full length L chain is shown in SEQ ID NO: 28 and the base sequence of full length L chain is shown in SEQ ID NO: 29 Hereinafter, the sequence of No. 6-2-5 obtained in Example 1 is referred to as wild type, and a sequence obtained by modifying the same in Example 2 below is described as a variant type sequence.
　上述の工程の模式図を図５に示す。図５は、抗体の作製方法における一工程を示す図であり二価抗体発現ベクターへの組換えによる二価抗体の作製の工程を示している。 A schematic view of the above-mentioned process is shown in FIG. FIG. 5 is a diagram showing one step in the method for producing an antibody, and shows steps for producing a bivalent antibody by recombination into a bivalent antibody expression vector.
　得られた精製抗体を５～２０％のc-Pagel（C520L、ATTO社）を用いてＳＤＳ－ＰＡＧＥ電気泳動を行い、ＣＢＢ染色（178-00551、Wako）によってその精製度を確認した。 The resulting purified antibody was subjected to SDS-PAGE electrophoresis using 5-20% c-Page (C520L, ATTO), and the degree of purification was confirmed by CBB staining (178-00551, Wako).
　結果を図６に示す。図６は、野生型の二価抗体タンパク質を電気泳動後にＣＢＢ染色した結果を示す図である。 The results are shown in FIG. FIG. 6 shows the results of CBB staining of wild-type bivalent antibody protein after electrophoresis.
　なお、上述の組換えに関しては、参考文献：“Tateishi et al., J Vet Med Sci. 2008Apr;70(4): 397-400”に記載の方法に従った。また、Ｈ鎖可変領域のＰＣＲに用いたプ
ライマー配列を配列番号１３および配列番号１４に、Ｌ鎖可変領域のＰＣＲに用いたプライマー配列を配列番号１５および配列番号１６にそれぞれ示す。 In addition, regarding the above-mentioned recombination, the method described in reference: "Tateishi et al., J Vet Med Sci. 2008 Apr; 70 (4): 397-400" was followed. The primer sequences used for PCR of the H chain variable region are shown in SEQ ID NO: 13 and SEQ ID NO: 14, and the primer sequences used for PCR of the L chain variable region are shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively.
　（１－２．Ｈ５Ｎ１亜型鳥インフルエンザウイルス抗体の活性測定）
　抗体価の測定については、以下の通り行った。５μｇ／ｍｌのrecombinant hemagglutinin（ｒＨＡ）（Ｈ５Ｎ１ウイルス由来、A/Vietnam/1203/2004、CT6450、Protein Sciences Corp.）またはウシ血清アルブミン（ＢＳＡ（bovine serum albumin））を含むＰＢＳを同様に固相化し、ブロッキング後にマウス／ニワトリキメラ抗体の精製抗体（No. 6-2-5）を、０～２μｇ／ｍｌの濃度で反応させた。二次抗体としてＨＲＰ標識Goat anti-mouse IgG (H+L)（474-1806、KPL社）を用い、上記２．と同様にして抗体の反応性を検出した。結果を図７に示す。図７は、野生型の二価抗体の抗体価を示す図である。 (1-2. Activity measurement of H5N1 avian influenza virus antibody)
The antibody titer was measured as follows. Similarly, PBS containing 5 μg / ml of recombinant hemagglutinin (rHA) (derived from H5N1 virus, A / Vietnam / 1203/2004, CT6450, Protein Sciences Corp.) or bovine serum albumin (BSA (bovine serum albumin)) was similarly immobilized After blocking, purified mouse / chicken chimeric antibody (No. 6-2-5) was reacted at a concentration of 0 to 2 μg / ml. As the secondary antibody, HRP-labeled Goat anti-mouse IgG (H + L) (474-1806, manufactured by KPL) is used. The reactivity of the antibody was detected in the same manner as in. The results are shown in FIG. FIG. 7 shows the antibody titer of a wild-type bivalent antibody.
　（１－３．抗体結合特異性の評価）
　異なる株のＨ５Ｎ１亜型鳥インフルエンザウイルス、およびＨ５以外の亜型鳥インフルエンザウイルスと本発明に係る抗体の反応性の測定を、以下の方法を用いて行った。 (1-3. Evaluation of antibody binding specificity)
The measurement of the reactivity of the antibody according to the present invention with different strains of H5N1 avian influenza virus and avian influenza viruses other than H5 was carried out using the following method.
　１０μｇ／ｍｌの各種ウイルス、ｒＨＡまたはＢＳＡのいずれかを含むＰＢＳを上記と同様に固相化し、ブロッキング後にマウス／ニワトリキメラ本発明に係る抗体の精製抗体（No. 6-2-5）を２μｇ／ｍｌの濃度で反応させた。二次抗体にＨＲＰ標識Goat anti-mouse IgG(H+L)を用い、上記２．と同様にして検出した。結果を図８に示す。図８は、野生型の二価抗体のＨ５Ｎ１亜型鳥インフルエンザウイルスの変異体および複数種類のＨ５以外の鳥インフルエンザウイルス亜型に対する抗体結合性を示す図である。図８に示されるように、本実施例で得られた野生型の精製二価抗体（No. 6-2-5）はＨ５亜型以外の鳥インフルエンザウイルスに対する結合性はほぼない一方、何れのＨ５亜型の鳥インフルエンザウイルスに対しても結合性が高く、特異性があることが示された。 PBS containing 10 μg / ml of various viruses, either rHA or BSA was immobilized in the same manner as above, and after blocking, mouse / chicken chimera 2 μg of purified antibody (No. 6-2-5) of the antibody according to the present invention The reaction was carried out at a concentration of / ml. HRP-labeled Goat anti-mouse IgG (H + L) is used as the secondary antibody, as described in 2. above. It detected in the same way. The results are shown in FIG. FIG. 8 shows antibody binding to wild-type bivalent antibody H5N1 avian influenza virus variants and to a plurality of H5 avian influenza virus subtypes other than H5. As shown in FIG. 8, the purified wild-type bivalent antibody (No. 6-2-5) obtained in this example has almost no binding ability to avian influenza virus except subtype H5, It also showed high binding ability and specificity to the H5 subtype avian influenza virus.
　〔実施例２：変異型抗体の作製および反応性の評価〕
　（２－１．抗体の作製）
　＜変異ライブラリーの作製＞
　上述のクローン番号No. 6-2-5のＨ鎖を鋳型として、Ｉｎｖｅｒｓｅ　ＰＣＲ法を用いてNo. 6-2-5のＨ鎖のＣＤＲ３に９塩基ずつランダムな変異を導入したランダム変異体を７種類作製した（SMK-101、TOYOBO）。用いたプライマーセットは配列番号３０～４３に示す。 Example 2 Preparation of Mutant Antibody and Evaluation of Reactivity
(2-1. Preparation of antibody)
<Preparation of mutant library>
Using the H chain of the above-mentioned clone No. 6-2-5 as a template, random mutations in which 9 base random mutations were introduced into CDR3 of the H chain of No. 6-2-5 using Inverse PCR method were used. Seven types were prepared (SMK-101, TOYOBO). The primer sets used are shown in SEQ ID NOS: 30-43.
　得られた７種類のランダム変異体のＨ鎖の可変領域およびＬ鎖の可変領域をそれぞれＰＣＲで増幅したものをリンカーでつなぎ合わせ、ｓｃＦｖファージライブラリーを作製した。ｓｃＦｖファージライブラリーの作製方法の詳細は、参考文献：“Nakamura et al., J Vet Med Sci. 2004 Ju;66 (7): 807-814”に記載の方法に従って行った。 The variable regions of the H chain and the variable region of the L chain of the seven random variants obtained were amplified by PCR, respectively, and joined with a linker to prepare a scFv phage library. The details of the method of preparing the scFv phage library were performed according to the method described in the reference: "Nakamura et al., J Vet Med Sci. 2004 Ju; 66 (7): 807-814".
　＜パニング選択＞
　ｓｃＦｖファージ抗体ライブラリーを用いて、ｒＨＡ（11700-V08H 、Sino Biological Inc.）に対するパニングを行った。パニングは参考文献：“Nakamura et al., J Vet Med Sci. 2004 Ju;66 (7): 807-814”に記載の方法に従って行った。４回パニングを実施した後、反応性の上昇をＥＬＩＳＡによって確認した。反応性が上昇してきた２回目のパニングライブラリからスクリーニングを実施した。スクリーニングは、〔実施例１〕における（１－１．抗体の作製）＜パニング選択＞に記載の方法と同様の方法を用いて行った。 <Panning selection>
The scFv phage antibody library was used to perform panning against rHA (11700-V08H, Sino Biological Inc.). Panning was performed according to the method described in the reference: "Nakamura et al., J Vet Med Sci. 2004 Ju; 66 (7): 807-814". After 4 rounds of panning, the increase in reactivity was confirmed by ELISA. Screening was performed from the second round of panning library whose reactivity has been increased. The screening was performed using the same method as that described in (1-1. Preparation of antibody) in <Example 1><Panningselection>.
　外部委託によって、配列確認を実施した（ユーロフィンジェノミクス）。その結果、新規な配列の抗体の取得が確認できた。新規取得クローンNo. 6-2-5-21の単鎖抗体全長のアミノ酸配列を配列番号４４に示す。新規に取得されたクローンであるNo. 6-2-5-21の配列は、No. 6-2-5と比較して、Ｈ鎖のＣＤＲ３の配列以外は同一の配列を有していた。No. 6-2-5-21のＨ鎖可変領域のアミノ酸配列を配列番号４５に、ＣＤＲ３のアミノ酸列を配列番号４６に、ＣＤＲ３の塩基配列を配列番号４７にそれぞれ示す。さらに、No. 6-2-5-21のＨ鎖におけるＣＤＲ３の配列を図９に示す。図９は、野生型抗体No. 6-2-5のＨ鎖のＣＤＲ３のアミノ酸配列と変異型抗体No. 6-2-5-21のＨ鎖のＣＤＲ３のアミノ酸配列とのアライメントを示す図である。 Sequence confirmation was performed by outsourcing (Eurofin Genomics). As a result, acquisition of an antibody of a novel sequence was confirmed. The amino acid sequence of full length single chain antibody of newly obtained clone No. 6-2-5-21 is shown in SEQ ID NO: 44. The sequence of No. 6-2-5-21, which is a newly obtained clone, had the same sequence as that of No. 6-2-5 except for the sequence of CDR3 of the H chain. The amino acid sequence of the H chain variable region of No. 6-2-521 is shown in SEQ ID NO: 45, the amino acid sequence of CDR3 in SEQ ID NO: 46, and the base sequence of CDR3 in SEQ ID NO: 47. Furthermore, the sequence of CDR3 in the H chain of No. 6-2-5-21 is shown in FIG. FIG. 9 is a diagram showing an alignment of the amino acid sequence of the CDR3 of the H chain of wild type antibody No. 6-2-5 with the amino acid sequence of the CDR3 of the H chain of mutant antibody No. 6-2-5-21. is there.
　＜二価抗体への組換え＞
　次に、新規に取得されたクローンであるNo. 6-2-5-21の二価抗体への組換えを行った。まず、新規に取得されたクローンであるNo. 6-2-5-21のｓｃＦｖ抗体をコードするＤＮＡ鎖を鋳型にして、ニワトリ由来抗体遺伝子Ｈ鎖可変領域およびＬ鎖可変領域のＰＣＲ増幅を行った後、ＳａｃＩＩ（R0157S、BioLabs）およびＮｈｅＩ（R0131S、BioLabs）で制限酵素処理したマウス/ニワトリキメラ抗体（ＩｇＧ１）発現ベクター（Ｈ鎖用発現ベクター：pcDNA4/myc-His、Ｌ鎖用発現ベクター：pcDNA3/myc-His、Invitrogen）に、GeneArt（登録商標）Seamless Cloning and Assembly（A14606、lifetechnologies）を用いて組換えた。実施例１と同様に、使用したマウスキメラ発現ベクターはTateishi et al., J Vet Med Sci. 2008 Apr;70(4): 397-400に記載されているベクターを使用した。可変領域増幅用プライマーの配列を配列番号４８～５１に示す。 <Recombination to bivalent antibody>
Next, recombination to a bivalent antibody of No. 6-2-5-21, which is a newly obtained clone, was performed. First, PCR amplification of the chicken-derived antibody gene H chain variable region and L chain variable region was performed using the DNA chain encoding the scFv antibody of No. 6-2-5-21 which is a newly obtained clone as a template Then, mouse / chicken chimeric antibody (IgG1) expression vector (H chain expression vector): restriction vector treated with SacII (R0157S, BioLabs) and NheI (R0131S, BioLabs) expression vector: pcDNA4 / myc-His, L chain expression vector: Recombination into pcDNA3 / myc-His, Invitrogen) using GeneArt® Seamless Cloning and Assembly (A14606, lifetechnologies). As in Example 1, the mouse chimeric expression vector used was the vector described in Tateishi et al., J Vet Med Sci. 2008 Apr; 70 (4): 397-400. The sequences of the variable region amplification primers are shown in SEQ ID NOs: 48 to 51.
　＜二価抗体の製造＞
　作製したＨ鎖およびＬ鎖のコンストラクトを、ほ乳類培養細胞にExpi293Expression system（Cat#A14635、Invitrogen）を用いてトランスフェクトした後、発現した抗体の精製をProtein G Sepharose 4 Fast Flow（17-018-02、GE healthcare）を用いて行った。以上により、本発明に係る変異型の二価抗体のクローンを得た（No. 6-2-5-21）。得られた精製抗体を各種結合活性の測定に用いた。なお、得られたNo. 6-2-5-21の二価抗体のＨ鎖全長のアミノ酸配列を配列番号５２に、Ｈ鎖全長の塩基配列を配列番号５３にそれぞれ示す。また、Ｌ鎖全長のアミノ酸配列および塩基配列は、野生型No. 6-2-5の配列と同一であった。 <Production of bivalent antibody>
After transfecting the prepared heavy chain and light chain construct into cultured mammalian cells using Expi293 Expression system (Cat # A14635, Invitrogen), purification of the expressed antibody is performed by Protein G Sepharose 4 Fast Flow (17-018-02). , GE healthcare). Thus, a clone of a mutated bivalent antibody according to the present invention was obtained (No. 6-2-5-21). The purified antibodies obtained were used for measurement of various binding activities. The amino acid sequence of the H chain full length of the obtained bivalent antibody No. 6-2-5-21 is shown in SEQ ID NO: 52, and the base sequence of the H chain full length in SEQ ID NO: 53. In addition, the amino acid sequence and the nucleotide sequence of the full-length L chain were identical to the sequence of wild type No. 6-2-5.
　（２－２．変異型抗体の活性測定）
　＜ＥＬＩＳＡを用いた二価抗体の反応性評価＞
　５μｇ／ｍｌのｒＨＡ（11700-V08H、Sino Biological Inc.）を含むＰＢＳを５０μｌ／ウェルの濃度で９６ウェルプレート（442404、Thermo）に入れ、一晩、４℃で抗原を固相化させた。固相化の後、25％Block Ace（UK-B80DS、ファーマバイオメディカ）を含むＰＢＳでウェルを３７℃で１時間ブロッキングした。ＰＢＳ－Ｔで１回洗浄した後、１μｇ／ｍｌの濃度から段階希釈をしたNo. 6-2-5またはNo. 6-2-5-21を含むＰＢＳを３７℃で１時間反応させた。二次抗体として、ＨＲＰ標識Goat anti-mouse IgG (H+L)（474-1806、KPL）を１０％Block Aceに１０００倍希釈した溶液に加え、基質として用いたＯＰＤの発色をプレートリーダー（Model 680、BIO-RAD）で４９０ｎｍおよび６３０ｎｍの吸光度を測定した。結果を図１０に示す。図１０は、変異型の二価抗体の抗体価を示す図である。 (2-2. Measurement of activity of mutant antibody)
<Evaluation of bivalent antibody reactivity using ELISA>
PBS containing 5 μg / ml rHA (11700-V08H, Sino Biological Inc.) was placed in a 96 well plate (442404, Thermo) at a concentration of 50 μl / well, and the antigen was immobilized overnight at 4 ° C. After immobilization, the wells were blocked for 1 hour at 37 ° C. with PBS containing 25% Block Ace (UK-B80DS, Pharma Biomedica). After washing once with PBS-T, PBS containing No. 6-2-5 or No. 6-2-5-21 serially diluted from a concentration of 1 μg / ml was reacted at 37 ° C. for 1 hour. Add HRP-labeled Goat anti-mouse IgG (H + L) (474-1806, KPL) as a secondary antibody to a solution diluted 1000-fold in 10% Block Ace, and use OPD color as a substrate as a plate reader (Model Absorbance at 490 nm and 630 nm was measured with 680, BIO-RAD). The results are shown in FIG. FIG. 10 is a diagram showing the antibody titer of a mutated bivalent antibody.
　＜Biacoreを用いた二価抗体の反応性評価＞
　Biacore（Biacore T200、GE Healthcare）を行い、No. 6-2-5およびNo. 6-2-5-21のｒＨＡ（11700-V08H、Sino Biological）に対する親和性を評価した。No. 6-2-5およびNo. 6-2-5-21のＦｃ部位はマウス型であるため、Mouse Antibody Capture Kit（BR-1008-38、GE Healthcare）を用いて、親和性を測定した。具体的には、メーカー提供の標準プロトコルに従い、ＮＨＳ／ＥＤＣを使用し、ＣＭ５チップ表面のフリーカルボキシル基を利用したアミンカップリング法によって、ウサギ抗マウスポリクローナル抗体をＣＭ５チップ表面に固定化した。次に、No. 6-2-5およびNo. 6-2-5-21をウサギ抗マウスポリクローナル抗体にキャプチャーした。Biacore T200に種々の濃度のｒＨＡを供し、カイネティクスセンサーグラムを作成し、結合定数を計算した。結果を以下の表１に示す。 <Evaluation of bivalent antibody reactivity using Biacore>
Biacore (Biacore T200, GE Healthcare) was performed to evaluate the affinity for No. 6-2-5 and No. 6-2-5-21 for rHA (11700-V08H, Sino Biological). Since the Fc site of No. 6-2-5 and No. 6-2-5-21 is a mouse type, the affinity was measured using Mouse Antibody Capture Kit (BR-1008-38, GE Healthcare) . Specifically, a rabbit anti-mouse polyclonal antibody was immobilized on the surface of the CM5 chip by amine coupling using free carboxyl groups on the surface of the CM5 chip using NHS / EDC according to a standard protocol provided by the manufacturer. Next, Nos. 6-2-5 and 6-2-5-21 were captured on rabbit anti-mouse polyclonal antibodies. Different concentrations of rHA were applied to Biacore T200 to generate kinetic sensorgrams and to calculate binding constants. The results are shown in Table 1 below.
Figure JPOXMLDOC01-appb-T000001
 　（２－３．抗体結合特異性の評価）
　Ｈ５亜型以外の亜型の鳥インフルエンザウイルスとNo. 6-2-5-21抗体の反応性の測定を、以下の方法を用いて行った。
Figure JPOXMLDOC01-appb-T000001
 (2-3. Evaluation of antibody binding specificity)
The measurement of the reactivity of the avian influenza virus of subtypes other than H5 subtype and No. 6-2-5-21 antibody was performed using the following method.
　５μｇ／ｍｌのＨ５亜型およびＨ５以外の各亜型のｒＨＡを含むＰＢＳを上記と同様に固相化し、ブロッキング後にマウス/ニワトリキメラ抗体No. 6-2-5-21を１μｇ／ｍｌの濃度で反応させた。二次抗体にＨＲＰ標識Goat anti-mouse IgG (H+L)を用い、＜ＥＬＩＳＡを用いた二価抗体の反応性評価＞と同様にして検出した。結果を図１１に示す。図１１は変異型の二価抗体の、各亜型鳥インフルエンザウイルスに対する抗体結合性を示す図である。 Immobilize PBS containing 5 μg / ml H5 subtype and rHA of each subtype except H5 in the same manner as above, and after blocking mouse / chicken chimeric antibody No. 6-2-5-21 at a concentration of 1 μg / ml It was made to react. HRP-labeled Goat anti-mouse IgG (H + L) was used as a secondary antibody, and detection was performed in the same manner as in <Evaluation of reactivity of bivalent antibody using ELISA>. The results are shown in FIG. FIG. 11 is a view showing antibody binding of each of the mutant bivalent antibodies to each of the avian influenza viruses.
　〔参考例〕
　（１．ＨＡ１抗原ポリペプチドの配列の解析）
　ＮＣＢＩのインフルエンザウイルスリソース（ウェブページ：http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html）から、１９７９５個の鳥インフルエンザウイルスのＨＡ遺伝子のアミノ酸配列を取得した。なお、これらの全ＨＡ遺伝子は、Ｈ１～Ｈ１６までの全亜型を含んでいる。遺伝子解析ソフトウェアであるBioEdit（ウェブページ：http://www.mbio.ncsu.edu/bioedit/bioedit.html）を用いて配列のアライメントを作成し、Ｈ１～Ｈ１６の各亜型のコンセンサス配列をそれぞれ作成した。立体構造解析ソフトウェアであるPymol（ウェブページ：http://www.pymol.org/）を用い、プロテインデータバンク（ウェブページ：http://pdbj.org/）に登録されているＨ５亜型のＨＡタンパク質（PDB ID:2IBX）の立体構造とＨ５亜型のアミノ酸の配列とを比較した。 [Reference example]
(1. Analysis of sequence of HA1 antigen polypeptide)
From the NCBI influenza virus resource (web page: http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html), the amino acid sequence of the HA gene of 19795 avian influenza viruses was obtained. These all HA genes contain all subtypes H1 to H16. Sequence alignments were made using the gene analysis software BioEdit (web page: http://www.mbio.ncsu.edu/bioedit/bioedit.html), and the consensus sequences for each of the H1 to H16 subtypes were Created. The three-dimensional structure analysis software Pymol (web page: http://www.pymol.org/) was used, and the H5 subtype registered in the protein data bank (web page: http://pdbj.org/) The three-dimensional structure of HA protein (PDB ID: 2IBX) was compared with the sequence of the amino acid of H5 subtype.
　図１２は、Ｈ５Ｎ１亜型鳥インフルエンザウイルスの抗原として用いた合成ポリペプチドの配列（右）およびＨＡタンパク質の立体構造（左）を示す図である。 FIG. 12 shows the sequence (right) of a synthetic polypeptide used as an antigen of H5N1 avian influenza virus, and the three-dimensional structure (left) of an HA protein.
　図１２に示すように、本実施例の抗体作製に用いた抗原ポリペプチドは、配列番号１０として示す１６個のアミノ酸からなるアミノ酸配列は、ＨＡ１領域のうち、Ｈ５Ｎ１亜型の異なるクレード０～９の株間で保存されていた。また、このアミノ酸配列は、ＮＣＢＩに登録されている１９９７年以降に分離された２８１２株のＨ５亜型の９９．４％において、保存されていた。 As shown in FIG. 12, in the antigen polypeptide used for antibody production of this example, the amino acid sequence consisting of 16 amino acids shown as SEQ ID NO: 10 is a clade 0-9 different in H5N1 subtype in the HA1 region. It was saved among the stocks of In addition, this amino acid sequence was conserved in 99.4% of H5 subtypes of 2812 strains isolated after 1997 registered in NCBI.
　また、図１２にも示されている通り、この１６個のアミノ酸からなるアミノ酸配列は、Ｈ５亜型の異なる株間において高く保持され、且つ立体構造上タンパク質の表面に出ていた。 In addition, as also shown in FIG. 12, the amino acid sequence consisting of 16 amino acids was highly retained between strains different in H5 subtype, and appeared on the surface of the protein in terms of conformation.
　（２．Ｈ１～Ｈ１６の亜型鳥インフルエンザウイルス間およびＨ５Ｎ１亜型鳥インフルエンザウイルスに存在しているすべてのクレード間の配列同一性）
　Ｈ１～Ｈ１６の亜型鳥インフルエンザウイルスにおいて、ＮＣＢＩのインフルエンザウイルスリソース（ウェブページ：http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html）から、Ｈ１亜型について８７８１個、Ｈ２亜型について２３１個、Ｈ３亜型について４５３６個、Ｈ４亜型について６０８個、Ｈ５亜型について２８１２個、Ｈ６亜型について６８７個、Ｈ７亜型について７８７個、Ｈ８亜型について６５個、Ｈ９亜型について７３９個、Ｈ１０亜型について１９０個、Ｈ１１亜型について１８７個、Ｈ１２亜型について８４個、Ｈ１３亜型について５３個、Ｈ１４亜型について４個、Ｈ１５亜型について１２個、Ｈ１６亜型について１９個の鳥インフルエンザウイルスのＨＡ遺伝子全長のアミノ酸配列を取得した。続いて、Ｈ１～Ｈ１６亜型それぞれのアミノ酸配列のコンセンサス配列を亜型毎に作成し、遺伝子解析ソフトウェアGENETYX-MAC ver.17（株式会社ゼネティクス）を用いて、それらのアミノ酸のコンセンサス配列について、Ｈ１～Ｈ１６のすべての亜型間の配列同一性を調べた。また、Ｈ５Ｎ１亜型鳥インフルエンザウイルスに存在するすべてのクレードについて、すべてのクレード間の配列同一性を調べた。ただし、各クレードの配列は、それぞれ図１３に記載した株のアミノ酸配列を用いた。結果を図１３に示す。図１３は、Ｈ１～Ｈ１６亜型鳥インフルエンザウイルスにおけるすべての亜型間およびＨ５Ｎ１亜型鳥インフルエンザウイルスに存在しているすべてのクレード間の配列同一性を示す図である。（ａ）はＨ１～Ｈ１６亜型鳥インフルエンザウイルスにおけるすべての亜型間の配列同一性を示しており、（ｂ）は、Ｈ５Ｎ１亜型鳥インフルエンザウイルスに存在しているすべてのクレード間の配列同一性を示している。 (Sequence identity between H1 to H16 subtype avian influenza virus and all clades present in H5N1 subtype avian influenza virus)
In H1 to H16 subtype avian influenza viruses, 8781 for H1 subtypes from NCBI influenza virus resources (web page: http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html) H2 subtype 231, H3 subtype 4536, H4 subtype 608, H5 subtype 2812, H6 subtype 687, H7 subtype 787, H8 subtype 65, 739 for H9 subtype, 190 for H10 subtype, 187 for H11 subtype, 84 for H12 subtype, 53 for H13 subtype, 4 for H14 subtype, 12 for H15 subtype, H16 The amino acid sequence of the full-length HA gene of 19 avian influenza viruses was obtained for the subtype. Subsequently, a consensus sequence of the amino acid sequence of each of the H1 to H16 subtypes is created for each subtype, and using the gene analysis software GENETYX-MAC ver. 17 (Genetics Co., Ltd.), the consensus sequence of those amino acids is H1. Sequence identity among all subtypes of H16 was examined. In addition, all the clades present in the H5N1 avian influenza virus were examined for sequence identity among all clades. However, the sequence of each clade used the amino acid sequence of the strain described in FIG. 13, respectively. The results are shown in FIG. FIG. 13 is a diagram showing sequence identity among all subtypes of H1-H16 subtype avian influenza virus and among all clades present in H5N1 subtype avian influenza virus. (A) shows sequence identity among all subtypes in H1-H16 subtype avian influenza virus, (b) shows sequence identity among all clades present in H5N1 subtype avian influenza virus Show the sex.

Claims (17)

  1. 　Ｈ５亜型鳥インフルエンザウイルスのヘマグルチニンＨＡ１領域のうち、配列番号１０に示されるアミノ酸配列からなるヘマグルチニンＨＡ１抗原ポリペプチドに特異的に結合することを特徴とする、抗体またはその可変領域を含む抗体断片。 An antibody or an antibody fragment comprising a variable region thereof, characterized in that it specifically binds to a hemagglutinin HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 in the hemagglutinin HA1 region of H5 subtype avian influenza virus.
  2. 　重鎖可変領域中に、配列番号４に示されるＣＤＲ１、配列番号５に示されるＣＤＲ２および配列番号６または配列番号４６に示されるＣＤＲ３のすべてを含んでおり、且つ、軽鎖可変領域中に、配列番号７に示されるＣＤＲ１、配列番号８に示されるＣＤＲ２および配列番号９に示されるＣＤＲ３のすべてを含んでいることを特徴とする、請求項１に記載の抗体またはその可変領域を含む抗体断片。 The heavy chain variable region comprises all of CDR1 shown in SEQ ID NO: 4, CDR2 shown in SEQ ID NO: 5 and CDR3 shown in SEQ ID NO: 6 or SEQ ID NO: 46, and in the light chain variable region The antibody according to claim 1, which comprises all of CDR1 shown in SEQ ID NO: 7, CDR2 shown in SEQ ID NO: 8 and CDR3 shown in SEQ ID NO: 9, or an antibody fragment containing the variable region thereof .
  3. 　重鎖可変領域が、配列番号２に示されるアミノ酸配列、または、配列番号２に示されるアミノ酸配列において、１～１３個のアミノ酸が置換、欠失、挿入、および／もしくは付加されたアミノ酸配列からなり、且つ
    　軽鎖可変領域が、配列番号３に示されるアミノ酸配列、または、配列番号３に示されるアミノ酸配列において、１～１０個のアミノ酸が置換、欠失、挿入、および／もしくは付加されたアミノ酸配列からなることを特徴とする、請求項１または２に記載の抗体またはその可変領域を含む抗体断片。     From the amino acid sequence as shown in SEQ ID NO: 2 or the amino acid sequence as shown in SEQ ID NO: 2, from 1 to 13 amino acids substituted, deleted, inserted and / or added And 1 to 10 amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 3 or the amino acid sequence shown in SEQ ID NO: 3 The antibody according to claim 1 or 2, which comprises an amino acid sequence, or an antibody fragment comprising a variable region thereof.
  4. 　重鎖可変領域が、配列番号４５に示されるアミノ酸配列からなり、且つ
    　軽鎖可変領域が、配列番号３に示されるアミノ酸配列からなることを特徴とする、請求項３に記載の抗体またはその可変領域を含む抗体断片。     The antibody according to claim 3 or the variable thereof, wherein the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 45, and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 3. Antibody fragment containing the region.
  5. 　以下の（１）～（４）の何れかに示す、Ｈ５亜型鳥インフルエンザウイルスのヘマグルチニンＨＡ１領域のうち、配列番号１０に示されるアミノ酸配列からなるヘマグルチニンＨＡ１抗原ポリペプチドに特異的に結合することを特徴とする、請求項１～４のいずれか一項に記載の抗体またはその可変領域を含む抗体断片：
    （１）配列番号１または４４に示されるアミノ酸配列からなる抗体またはその可変領域を含む抗体断片、
    （２）配列番号１または４４に示されるアミノ酸配列において１～３５個のアミノ酸が置換、欠失、挿入、および/または付加されたアミノ酸配列からなる抗体またはその可変領域を含む抗体断片、
    （３）配列番号１または４４に示されるアミノ酸配列に対して９０％以上の配列同一性を有する抗体またはその可変領域を含む抗体断片、
    （４）上記（１）に記載の抗体またはその可変領域を含む抗体断片をコードするポリヌクレオチドと相補的な配列からなるポリヌクレオチドに対して、ストリンジェントな条件下においてハイブリダイズするポリヌクレオチドによってコードされるアミノ酸配列からなる抗体またはその可変領域を含む抗体断片。     Specifically binding to a hemagglutinin HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 in the hemagglutinin HA1 region of H5 subtype avian influenza virus shown in any of the following (1) to (4) An antibody fragment according to any one of claims 1 to 4 characterized by: or an antibody fragment comprising a variable region thereof:
    (1) An antibody consisting of the amino acid sequence shown in SEQ ID NO: 1 or 44 or an antibody fragment containing the variable region thereof
    (2) An antibody fragment comprising an amino acid sequence in which 1 to 35 amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 1 or 44, or an antibody fragment comprising the variable region thereof
    (3) An antibody fragment having a sequence identity of 90% or more to the amino acid sequence shown in SEQ ID NO: 1 or 44, or an antibody fragment comprising the variable region thereof
    (4) A polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of a sequence complementary to a polynucleotide encoding an antibody described in (1) above or an antibody fragment containing the variable region thereof Or an antibody fragment comprising the variable region thereof.
  6. 　下記の（ａ）～（ｃ）の工程を含む製造方法によって得られたことを特徴とする、請求項１～５のいずれか一項に記載の抗体またはその可変領域を含む抗体断片：
    　（ａ）配列番号１０に示されるアミノ酸配列からなるヘマグルチニンＨＡ１抗原ポリペプチドをトリに免疫する工程；
    　（ｂ）上記工程（ａ）で免疫したトリから上記ヘマグルチニンＨＡ１抗原ポリペプチドに特異的に結合するファージ抗体を含むファージ抗体ライブラリーを得る工程；および
    　（ｃ）上記工程（ｂ）で得られたファージ抗体のうち、上記ヘマグルチニンＨＡ１抗原ポリペプチドに特異的に結合する抗体を濃縮および選抜する工程。     The antibody or the antibody fragment containing the variable region thereof according to any one of claims 1 to 5, which is obtained by a production method comprising the following steps (a) to (c):
    (A) immunizing avian with a hemagglutinin HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10;
    (B) obtaining a phage antibody library comprising a phage antibody which specifically binds to the hemagglutinin HA1 antigen polypeptide from the birds immunized in the step (a); and (c) obtained in the step (b) A step of concentrating and selecting an antibody that specifically binds to the hemagglutinin HA1 antigen polypeptide among phage antibodies.
  7. 　一本鎖可変領域断片または二価抗体であることを特徴とする、請求項１～６のいずれか一項に記載の抗体またはその可変領域を含む抗体断片。 The antibody according to any one of claims 1 to 6, which is a single-chain variable region fragment or a bivalent antibody, or an antibody fragment containing the variable region thereof.
  8. 　重鎖が配列番号２６または配列番号５２に示されるアミノ酸配列からなり、且つ、
    　軽鎖が配列番号２８に示されるアミノ酸配列からなる二価抗体であることを特徴とする、請求項１～７のいずれか一項に記載の抗体またはその可変領域を含む抗体断片。     The heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 26 or SEQ ID NO: 52, and
    The antibody fragment according to any one of claims 1 to 7, characterized in that the light chain is a bivalent antibody consisting of the amino acid sequence shown in SEQ ID NO: 28.
  9. 　請求項１～８のいずれか一項に記載の抗体またはその可変領域を含む抗体断片をコードすることを特徴とする、ポリヌクレオチド。 A polynucleotide encoding the antibody according to any one of claims 1 to 8 or an antibody fragment comprising the variable region thereof.
  10. 　請求項９に記載のポリヌクレオチドを有することを特徴とする、組換えベクター。 A recombinant vector comprising the polynucleotide according to claim 9.
  11. 　請求項１０に記載のポリヌクレオチドまたは請求項１０に記載の組換えベクターが導入されていることを特徴とする、形質転換体。 A transformant, wherein the polynucleotide according to claim 10 or the recombinant vector according to claim 10 is introduced.
  12. 　Ｈ５亜型鳥インフルエンザウイルスのヘマグルチニンＨＡ１領域のうち、配列番号１０に示されるアミノ酸配列からなるヘマグルチニンＨＡ１抗原ポリペプチドに特異的に結合する抗体を製造する方法であって、
    　下記の（ａ）～（ｃ）の工程を含むことを特徴とする、方法：
    　（ａ）配列番号１０に示されるアミノ酸配列からなるヘマグルチニンＨＡ１抗原ポリペプチドをトリに免疫する工程；
    　（ｂ）上記工程（ａ）で免疫したトリから上記ヘマグルチニンＨＡ１抗原ポリペプチドに特異的に結合するファージ抗体を含むファージ抗体ライブラリーを得る工程；および
    　（ｃ）上記工程（ｂ）で得られたファージ抗体のうち、上記ヘマグルチニンＨＡ１抗原ポリペプチドに特異的に結合する抗体を濃縮および選抜する工程。     A method for producing an antibody which specifically binds to a hemagglutinin HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 in the hemagglutinin HA1 region of H5 subtype avian influenza virus,
    Method comprising the following steps (a) to (c):
    (A) immunizing avian with a hemagglutinin HA1 antigen polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10;
    (B) obtaining a phage antibody library comprising a phage antibody which specifically binds to the hemagglutinin HA1 antigen polypeptide from the birds immunized in the step (a); and (c) obtained in the step (b) A step of concentrating and selecting an antibody that specifically binds to the hemagglutinin HA1 antigen polypeptide among phage antibodies.
  13. 　上記抗体は、一本鎖可変領域断片または二価抗体であることを特徴とする、請求項１２に記載の方法。 The method according to claim 12, wherein the antibody is a single chain variable region fragment or a bivalent antibody.
  14. 　配列番号１０に示されるアミノ酸配列からなることを特徴とする、ポリペプチド。 A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 10.
  15. 　試料中に含まれるＨ５亜型鳥インフルエンザウイルスを検出するためのキットであって、請求項１～８のいずれか一項に記載の抗体またはその可変領域を含む抗体断片を備えていることを特徴とする、キット。 A kit for detecting H5 subtype avian influenza virus contained in a sample, characterized in that the kit comprises an antibody according to any one of claims 1 to 8 or an antibody fragment comprising a variable region thereof. To make a kit.
  16. 　試料中に含まれるＨ５亜型鳥インフルエンザウイルスを検出する方法であって、
    　請求項１～８のいずれか一項に記載の抗体またはその可変領域を含む抗体断片と、生体から調製した試料とを反応させる工程を含むことを特徴とする、方法。     A method for detecting H5 subtype avian influenza virus contained in a sample, comprising:
    A method comprising the step of reacting an antibody according to any one of claims 1 to 8 or an antibody fragment comprising a variable region thereof with a sample prepared from a living body.
  17. 　上記試料は、気管、咽喉頭、口腔または総***腔の、拭い液であることを特徴とする、請求項１６に記載の方法。 17. The method according to claim 16, wherein the sample is a swab of the trachea, larynx, oral cavity or cloacal cavity.
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