WO2006093080A1 - PROCEDE DESTINE A PRODUIRE UN ANTICORPS BIVALENT RECOMBINE DE POULE A PARTIR D’UN FRAGMENT VARIABLE A CHAINE UNIQUE (scFv) DE POULE ET ANTICORPS OBTENU A LA SUITE DE CE PROCEDE - Google Patents

PROCEDE DESTINE A PRODUIRE UN ANTICORPS BIVALENT RECOMBINE DE POULE A PARTIR D’UN FRAGMENT VARIABLE A CHAINE UNIQUE (scFv) DE POULE ET ANTICORPS OBTENU A LA SUITE DE CE PROCEDE Download PDF

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WO2006093080A1
WO2006093080A1 PCT/JP2006/303583 JP2006303583W WO2006093080A1 WO 2006093080 A1 WO2006093080 A1 WO 2006093080A1 JP 2006303583 W JP2006303583 W JP 2006303583W WO 2006093080 A1 WO2006093080 A1 WO 2006093080A1
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Prior art keywords
primer
antibody
seq
heavy chain
light chain
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PCT/JP2006/303583
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English (en)
Japanese (ja)
Inventor
Haruo Matsuda
Syuichi Furusawa
Hiroyuki Horiuchi
Nahoko Nishibori
Toshi Shimamoto
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National University Of Corporation Hiroshima University
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Priority to JP2007505915A priority Critical patent/JP4830115B2/ja
Publication of WO2006093080A1 publication Critical patent/WO2006093080A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2872—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against prion molecules, e.g. CD230
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/23—Immunoglobulins specific features characterized by taxonomic origin from birds

Definitions

  • the present invention relates to a method for producing a chicken bivalent antibody by dimerizing a single-chain variable region fragment (scFv) obtained by the phage display method, the antibody obtained by the method, and the antibody This is related to a typical usage example.
  • scFv single-chain variable region fragment
  • the present inventors have so far focused on a chicken-type antibody as a non-mammalian antibody.
  • -Avian birds are phylogenetically lower than mammals, but they are animals with an elaborate immune system similar to mammals.
  • it is useful for producing specific antibodies against proteins conserved in many mammals.
  • specific antibodies against proteins (antigens) that are difficult to produce using mice and rats can be produced in -bird birds.
  • N-glycolylneuraminic acid hereinafter referred to as “NeuGc”
  • an antigen that serves as a human cancer marker is present in most mammals except humans.
  • prion protein (hereinafter referred to as “PrP” t ⁇ ⁇ ), which is a pathogen of Creutzfeldt's Jacob disease and mad cow disease, has 90% or more homology among mammalian animals, so it produces antibodies in mammals. Although it is difficult, since the homology between mammals and -birds is around 30%, it is possible to produce antibodies in -birds. In fact, the inventors of the present invention have succeeded in producing a chicken-type monoclonal antibody against NeuGc and PrP by the cell fusion method. The other merit of the chicken-type antibody is that it has no cross-reactivity with mammalian-type antibodies. It is possible to establish an antigen detection system.
  • Non-Patent Document 1 Non-Patent Document 1
  • the non-patent document 2 the phage display method
  • the present inventors have succeeded in producing a recombinant-avian avian antibody by introducing a gene recombination technique for the purpose of mass production, structural modification, functional modification, etc. of a -avian avian antibody.
  • Patent Document 2 Japanese Patent Application No. 2004-325658 (filed on Nov. 9, 2004), specification and drawings)
  • the above phage display method is a simple method for producing an avian avian monoclonal antibody.
  • CH3 region heavy chain constant region
  • human scFv antibodies have been converted to human monoclonal antibodies.
  • the method involves amplifying the variable region by PCR and connecting it to the leader region and the constant region with a restriction enzyme. (See Non-Patent Document 5).
  • the above-described recombinant chickenpox-type antibody uses the gene of a chicken-pork type monoclonal antibody secreted by hypridoma, and as described above, it is difficult to prepare a hyperidoma, and this is not always a simple method.
  • the present invention has been made in view of the above problems, and the object thereof is to provide a single-chain variable region fragment (scFv) obtained by the phage display method as a simple method for producing a recombinant-avian bivalent antibody. It is intended to provide a method for producing a chicken-type antibody by dimerization, an antibody obtained by the method, and a typical use example of the antibody.
  • scFv single-chain variable region fragment
  • Boei E. et al "junctional human monoclonal antibodies of all isotypes constructed from phage display library-derived single-chain Fv antibody faragments, J. Immunol.Methods 239 (2000), 153-166.
  • the present inventors have intensively studied in order to solve the above problems, and have completed the present invention. That is, the method according to the present invention is a method for producing a recombinant neutral bivalent antibody that solves the above-described problems, and comprises a polynucleotide encoding a single-chain variable region fragment of a chicken type as a light chain with a polynucleotide as a cage.
  • the above-described amplification step for solving the above-mentioned problem is performed in SEQ ID NO: Amplify the light chain variable region gene using the first primer having the base sequence shown in 1 and the second primer having the base sequence shown in SEQ ID NO: 2, and the base sequence shown in SEQ ID NO: 7 A step of amplifying the heavy chain variable region gene using the seventh primer having the above and the eighth primer having the base sequence represented by SEQ ID NO: 8.
  • the light chain leader sequence that solves the above-mentioned problems includes a third primer having the base sequence shown in SEQ ID NO: 3, and SEQ ID NO: 4. It may be a polynucleotide amplified using a fourth primer having the nucleotide sequence shown.
  • the light chain constant region gene which should solve the above-mentioned problems, includes a fifth primer having the base sequence shown in SEQ ID NO: 5, and a sequence. It may be a polynucleotide amplified using the sixth primer having the base sequence shown in No. 6.
  • the light chain leader sequence for solving the above-mentioned problems is characterized in that the third primer having the base sequence shown in SEQ ID NO: 3 and SEQ ID NO: 4 A polynucleotide amplified using a fourth primer having the nucleotide sequence shown, and the light chain constant region gene is shown in SEQ ID NO: 5 and the fifth primer having the nucleotide sequence shown in SEQ ID NO: 5 It may be a polynucleotide amplified using a sixth primer having a base sequence.
  • the heavy chain leader sequence which should solve the above problems, has a ninth primer having the base sequence shown in SEQ ID NO: 9 and the base sequence shown in SEQ ID NO: 10. A polynucleotide amplified with the 10th primer.
  • the heavy chain constant region gene which should solve the above-mentioned problem, comprises the 11th primer having the base sequence shown in SEQ ID NO: 11, and the base sequence shown in SEQ ID NO: 12.
  • the polynucleotide may be amplified using the twelfth primer.
  • the heavy chain leader sequence for solving the above problems is A polynucleotide amplified using a ninth primer having the base sequence shown in SEQ ID NO: 9 and a tenth primer having the base sequence shown in SEQ ID NO: 10, and the heavy chain constant region gene is SEQ ID NO:
  • the polynucleotide may be amplified using the eleventh primer having the base sequence shown in 11 and the twelfth primer having the base sequence shown in SEQ ID NO: 12.
  • the light chain expression gene fragment preparation step which should solve the above problems, includes the light chain leader sequence; the light chain variable region gene; and the light chain constant region gene.
  • the vertical type may be a step of performing an amplification reaction using the third primer and the sixth primer.
  • the heavy chain expression gene fragment preparation step which solves the above problems, includes the heavy chain leader sequence; the heavy chain variable region gene; and the heavy chain constant region gene.
  • a step of performing an amplification reaction using the ninth primer and the twelfth primer may be used.
  • the antibody according to the present invention is an antibody produced by the method according to the present invention, which should solve the above problems.
  • the antibody according to the present invention should solve the above problems (a) the amino acid sequence represented by SEQ ID NO: 13; or (b) the amino acid sequence represented by SEQ ID NO: 13, An amino acid sequence with several amino acid substitutions, deletions, insertions or additions, a light chain of force, and (c) the amino acid sequence shown in SEQ ID NO: 14; or (d) the amino acid shown in SEQ ID NO: 14
  • the sequence is characterized by having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted, or added, a heavy chain, and has an activity of binding to a prion protein.
  • the antibody according to the present invention comprises a light chain consisting of the amino acid sequence shown in SEQ ID NO: 13 and a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 14 to solve the above problems. It may be a featured antibody.
  • the antibody according to the present invention may be an antibody labeled with an enzyme or a radioisotope that can be used as a marker for detection to solve the above-mentioned problems!
  • the enzyme that can be used as the detection marker is an enzyme that reacts with a substrate and develops color. preferable.
  • peroxidase, galactosidase, and the like are used as enzymes that can be used as detection markers.
  • radioactive isotopes that can be used as the detection marker include 14 C, 3 H, 32 P, 35 S, 9Q Tc, m In, 125 I, and I.
  • antigen prion protein
  • the labeling method should be performed by a conventionally known method.
  • a detection kit for a prion protein according to the present invention is characterized by comprising an antibody that can be used in the present invention to solve the above problems.
  • the prion protein detection kit according to the present invention is characterized by including a step of detecting the prion protein using the antibody according to the present invention, which solves the above-mentioned problems.
  • the prion disease diagnostic kit according to the present invention is characterized by including the antibody according to the present invention.
  • the method for diagnosing prion disease according to the present invention is characterized by including a step of detecting an abnormal prion protein in a sample whose biological strength is also prepared using the antibody according to the present invention.
  • a recombinant chicken bivalent antibody having two antigen-binding sites is produced from a single-chain variable region fragment (scFv) obtained by the phage display method.
  • scFv single-chain variable region fragment
  • the antibody obtained by the above method is a bivalent antibody having two antigen-binding sites, so it has a high affinity with the antigen and has an Fc region, so that the secondary antibody for detection binds. If the antigen can be detected with high sensitivity due to the large number of regions, the effect can be obtained.
  • the antibody that is useful in the present invention is a recombinant-avian avian bivalent antibody. Therefore, it is possible to establish a highly sensitive antigen detection system free from non-specific reactions. Furthermore, it is possible to provide diagnostic agents and diagnostic methods for various diseases using the sensitive antigen detection system. Has an effect.
  • a highly sensitive detection system and a highly sensitive detection method for prion protein can be established.
  • prion diseases such as Creutzfeldt / Jakob disease or Ushi spongiform encephalopathy
  • FIG. 1 is a diagram for explaining an outline of a construction method of pcCKL-3-15.
  • FIG. 2 (a) is a diagram for explaining the outline of the construction method of pcCKH-2.
  • FIG. 2 (b) shows the nucleotide sequence of the oligonucleotide used for the construction of pcCKH-2.
  • FIG. 3 is a diagram for explaining the outline of the construction method of pcDHF3-15.
  • FIG. 4 shows the results of detection of abnormal prion protein (BSE—UK10PK) using each antibody in Example 2, and (a) shows the results of detection with 44B1.
  • B shows the results of detection with T2
  • C shows the results of detection using a 3-15 bivalent antibody
  • d shows the results of detection using a 3-15-valent antibody. Results are shown.
  • FIG. 5 shows the results of BSE detection using an HRP-labeled secondary antibody in Example 3, and (a) shows the results of BSE detection by the direct method using HRP-labeled Ab3_15. (B) shows the detection results of BSE by the direct method using HRP-labeled T2.
  • the method of the present invention is a method for producing a recombinant-avian avian bivalent antibody, comprising:-a polynucleotide encoding a avian avian single-chain variable region fragment as a cage, and a light chain variable region gene; Amplification step for amplifying the heavy chain variable region gene; preparation of a light chain expression gene fragment linking the light chain leader sequence functioning in the host cell, the light chain variable region gene, and the light chain constant region gene of the chicken antibody A step of preparing a heavy chain expression gene fragment by linking a heavy chain leader sequence that functions in a host cell, the heavy chain variable region gene, and a heavy chain constant region gene of a chicken antibody; the light chain expression gene Fragments and And a step of introducing a gene fragment for expressing a heavy chain into a host cell; and a step of culturing the host cell.
  • a chicken bivalent antibody means a -bird avian antibody having two antigen-binding sites per molecule.
  • it means an antibody having a bivalent valence with an antigen. That is, two homologous light chains (light chain variable region and light chain constant region) and two heavy chains (heavy chain variable region and heavy chain constant region), respectively, are disulfide bonds (SS bonds)
  • SS bonds disulfide bonds
  • obtained by the phage display method obtained by the phage display method
  • phage display antibody has a light chain variable region and a heavy chain variable region connected by a linker, and the two variable regions are close together to form one antigen-binding site.
  • a phage display antibody is an antibody having one antigen binding site per molecule.
  • the phage display antibody in which the light chain and the heavy chain are connected by a linker may be called a single chain variable region fragment (single chain FV (scFv)) or a single chain antibody.
  • the term "bird-type antibody” refers to an antibody (IgM, IgA, IgY) produced by a chicken bird against an antigen by an immune function of the chicken bird.
  • a single antibody that recognizes only a specific portion of an antigen is called a “-avian monoclonal antibody”.
  • the “bird-type antibody” means an antibody having the same structure as the natural antibody produced by the chicken itself, for example, a gene encoding a chicken-type antibody in a host cell other than chicken.
  • some of the amino acids of the antibody may be deleted or substituted, or may be added with amino acids (structure modification-avian antibody).
  • the amplification step is a step of amplifying a light chain variable region gene and a heavy chain variable region gene using a polynucleotide encoding a ⁇ bird-type single-stranded variable region fragment as a cocoon.
  • the "native single-chain variable region fragment” means a single-chain variable region fragment in which a light chain variable region and a heavy chain variable region of a chicken antibody are connected by a linker. In the present specification, for convenience, the single-chain variable region fragment is referred to as “scFv”.
  • the scFv-encoding polynucleotide (hereinafter referred to as "scFv polynucleotide" t ⁇ ), which is the above-mentioned scissors, can be obtained, for example, by recovering phage force DNA that displays a desired scFv.
  • the scFv polynucleotide may be a polynucleotide fragment or a plasmid, and its sequence information power may be obtained by chemical synthesis.
  • the scFv polynucleotide includes a light chain variable region that binds to a desired antigen and a region encoding a heavy chain variable region. Therefore, the light chain variable region gene (in other words, “polynucleotide encoding the light chain variable region”) and the heavy chain variable region gene (in other words, “polynucleotide encoding the heavy chain variable region”) are used in the scFv polynucleotide. Using a known DNA amplification method such as the PCR method and its modification method, it can be amplified.
  • primers for amplifying the light chain variable region gene and the heavy chain variable region gene can be designed based on the base sequence information of each primer used to amplify various light chain variable region genes and heavy chain variable region genes during the phage display method.
  • each primer designed as described above that is, a primer used for amplifying the light chain variable region and a primer used for amplifying the heavy chain variable region are used. If one set of each is prepared, the same phage library can be used for almost all of the selected phage-derived scFv polynucleotides. That is, almost all of the light chain variable region gene or heavy chain variable region gene of an antibody against various antigens can be amplified with one set of primers.
  • a primer for amplifying a light chain variable region gene applicable to the method of the present invention for example, a first primer having a base sequence of GCAGGCAGCGCTGACTCAGCC (SEQ ID NO: 1), and CTGGCCGAGGACGGTCAGGGTT (SEQ ID NO: 2)
  • a primer for amplifying a heavy chain variable region gene applicable to the method of the present invention for example, a seventh primer having a base sequence of CTGATGGCGGCCGTG ACGTT (SEQ ID NO: 7), and GGAGGAGACGA TGACTTCGGT (SEQ ID NO: 8)
  • the 8th primer which has a base sequence is mentioned.
  • the primer applicable to the present invention is not limited to the above primer, and may have a base sequence in which one or several bases are substituted, deleted, inserted or added. May be a primer which also has a complementary sequence power.
  • the first primer, the second primer, the seventh primer, and the eighth primer were designed with the following viewpoints.
  • the primer design method is not limited to this.
  • -Phosphorus-type phage display antibody expression plasmids were immunized with the desired antigen-cDNA was synthesized from mRNA extracted from the spleen of chickens by reverse transcriptase, and the following primers (light chain variable region amplification) Primers (CLSB, CLF), heavy chain variable region amplification A light chain variable region gene and a heavy chain variable region gene are amplified using a primer (CHB, CHSF) and inserted into a plasmid pPDS (for details, see Yamanaka. H ⁇ et al and hicken monoclonal antioody isolated by a phage display system. See J. Immunol. 1996, 157: 1156-1162).
  • the base sequence of CLBSB is TCTGACG GTCGCGCTGACTCAGCC (SEQ ID NO: 15)
  • the base sequence of CLF is ATTAGCGCTT AAGGACGGTCAGGGTT (SEQ ID NO: 16)
  • the base sequence of CHB is CTGATGGCGGCC GTGACGTT (SEQ ID NO: 17)
  • the base sequence of CHSF is TCCACCTGTCGACACGATGA CTTCGGT Number 18).
  • The- ⁇ avian phage display antibody obtained by the above method amplifies the light chain variable region gene and the heavy chain variable region gene constituting the antibody using the primers (CLSB, CLF, CHB, CHSF). Therefore, it always has the base sequence of the primer (CLSB, CLF, CHB, CHSF). Therefore, if a primer to be applied in the present invention is designed based on the base sequences of the above primers (CLS B, CLF), a light chain variable region gene of a chicken antibody against any antigen can be amplified. By designing a primer to be applied to the present invention based on the base sequences of the primers (CHB, CHSF), it is possible to amplify the light chain variable region gene of a chicken antibody against any antigen.
  • the first primer is designed based on a partial base sequence of CLSB
  • the second primer is designed based on a partial base sequence of CLF
  • the seventh primer Uses the base sequence of CHB as it is
  • the 8th primer is designed based on the partial base sequence of CHSF.
  • a base sequence that does not exist in the base primer is added to the base sequence of a part of the base primer. Used to link the light chain variable region gene and the leader sequence or the light chain variable region gene and the light chain constant region gene in the light chain expression gene fragment preparation step and heavy chain expression gene fragment preparation step It is a base sequence and is a base sequence that exists as a complementary sequence in the two nucleotide chains to be linked.
  • This step is a step of preparing a gene fragment used for light chain expression by linking a light chain leader sequence that functions in a host cell, the light chain variable region gene, and a light chain constant region gene of a chicken antibody. is there.
  • leader sequence is a nucleotide chain that encodes the amino acid terminal domain of a secreted protein, and is a nucleotide chain that encodes a secretion signal when a protein synthesized in the cell is secreted outside the cell.
  • the above “light chain leader sequence” means a nucleotide chain that encodes a secretion signal of an antibody light chain synthesized in a host cell.
  • the leader sequence used in the present invention is not particularly limited as long as the polypeptide encoded by the host functions as a secretion signal in the host. , You can use.
  • the light chain leader sequence for example, the leader sequence of an anti-PrP- ⁇ tri-monoclonal antibody (HUC2-13, see Patent Document 2 for details) prepared by the present inventors using the cell fusion method, pSecTag2 ( The leader sequence of Murine Ig kappa-chain V-J2-C from Invitrogen is available.
  • the method for obtaining the light chain leader sequence is not particularly limited, and the leader sequence may be obtained by chemical synthesis based on the above known base sequence information, or any of the above monoclonal antibodies may be obtained.
  • the mRNA synthesized from the hybridoma to be produced may be obtained by PCR, etc. using the synthesized cDNA as a saddle type.
  • the third primer having the base sequence of GCCATGGCCTGGGCTCCTCTCCT (SEQ ID NO: 3) and the base sequence of GGCTGAGTCAGCGCTGCCTGC (SEQ ID NO: 4)
  • a fourth primer having The third primer may contain a base sequence (AAGCTT) to add a restriction enzyme site (Hindlll) used for cloning. That is, it may be AAGCTTGCCATGGCCTGGGCTCCTCTCCT (SEQ ID NO: 19).
  • the third primer may further have a base sequence unrelated to the leader sequence added to the 5 ′ end. For example, the base shown in SEQ ID NO: 19 Primer with a base sequence (ATATAT) added to the 5 'end of the third primer
  • a primer having a base sequence ability of The nucleotide sequence is a sequence in which the light chain leader sequence is also cleaved by a restriction enzyme (Hindlll) upon introduction into the betater, and the type of base and the combination of bases are not particularly limited. Yes.
  • Hindlll is added as described above.
  • the type of the cloning site of the vector to be introduced, the light chain is not limited thereto. It may be adopted after appropriate selection based on the restriction enzyme site information contained in the leader sequence.
  • the “light chain constant region gene of a chicken antibody” means a polynucleotide encoding the light chain constant region of a chicken antibody.
  • the light chain constant region of the chicken-type antibody is common to almost all of the chicken-type antibodies, and the nucleotide sequence information of the conventionally-known chicken-type monoclonal antibody is obtained.
  • the normal region gene may be obtained by chemical synthesis or the like.
  • the base sequence information of the light chain constant region for example, the germline base sequence information (gene bank Accession No. M24403) of the avian avian antibody gene light chain prepared by the present inventors using the cell fusion method can be used. is there.
  • the method for obtaining the light chain constant region gene is not particularly limited, and the above-mentioned conventionally known nucleotide sequence information may be obtained by chemical synthesis, or a hybridoma producing any one of the above monoclonal antibodies.
  • cDNA synthesized from mRNA may be obtained by PCR or the like in a saddle type.
  • the light chain constant region to be amplified only needs to have a region capable of forming an S—S bond with the heavy chain, and is not particularly required to have a full length.
  • Primers used for obtaining the light chain constant region gene by PCR or the like include, for example, a fifth primer having the base sequence of AACCCTGACCGTCCTCGGCCA (SEQ ID NO: 5), and the base sequence of TTAGCACTCGGACCTCTTCAG (SEQ ID NO: 6)
  • a sixth primer having The sixth primer may contain a base sequence (TCTAGA) to add a restriction enzyme site (Xbal) used for cloning. That is, it may be TCTAGATTAGCACTCGGACCTCTTCAG (SEQ ID NO: 21)! /.
  • the sixth primer having the base sequence ability shown in the above SEQ ID NO: 21 may be added to the 5 ′ end of the base sequence ability unrelated to the light chain constant region gene.
  • a primer having a base sequence (TC) added to the 5 ′ end of the sixth primer that is, a primer comprising the base sequence of TCTCTAGATTAGCACTCGGACCTCTTCAG (SEQ ID NO: 22) may be used.
  • the base sequence (TC) is a sequence in which the light chain constant region gene force is also cleaved by a restriction enzyme (Xbal) upon introduction into a vector, and the type of base and the combination of bases are not particularly limited. Absent.
  • the force to which Xbal is added as described above.
  • the present invention is not limited to this. It is only necessary to select them based on the information on restriction enzyme sites contained in the gene.
  • This step includes, for example, the light chain leader sequence amplified using the third and fourth primers, the light chain constant region gene amplified using the fifth and sixth primers, and the first and second sequences.
  • a light chain variable region gene amplified using a primer can be used as a saddle type, and an amplification reaction such as PCR can be performed using a third primer and a sixth primer.
  • the first primer and the fourth primer are in a complementary relationship, and it can be said that the amplified fragments amplified using both primers are in a state where annealing can occur through the complementary sequence. That is, it can be said that the 3 ′ end of the light chain leader sequence and the 5 ′ end of the light chain variable region can be linked by annealing.
  • the second primer and the fifth primer are partially complementary, and it can be said that the amplified fragments amplified using both primers are in a state where annealing can occur via the complementary sequence.
  • the 3 'end of the light chain variable region and the 5' end of the light chain constant region are joined by annealing. It can be said that it is possible.
  • the light chain leader sequence amplified using the third and fourth primers, the light chain constant region gene amplified using the fifth and sixth primers, and amplified using the first and second primers When the light chain variable region genes thus prepared are mixed and subjected to an annealing reaction, the above-mentioned polynucleotides linked to each other, that is, light chain expression gene fragments can be prepared. Furthermore, the base sequence of the third primer is present at the 5 ′ end of the light chain expression gene fragment, and the base sequence complement sequence of the sixth primer is present at the 3 ′ end of the light chain expression gene fragment. If an amplification reaction is carried out using both primers, the gene fragment for light chain expression can be amplified in large quantities.
  • the light chain leader sequence amplified using the 3rd and 4th primers and the 1st and 2nd primers are used for amplification.
  • the light chain variable region gene is used as a saddle type, and the light chain leader sequence and the light chain variable region gene are ligated by performing an amplification reaction such as PCR using the third primer and the second primer.
  • the light chain variable region gene amplified using the first and second primers and the light chain constant region gene amplified using the fifth and sixth primers are in a saddle type, and the first primer and the first primer After performing an amplification reaction such as a PCR method using 6 primers to link the light chain variable region gene and the light chain constant region gene, a polynucleotide further connecting the light chain variable region gene and the light chain constant region gene, The amplification reaction may be carried out using the third and sixth primers, using the third and fourth primers as V and the light chain leader sequence amplified using the third and sixth primers.
  • the light chain leader sequence, the light chain variable region gene, and the light chain constant region gene may be linked by appropriately selecting a normal genetic engineering technique that is not limited to the above method.
  • the primer applicable to this process is the target genetic It is not limited to the above primer as long as it can amplify a molecule (polynucleotide), and has a base sequence in which one or several bases are substituted, deleted, inserted or added. It may also be a primer that also has a complementary sequence ability.
  • a heavy chain leader sequence that functions in a host cell, the heavy chain variable region gene, and a heavy chain constant region gene of a rabbit-type antibody are linked to prepare a gene fragment used for heavy chain expression. It is.
  • a "leader sequence” is a nucleotide chain that encodes the amino acid terminal domain of a secreted protein, and is a nucleotide chain that encodes a secretory signal when a protein synthesized in the cell is secreted outside the cell.
  • the “heavy chain leader sequence” means a nucleotide chain encoding a secretion signal of an antibody heavy chain synthesized in a host cell.
  • the leader sequence used in the present invention is not particularly limited as long as the polypeptide encoded by the host functions as a secretion signal in the host. , You can use.
  • the heavy chain leader sequence for example, the leader sequence of the anti-PrP- ⁇ tri-monoclonal antibody (HUC2-13, see Patent Document 2 for details) prepared by the present inventors using the cell fusion method can be used. It is.
  • the method for obtaining the heavy chain leader sequence is not particularly limited, and the leader sequence may be obtained by chemical synthesis from the above-mentioned conventionally known base sequence information, or any of the above monoclonal antibodies is produced. Alternatively, cDNA synthesized from the mRNA of the hybridoma to be obtained can be obtained as a template by PCR or the like.
  • Primers used when the heavy chain leader sequence is obtained by PCR or the like are, for example, the 9th primer having the base sequence of ACCATGAGCCCACTCGTCTCC (SEQ ID NO: 9) and the base sequence of AACGTCACGGCCGCCATCAG (SEQ ID NO: 10)
  • the 10th primer which has is mentioned may contain a base sequence (GGTACC) for adding a restriction enzyme site (Kpnl) used for cloning. In other words, it may be GGTACCACCATGAGCCCACTCGTCTCC (SEQ ID NO: 23)! /.
  • the above 9th primer includes a base sequence unrelated to the leader sequence! /, May! / ⁇ ⁇ .
  • the base sequence (TT) is a sequence that is cleaved from the heavy chain leader sequence by a restriction enzyme (Kpnl) upon introduction into a vector, and the type of base and the combination of bases are not particularly limited. Absent.
  • Kpnl is added as described above.
  • the present invention is not limited to this.
  • the cloning site type and heavy chain leader sequence of the vector to be introduced are not limited thereto. It is only necessary to select and use the information based on the information on the restriction enzyme sites included.
  • the "heavy chain constant region gene of a chicken antibody” means a polynucleotide encoding the heavy chain constant region of a chicken antibody.
  • the heavy chain constant region of a chicken antibody is common to almost all of the chicken-type antibodies, and the base sequence information of the heavy chain constant region is obtained from the base sequence information of a conventionally known chicken-type monoclonal antibody.
  • the normal region gene may be obtained by chemical synthesis or the like.
  • the nucleotide sequence information of the heavy chain constant region for example, the germline nucleotide sequence information (gene bank Accession No. M30319 and X07174) of the avian avian antibody gene heavy chain prepared by the present inventors using the cell fusion method is used.
  • the method for obtaining the heavy chain constant region gene is not particularly limited, and the above-mentioned conventionally known base sequence information ability may be obtained by chemical synthesis, or any one of the above monoclonal antibodies is produced.
  • the cDNA synthesized by the hybridoma can be obtained by PCR or the like using the synthesized cDNA as a cage.
  • the heavy chain constant region gene to be amplified is difficult to amplify the full length because of its large base size. Therefore, it is not necessary to amplify the full length.
  • the 11th primer having the base sequence of ACCGAAGTCATCGTCTCCTCC (SEQ ID NO: 11) and CAAACACAACAGCTCCACC ( A 12th primer having the base sequence of SEQ ID NO: 12) can be mentioned.
  • the heavy chain constant region gene (part) that is amplified can be used for introduction into a vector.
  • the eleventh and twelfth primers are designed so that the heavy chain constant region gene (part) obtained by these primers contains a Hindlll site. Therefore, if the primer is designed so that the heavy chain constant region gene obtained by amplification contains the Hindlll !, the primer is not limited to the primers having the nucleotide sequences of SEQ ID NOS: 11 and 12 above. However, it is preferable to design the primer so that the terminal force of the Hindlll site is added at least 6 bp or more.
  • restriction enzyme site contained in the heavy chain constant region gene obtained by amplification is not limited to Hindlll, but the type of cloning site of the vector to be introduced, and other restriction sites contained in the heavy chain constant region gene. Appropriate selection may be made based on information on enzyme sites.
  • This step includes, for example, the heavy chain leader sequence amplified using the ninth and tenth primers, the heavy chain constant region gene amplified using the eleventh and twelfth primers, and the seventh and eighth primers.
  • the heavy chain variable region gene amplified by using the 9th primer and the twelfth primer as a saddle can be used for amplification reaction such as PCR.
  • the 10th and 7th primers are in a complementary relationship, and it can be said that the amplified fragments amplified using both primers are in a state where annealing can occur via the complementary sequences. That is, it can be said that the 3 ′ end of the heavy chain leader sequence and the 5 ′ end of the heavy chain variable region gene can be linked by annealing.
  • the eighth primer and the eleventh primer are in a complementary relationship, and amplification fragments amplified using both primers can be said to be in a state where annealing can occur via their complementary sequences. That is, annealing the 3 'end of the heavy chain variable region and the 5' end of the heavy chain constant region It can be connected by
  • the heavy chain leader sequence amplified using the ninth and tenth primers the heavy chain constant region gene amplified using the eleventh and twelfth primers, and the seventh and eighth primers
  • the amplified heavy chain variable region genes are mixed and subjected to an annealing reaction
  • the above-ligated polynucleotides that is, heavy chain expression gene fragments can be prepared.
  • the base sequence of the 9th primer is present at the 5 'end of the heavy chain expression gene fragment
  • the base sequence complementation sequence of the 12th primer is present at the 3' end of the heavy chain expression gene fragment. If the amplification reaction is performed using both primers, the gene fragment for heavy chain expression can be amplified in large quantities.
  • the heavy chain leader sequence amplified using the ninth and tenth primers and the heavy chain amplified using the seventh and eighth primers After linking the heavy chain leader sequence and the heavy chain variable region gene by performing an amplification reaction such as PCR using the 9th and 8th primers using the variable region gene as a saddle, the heavy chain leader sequence And a heavy chain constant region gene amplified using the 11th and 12th primers, and a 9th and 12th primer for further amplification reaction. What should I do?
  • the heavy chain variable region gene amplified using the seventh and eighth primers and the heavy chain constant region gene amplified using the eleventh and twelfth primers are used as saddles, and the seventh primer and the A polynucleotide in which a heavy chain variable region gene and a heavy chain constant region gene are linked by performing an amplification reaction such as a PCR method using 12 primers and then linking the heavy chain variable region gene and the heavy chain constant region gene.
  • the amplification reaction may be performed using the ninth and twelfth primers, with the heavy chain leader sequence amplified using the ninth and tenth primers as a saddle.
  • the heavy chain variable region gene amplified using the eleventh and twelfth primers is a part of the heavy chain variable region gene. Therefore, the heavy chain constant region gene (part), the heavy chain leader sequence amplified using the ninth and tenth primers, and the heavy chain expression gene fragment ligated with the seventh and eighth primers are: A heavy chain of an avian avian antibody It codes the part, not the full length. When a heavy chain expression gene fragment that encodes a part of the heavy chain of the avian species antibody is to encode the full length
  • the Hindlll digested fragment of the full-length heavy chain constant region gene and the Hindlll digested fragment of the heavy chain expression gene fragment may be ligated. As a result, a gene fragment for heavy chain expression encoding the full length of the heavy chain of a rabbit-type antibody can be obtained.
  • the conditions such as the PCR method carried out in this step may be adopted after appropriate examination.
  • the heavy chain leader sequence, heavy chain variable region gene, and heavy chain constant region gene may be linked by appropriately selecting a normal genetic engineering technique that is not limited to the above method.
  • the primer applicable to the present invention is not limited to the primer as long as it can amplify the target gene (polynucleotide). One or several bases are substituted, deleted, inserted or added.
  • the primer may also have a base sequence, or may be a primer having a complementary sequence ability.
  • the method according to the present invention includes a step of obtaining a transformant by introducing the gene fragment for light chain expression and the gene fragment for heavy chain expression obtained in the above step into a host cell (transformation step). Included, prefer to be.
  • light chain expression vector for introducing a light chain expression gene fragment and a heavy chain expression gene fragment into a host cell and expressing the light chain and heavy chain in the cell.
  • This will be referred to as “heavy chain expression vector”) and the construction method thereof.
  • the light chain expression vector or the heavy chain expression vector is not particularly limited as long as it can express the light chain or heavy chain of a rabbit-type monoclonal antibody in a host cell. It may be a circular vector or a linear shape. Therefore, basically, the light chain expression gene fragment or heavy chain expression gene fragment obtained may be constructed to be controllably linked downstream of the promoter.
  • the type of plug motor to be used is not particularly limited as long as it functions in the host cell.
  • SV40 Yasushi 'Papilloma' Will (BPV) promoter includes human cytomegalovirus promoter (CMV).
  • the above light chain expression vector or heavy chain expression vector may contain various DNA segments other than the promoter.
  • Examples of the DNA segment include a sequence to which a drug resistance gene (neomycin resistance gene, zeocin resistance gene, etc.), a terminator, and a purification tag (histidine tag, etc.) that serve as markers for gene transfer are added.
  • a commercial vector suitable for the host cell to be introduced may be used.
  • pcDNA3.1 / myc-His (A) (Invitrogen) and pcDNA4 / myc-His (A) (Invitrogen) are used as a base, light chain expression vector, and heavy chain An expression vector has been constructed.
  • the force pEFl / myc-His (Invitrogen), pSecTag2 / HygroOnvitrogen) can be used as a known vector.
  • each expression vector is not particularly limited, and it is only necessary to link necessary gene sequences using ordinary genetic engineering techniques.
  • each of the above expression vectors may be constructed without subcloning the light chain expression gene fragment or the heavy chain expression gene fragment obtained by PCR, or a subcloning vector such as pUC19 or pBluescript II may be used.
  • Each of the above expression vectors may be constructed after subcloning using Escherichia coli and the like.
  • Host cell transformation is performed using the light chain expression vector and heavy chain expression vector.
  • An antibody molecule is a dimer in which two molecules of a complex in which a light chain and a heavy chain are formed by S—S bonds (light chain and single chain complex) are combined. Therefore, it is necessary to introduce both the light chain expression gene fragment and the heavy chain expression gene fragment into the host cell.
  • the host cell to be transformed is not particularly limited, and a host corresponding to each expression vector constructed as described above may be selected and used. That is, the host cell may be an animal-derived cell or a plant-derived cell.
  • Animal-derived cells and plant-derived cells are meant to include cells, tissues, and organs.
  • cells derived from animals having an immune system are preferred, and cells (cultured cells) that can be cultured in a liquid medium or the like are preferred.
  • Examples of animal-derived cultured cells include Chinese, Muster ovary cells (CHO cells), Hela cells, melanoma cells, mouse 3T3 cells, etc.
  • plant-derived cultured cells examples include tobacco BY2 cells. It is done. In the examples described later, CHO cells are used as host cells. This is because the cells can be cultured in suspension and are suitable for mass production of antibodies due to their short growth time of 12 hours and high proliferation ability.
  • the transformation method is not particularly limited, and a method suitable for the host cell and the expression vector may be selected and used.
  • a conventionally known method such as an electopore position method, a particle gun method, a calcium phosphate method, a protoplast z spheroplast method, a liposome method, or a DEAE dextran method can be suitably used.
  • a general transformation method for plant-derived cells includes a transformation method using agrobacterium (agrobacterium).
  • the light chain expression gene fragment and the heavy chain expression gene fragment are preferably integrated into the genome of the host cell. By incorporating the antibody gene into the genome, it is possible to reliably transmit the gene contained in the vector structure to daughter cells after cell division, and to maintain the production efficiency of -avian type antibodies. It is the power to become.
  • the method for confirming whether or not the light chain expression gene fragment and the heavy chain expression gene fragment have been introduced into the host cell is not particularly limited, and various known methods may be used. it can. Specifically, various markers may be used. For example, a gene that is deleted in the host cell is used as a marker, and a plasmid containing this marker and a recombinant plant virus gene is introduced into the host cell as an expression vector. As a result, the introduction of the gene of the present invention can be confirmed based on the expression power of the marker gene.
  • CHO cells are transformed, and CHO cells are transformed with a drug resistance marker (neomycin resistance gene, zeocin resistance gene) in a medium containing neomycin and zeocin.
  • a drug resistance marker neomycin resistance gene, zeocin resistance gene
  • Other markers include the puromycin metabolite, the bleomycin metabolite, the XGPRT gene, the DHFR gene, and the thymidine kinase gene. Effective for selection, bialaphos resistance marker, kanamycin resistance marker, etc. are effective for selection of plant cells.
  • a so-called dienomic PCR method can be used in which the genomic DNA prepared for host cell strength is used as a cage and the entire gene of the introduced protein (transcription factor) is specifically amplified. If it can be confirmed by electrophoresis or the like that the gene encoding the target protein (transcription factor) is amplified by this method, the introduction of the gene can be confirmed.
  • the method according to the present invention preferably further includes a step of culturing the above transformant to produce a recombinant-avian bivalent antibody.
  • the above transformant may be cultured, and the desired recombinant-pork bivalent antibody may be purified.
  • the culture method and culture conditions of the transformant are not particularly limited as long as a suitable method for culturing the transformant is used.
  • a suspension culture method, a carrier adhesion culture method, a holo-fiber culture method and the like are suitable.
  • the suspension culture method is more preferable because it can utilize a force jar mentor, in which applicable cells are limited to lymphoid cells and the like, and can be easily scaled up.
  • the transformed CHO cells employed in the examples described later are cells that can be cultured in suspension as described above.
  • the medium for culturing animal cells is not limited, but serum may be added to amino acids, vitamins, glucose, and salts.
  • bicarbonate Z carbon dioxide buffer is used as the buffer, and CO is used as the incubator.
  • the culture conditions are generally 28 ° C and 40 ° C depending on the power cell line cultured at 37 ° C.
  • F12 media GIBCO BRL
  • FBS fetal bovine serum
  • the medium for culturing plant cells is not limited, but may contain inorganic salts, carbon sources, vitamins, and amino acids. Sarako, coconut milk Or yeast extract may be added to promote growth. In addition, plant hormones such as auxin and cytokinin, gibberellin, abscisic acid and ethylene may be added. As for the culture conditions, the optimum one may be adopted depending on the cells to be cultured, such as light, temperature, presence or absence of aeration.
  • the transformant introduced with the light chain expression gene fragment and the heavy chain expression gene fragment produces the light chain and heavy chain of the target recombinant chicken bivalent antibody in the cell.
  • the produced light chain and heavy chain form a light chain single chain complex by S—S bond in the cell, and further, a dimer of the light chain single chain complex is formed to become an antibody molecule.
  • the antibody molecules produced at this time are secreted into the culture medium or accumulate in the cells. Whether the antibody molecule produced is secreted or accumulates in the cell depends on the type of transformant cell and the culture method.
  • the culture supernatant is prepared by centrifugation, filtration, etc. do it.
  • the recombinant-avian type bivalent antibody accumulates in the cells, the cells are disrupted by a known cell disruption method using glass beads or the like, and the recombinant-avian type is obtained from the disrupted cells. If you get a bivalent antibody.
  • the recombinant-avian avian divalent antibody obtained by the above method may be purified by a method using affinity chromatography or a resin for purification.
  • the histidine tag is designed to be added to the C-terminus of the produced recombinant chickenpox bivalent antibody heavy chain. Since histidine has the property of adsorbing to nickel, the target recombinant-avian bivalent antibody can be easily purified by using a nickel column.
  • the antibody according to the present invention is a recombinant-bird avian bivalent antibody obtained by the method according to the present invention.
  • Such antibodies include, for example, anti-PrP fuchons ⁇ ???
  • the antibody encoding the mammalian prion protein J. Vet. Med. Sci. 66 807-814) is used as a serotype and the antibody obtained by the method of the present invention described above (hereinafter referred to as “3-15”).
  • a bivalent antibody The details of the method for obtaining 3-15 bivalent antibody will be explained in the examples.
  • the 3-15 bivalent antibody has (a) an amino acid sequence represented by SEQ ID NO: 13; or (b) one or several amino acids substituted or deleted in the amino acid sequence represented by SEQ ID NO: 13, Inserted or added amino acid sequence, forceful light chain, and (c) the amino acid sequence shown in SEQ ID NO: 14; or (d) one or several amino acids in the amino acid sequence shown in SEQ ID NO: 14
  • An antibody comprising a heavy chain consisting of an amino acid sequence in which an amino acid is substituted, deleted, inserted, or added, and having an activity of binding to a prion protein.
  • SEQ ID NO: 27 shows the base sequence of the polynucleotide encoding the light chain having the amino acid sequence ability shown in SEQ ID NO: 13.
  • the nucleotide sequence of a polynucleotide encoding a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 14 is shown in SEQ ID NO: 28.
  • the polynucleotide encoding the light chain and the polynucleotide encoding the heavy chain of the above 3-15 bivalent antibody are not limited to SEQ ID NOS: 27 and 28, and each base can be appropriately changed according to the codon table. is there.
  • Such a 3-15 bivalent antibody is a bivalent antibody prepared using the anti-PrP phage display antibody 3-15 (hereinafter referred to as "3-15-valent antibody” as a saddle type), and has binding activity to PrP. As shown in the examples described later, the 3-15 bivalent antibody has a much higher PrP binding capacity than the 3-15-valent antibody, and is currently a definitive test for BSE. PrP binding ability similar to that of PrP antibodies [44B1 (see Virology 320 (2004) p40-51) and T2] used in C.2) and lower detection knockdown than 44B1. I was able to detect Pr P with high sensitivity. [0092] ⁇ Utilization of 3-15 bivalent antibody>
  • the above 3-15 bivalent antibody can detect PrP in a sample with high sensitivity. Moreover, prion disease can be diagnosed by detecting abnormal PrP using a 3-15 bivalent antibody.
  • Primary disease as used herein is a general term for diseases caused by PrP (particularly abnormal PrP). It is caused by Creutzfeld's Jacob disease, bovine spongiform encephalopathy (BSE), and Higgiyagi. Scrapie, Grestmann- Straussle syndrome (GSS), Crewe disease, etc. are known.
  • the detection method of PrP is not particularly limited, and a known method such as ELISA method, Western blot method, RIA method may be adopted. Further, the conditions in the above method may be performed under standard conditions, and the optimal amount of antibodies used for detection may be selected as appropriate.
  • Examples of a method for detecting PrP by Western plotting and a method for diagnosing prion disease include the following methods.
  • a sample obtained from a living body e.g., brain crush fluid, cerebrospinal fluid, spinal fluid, etc.
  • a sample obtained from a living body e.g., brain crush fluid, cerebrospinal fluid, spinal fluid, etc.
  • samples obtained from living organisms e.g, brain crush fluid, cerebrospinal fluid, spinal fluid, etc.
  • samples obtained from living organisms eg, brain crush fluid, cerebrospinal fluid, spinal fluid, etc.
  • the prion protein may be detected by Western blotting.
  • Abnormal PrP is resistant to proteinase K. If a sample treated with proteinase K as described above is detected using a 3-15 bivalent antibody, will the sample contain abnormal PrP? It is possible to diagnose whether or not it is a prion disease.
  • the sample to which the above method can be applied is not particularly limited as long as it is a biologically derived cell that expresses PrP, and examples thereof include human, urchin, horse, hidge and goat-derived cells. .
  • the sample is preferably in the form of a cell extract.
  • the 3-15 bivalent antibody can be used in a PrP detection kit or a prion disease diagnostic kit.
  • the PrP detection kit or the prion disease diagnosis kit may contain PrP or a force capable of detecting abnormal PrP and other components as long as at least a 3-15 bivalent antibody is contained.
  • Other configurations include, for example, proteinase, electric Examples include electrophoresis gels, electrophoresis reagents, western blotting reagents, normal PrP, and abnormal PrP.
  • a recombinant-avian avian divalent antibody against human prion protein (PrP) was produced.
  • the light chain variable region gene was prepared by the expression plasmid for anti-PrP phage antibody 3-15 ph Ab3-lb (Nakamura et al Establishment of a chicken Monoclonal antibody panel a gainst mammalian prion protein J. Vet. Med. Sci. 66 807 -See Fig. 814) Using the first primer (SEQ ID NO: 1) and the second primer (SEQ ID NO: 2) by the PCR method as a vertical type o
  • the light chain constant region gene was prepared by using a recombinant anti-PrP- ⁇ avian antibody light chain expression plasmid (pcCKL-1; for its construction method, see Patent Document 2 (Japanese Patent Application No. 2004-325658 (2004 (Applied on Nov. 9, 1))) (See the description and drawings), and the PCR was carried out by PCR using the 5th primer (SEQ ID NO: 5) and 6th primer (SEQ ID NO: 22).
  • the light chain leader sequence was prepared by the recombinant anti-PrP-type avian antibody expression plasmid constructed by the inventors (pcCKL-4, where the V region of pcCKL-1 is the HUNN of the -type avian monoclonal antibody. — Converted to 1.
  • HUNN 1, see “Nakamura et al Establis hment of a chicken monoclonal antibody panel against mammalian prion protein j. V et. Med. Sci. 66 807-814”.
  • PCR was carried out using the third primer (SEQ ID NO: 20) and the fourth primer (SEQ ID NO: 4).
  • PCR was performed under the following conditions.
  • the composition of the PCR reaction solution is 10 X PCR buffer (Toyo 5 ⁇ l; 2 mM dNTP 5 l; 25 mM MgS04 3.2 ⁇ Kfinal cone. 1.6 mM); Primer (10 ⁇ M) 2.5 ⁇ Kfinal cone. 0.5 ⁇ M) DNA1 l; KOD-plus (manufactured by Toyobo Co., Ltd.) 1 ⁇ 1, and finally made up to 50 1 with distilled water.
  • the reaction was carried out under the conditions of (1) 94 ° C for 2 minutes; (2) 94 ° C for 15 seconds; (3) 55 ° C for 30 seconds; (4) 68 ° C for 2 minutes (1 ), (2) to (4) were performed 30 cycles.
  • PCR using the third primer (SEQ ID NO: 20) and the sixth primer (SEQ ID NO: 22) using the light chain variable region gene, light chain constant region gene, and light chain leader sequence obtained above as a saddle type was done.
  • the PCR conditions were the same as those described in (1) except that 0.5 ⁇ 1 of the light chain variable region gene, the light chain constant region gene, and the light chain leader sequence were added as cocoon-type DNA per 50 1 reaction solution. According to the method of (4).
  • the amplified fragment (about 700 bp) obtained by PCR was cleaved with HindIII and Xbal, inserted into the HindIII and Xbal sites of pcDNA3.1 / myc-His (A) (Invitrogen), and pcCKL— 3—1 5 was built.
  • the pcCKL-3-15 was confirmed to be free of mutation by sequencing.
  • PcCKL-3-15 also has a neomycin resistance gene, CMV (human cytomegalovirus) fluoro- ⁇ ta ' ⁇ , BGH (bovine growth hormone) poly A signal. .
  • pcDNA4 / myc-His (A) (Invitrogen) was cleaved with Hindlll, blunt-ended with Klenow fragment, and the Hindlll site present in the original was deleted by self-ligation.
  • a plasmid for expression of heavy chain of recombinant anti-PrP-type avian antibody constructed by the inventors (pcCKH-1, for its construction method, patent document 2 (Japanese Patent Application No. 2004-325658 (filed on Nov.
  • Anti-PrP-Avian type antibody obtained by digesting Kpnl and PinAI The gene fragment was inserted into the Kpnl and PinA sites of the above plasmid. Oligonucleotides (SEQ ID NOs: 25 and 26) shown in FIG. 2 (b) were synthesized and inserted into the Kpnl-Hindlll site of the above plasmid to construct pcCKH-2.
  • Figure 3 shows the outline of pcDHF3-15 construction method.
  • the heavy chain variable region gene was prepared by the expression plasmid for anti-PrP phage antibody 3-15 ph Ab3-lb (Nakamura et al Establishment of a chicken Monoclonal antibody panel a gainst mammalian prion protein J. Vet. Med. Sci). 66 807-814) was performed by PCR using the 7th primer (SEQ ID NO: 7) and 8th primer (SEQ ID NO: 8).
  • the heavy chain constant region gene (partially) was prepared using a recombinant anti-PrP-type antibody heavy chain expression plasmid (pcCKH-1, constructed by the present inventors, and a patent document for its construction method). Using the eleventh primer (SEQ ID NO: 11) and the twelfth primer (SEQ ID NO: 12) as a saddle, refer to item 2 (Japanese Patent Application No. 2004-325658 (filed on Nov. 9, 2004)). This was done by PCR.
  • the light chain leader sequence was prepared by PCR using the ninth primer (SEQ ID NO: 24) and the tenth primer (SEQ ID NO: 10) using pcCKH-1 as a saddle.
  • PCR was performed under the following conditions.
  • the composition of the PCR reaction solution is 10 X PCR buffer (Toyobo Co., Ltd.) 5 ⁇ 1; 2 mM dNTP 5 1; 25 mM MgSO 3.2 ⁇ Kfinal cone. 1.6 mM);
  • the heavy chain variable region gene, the heavy chain constant region gene, and the heavy chain leader sequence obtained above as a saddle type PCR was performed.
  • the composition of the PCR reaction solution was 10 X PCR buffer (Toyobo) 5 ⁇ 1; 2 ⁇ dNTP 5 ⁇ l; 25 mM MgSO 2 Kfinal cone. ImM); primer (10 ⁇ M) 1.5 ⁇ Kfinal cone. 0.3 ⁇ M); vertical DNA 0.5 l each; KOD—plus (Toyobo Co., Ltd.) 1 ⁇ 1 and finally with distilled water 50 It was up to 1.
  • the reaction was carried out under the conditions of (1) 94 ° C for 2 minutes; (2) 94 ° C for 15 seconds; (3) 55 ° C for 30 seconds; (4) 68 ° C for 2 minutes (1 ), (2) to (4) were performed 30 cycles.
  • PcDHF3-15 has a zeocin metagene, a CMV (human cytomegalovirus) promoter, and a BGH (bovine growth hormone) poly A signal.
  • the drug-resistant cells were selected using 400 ⁇ g / ml Geneticin (Sigma) and 200 ⁇ g / ml Zeocine dnvitrogen). Further, the above-mentioned drug metastatic cells were cultured for 3 days under conditions of 5% CO and 37 ° C. using F12 media (GIBCO BRL) containing 10% fetal bovine serum (FBS). Obtained in the culture
  • the ELISA method was performed as follows. Each clone was cultured, arranged in about 1 ⁇ 10 V plate and cultured for 3 days. The culture supernatant was examined for the reactivity to the antigen by ELISA. Antigen recombinant mouse prion protein (prepared by the inventors) was immobilized on an ELISA plate at 4 ° C overnight. Blocking was performed with PBS containing 25% Block Ace (manufactured by Snow Brand Milk Products) at 37 ° C for 1 hour. After washing, each cell culture supernatant was added and incubated at 37 ° C for 1 hour.
  • a medullary part was collected from a BSE sushi brain (BSE-UK10) imported from the United Kingdom, and this was crushed with glass beads and then treated with proteinase K.
  • Abnormal PrP sample was prepared with sample buffer for SDS-PAGE. The sample solution was boiled for 6 minutes and subjected to SDS-PAGE using a 12% gel.
  • BSE brain emulsion prepared using distilled water from the brain of BSE rush imported from the UK (BSE—UK10) (Source: National Institute of Animal Health, Prion Disease Center) ) TN Buffer (100 mM NaCl, 50 mM Tris—Hcl (pH 7.6)) was added to prepare a 20% (w / v) BSE brain emulsion.
  • sample buffer 1001 for IX SDS-PAGE was added and boiled at 100 ° C for 6 minutes to obtain a sample solution.
  • Electrophoresis was carried out on a Western precast gel (NuPAGE 12% Bis-TrisGel, Invitrogen) at a constant voltage of 200 V for 40 minutes.
  • the mixture was transferred onto a polyvinyl membrane (Immuno-Blot PVDF membrane (manufactured by Bio-RAD)) using a wet type blotter at a constant voltage of 90 v for 40 minutes. After the transfer, blocking was performed with 5% skim milk (Block Ace (manufactured by Snow Brand Milk Products)) and PBS containing 0.05% Tween 20. After washing the membrane, abnormal PrP (BSE-UK10PK) was detected using 3-15 bivalent antibody as the primary antibody. For comparison, the 3-15-valent antibody, 44B1 (obtained from Mr.
  • the secondary antibody reaction uses 3 to 15 dilute HRP anti chicken IgG (H + L) to detect 3-15 bivalent antibodies, and 44B1 and T2 to detect 44B1 and T2.
  • HRP anti mo use IgG (H + L) diluted 3000 times was used.
  • the culture supernatant of transformed CHO cells was used, and the amount of the antibody was measured by ELISA.
  • the 3-15-valent antibody used was expressed as a soluble form, subjected to affinity purification with a histidine tag, and further subjected to gel filtration purification. The amount of protein was measured and used as the amount of antibody. Color was emitted using Super Signal West Dura Extended Duration Substrate (PIERCE) and then detected using FluorChem IS-8044 (Invitrogen).
  • FIG. Fig. 4 (a) shows the results of detection with 44B1
  • Fig. 4 (b) shows the results of detection with T2
  • Fig. 4 (c) shows the results of using 3-15 bivalent antibody.
  • the detection results are shown
  • Fig. 4 (d) shows the detection results using a 3-15-valent antibody.
  • Each lane shows the results of electrophoresis of abnormal PrP (BSE-UK10PK) from the left at concentrations of 5 mg / lane, 2.5 mg / lane, and 1.3 mg / lane.
  • Each band in FIG. 4 shows a glycan type abnormal PrP, a monosaccharide chain type abnormal PrP, and a sugar-free chain type abnormal PrP.
  • the 3-15 bivalent antibody has an abnormal PrP binding ability almost equal to the antibody (44B1, T2) currently used for BSE confirmation test. Furthermore, it was found that PrP can be detected with higher sensitivity than 44B1, which has a lower detection background.
  • the 3-15-valent antibody had low detection sensitivity for abnormal PrP, whereas the 3-15 bivalent antibody clearly had high detection sensitivity. Therefore, it was proved that the antigen can be clearly detected with high sensitivity by converting the monovalent antibody (scFv) obtained by the phage display method into a bivalent antibody using the method of the present invention.
  • HRP-labeled 3-15 bivalent antibody hereinafter referred to as “HRP-labeled 3-15 bivalent antibody”
  • HRP-labeled T2 hereinafter “HRP-labeled T2” t
  • the antibody was labeled with HRP using Peroxidase-Labeling-Kit (manufactured by Doujin Chemical Laboratory Co., Ltd.) according to the attached manual.
  • the antibody was diluted 3000 times and used for detection of abnormal PrP (BSE-UK10PK).
  • PBS-T (0.2% Tween 20-PBS) was used for antibody dilution.
  • FIG. Fig. 5 (a) shows the detection result of abnormal PrP (BSE-UK10PK) by the direct method using HRP-labeled 3-15 bivalent antibody
  • Fig. 5 (b) shows direct detection using HRP-labeled T2. Show the detection result of abnormal PrP (BSE—UK10PK) by the law.
  • Each lane in Fig. 5 (a) and (b) shows abnormal PrP (BSE—UK10PK) 2.5 mg / la from the left.
  • the results of electrophoresis performed at concentrations of ne, 1.3 mg / lane, 0.6 mg / lane, 0.3 mg / lane, and 0.15 mg / lane are shown.
  • a recombinant chicken bivalent antibody having two antigen-binding sites is produced from a single-chain variable region fragment (scFv) obtained by the phage display method.
  • scFv single-chain variable region fragment
  • the antibody obtained by the above method is a bivalent antibody having two antigen-binding sites, and thus has high affinity with the antigen. It also has an Fc region, and there is a region to which a secondary antibody for detection binds. Because of the large amount, the antigen can be detected with high sensitivity.
  • the antibody that is useful in the present invention is a recombinant-avian bivalent antibody. Therefore, it is possible to establish a highly sensitive antigen detection system free from non-specific reactions. Furthermore, the present invention has an effect of providing diagnostic agents and diagnostic methods for various diseases using the sensitive antigen detection system.
  • a recombinant chicken bivalent antibody is produced from an scFv that binds to a prion protein by the method according to the present invention
  • a highly sensitive detection system and a highly sensitive detection method for the prion protein can be established.
  • prion diseases such as Creutzfeldt / Jakob disease or Ushi spongiform encephalopathy
  • the present invention can be effectively used in a wide range of industries including antibodies, such as the pharmaceutical industry, the test chemical industry, and the food industry. Furthermore, it can be applied to the experimental research industry.

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Abstract

L’invention concerne un procédé destiné à la seule production d’un anticorps bivalent recombiné de poule en dimérisant un fragment variable à chaîne unique (scFv) obtenu par un procédé d’expression phagique destiné à produire un anticorps de poule. L’invention a également pour objet un anticorps obtenu grâce à ce procédé, ainsi qu’une application représentative de l’anticorps. Le procédé destiné à produire un anticorps bivalent recombiné de poule comprend les étapes suivantes : l’amplification d’un gène de la région variable à chaîne légère et d’un gène de la région variable à chaîne lourde en utilisant, en tant que matrice, un polynucléotide codant pour un fragment variable à chaîne unique de poule ; la préparation d’un fragment de gène de l’expression d’une chaîne légère en ligaturant une séquence de tête à chaîne légère active dans une cellule hôte, un gène de la région variable à chaîne légère et un gène de la région constante à chaîne légère d’un anticorps de poule ; et enfin, la préparation d’un fragment de gène de l’expression d’une chaîne lourde en ligaturant une séquence de tête à chaîne lourde active dans une cellule hôte, un gène de la région variable à chaîne lourde et un gène de la région constante à chaîne lourde d’un anticorps de poule.
PCT/JP2006/303583 2005-03-01 2006-02-27 PROCEDE DESTINE A PRODUIRE UN ANTICORPS BIVALENT RECOMBINE DE POULE A PARTIR D’UN FRAGMENT VARIABLE A CHAINE UNIQUE (scFv) DE POULE ET ANTICORPS OBTENU A LA SUITE DE CE PROCEDE WO2006093080A1 (fr)

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JP2007505915A JP4830115B2 (ja) 2005-03-01 2006-02-27 ニワトリ型一本鎖可変領域断片（ｓｃＦｖ）から組換えニワトリ型二価抗体を製造する方法によって得られた抗体

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JP2005056670 2005-03-01

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JP2019531091A (ja) * 2016-09-15 2019-10-31 オーグメンタ・バイオワークス・インコーポレーテッド 免疫レパートリー配列増幅法および適用
CN114014927A (zh) * 2021-12-13 2022-02-08 东北农业大学 抗鸡传染性法氏囊病毒的重链高亲和力抗体的制备和应用

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* Cited by examiner, † Cited by third party
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JP2006282521A (ja) * 2005-03-31 2006-10-19 Hiroshima Univ ニワトリキメラ抗体およびその利用
JP2019531091A (ja) * 2016-09-15 2019-10-31 オーグメンタ・バイオワークス・インコーポレーテッド 免疫レパートリー配列増幅法および適用
CN114014927A (zh) * 2021-12-13 2022-02-08 东北农业大学 抗鸡传染性法氏囊病毒的重链高亲和力抗体的制备和应用

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