WO2005035751A1 - Method of quickly and efficiently selecting high-functioning protein, high-functioning protein thus obtained, method of producing the same and method of using the same - Google Patents

Method of quickly and efficiently selecting high-functioning protein, high-functioning protein thus obtained, method of producing the same and method of using the same Download PDF

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WO2005035751A1
WO2005035751A1 PCT/JP2004/015290 JP2004015290W WO2005035751A1 WO 2005035751 A1 WO2005035751 A1 WO 2005035751A1 JP 2004015290 W JP2004015290 W JP 2004015290W WO 2005035751 A1 WO2005035751 A1 WO 2005035751A1
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protein
library
antibody
minutes
molecule
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PCT/JP2004/015290
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French (fr)
Japanese (ja)
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Hiroshi Yanagawa
Noriko Tabata
Kanehisa Kojoh
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Keio University
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Priority to JP2005514660A priority Critical patent/JP4729764B2/en
Publication of WO2005035751A1 publication Critical patent/WO2005035751A1/en

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803General methods of protein analysis not limited to specific proteins or families of proteins
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/10Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034Isolating an individual clone by screening libraries
    • C12N15/1062Isolating an individual clone by screening libraries mRNA-Display, e.g. polypeptide and encoding template are connected covalently

Definitions

  • the present invention relates to a method for selecting a highly functional protein, a highly functional protein obtained thereby, and a method for producing and using the same.
  • Non-Patent Document 4 a method for single-chain antibodies (Non-Patent Document 4) in which the C-terminal of one chain is linked to the N-terminal of the other with a very flexible peptide linker! / ⁇ You. [0004]
  • these techniques still have some disadvantages.
  • the phage display uses a realistic DNA library with an upper limit of about 10 9 (for antibodies), small library size, and the use of biological systems (e.g., E. coli infection, amplification, etc.). Will have an unexpected bias (mutation, etc.).
  • coli cannot be used. Since ribosome display is a complete in vitro experimental system using a cell-free translation system, these biases are ineffective, and the upper limit of the library can be 10 1 Q -10 U. However, the formation efficiency of the mRNA-protein-ribosome ternary complex is extremely low at 0.2% (Non-patent document 3), and is several hundredths of that of the in vitro viru S (IVV) method (Non-patent document 5). It is. In addition, since the complex is very unstable, there is a problem that a stronger selection pressure cannot be applied during the biobanning operation.
  • the selection pressure in the banner is the most important factor in the antibody concentration, and if the selection pressure is not strongly applied, it becomes necessary to repeat more selection cycles.
  • the selection pressure since a wide variety of antibodies with different stability and affinity can be obtained for one type of antigen, it is necessary to clone, express, and analyze many samples in order to obtain more functional ones. And This is not only a lot of hard work, but also time and money.
  • Non-Patent Document 6 As cell-free translation systems for synthesizing proteins in vitro, large-scale expression of proteins has been studied in a wheat germ system (Non-Patent Document 6) and an E. coli system (Non-Patent Document 7). ing. Along with this, 3'UTR is generally used to improve the stability and translation efficiency of mRNA as a stable translation template capable of expressing a large amount of protein (Non-Patent Document 8). A method such as substitution or modification of the chemical structure of mRNA (Non-Patent Document 9) is used.
  • RNA-protein linked molecule (IW) in which protein and mRNA are covalently linked via puromycin by performing a cell-free translation reaction using type III mRNA containing a piuromycin bound to the 3 'end.
  • Library can be constructed. After screening in vitro proteins that bind to the target molecules of this library of mapping molecules, the genes can be amplified and decoded by reverse transcription PCR. After that, we performed a proteome analysis of the IW method.
  • Non-Patent Document 5 a type II DNA that can express IVV and C-terminally labeled proteins with high efficiency using a cell-free translation system of wheat germ extract.
  • the present inventors have previously described a method for efficiently constructing IW, an assigning molecule, by linking mRNA (genotype) and protein (phenotype) to a cell-free translation system via pure bitemycin.
  • a large amount of protein is required for detailed function and three-dimensional structure analysis of proteins and production of antibodies. For this reason, it is difficult to purify the endogenous target protein and obtain a required amount thereof. Therefore, a recombinant protein in which cDNA of the target protein is expressed in a suitable host and used is used.
  • Various hosts for expressing a single-chain antibody can be considered, such as Escherichia coli, yeast, insect cells, and animal-derived cultured cells. Among them, E. coli is convenient because a large amount of protein can be obtained in a short period of time at low cost.
  • the use of a system that infects insect cells with a recombinant baculovirus allows expression of proteins that is difficult with the E. coli system.
  • Antibodies play an important role in various analytical tools.
  • One of them is the detection and quantification of proteins.
  • Methods for detecting proteins using antibodies include in vitro methods such as Western blotting, immunostaining, fluorescent antibody staining, and antibody chip methods, and in vivo methods such as immunoprecipitation.
  • the antibody In order to detect proteins using such a technique, the antibody must be pre- An emitted protein, for example, GFP is fused, or an antibody is fused to an enzyme protein, for example, horseradish peroxidase or alkaline phosphatase, and the enzyme activity is used as an index.
  • Methods for detecting protein-protein interaction using antibodies include surface plasmon resonance, fluorescence resonance energy transfer, fluorescence depolarization, evanescent field imaging, fluorescence correlation spectroscopy, fluorescence imaging, and solid-phase imaging.
  • enzyme immunoassays and the like Fluorescence Correlation Spectroscopy (FCS) requires a small amount of sample (approximately femtoliter), requires a short measurement time (approximately 10 seconds), and is easy to automate for HTS.
  • FCS Fluorescence Correlation Spectroscopy
  • EVOTEC is developing a device aiming for Ultra HTS, which screens more than 100,000 samples per day), and is superior as a detection system (Non-Patent Document 13).
  • the IW method is a method for constructing an mRNA-protein linking molecule in which mRNA and a protein are chemically linked via puromycin on the ribosome when mRNA is expressed in a cell-free translation system or the like ( Non-patent document 10; Non-patent document 12; Non-patent document 5). Furthermore, the mRNA-protein-linked molecule can be obtained by in vitro selection, and the mRNA portion of the selected assigning molecule can be amplified by reverse transcription PCR.
  • DTT dithiothreitol
  • a protein having an SS bond represented by an antibody since various commercially available cell-free translation systems contain DTT (dithiothreitol) as a reducing agent, they are not suitable for expression of a protein having an SS bond represented by an antibody.
  • DTT is essential for the expression of the wheat germ cell-free translation system, screening and expression examples of antibodies with high binding activity from single-chain antibody cDNA libraries have been Absent. Therefore, it is desired to establish a wheat embryo cell-free translation system capable of screening for a desired antibody having a very high binding activity even in the presence of DTT.
  • Non-Patent Document 2 Smith, G.P. (1985) Science 228, 1315-1317
  • Non-Patent Document 3 Hanes, J. and Pluckthun, A. (1997) Proc. Natl. Acad. Sci. USA 94,
  • Non-Patent Document 4 Huston, J.S., Margolies, M.N., Haber, E. (1996) Adv. Protein Chem., 49, 329.
  • Non-patent document 5 Miyamoto-Sato, E., et al., (2003) Nucleic Acids Res., 31, e78
  • Non-patent document 6 Madin K, et al. (2000) Proc. Natl. Acad. Sci. USA ., 97, 559-564
  • Non-Patent Document 7 Shimizu, Y. et al. (2001) Nat.BiotechnoL, 19, 751-755.
  • Non-Patent Document 8 Sachs.A.B., Et al. (1997) Cell 89, 831-838
  • Non-Patent Document 9 Ueda. T "et al. (1991) Nucleic Acids Symp Ser. 25, 151-152
  • Non-Patent Document 10 Nemoto, N., et al., (1997) FEBS Lett., 414, 405- 408
  • Patent Document 1 JP-A-10-816636
  • Patent Document 2 International Publication No. W098 / 16636 pamphlet
  • Patent Document 3 Japanese Patent Application Laid-Open No. 2002-176987
  • Patent Document 4 International Publication No. WO02 / 48347 pamphlet
  • Non-patent literature l l Nemoto, N., et al. (1999) FEBS Lett. 462, 43-46
  • Non-patent Document 12 Miyamoto-Sato, E "et al. (2000) Nucleic Acids Res. 28, 1176-1182
  • Patent Document 5 JP-A-11-322781
  • Patent Document 6 JP-A-2000-139468
  • Patent Document 7 International Publication WO02 / 46395 pamphlet
  • Non-Patent Document 13 Masataka Kaneshiro (1999) Protein nucleic acid enzyme 44: 1431-1438
  • the present inventors believe that, when a library of nucleic acids encoding a protein is prepared by the IW method, the target is not obtained conventionally by inactivation of the protein in the selection of the protein. As a result, they have found that heat treatment at such a high temperature can apply a high selection pressure, and as a result, a high-functional protein can be selected quickly and efficiently. In addition, they have found that a high-functional protein can be selected even when a protein having an SS bond is translated by a cell-free translation system that requires a reducing agent. The present invention has been completed based on the above findings.
  • the present invention provides the following.
  • a method for selecting a protein that interacts with a target molecule or a nucleic acid encoding the same comprising the following steps (a) to (d).
  • a method for producing a protein that interacts with a target molecule comprising: (1) selecting a nucleic acid encoding a protein that interacts with the target molecule by any one of the selection methods (1) and (9); A production method comprising a step of producing a protein by translating a selected nucleic acid.
  • step of producing a single-chain antibody comprises translating the selected nucleic acid with a cell-free translation system containing a thiol compound.
  • the step of producing a single-chain antibody includes producing as a fusion protein a single-chain antibody encoded by the selected nucleic acid and an enzyme or green fluorescent protein (GFP). (11) Manufacturing method.
  • a single-chain antibody having angiotensin II-binding activity which has the amino acid sequence shown in the following (A) or (B):
  • SEQ ID NO: 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103 or Pamino 105 is an amino acid rooster.
  • a single-chain antibody having binding activity to Lewis X which is represented by the following (A) or (B): A single-chain antibody having an amino acid sequence.
  • Fluorescence correlation spectroscopy Fluorescence correlation spectroscopy, fluorescence imaging analysis, fluorescence resonance energy transfer, evanescent field molecular imaging, fluorescence depolarization, surface plasmon resonance, or enzyme-linked immunosorbent assay (25) Method.
  • a therapeutic agent comprising the antibody of (27) as an active ingredient.
  • FIG. 1 Construction of a single-chain antibody cDNA library used in the present invention.
  • FIG. 3 Chemical structural formula of angiotensin II-pyotin.
  • MI2 MI1 was reacted in [18] at 50 ° C for 30 minutes, then at 99 ° C for 5 minutes, and angiotensin II was selected as an antigen and collected in [25].
  • MI3 A library collected by reacting MI2 in [18] at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes, selecting angiotensin II as an antigen, and collecting in [25].
  • FIG. 6 The translation solution obtained in [15], in which the corresponding molecules were confirmed by 5% polyacrylamide electrophoresis in the presence of 8M urea (electrophoresis photograph).
  • MHO Library obtained in [11]; MK1:
  • the library obtained in [11] was reacted in [18] at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes, and Lewis X was selected as an antigen, and the library recovered in [25]; MK2: MK1 In [18], the library was reacted at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes, and Lewis X was selected as an antigen, and the library recovered in [25].
  • FIG. 7 The translation solution obtained in [15], in which the corresponding molecules were confirmed by 5% polyacrylamide electrophoresis in the presence of 8M urea (electrophoresis photograph).
  • MM1 The library obtained in [11] was reacted at 50 ° C for 30 minutes in [18], and angiotensin II was selected as an antigen, and the library was recovered in [25];
  • MM2 MM1 was recovered in [18].
  • FIG. 8 The eluate obtained in [21] was subjected to PCR in [22] and subjected to 1% agarose gel electrophoresis. The top is an electrophoresis gel (electrophoresis photograph). The lower bar graph shows the absorption of ethidium bromide in the upper electrophoresis gel measured and quantified using MOLECULAR IMAGER FX (Bio-rad).
  • FIG. 11 A phylogenetic tree created by TreeViewPPC after sequence alignment by clustak based on amino acid sequences for in-frame results obtained by performing sequence analysis in [31].
  • a surrounded by a line indicates the one converged by the selection of Angiotensin II, and B surrounded by a line indicates the one converged by the selection of Lewis X.
  • the number of the clone was shown after the library abbreviation.
  • FIG. 12 is an enlarged view of A and B surrounded by the line in FIG. A: Converged by Angiotensin II selection, B: Converged by Lewis X selection.
  • FIG. 13 Western blot results (electrophoresis photograph). The right two are Western blots of MI3-55 [37]. Left three are Western blot controls.
  • FIG. 14 A bar graph showing the results obtained by translating the sequence obtained by selection using angiotensin II as an antigen in [34] and then analyzing it in Biacore in [39].
  • FIG. 15 A bar graph showing the result obtained by translating the sequence obtained by selection using Lewis X as an antigen with [34] and then analyzing with Biacore [39].
  • FIG. 16 A bar graph showing the results of translating MI3-55 at [34], treating it at 4 ° C. or 60 ° C. or 99 ° C. for 5 minutes, and analyzing with Biacore of [39].
  • FIG. 17 A bar graph showing the result of translating MI3-55 at [34], treating it at 4 ° C. or 60 ° C. or 99 ° C. for 5 minutes, and analyzing the result by ELISA [40].
  • the method for selecting a protein that interacts with a target molecule or a nucleic acid that encodes the protein according to the present invention is characterized in that the nucleic acid portion of the assigning molecule in the library encodes a protein that interacts with the target molecule; Except for subjecting the library of molecules to heat treatment, the method can be performed according to a conventional nucleic acid selection method using a corresponding molecule (hereinafter, also referred to as "IW method").
  • Target molecule means a molecule that interacts with a protein to be selected, and specifically includes proteins, nucleic acids, sugar chains, low molecular weight compounds, and the like.
  • selection vs. As long as it has the ability to interact with the elephant protein, there is no particular limitation on the full length of the protein or a partial peptide containing a binding active site.
  • the protein may be a protein whose amino acid sequence and function are known or unknown. These can be used as a target molecule even with a synthesized peptide chain, a protein purified from a living body, or a protein translated using an appropriate translation system such as a cDNA library, and purified.
  • the synthesized peptide chain may be a glycoprotein having a sugar chain bonded thereto.
  • a protein which is translated and purified using a suitable method for a purified protein having a known amino acid sequence or a cDNA library can be used.
  • the nucleic acid any DNA or RNA can be used as long as it has an ability to interact with the protein to be selected.
  • the nucleic acid may have a known nucleotide sequence or function, or may have an unknown nucleic acid.
  • those having a function as a nucleic acid capable of binding to a protein and having a known base sequence, or those that have been cut and isolated using a genomic library first-class restriction enzyme or the like can be used.
  • the sugar chain is not particularly limited as long as it has the ability to interact with the protein to be selected, and the sugar sequence or function may be a known sugar chain or an unknown sugar chain.
  • a sugar chain which has already been separated and analyzed and whose sugar sequence or function is known is used.
  • the low molecular weight compound is not particularly limited as long as it has the ability to interact with the protein to be selected. Either those whose functions are unknown or those whose ability to bind to a protein is already known can be used.
  • reaction between these target molecules and the target protein of the present invention or “interaction” between the target molecules usually means a covalent bond, a hydrophobic bond, a hydrogen bond, a Van der Waals between two molecules. At least one of a bond and a bond by electrostatic force. A force indicating an action by a force acting between molecules. This term should be interpreted in the broadest sense, and should not be interpreted restrictively in any sense.
  • the covalent bond includes a coordinate bond and a dipole bond.
  • the coupling by electrostatic force includes not only electrostatic coupling but also electric repulsion.
  • a binding reaction, a synthesis reaction, and a decomposition reaction resulting from the above-described actions are also included in the interaction.
  • Specific examples of the interaction include binding and dissociation between an antigen and an antibody, and a protein receptor. Binding and dissociation between a molecule and a ligand, binding and dissociation between an adhesion molecule and a partner molecule, binding and dissociation between an enzyme and a substrate, binding and dissociation between a nucleic acid and a protein bound thereto, proteins in a signal transduction system Binding and dissociation between glycoproteins and proteins, binding and dissociation between sugar chains and proteins, or binding and dissociation between low molecular weight compounds and proteins. Can be
  • the IW method is much more stable than ribosome display or phage display because the mRNA is covalently bound to the protein via the protein synthesis inhibitor puromycin or a derivative thereof. Therefore, it is possible to perform a higher selective pressure baning than the two techniques of ribosome display and phage display, thereby selecting not only specificity but also a protein that interacts with a more stable target molecule. In addition, the desired protein can be obtained in a smaller number of selection cycles because of the higher and higher concentration effects. Furthermore, it is not necessary to analyze many samples because it is concentrated to almost single. With these, the operation is simple, and the time and cost can be greatly reduced.
  • the establishment of highly functional antibodies (specificity, affinity, stability, etc.) in the library depends on the size (diversity) of the library.
  • the upper limit of the selectable library of the IW method can be 10 13 or more if conditions are satisfied, and a library larger than any other method can be targeted.
  • unnecessary bias is not applied.
  • the obtained protein can be expressed as a functional protein in a cell-free translation system containing a reducing agent such as DTT, it is possible to prepare a large number of samples, thereby facilitating high-throughput filtration.
  • the protein thus selected can be used in various fields depending on its function.
  • the high affinity single-chain antibody thus selected can be used for extracellular or intracellular protein detection and interaction detection.
  • the high-affinity single-chain antibody which has been selected for the mouse antibody cDNA library, is replaced with a human IgG antibody variable region and CDR region (complementarity determining region) to produce a chimeric IgG antibody or a humanized mouse antibody.
  • Make IgG antibodies These antibodies produce little or no human anti-mouse antibody when elicited when administered to humans .
  • a high-affinity single-chain antibody which has been selected as a human antibody cDNA library, can produce a fully human monoclonal IgG antibody by replacing the variable region of the human HgG antibody. This can be used as an antibody drug that does not cause anaphylactic symptoms.
  • the single-chain antibody used as the protein to be selected in the present invention has a V chain and a V chain
  • the DNA library encoding a single-chain antibody used in the present invention is not particularly limited as long as it is a library containing DNAs encoding a large number of single-chain antibodies. In particular, it is preferable to use one containing DNA encoding more than 10 9 different antibodies, which can correspond to any antigen.
  • Powerful antibody DNA libraries include, but are not limited to, those derived from higher vertebrates, preferably mouse, human, -petri, goat, camel spleen and B cells, used in conventional in vitro antibody selection systems, including commercially available ones. Any natural antibody DNA library may be used.
  • a mouse native antibody cDNA library (Krebber, A. et al. (1997)) J. Immun. Methods. 201, 35-55: Engberg, J. (1996) Molecular Biotech. 6, 287-310) MRNA was extracted from the spleen of the mouse, and the gene fragments encoding the variable regions (V, V) of the antibody H and L chains were analyzed by RT-PCR.
  • the obtained fragment force is also the C-terminal of V
  • the mouse single-chain antibody cDNA library is completed.
  • any linker sequence can be used as long as it has a relatively high degree of freedom, but it is generally preferable to use a (Gly Ser) sequence (SEQ ID NO: 120).
  • the order of joining is V
  • any mutant type antibody DNA library can be advantageously used.
  • any mutant type antibody DNA library can be advantageously used.
  • any mutant type antibody DNA library can be advantageously used.
  • n-CoDeR A library that extracts only CDRs (three H chains and three L chains) from antibody cDNA by PCR and incorporates them into an antibody frame that is easy to express in E. coli and phage (n-CoDeR: diverse Sex is 2 X 10 9 ) (Jirholt, P. et al. (1998) GENE 215, 471-476:
  • Step (a) is a step of preparing a library of DNAs encoding proteins that interact with the target molecule.
  • a library of DNAs encoding this protein can be prepared according to a conventional method.
  • step (b) the DNA of the library prepared in (a) is transcribed, and a spacer containing puromycin is ligated to the 3 'end of the transcribed RNA (hereinafter, prepared here). Is sometimes referred to as a “translation template”), and then a library of mapping molecules is prepared in a cell-free translation system by preparing mapping molecules for genotypes and phenotypes. You.
  • This step can be performed in the same manner as in the ordinary IW method.
  • the force spacer which will be described in more detail below, preferably contains polyethylene glycol.
  • the step (c) is a step of subjecting the library of assigning molecules to heat treatment.
  • the heat treatment means exposure to heating conditions that would not be imposed by the treatment in the normal IW method.
  • the assigning molecule is directly bound to the target molecule after translation, it is necessary to expose the subject to a heating condition that does not exist during translation, and the RNA portion of the assigning molecule is converted into an RNA-DNA hybrid by reverse transcription.
  • exposure is to heating conditions that are not between translation and reverse transcription.
  • This temperature is typically 50-100 ° C for 1-30 minutes, depending on the process of translation and force binding to the target molecule. Selected from a range.
  • heat treatment is preferably performed at a temperature of 80 ° C or higher, more preferably 90 ° C or higher, regardless of whether there is only translation or translation and reverse transcription.
  • the RNA portion of the assigning molecule be converted into an RNA-DNA hybrid by reverse transcription before the step of (U. This prevents nonspecific binding of the RNA to the carrier and the target molecule.
  • reverse transcription it is preferable to remove a substance derived from a cell-free translation system that inhibits the reverse transcription reaction before reverse transcription. , A spin column, a nickel column and the like.
  • the order of the heat treatment, the reverse transfer, and the fractionation is not particularly limited, but usually, the order of the heat treatment, the fractionation, and the reverse transfer, or the order of the fractionation, the reverse transfer, and the heat treatment is generally. preferable.
  • Step (d) is a step in which the assigning molecule is bound to the target molecule, washed sufficiently, then eluted, and the nucleic acid portion of the assigning molecule is amplified by reverse transcription-PCR or PCR. This step can be performed in the same manner as in the ordinary IW method. Elution can usually be performed by elution with a solution containing a free target molecule.
  • a process for using the DNA amplified in (d) as a library in (a) is performed.
  • a 5 'UTR sequence amplification primer sequence-SP6 promoter sequence- ⁇ 29 enhancer sequence
  • a 5 'UTR sequence amplification primer sequence-SP6 promoter sequence- ⁇ 29 enhancer sequence
  • a 5 'UTR sequence amplification primer sequence-SP6 promoter sequence- ⁇ 29 enhancer sequence
  • a 5 'UTR sequence amplification primer sequence-SP6 promoter sequence- ⁇ 29 enhancer sequence
  • a T7 tag MASMTGGQQMG (SEQ ID NO: 118)
  • This tag may be any commonly used tag.
  • FLAG tag (DYKDDDDK (SEQ ID NO: 119))
  • STOP codon, insertion, deletion some undesired mutations (STOP codon, insertion, deletion) by PCR amplification, chemical synthesis, etc. Therefore, the C-terminal tag allows you to select only in-frame tags, which can also be used with any commonly used tag. Absent.
  • the protein is a single chain antibody.
  • H chain and L chain of mouse spleen-derived antibody (IgG) mRNA These variable regions were extracted by RT-PCR, and the C-terminus of the H chain and the N-terminus of the L chain were
  • FIG. 2 shows an outline of the steps of the selection method of the present invention when a single-chain antibody DNA library is used.
  • the desired antibody is enriched by such a selection cycle, ie, by repeating selection, amplification, library restructuring, and translation.
  • a certain selection pressure at the time of selection the properties of the obtained antibody and the concentration efficiency are determined.
  • the target antibody can be selected even when a high selection pressure is applied so that the target antibody cannot be obtained by the conventional method.
  • the coding part of the protein to be selected comprises a 5 'terminal region, an ORF region, and a 3' terminal region, and may or may not have a Cap structure at the 5 'terminal.
  • the 3 'terminal region of the coding portion is a poly Ax8 sequence as an A sequence, an Xhol sequence as an X sequence, a sequence having (C or G) NN (C or G) with 4 or more bases, and an A sequence.
  • a configuration comprising an A sequence, an X sequence, or a Flag-tag sequence as an affinity tag sequence upstream of the XA sequence can be considered.
  • the affinity tag sequence is a sequence using any means capable of detecting or purifying a protein, such as one utilizing an antigen-antibody reaction such as HA-tag or IgG protein A (z domain) or His-tag. But I can't help.
  • the combination of XA sequences is important.
  • the base is important, and one having a (C or G) NN (C or G) sequence is preferred.
  • the 5 'terminal region is composed of a transcription promoter and a translation enhancer.Transcription promoters are T7 / T3, SP6, etc. can be used, and there is no particular limitation!
  • a part of the omega sequence (029) of the translation enhancer includes a part of the omega sequence of TMV (Gallie DR, Walbot V. (1992) Nucleic Acids Res., 20, 4631-4638).
  • the polyethylene glycol (PEG) portion is a CCA region, a PEG region, and a donor region.
  • the minimum required configuration is the donor region. From the viewpoint of translation efficiency, those having a PEG moiety as well as the donor region are more preferable, and those having a pure mouth mycin which has no binding ability to amino acids are preferable!
  • the molecular weight range of the polyethylene glycol in the PEG region is from 400 to 30,000, preferably from 1,000 to 10,000, and more preferably from 2,000 to 6,000.
  • the CCA domain can be configured with or without Puromycin!
  • a configuration containing puromycin is used in a cell-free translation system so that a spacer can bind to the C-terminus of the protein translated there.
  • Including puromycin includes including derivatives thereof. Examples of derivatives include the following. 3 N-aminoacylpuromycin aminonucleosides (3'- ⁇ —Aminoacylpuromycin aminonucleoside, PANS-amino acids), such as PANS-Gly for glycine in the amino acid portion, PANS-Val for phosphorus, PANS-Ala for alanine, etc. PANS-all amino acids corresponding to all amino acids can be used.
  • 3'-N-aminoacyl adenosine aminonucleoside an amide bond formed as a result of dehydration condensation of the amino group of 3'-aminoadenosine and the carboxyl group of amino acid as a chemical bond
  • AANS-amino acid 3'-Aminoacyladenosine aminonucleoside
  • AANS-amino acid for example, AANS-Gly of glycine, AANS-Val of norin, AANS-Ala of alanine, and other AANS-amino acids corresponding to all amino acids
  • nucleosides or those in which a nucleoside and an amino acid are ester-linked can also be used.
  • Other substances that have a chemical structural skeleton similar to nucleosides or nucleosides Any substance can be used as long as the substance has a chemical structure and can be chemically bonded.
  • CCA CCA region
  • a structure containing no puromycin or a structure containing puromycin having no binding ability to amino acids is preferable. Any substance in which the amino group of the above puromycin derivative lacks the ability to bind to amino acids, and the CCA region lacking puromycin may also be a potential force.Includes puromycin, which cannot bind proteins on the ribosome. Efficiency can be increased. The reason is unclear, but puromycin, which cannot bind to proteins, may stimulate turnover by stimulating liposomes. In the case of uncombined piuromycin, the above puromycin is made uncombinable with amino acids in an appropriate manner.
  • the PEG portion can be configured to have a modifying substance.
  • the translation template can be recovered, reused by purification, or used as a tag for fixing.
  • a modifying substance a fluorescent substance, biotin, or various separation tags such as His-tag may be introduced into at least one base of DNA and / or RNA.
  • each length is about 60 bp in the 5 'end region. It is about 40 bp in the 3 'end region, and has a length that can be designed as an adapter region in a PCR primer. This has created a new effect.
  • any vector, plasmid or cDNA library PCR can easily create a coding part having a 5'-terminal region and a 3'-terminal region.
  • a translation template with high translation efficiency can be obtained.
  • the ligation of the PEG part and the code part is performed by a general DNA ligase method.
  • the method is not particularly limited, and any method such as a method using a compound or a photoreaction can be used.
  • the A sequence in the 3'-terminal region is important as the range that affects the ligation efficiency in the coding region, and at least 2 residues of dA and / or rA mixed or single It is a poly A continuous chain, preferably a poly A continuous chain of 3 or more residues, more preferably 6 to 8 residues or more.
  • the DNA and / or RNA sequence at the 5 'end of the donor region of the PEG part affects ligation efficiency.
  • dC dihydroxycytidylic acid
  • dCdC diideoxycytidylic acid
  • the type of the base is preferably in the order of C> (U or T)>G> A. Further, it is preferable to add polyethylene glycol having the same molecular weight as the PEG region during the ligation reaction.
  • the protein to be selected which is synthesized by the translation of the protein to be selected with the translation template in the presence of the modifying agent, and is C-terminally modified with the modifying agent, and also serves as the translation template and the modifying agent.
  • the feature lies particularly in the configuration of the code part of the translation template. The details are described below.
  • the PEG portion of the translation template is characterized in that puromycin cannot be linked to amino acids.
  • the 3 ′ terminal region is an XA sequence, and the first 4 bases are important in the X sequence, and (C or G ) Those having the sequence of NN (C or G) are preferred.
  • the length is about 60 bp and about 40 bp in the 3'-terminal region, and it can be designed as an adapter region for PCR primers. This makes it possible to easily create a code portion having a 5'-terminal region and a 3'-terminal region by PCR, and ligating a PEG portion instead of the 3'UTR to this code portion to obtain a C-terminal portion.
  • a translation template with high translation efficiency suitable for labeling can be obtained.
  • the modifying agent is an axepept having a group (including a residue) capable of binding to a protein by a transpeptidation reaction in a protein translation system, ie, a transpeptidation reaction on a ribosome.
  • the linker has a configuration in which the linker is linked to the modifier via a nucleotide linker.
  • the fluorescent substance examples include fluorescent dyes such as fluorescein series, rhodamine series, Cy3, Cy5, eosin series, and NBD series, and fluorescent proteins such as green fluorescent protein (GFP).
  • any compound may be used as a marker, such as a coenzyme such as biotin, a protein, a peptide, a saccharide, a lipid, a dye, and polyethylene glycol.
  • the modifying portion preferably has a fluorescent group, a group that binds to a protein, or both.
  • the receptor section has a group capable of binding to a protein by a transpeptidation reaction in a protein translation system, and preferably has a residue of pure mouth mycin or a derivative thereof.
  • Puromycin has a structure similar to aminoacyl-tRNA and is known as an antibiotic that inhibits protein synthesis, but is known to bind to the C-terminus of proteins at low concentrations (Miyamoto-Sato, E. et. al. (2000) Nucleic Acids Res. 28: 1176-1182).
  • the puromycin derivative that can be used is any substance having a structure similar to puromycin and capable of binding to the C-terminus of the protein.
  • nucleotide linker that connects between the modification part and the receptor part is, specifically, a nucleic acid or a nucleic acid derivative in which one or more ribonucleotides or deoxyribonucleotides are connected, and particularly preferred examples include:
  • An example is a conjugate having one ribonucleotide (-rC-) or one deoxyribonucleotide (_dC-) containing a cytosine base.
  • the nucleotide linker is deoxycytidylic acid, 2'-deoxycitidyl- (3 ', 5')-2'-deoxytidylic acid, ribocytidylic acid, or ribocytidyl- (3 ', 5')- Preferably it is ribocytidylic acid! / ,.
  • the modifying agent can be produced by binding the above-mentioned modified portion and the acceptor portion via a desired nucleotide linker by a chemical bonding method known per se.
  • the above-mentioned receptor portion protected with an appropriate protecting group is bound to a solid-phase carrier, and a nucleotide phosphoramidite and a deoxynucleotide phosphoramidite are used as a nucleotide linker using a nucleic acid synthesizer. It can be produced by sequentially binding a phosphoramidite to which a fluorescent substance or biotin is bound as a functional modifier, and then performing deprotection.
  • the above components or the type of bonding can be combined by a liquid phase synthesis method, or both can be used in combination.
  • a metal ion such as nickel
  • the metal ion can be coordinated with a chelating reagent such as triacetate triacetic acid or iminodiacetic acid, and then the metal ion is distributed in the next step. Can be placed.
  • the PEG portion of the translation template is the same as described above, except that puromycin can be linked to amino acids.
  • the coding portion is the same as that described above.
  • the 3 ′ end region is an A sequence, and the efficiency of association of total proteins is significantly improved. It was confirmed that the amount of free protein was drastically reduced.
  • PEG-C-terminally modified proteins can be used with RNase A when the coding part is not used for protein detection or interaction detection, for example, when applied to FCCS measurement, fluorescent readers, protein chips, etc. It is preferable to cut intentionally. Cleavage can eliminate the difficulty of detecting protein-protein interactions due to interference with the coding region.
  • a cell-free translation system In order to form IW from the protein cDNA library, a cell-free translation system is used. Specifically, thiolation such as dithiothreitol (DTT) and ⁇ -mercaptoethanol A wheat germ extract containing or not containing the compound, a heron reticulocyte extract, and an Escherichia coli S-30 extract are used. In particular, it has been difficult to express an active antibody as a protein in the cell-free translation system containing DTT, but in the present invention, it is possible to select an active antibody by using IW even in the presence of DTT. I was able to do it.
  • DTT dithiothreitol
  • ⁇ -mercaptoethanol A wheat germ extract containing or not containing the compound, a heron reticulocyte extract, and an Escherichia coli S-30 extract are used.
  • DTT dithiothreitol
  • ⁇ -mercaptoethanol A wheat germ extract containing or not containing the compound,
  • the translation template of the above protein is caloried, and incubated at 25-37 ° C for several hours to form IW.
  • a C-terminal modified protein is synthesized.
  • the synthesized IVV is directly used for the next heat treatment step or reverse transcription step.
  • the assigning molecule also referred to as “IW”
  • the cell-free translation system contains a substance that inhibits the reverse transcription reaction.
  • gel filtration preferably Sephadex G200 (manufactured by Amersham Bioscience), or spin column, preferably Ultrafree MC, 100,000 NMWL (manufactured by Millipore), or PURESYSTEM (post-genome) of E. coli cell-free translation system (Manufactured by Research Institute), use nickel resin.
  • RNA-DNA hybrid By making the mRNA portion of the IW into an RNA-DNA hybrid by the reverse transcription reaction (RT), nonspecific adsorption of the mRNA portion to the carrier and the antigen can be prevented.
  • RT reverse transcription reaction
  • mRNA is denatured by heating at 65 ° C, then cooled to 4 ° C to anneal, ReverTra Ace (TOYOBO) is added, and the reaction is performed at 50 ° C for 30 minutes.
  • the reverse transcriptase may be any enzyme under any conditions as long as it performs a reverse transcription reaction. It is not limited to the above conditions.
  • the heat treatment conditions of IVV with mRNA part as RNA-DNA hybrid are also usually in the range of 1 to 30 minutes at 50-100 ° C. Force that is the condition selected is higher than the maximum temperature of reverse transcription. .
  • the DNA polymerase used for RT-PCR and PCR is not particularly limited as long as it is used for the PCR reaction, but the high amplification efficiency and the fidelity of the PCR are not too high. Are more preferred.
  • the KOD Dash (TOYOBO product) used in the present invention can be efficiently amplified even from a very small amount of type I DNA, and can be evolved into a more highly functional one because appropriate mutations are introduced.
  • the target molecule is a peptide (which may be chemically synthesized or isolated from nature or may be a partial digest of a protein), a protein, a nucleic acid (DNA or RNA), a saccharide, various small molecules. All compounds and substances, such as compounds, metals and metal compounds, are applicable.
  • the target molecule used in the present invention may be a force capable of binding to a solid phase such as a resin or a bead.
  • a solid phase such as a resin or a bead.
  • One that is bound by an outer portion is included.
  • the modifying substance used in the case of binding via the modifying substance is usually a molecule that specifically binds to a specific polypeptide (hereinafter, may be referred to as "ligand”).
  • a specific polypeptide hereinafter, sometimes referred to as “adaptor protein” that binds to the ligand is bound to the phase surface.
  • the adapter protein also includes a binding protein, a receptor protein constituting the receptor, an antibody, and the like.
  • adapter protein Z ligands examples include biotin-binding protein Z-biotin such as avidin and streptavidin, maltose-binding protein Z-maltose, G-protein Z guanine nucleotide, polyhistidine peptide Z-nickel, and cobalt.
  • biotin-binding protein Z-biotin such as avidin and streptavidin
  • maltose-binding protein Z-maltose G-protein Z guanine nucleotide
  • polyhistidine peptide Z-nickel examples of the combination of adapter protein Z ligands include biotin-binding protein Z-biotin such as avidin and streptavidin, maltose-binding protein Z-maltose, G-protein Z guanine nucleotide, polyhistidine peptide Z-nickel, and cobalt.
  • Various receptors such as metal ion, glutathione S-transferase Z glutathione
  • combinations of adapter protein Z ligands include a biotin binding protein such as avidin and streptavidin, a maltose binding protein Z maltose, a polyhistidine peptide Z a metal ion such as nickel or cobalt, and glutathione. Particularly preferred is a combination of streptavidin z-biotin, in which s-transferase Z daltathione is preferred.
  • the binding of the adapter protein to the surface of the solid phase can be carried out by a method known per se, and specifically, for example, tannic acid, formalin, dartalaldehyde, pyrvicaldehyde, benzodiazobenzidine , Toluene-2,4-diisocyanate, amino group, carboxyl group convertible to active ester, or water convertible to phosphoramidite
  • a method using an acid group or an amino group can be used.
  • a known method usually used to bind proteins, nucleic acids, sugar chains, and low molecular weight compounds to the solid phase specifically, for example, tannic acid , Formalin, glutaraldehyde, pyrvicaldehyde, bis-diazodyl benzidine, toluene-2,4-diisocyanate, amino group, hydroxyl group which can be converted to active ester, or hydroxyl or amino group which can be converted to phosphoamidide Can be used.
  • the solid phase carrier is preferably agarose beads or agarose beads in which a magnetic substance is embedded.
  • the distance between the solid surface and the antigen molecule should be more than 30 angstroms from a steric point of view!
  • a high affinity antibody There are generally two methods for obtaining a highly functional protein.
  • a high affinity antibody will be described as an example.
  • One is to set the antigen concentration to be low in the selection experiment and select only the antibody at that concentration.
  • the amount of antibody to be recovered is naturally small, and the efficiency is low. Accordingly, the antigen concentration that can be set is limited to the concentration that can be recovered.
  • the second is off-rate selection (Hawkins, R. (1992) J. Mol. Biol. 226, 889-896; Boder, E. (1997) Nat. Biotechnol. 15, 553-557; Jermutus, L. (2001 Natl. Acad. Sci. USA 98, 75-80), which uses the dissociation rate of antigen and antibody.
  • dissociation constant (Kd) is represented by the ratio of the association rate constant (on-rate) and dissociation rate constant (off-rate), association constant of biopolymers substantially within a certain range (10 4 - 10 6 M — Because of the ⁇ , the real Kd is determined by the dissociation rate constant, so antibodies with a slower dissociation rate have a higher affinity, so the longer the elution time with the antigen, the more concentrated the antibody with higher affinity
  • this method has the disadvantage that it takes longer to perform one cycle of a selection experiment, and a solution to this problem is to use an antigen or antibody sequence-specific instead of eluting with the antigen.
  • protease is eluted by quenching with proteinase, which has a very high substrate specificity, such as TMV protease factor Xa, from trypsin, proteinase K, and the like. Specificity The above methods are also applicable to the present invention, and are not limited by any proteases including, but not limited to, Is an extremely effective method.
  • a method for producing a protein using the nucleic acid selected by the selection method of the present invention that is, a step of selecting a nucleic acid encoding a protein that interacts with a target molecule by the selection method of the present invention, and A method for producing a protein by translating a selected nucleic acid is also provided.
  • the step of selecting a nucleic acid is as described for the selection method of the present invention.
  • the nucleic acid may be translated by a cell-free translation system, or a living cell such as Escherichia coli may be transformed with the nucleic acid by using a plasmid into which the nucleic acid has been introduced, and the protein may be expressed in the living cell. You may let them.
  • a cell-free translation system a cell-free translation system containing a thiol compound such as dithiothreitol or j8-mercaptoethanol, preferably, a wheat germ extract, a rabbit egret reticulocyte extract, or an Escherichia coli S-30 extract Liquid.
  • the protein When expressing the protein, the protein may be expressed as a fusion protein of a protein encoded by the selected nucleic acid and an enzyme or a green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • Examples of the protein obtained by the production method of the present invention include a single-chain antibody.
  • a single-chain antibody having angiotensin II binding activity which has the amino acid sequence shown in the following (A) or (B).
  • SEQ ID NO: 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103 or Pamino 105 is an amino acid rooster.
  • a single-chain antibody having binding activity to Lewis X having the amino acid sequence shown in the following (A) or (B).
  • Cell-free translation systems include wheat germ extracts, egret reticulocyte extracts, and E. coli S-30 extracts.
  • Specific examples of expressing proteins in large amounts using living cells include bacteria such as Escherichia coli, Bacillus subtilis, thermophiles, and yeast, cultured cells such as insect cells and mammals, as well as nematodes, Drosophila, Any cells, such as zebrafish and mice, may be used.
  • bacteria such as Escherichia coli, Bacillus subtilis, thermophiles, and yeast
  • cultured cells such as insect cells and mammals, as well as nematodes, Drosophila, Any cells, such as zebrafish and mice, may be used.
  • the above-mentioned C-terminal labeled ⁇ can directly introduce both of the associated modified proteins, or, if the above-described translation template is introduced and the C-terminal labeling is carried out, simultaneously.
  • a modified protein is synthesized by introducing a 100 / zM modifier into cells by electroporation, microinjection, or the like, and keeping the cells at the optimal growth temperature for several hours.
  • mapping the mapping molecule is synthesized by introducing the above translation template and keeping the cells at the optimal growth temperature for several hours. Both synthesized modified proteins can be recovered by disrupting the cells and subjected to subsequent purification or detection processes. In addition, subject to the detection process in the cell as it is Is also possible. Select an appropriate translation template according to the translation system used
  • RNA promoters are used for transcription into RNA. Since the protein to be expressed may be toxic to the host Escherichia coli, most vectors have some form of expression control. This control is also important so that the growth of E. coli is not hindered until E. coli is transformed with a vector containing the gene encoding the expression protein and expression is induced.
  • a ribosome binding site Immediately downstream of the promoter and immediately upstream of the claw site, a ribosome binding site,
  • the higher-order structure of mRNA near the translation start point is important.
  • the expression system in Escherichia coli is performed in the cytoplasm.
  • proteins having an SS bond, such as antibodies become inclusion bodies. Therefore, in the case of an antibody, E. coli expression is generally performed in periplasm under an irritating environment. In this case, it is achieved by introducing a signal sequence (for example, pel B leader or ompT) into the N-terminus of the antibody.
  • a signal sequence for example, pel B leader or ompT
  • the expression level in periplasm is extremely low compared to the cytoplasm.
  • DTT exists in the antibody obtained in the present invention. Since it has been selected under a reducing environment, it can be expressed as a water-soluble functional antibody even when expressed in the cytoplasm of Escherichia coli.
  • the single-chain antibody obtained by the production method of the present invention can be used for immunological detection of a protein.
  • the immunological detection method include a Western blot method, an immunostaining method, a fluorescent antibody staining method, an antibody chip method, and an immunoprecipitation method.
  • the protein interaction is detected by bringing the single-chain antibody obtained by the production method of the present invention into contact with a protein and detecting the interaction between the single-chain antibody and the protein.
  • Methods for detecting protein interactions include fluorescence correlation spectroscopy, fluorescence imaging analysis, fluorescence resonance energy transfer, evanescent field molecular imaging, fluorescence depolarization, surface plasmon resonance, and enzyme-linked immunosorbent assay.
  • the single-chain antibody, the peptide, and the protein are modified with some modifying agent, for example, a fluorescent dye, or an enzyme or a fluorescent protein ( It is necessary to construct a fusion protein with GFP) and detect it.
  • some modifying agent for example, a fluorescent dye, or an enzyme or a fluorescent protein
  • the method for modifying single-chain antibodies, peptides and proteins with fluorescent dyes has been described above. Construction of the fusion protein involves fusing GFP or an enzyme, preferably alkaline phosphatase ⁇ horseradish rust) peroxidase, to the C-terminus or N-terminus of the single-chain antibody.
  • Such a single-chain antibody fusion protein is obtained by expression and purification in a cell-free translation system or a system of Escherichia coli, baculovirus, or animal cells. Fluorescence is detected for GFP, visible light for alkaline phosphatase, and chemiluminescence for horseradish peroxidase with a luminometer.
  • a fusion protein of a secondary antibody of a single-chain antibody tag Flag-tag, T7-tag, HA-tag, etc.
  • GFP or alkaline phosphatase ⁇ horseradish peroxidase is used. May be.
  • tissue staining using antibodies is relatively simple in operation, and only an antibody that recognizes the target molecule is prepared.
  • antibodies that recognize that molecule are often obtained in some way, so even the first person can start the experiment immediately.
  • Another advantage of the present method is that the presence of a protein can be directly confirmed in the form of gene expression.
  • not all antibodies can be used for tissue staining. Good results cannot be expected with antibodies because they cannot recognize the structure of the molecule in vivo. The determination of the ability to prepare antibodies suitable for tissue staining is a key game.
  • the basic principle is the same as that of the Western plot, but in the case of tissue staining, the protein is fixed in the tissue with formaldehyde or the like, and its localization is detected.
  • Ability to detect immobilized target protein using primary and secondary antibodies If the signal is weak and difficult to detect, a method has been developed to specifically amplify only the target signal. ing.
  • observing the localization of endogenous proteins in cells using an antibody that specifically recognizes that molecule can provide very useful information. Many,. Unlike when expressed as a GFP fusion protein, etc., it is not possible to observe the movement of the molecule as it is in a living cell, but the localization of the target protein in various states such as response to stimulation and treatment of drugs and cell motility. You can observe the change in location. Even when there is no antibody against the protein of interest, localization of the molecule of interest using the antibody against the tag can be achieved by using a method such as transiently expressing the protein in a cell with the tag attached. Can be observed.
  • the localization of the tagged protein observed in this case does not always coincide with the actual localization of the protein, and is often the result of overexpression.
  • the operation of the experiment is relatively easy, it is necessary to examine the conditions of the immobilization method depending on the localization of the antibody and the target protein, and above all, prepare a specific antibody against the protein of interest Is an important point.
  • the membrane is permealized (permealized; increased permeability) using a surfactant or the like, and a specific antibody (primary antibody) against the target molecule is used.
  • a complex of the target protein and antibody is formed in the cells.
  • non-specifically bound antibodies Since many non-specifically bound antibodies remain in the cells, they are removed by washing, and then an antibody that recognizes the primary antibody and is labeled with a fluorescent substance (secondary antibody) A complex of protein-primary antibody-secondary antibody is formed. Like the primary antibody, the non-specifically bound secondary antibody is washed away, encapsulated, and observed under a microscope.
  • the Western plot refers to an operation of electrically transferring a protein after electrophoresis to a membrane, or a series of experiments including such an operation.
  • a commonly used method for protein electrophoresis is SDS-PAGE.
  • SDS as a reducing agent and 2-mercaptoethanol having a negative charge and heat-treated
  • the higher order structure of the protein is almost completely destroyed.
  • SDS binds to the protein at a protein molecular weight ratio (1: 1.4)
  • the charge state becomes uniform.
  • this is electrophoresed in a polyacrylamide gel, it can be separated according to the molecular weight of the protein.
  • the protein developed in the gel is electrophoretically transferred to a membrane, and reacted with an antibody specific to the target protein in the sample on the membrane to immunologically target the target protein. To be detected.
  • the method of Western blot using SDS-PAGE and enzyme activity is described, but it is not particularly limited to this protocol.
  • the flow of the Western blot operation is shown below. (1) After denaturing the protein sample with a sample buffer containing SDS, etc., separate proteins according to molecular weight by SDS-PAGE. (2) After electrophoresis, the gel is placed on the transfer device with the gel layered on it, and the protein band developed in the gel Is electrically transferred (plotted) onto the membrane. (3) After blocking to prevent nonspecific adsorption to the protein, a primary antibody reaction is performed with an antibody that specifically recognizes the target protein.
  • the secondary antibody reacts with a secondary antibody (with a chromogenic enzyme) that specifically recognizes the primary antibody molecule.
  • a color reaction is performed using the enzyme activity of the chromogenic enzyme attached to the secondary antibody, for example, a peroxidase activity or an alkaline phosphatase activity, and the target protein is detected.
  • Immunoprecipitation refers to a specific antigen-antibody reaction and modification of the C-terminal portion of a single-chain antibody with biotin, various tags such as Flag-tag, T7-tag, By using HA-tag, His-tag, etc. (this binds to streptavidin or tag antibody covalently bound to agarose beads, it can be easily separated from the solution by centrifugation), and the antigen corresponding to the used antibody can be obtained. It is a method to separate and purify only proteins. However, at present, not only is the antigen protein separated and purified, but rather the complex with the antigen protein that directly binds to the antibody by appropriately weakening the washing strength and the lysate detergent used.
  • the high-affinity antibodies selected in the present invention serve the purpose. Commonly used antibodies require higher titers (10 7 -IOVOI 1 ) than Western blots, and require a certain degree of specificity.
  • the aspect of the protein complex that can be separated changes depending on the buffer used for washing, the number of washings, the composition of the solubilization buffer, and the like.
  • the protocol is shown below, but is not particularly limited.
  • (1) Recovery of lysate As a preparation step for performing immunoprecipitation, it is necessary to first obtain a cell-free protein solution from cells. Depending on the concentration and composition of the solution, the results of subsequent immunoprecipitation will also vary. The lysate contains various proteins and various proteins.
  • Antigen-antibody reaction An antigen-antibody reaction is caused by adding a single-chain antibody that recognizes the target protein to the lysate.
  • the modification obtained above is usually used.
  • the functional protein and the target molecule are brought into contact with each other by appropriately combining them according to the type of the modifying substance, the type of the reaction system, etc., and the signal generated by the modified functional protein or the target molecule is based on the interaction between the two molecules. ! / Analyze the interaction by measuring the change in the signal generated.
  • the analysis of the interaction may be performed, for example, by fluorescence correlation spectroscopy, fluorescence imaging analysis, fluorescence resonance energy transfer, evanescent field molecular imaging, fluorescence depolarization, surface plasmon resonance, or solid-phase enzyme immunoassay. It is done by law. Further, the two sets of the modified functional protein obtained above and the target molecule are brought into contact with each other by appropriately combining them according to the type of the modifying substance, the type of the reaction system, and the like. The interaction is analyzed by measuring a change in the signal generated based on the interaction between the antigen molecules.
  • Interaction analysis can be performed, for example, by fluorescence correlation spectroscopy, fluorescence imaging analysis, fluorescence resonance energy transfer, evanescent field molecular imaging, fluorescence depolarization, surface plasmon resonance, or enzyme-linked immunosorbent assay. Is performed by The details of these methods are described below. The target molecules and interactions are as described above.
  • the functional protein to be used may be used after being modified with a modifying substance according to the embodiment. Can do.
  • the modifier is usually selected from non-radioactive modifiers such as fluorescent substances.
  • the fluorescent substance various fluorescent dyes having free functional groups (for example, carboxyl group, hydroxyl group, amino group, etc.) and capable of being linked to the above-mentioned single-chain antibodies such as proteins and nucleic acids, for example, fluorescein series, rhodamine series, Any of Cy3, Cy5, eosin series, NBD series and the like may be used.
  • the type and size of the compound are not limited as long as the compound can be modified such as a dye.
  • these modifying substances those suitable for a method of measuring or analyzing a change in a signal generated based on an interaction between a target molecule and a modified functional protein are appropriately used.
  • the above-mentioned modifying substance can be bound to the functional protein using an appropriate method known per se. Specifically, for example, the above-described method of modifying the C-terminus can be used.
  • the modified functional protein or the target molecule used in the present invention may be bound to a solid phase depending on the embodiment. Those that are combined by other parts are listed.
  • the modifying substance used in the case of binding via a modifying substance is usually a molecule that specifically binds to a specific polypeptide (hereinafter, may be referred to as "ligand”).
  • a specific polypeptide hereinafter, sometimes referred to as “adaptor protein” that binds to the ligand is bound to the phase surface.
  • the adapter protein also includes a binding protein, a receptor protein constituting the receptor, an antibody, and the like.
  • adapter protein Z ligands examples include biotin-binding protein Z-biotin such as avidin and streptavidin, maltose-binding protein Z-maltose, G-protein Z guanine nucleotide, polyhistidine peptide Z-nickel, and cobalt.
  • biotin-binding protein Z-biotin such as avidin and streptavidin
  • maltose-binding protein Z-maltose G-protein Z guanine nucleotide
  • polyhistidine peptide Z-nickel examples of the combination of adapter protein Z ligands include biotin-binding protein Z-biotin such as avidin and streptavidin, maltose-binding protein Z-maltose, G-protein Z guanine nucleotide, polyhistidine peptide Z-nickel, and cobalt.
  • Various receptors such as metal ion, glutathione S-transferase Z glutathione
  • adapter protein Z ligands include biotin-binding proteins such as avidin and streptavidin, and maltose-binding protein Z-matrix. Particularly preferred is a combination of streptavidin Z-biotin, which is preferably a metal ion such as lactose, polyhistidine peptide Z nickel or cobalt, glutathione s-transferase Z daltathione, and the like. These binding proteins are known per se, and the DNA encoding the protein has already been closed.
  • the binding of the adapter protein to the solid phase surface can be carried out by a method known per se, and specifically, for example, tannic acid, formalin, dartalaldehyde, pyrvicaldehyde, benzodiazobenzidine
  • a method utilizing a toluene-2,4-diisocyanate, an amino group, a carboxyl group convertible to an active ester, or a hydroxyl group or amino group convertible to a phosphoramidide can be used.
  • a known method usually used for binding proteins, nucleic acids, sugar chains, and low molecular weight compounds to a solid phase specifically, for example, tannic acid , Formalin, glutaraldehyde, pyrvicaldehyde, bis-diazoi di-benzizone, toluene-2,4-diisocyanate, amino group, hydroxyl group that can be converted to active ester, or hydroxyl group or amino group that can be converted to phosphoamidide
  • a method can be used.
  • Measurement is a means for collecting changes in signals used for analysis, and should not be construed as limiting in any sense.
  • Examples of the measuring method used include fluorescence correlation spectroscopy, fluorescence resonance energy transfer, evanescent field molecular imaging, fluorescence depolarization, fluorescence imaging analysis, surface plasmon resonance, and enzyme-linked immunosorbent assay. Any system that can detect intermolecular interactions is available.
  • Proc. Natl. Acad. Sci. USA, 91, 5740-5747 (1994)) is a method for measuring the flow velocity, diffusivity, volume contraction, etc. of particles under a confocal laser microscope or the like.
  • the interaction between the modified functional protein (C-terminal modified functional protein) and the antigen molecule is measured by measuring the change in the translational Brownian motion of one original modified molecule, thereby measuring the interacting molecules. can do.
  • the sample particles are excited by the excitation light, emit fluorescence in a part of the sample solution volume, and the emitted light is measured and Get the percentage. This value varies with the number of particles present in the volume of space observed at a particular time.
  • FCCS fluorescence cross-correlation spectroscopy
  • FRET fluorescence resonance energy transfer
  • the FCCS method when compared with other detection systems such as the fluorescence depolarization method, the FCCS method has advantages such as a small sample amount required, a short detection time, and easy automation for HTS. Furthermore, the FCCS method can provide very basic information such as the size and number of fluorescently labeled molecules, and may be used for general-purpose applications such as surface plasmon resonance. The difference between the two is that the surface plasmon resonance method detects the interaction in a state where the protein is immobilized, whereas the FCCS method allows the interaction in a solution that is closer to the natural state to be observed. is there. In the FCCS method, instead of the necessity of immobilizing proteins, it is necessary to label proteins with fluorescent dyes. The present invention has made it possible to overcome this problem.
  • a method for bringing the two into contact with each other is sufficient to allow the two target molecules to interact with each other. Any method may be used as long as it comes into contact with the substrate, but preferably, the emission wavelengths do not overlap at a concentration appropriate for a buffer or the like normally used for biochemistry in a measuring well of a commercially available FCCS device.
  • a solution in which two types of C-terminal modified functional proteins labeled with a fluorescent dye are dissolved, and a solution in which two unlabeled target molecules are dissolved in the same buffer at an appropriate concentration are added. Be done.
  • a method of performing a large number of analyzes at the same time for example, a plurality of different C-terminal modified functional proteins are respectively injected into each measurement well of the above-described measurement device for FCCS, and the function of each protein is added to each.
  • a method is used in which a target molecule solution capable of binding to a functional protein is charged, or a specific C-terminal modified functional protein is charged, and a plurality of different target molecule solutions are charged into each well.
  • a modified molecule is brought into contact with a solid-phased molecule, and emitted from the modified molecule remaining on the solid-phased molecule due to the interaction between both molecules.
  • This is a method of measuring or analyzing fluorescence using a commercially available fluorescence imaging analyzer.
  • one of the C-terminal modified functional protein and the target molecule may be immobilized by the method described above. Needs to be When the target molecule is used in the form of a solid phase, it can be modified, and can be used. In addition, when used without being immobilized, it is necessary to be modified with the above-mentioned modifying substance.
  • the C-terminal modified functional protein even if it is immobilized via the modified part, is immobilized with a part other than the modified part, for example, a fusion protein such as GST, Flag-tag, His-tag, etc. Can also be used.
  • a substrate for immobilizing a C-terminal modified functional protein or a target molecule is usually used for immobilizing proteins, nucleic acids, or the like-trocellulose membrane, nylon membrane, slide glass, or the like. Plastic microplates can also be used.
  • a modified target molecule ! a method for bringing a functional protein into contact with a solid-phased molecule as long as the two molecules can be brought into contact with each other to an extent sufficient for interaction. It is possible to use a modified target molecule! /, To prepare a solution in which a C-terminal modified functional protein is dissolved at an appropriate concentration in a buffer commonly used in biochemistry. However, a method of bringing this into contact with the surface of the solid phase is preferred.
  • a step of washing the excessively present modified target molecule or C-terminal modified functional protein with the same buffer or the like is carried out, and the target molecule or c-terminal remaining on the solid phase is removed.
  • a fluorescent signal that also emits the power of the modifying substance of the terminally modified functional protein, or a signal in which the fluorescent light emitted by the modified molecule that is immobilized on the solid phase and the fluorescent light that is emitted from the modifying molecule that remains on the solid phase are used as a commercially available signal.
  • Molecules that interact with immobilized molecules can be identified by measurement or analysis using an imaging analyzer.
  • this method as a method for performing a large number of analyzes at the same time, for example, a method in which a plurality of c-terminal modified functional proteins or modified or unmodified target molecules are addressed and immobilized on the solid phase surface, Alternatively, a method of immobilizing one kind of C-terminal modified functional protein or a modified or unmodified target molecule on a solid phase and contacting a plurality of kinds of c-terminal modified functional proteins or modified target molecules is used. .
  • a plurality of types of C-terminal modified functional proteins or modified target molecules are brought into contact with each other, the molecules remaining on the solid phase are obtained by dissociation due to a difference in buffer concentration or the like, and this is obtained by a known method. It can be identified by analysis.
  • FRET fluorescence resonance energy transfer
  • FRET does not occur because the distance is large, and the force of observing the fluorescence spectrum of the donor
  • the fluorescence spectrum of the acceptor is observed by FRET.
  • Fluorescent spark The presence or absence of protein-protein interaction can be determined from the difference in the wavelength of the torr.
  • a fluorescent dye a combination of fluorescein as a donor and rhodamine as an acceptor is often used.
  • GFP fluorescent green protein
  • a substrate for immobilizing the C-terminal-modified functional protein or the target molecule a substrate made of a material such as glass is used, and quartz glass is preferably used. It is also preferable that the surface is ultrasonically cleaned to prevent scattering of laser light.
  • a method for bringing a C-terminal modified functional protein or a modified target molecule into contact with an immobilized molecule by immobilizing the molecule to the immobilized molecule is sufficient to allow both molecules to interact with each other. Any method may be used as long as it can be contacted with each other, but it is preferable that the non-immobilized C-terminal modified functional protein or the modified target molecule is added at a concentration appropriate for a buffer commonly used in biochemistry. A preferred method is to prepare a dissolved solution and drop it on the surface of the solid phase. [0108] After the two molecules are brought into contact, the fluorescence excited by the evanescent field illumination is measured using a detector such as a CCD camera to identify the molecules that interact with the immobilized molecules. Can be.
  • a detector such as a CCD camera
  • a method of performing a large number of analyzes at the same time for example, a method of addressing a plurality of C-terminal modified functional proteins or modified target molecules on the substrate and immobilizing the same on the substrate is used.
  • the fluorescence polarization method uses a method in which a fluorescent molecule excited by fluorescence polarization emits light during the excited state. When the steady state is maintained, fluorescence is emitted in the same plane of polarization.However, when the excited molecule performs a rotating Brownian motion or the like during the excited state, the emitted fluorescence is different from the excitation light. This is a method that utilizes the fact that it becomes a plane.
  • the motion of a molecule is affected by its size, and when the fluorescent molecule is a macromolecule, the motion of the molecule during the excited state is almost negligible, whereas the emitted light remains polarized.
  • the polarization of the emitted light is eliminated because the moving speed is high. Therefore, the intensity of the fluorescence emitted from the fluorescent molecule excited by the plane-polarized light is measured on the original plane and a plane perpendicular to the original plane. The information about is obtained.
  • the behavior of a target molecule that interacts with a fluorescence-modified molecule without being affected by contaminants can be tracked. This is also a force that is measured as a change in the degree of polarization only when the target molecule interacts with the fluorescently modified molecule.
  • BECON manufactured by Panyera
  • this method can also be performed by using these apparatuses.
  • any method of contacting the two sets of C-terminal modified functional protein target molecules can be used as long as the two molecules can be brought into contact enough to interact with each other.
  • a solution prepared by dissolving the c-terminal modified functional protein at an appropriate concentration in a buffer or the like normally used in biochemistry is added to a measuring well of a commercially available fluorescence depolarizer.
  • the method is carried out by injecting a solution in which the target molecule is dissolved at an appropriate concentration in the same buffer.
  • the degree of the interaction In order to detect the optimal combination of the interaction between two targets measured in the present method, it is effective to numerically indicate the degree of the interaction.
  • an index indicating the degree of interaction for example, a value of a minimum target substance concentration that gives a maximum fluorescence polarization degree to a certain concentration of a C-terminal modified functional protein can be used.
  • the surface plasmon resonance method is a method in which surface plasmons are excited by molecules interacting at the metal-Z liquid interface, and are measured by changes in the intensity of reflected light (Cullen, DC, et al., Biosensors). , 3 (4), 211-225 (1987-88)).
  • the C-terminal modified protein must be immobilized by the method described above, but the target molecule does not need to be modified. .
  • a transparent substrate such as glass on which a metal thin film of gold, silver, platinum or the like is formed is used.
  • the transparent substrate is generally made of glass, etc., as long as it has a material power that is transparent to laser light, regardless of what is usually used for a surface plasmon resonance device. Its thickness is about 0.1-5 mm. The appropriate thickness of the metal thin film is about 100-2000A. A commercially available solid substrate for such a surface plasmon resonance device can also be used.
  • the C-terminal modified functional protein can be immobilized on the substrate by the above-described method.
  • the method of bringing the target molecule into contact with the C-terminal-modified functional protein may be any method as long as the two molecules come into contact with each other to an extent sufficient for interaction.
  • a method can be used in which the C-terminal-modified functional protein immobilized on a solid phase is brought into contact with a solution in which a target molecule is dissolved in a buffer commonly used in biochemistry at an appropriate concentration.
  • a solid-phase enzyme immunoassay (Enzyme Linked Immunosorbent Assay (ELISA): Crowther, JR, Methods in Molecular Biology, 42 (1995)) uses a solution containing an antibody to an antigen immobilized on a solid phase. The molecules are brought into contact with each other, and the interaction (antigen-antibody reaction) between the two molecules causes the antibody remaining on the immobilized antigen to bind to the modified molecule (such as IgG) that specifically binds to the fluorescent or modified molecule.
  • This is a method of measuring or analyzing a signal emitted from a dye as a substrate using a commercially available detector (ELISA reader).
  • a plastic microplate or the like usually used for ELISA can also be used as a substrate for immobilizing a C-terminal modified functional protein serving as an antigen.
  • the method for bringing the modified target molecule to be an antibody into contact with the solid phase molecule in the present method may be any method as long as the two molecules are brought into contact with each other to an extent sufficient for interaction.
  • a method is preferred in which a solution is prepared by dissolving a molecule in an appropriate concentration in a buffer commonly used in biochemistry, and the solution is injected into a microplate.
  • a step of washing the excessively present modified molecules that are not bound to the immobilized molecules with the same buffer or the like is performed to remove the modified molecules remaining on the solid phase.
  • a molecule that interacts with the immobilized antigen molecule can be identified.
  • the target molecule which has been measured by each of the above methods and has been found to have an interaction, is identified by an appropriate method known per se when the primary structure of the molecule is unknown.
  • the primary structure can be analyzed. Specifically, when the target molecule for which interaction has been recognized is a protein, the primary structure can be identified by analyzing the amino acid sequence using an amino acid analyzer or the like. Further, when the target molecule is a nucleic acid, the base sequence can be determined by a base sequence determination method using an auto DNA sequencer or the like.
  • the above-described method for immobilizing a C-terminal modified functional protein or target molecule on a solid phase via a modified portion is used.
  • the device can be constructed by combining known suitable means. Each means in the apparatus is known, and operations such as holding a substrate, adding a C-terminal modified protein solution, and washing may be performed by a method known per se. By combining these operations, a fully automatic or semi-automatic device for immobilizing a C-terminal modified protein can be constructed.
  • an apparatus can be constructed by combining known appropriate means for performing the above-described protein-target molecular interaction measurement.
  • the respective means in the present apparatus are known, and the respective operations such as holding of the substrate, addition of antigen molecules, washing, and signal detection in these means may be performed by methods known per se. By combining these operations, a fully-automatic or semi-automatic device for protein-protein interaction measurement can be constructed.
  • High-affinity single-chain antibody selected by the method of the present invention can be used as a chimeric IgG antibody by replacing the variable region of human IgG antibody or CDR (complementarity determining region). And humanized mouse IgG antibodies. These antibodies produce little or no human anti-mouse antibody when elicited when administered to humans. Furthermore, a highly human single-chain antibody selected by the method of the present invention, which is a human antibody cDNA library, can produce a fully human monoclonal antibody by replacing the variable region of the human HgG antibody. This is an antibody that does not cause anaphylactic symptoms It can be used as a medicine.
  • a human or other animal-derived DNA library is substituted with a variable region encoded by the nucleic acid and a human IgG variable region.
  • a human or humanized antibody constructed thereby.
  • a therapeutic agent comprising the antibody as an active ingredient.
  • Antibodies that bind to angiotensin II could be used as neutralizing antibodies in the treatment of hypertension. That is, since the physiological action of angiotensin II is a blood pressure increasing action, it is considered that the antibody binds to angiotensin II and suppresses the blood pressure increasing action.
  • Antibodies that bind to Lewis X could also be used to treat cancer.
  • Lewis X When cells become cancerous, Lewis X is expressed on the cell surface, and antibodies can be used to target it.
  • an anticancer drug that kills cancer cells or a toxin such as ricin is bound and administered systemically or locally, and the cancer cells are intensively attacked like antibody-powered S missiles, resulting in cancer-specific and effective missiles Treatment can be provided.
  • an immunoribosome preparation in which a drug is put in a lipid bilayer vesicle and an antibody is bound to the surface thereof is used as a kind of preparation used for missile treatment.
  • [0131] [2] cDNA-H solution 51 synthesized in [1] 51, each HB primer (lpmol / ⁇ 1) 2.5 1 shown in the following HB primer list, 10X PCR buffer ( ⁇ ) 2.5 ⁇ l 1, MulgGl / 2 forward primer (SEQ ID NO: 48) (lpmol / ⁇ 1) 1.25 ⁇ 1, MulgG3 forward primer (SEQ ID NO: 47) (lpmol / ⁇ 1) 1.25 ⁇ 1, KOD DASH polymerase (TOYOBO) 0.25 ⁇ 1 After mixing, RNase-Free water was added to make a total volume of 25 ⁇ l, and each was subjected to PCR reaction.
  • PCR was performed at 96 ° C for 5 minutes, followed by 25 cycles of 96 ° C, 30 seconds, 50 ° C, 30 seconds, 72 ° C, and 1 minute, followed by reaction at 72 ° C for 5 minutes.
  • Amplified gene respectively check the Pando of 500-900B P by 2% Agarosugeru electrophoresis movement, Gyotsu a phenol / black port Holm process It was. That is, add the same volume of phenol: cloth form: isoamyl alcohol (25: 24: 1), mix well, centrifuge at 4 ° C for 13,200 rpm for 5 minutes, transfer only the aqueous layer to a new tube, and re-equivalent volume.
  • Phenol cloth form: isoamyl alcohol (25: 24: 1) was mixed well and centrifuged at 13,200 rpm for 5 minutes at 4 ° C, and only the aqueous layer was transferred to a new tube. The resulting solution was subjected to ethanol precipitation. That is, add a 20 mg / ml glycogen solution (Nacala Tester Co., Ltd.) 0.1 volume of 1,3 M sodium acetate (pH 5.2) and 3 volumes of 100% ethanol, leave on ice for 15 minutes, and continue at 13,200 rpm for 20 minutes.
  • a 20 mg / ml glycogen solution Nacala Tester Co., Ltd.
  • 1,3 M sodium acetate pH 5.2
  • [3] [3] 1 ⁇ l of each DNA solution (19 types) synthesized in [2], ⁇ each corresponding ⁇ ⁇ primer (lOpmol / 1) 21 shown in the primer list, 10 X PCR buffer (TOYOBO) ) 10 ⁇ 1, (2 mM) dNTPs (TOYOBO) 101, VH forward primer HF1: HF2: HF3: HF4 (1: 1: 1: 1) mixture shown in the HF primer list below (lOpmol / ⁇ ⁇ ) 2 ⁇ KOD DASH polymerase (TOYOBO) 0.5 ⁇ l was mixed, RNase-Free water was added to make a total volume of 100 ⁇ l, and each was subjected to PCR reaction.
  • PCR was performed at 96 ° C for 5 minutes, followed by 20 cycles of 96 ° C, 30 seconds, 50 ° C, 30 seconds, 72 ° C, and 1 minute, followed by a reaction at 72 ° C for 5 minutes.
  • the amplified gene was confirmed to have a band of 500-900 bp by 2% agarose gel electrophoresis, and was subjected to phenol / cloth form treatment and ethanol precipitation. Then, after centrifuging and drying for about 15 minutes, each DNA (19 species) was dissolved in 10 ⁇ l of RNase-free water.
  • [6] 5 ⁇ l of the cDNA-L solution synthesized in [5] was added to each of the LB primers (lpmol / ⁇ 1) 2.5 1, 10X PCR buffer (TOYOBO) shown in the LB primer list below.
  • 2.5 ⁇ 1, MuCK forward primer (SEQ ID No. 49) (lpmol / ⁇ 1)
  • Add 2.5 ⁇ 1, KOD DASH polymerase (TOYOBO) 0.25 ⁇ 1 add RNase-Free water, add 25 ⁇ l, and reduce the total volume to 25 ⁇
  • the PCR reaction was performed as 1 for each.
  • PCR was performed at 96 ° C for 5 minutes, followed by 25 cycles of 96 ° C, 30 seconds, 48 ° C, 30 seconds, 72 ° C, and 1 minute, followed by reaction at 72 ° C for 5 minutes. went.
  • the amplified gene was confirmed to have a band of 500-900 bp by 2% agarose gel electrophoresis, and was subjected to phenol / clonal form treatment.
  • the obtained solution was subjected to ethanol precipitation. Then, after centrifugal drying for about 15 minutes, each DNA (18 species) was dissolved in 20 ⁇ l of RNase-free water.
  • PCR was performed at 96 ° C for 5 minutes, followed by 20 cycles of 96 ° C, 30 seconds, 48 ° C, 30 seconds, 72 ° C, and 1 minute, followed by a reaction at 72 ° C for 5 minutes.
  • the amplified gene was confirmed to have a band of 500-900 bp by 2% agarose gel electrophoresis, and was subjected to phenol / cloth form treatment and ethanol precipitation. Then, after centrifugal drying for about 15 minutes, each DNA (18 kinds) was dissolved in 10 ⁇ l of RNase-free water.
  • the mixture was mixed at a ratio to obtain a total 0.5 pmol L chain DNA solution.
  • PCR was further performed to ligate one H chain and one L chain.
  • 0.5 pmol of H chain DNA solution synthesized in [4] 0.5 pmol of L chain DNA solution synthesized in [8], 5'UTR (SEQ ID NO: 57) (lpmol / 1) 0.5 ⁇ 1, McD—Linker + (SEQ ID NO: 56) (lpmol / ⁇ 1) 0.5 ⁇ 1, McD 3'UTR (His Tag) (SEQ ID NO: 55) (lpmol / ⁇ 1) 0.5 1, 10 X PCR buffer (TOYOBO) 51, 2 mM
  • PCR was performed at 96 ° C for 5 minutes, followed by 96 ° C for 30 seconds, followed by ramping to 58 ° C for 5 minutes, and 58 ° C for 30 seconds, 72 ° C for 1 minute. After 10 cycles, the reaction was performed at 72 ° C for 5 minutes.
  • PCR was performed at 96 ° C for 5 minutes, followed by 15 cycles of 96 ° C, 30 seconds, 58 ° C, 30 seconds, 72 ° C, and 1 minute, followed by reaction at 72 ° C for 5 minutes. Was done.
  • RNase-free water 100 1 was prepared, heated at 70 ° C. for 15 minutes, and then treated with TE-saturated phenol. The obtained aqueous layer was treated with phenol / chloroform, followed by ethanol precipitation using the resulting solution. The obtained pellet is centrifuged and dried for about 15 minutes,
  • RNA obtained in [13] and [12] was purified using the RNeasy Mini kit (Qiagen). That is, RNase-Free water was added to the transcription reaction solution to make the total volume 100 ⁇ l, and 350 ⁇ l of RLT buffer (Qiagen), 51 of 2-mercaptoethanol 51, and 250 ⁇ l of 100% ethanol were added, and the RNeasy mini Spin the column, centrifuge at 4 ° C, 12,000 rpm for 16 seconds, remove the discharged solution, add RPE buffer (Qiagen ⁇ OO / zl to the column, centrifuge at 4 ° C, 12,000 rpm, 16 seconds) After that, remove the discharged solution, add RPE buffer (Qiagen ⁇ OO / z 1) to the column, centrifuge at 4 ° C, 12,000 rpm for 2 minutes, remove the discharged solution, and replace the column with a new one.
  • RPE buffer Qiagen ⁇ OO / z 1
  • TOYOBO polyethylene glycol
  • PEG polyethylene glycol
  • RNA to which one spacer molecule was bound was purified using the RNeasy Mini kit (Qiagen).
  • RNA bound to one spacer molecule obtained in [14], wheat germ extract (containing 5 mM DTT) (Promega) 12.5 ⁇ 1, Amino Acid mixture, Complete (ImM) (Promega) 2 1, RNase inhibitor (TOYOBO) 2 1, CH COOK (1M) (Promega) 2 1
  • angiotensin II-biotin (0.4 M) angiotensin II-biotin or (0.4 M) Lewis X-sp-biotin dissolved in an ELISA buffer having a composition of 100 mM Tris-HC1, 150 mM NaCl, pH 7.5, 0.1% Tween 20 The mixture was rotated and stirred with a rotary mixer (Nissin) at 500 ° C for 1 hour at 25 ° C.
  • FIG. 3 shows the chemical structural formula of angiotensin II-biotin
  • FIG. 4 shows the chemical structural formula of Lewis X-sp-biotin. 5 mM biotin 71 dissolved in the ELISA buffer was added and further stirred at 25 ° C. for 30 minutes with a rotary mixer (Nissin).
  • Blocking agent Add 400 / zl of the reverse-transcribed library obtained in [18] to the suspended resin in 600 ⁇ l of the suspended resin, and use a mini disc rotor (Biocraft) at 4 ° C for 15 minutes. The rotation was stirred.
  • PCR was performed at 96 ° C for 5 minutes, followed by 25-30 cycles of 96 ° C, 30 seconds, 58 ° C, 30 seconds, 72 ° C, 1 minute, and then 72 ° C, 5 minutes. The reaction was performed. The amplified genes were confirmed to have 900-1000 bp bands by 1% agarose gel electrophoresis.
  • [0163] The amplified gene obtained in [22] was purified using Wizard Plus Minipreps DNA Purification System (Promega). That is, transfer the PCR reaction product to a 1.5 ml tube, mix 100 ⁇ l of Direct purification buffer (3 ⁇ 4r ⁇ Promega) and 1 ml of DNA purification resin (Promega), mix with 1 ml of syrup, and use a 2.5 mL syringe (Terumo) for Wizard Minicolumn (Promega). Was served. The solution was extruded with a syringe and discarded (80%), and 2 ml of isopropanol was extruded again and discarded.
  • Wizard Plus Minipreps DNA Purification System Promega
  • the concentration of the DNA solution was estimated by measuring the absorption at 260 nm.
  • PCR was performed at 96 ° C for 5 minutes, followed by 96 ° C, 30 seconds, 58 ° C, 30 seconds, 72 ° C, 1 minute for 7-10 cycles, and then at 72 ° C, 5 minutes. The reaction was performed for minutes.
  • the amplified gene was confirmed to have a band of about 1000 bp by 1% agarose gel electrophoresis, and was subjected to phenol / cloth-form treatment and ethanol precipitation. After centrifugation and drying for about 15 minutes, the DNA was dissolved in RNase-free water (101).
  • RNase-free water 100 1 was prepared, heated at 70 ° C. for 15 minutes, and then treated with TE-saturated phenol. The obtained aqueous layer was subjected to phenol / chloroform-form treatment, and then the obtained solution was subjected to ethanol precipitation. The obtained pellet was centrifuged and dried for about 15 minutes, and the DNA was dissolved in 10 ⁇ l of RNase-free water.
  • FIG. 5 shows the results obtained by subjecting each translation solution obtained in the above steps to 5% polyacrylamide electrophoresis in the presence of 8-urea to identify the associating molecules.
  • the lower bar graph shows the results of measuring and quantifying the fluorescence of FITC of the upper electrophoresis gel using MOLECULAR IMAGER FX (Bio-rad).
  • Nega is one of the clones of library MH0 obtained in [11], MH0-15
  • Posi is one of the clones of library MI3, MI3-55
  • MHO is the live one obtained in [11].
  • MHO contains a considerable proportion of proteins that are not translated into proteins due to stop codons or mutations.
  • the efficiency of force association is as low as 15%, whereas that of Mil is 25% and that of MI2 is 25%.
  • the efficiency of association has increased to 37% and 41% for MI3.
  • Nega and Posi are in-frame clones, and the association efficiency in this case is 34% and 40%, respectively.
  • MI2 or MI3 has almost the same value as the association efficiency. Therefore, the selection at the protein level was successful, indicating that the library had a higher in-frame rate! /
  • FIG. 6 shows the results obtained by subjecting each translation solution obtained in the above steps to 5% polyacrylamide electrophoresis in the presence of 8M urea in the same manner as in FIG. 5, and confirming the corresponding molecules.
  • MHO is the library obtained in [11]
  • MK1 is the library obtained in [11] at [18] at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes
  • Lewis X is used as an antigen.
  • MK2 was prepared by reacting MK1 in [18] at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes, selecting the library using Lewis X as an antigen and recovering the library collected in [25]. Is shown.
  • a significant percentage of non-translated proteins due to stop codons or mutations contained a low efficiency of 15%, whereas 41% for MK1 and 41% for MK2. Efficiency of association increased with 36%!
  • FIG. 7 shows the results obtained by subjecting each translation solution obtained in the above steps to 5% polyacrylamide electrophoresis in the presence of 8 M urea and confirming the assigning molecules as in FIG. From the left, MM1 is the library obtained in [11], reacted at 50 ° C for 30 minutes in [18], selected angiotensin II as an antigen, and recovered in [25]. MM2 was MM1 in [18]. The library obtained in [25], MP1 was reacted with the library obtained in [11] at 99 ° C for 5 minutes at [15].
  • [18] Shows the library recovered in [25] after performing the reaction from [22] after reacting at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes.
  • the efficiency of association is 25% for MM1 and 33% for MM2.
  • MM1 has the same value as [18] compared to Mil that was reacted at 50 ° C for 30 minutes and then reacted at 99 ° C for 5 minutes at [18], and MM2 also shows an association efficiency that is inferior to MI2 This suggests that the selection at the protein level has been successful.
  • MP1 has an extremely low assignment efficiency of 8%, and it is possible that some denaturation of RNA or the assignment molecule may have occurred by reacting at 99 ° C for 5 minutes in [15].
  • FIG. 8 shows the results of PCR of the eluate obtained in [21] and PCR of the eluate obtained in [22] and 1% agarose gel electrophoresis.
  • the lower bar graph shows ethidium bromide in the upper electrophoresis gel.
  • the figure shows the results obtained by measuring and quantifying the absorption of MOLECULAR IMAGER FX (Bio-rad). From left, Nega is a library obtained in [11], MH0-15, one of the clones of MHO, Posi is a library, MI3-55, one of the clones in library MI3, and Mil is a library obtained in [11].
  • FIG. 9 shows that the flow obtained in [20] is diluted 1000-fold, the wash obtained in [20] and the eluate (Elute) obtained in [21] are diluted 1- and 10-fold. This is the result of PCR of [22] followed by 1% agarose gel electrophoresis.
  • 1 1x dilution of eluate
  • X 0.1 Abbreviation of 10-fold dilution of eluate.
  • MK1 is a library obtained by reacting the library obtained in [11] at 50 ° C for 30 minutes and then 99 ° C for 5 minutes in [18], selecting Lewis X as an antigen and collecting in [25], and MK2 is MK1. Is reacted at 50 ° C.
  • FIG. 10 shows that the flow obtained in [20] was diluted 1000-fold, and the Wash and [20] obtained in [20] were diluted.
  • the eluate obtained in [21] was diluted 1-fold and 10-fold and subjected to PCR in [22] and subjected to 1% agarose gel electrophoresis, and the absorption of ethidium bromide in the electrophoresis gel was measured.
  • [0180] [29] Colony suspension obtained in [28] 11; 10X PCR buffer (TOYOBO) 51; 2 mM dNTPs (TOYOBO) 51; M13FII (Torigami [J number 58] (10 pmol / 1) 1 1, M13RII (Toroki Column No. 59) (10 pmol / 1) 11 1, KOD DASH polymerase (TOYOBO) 0.25 ⁇ l, mix, add RNase-Free water and reduce the total volume PCR reaction was carried out with 50 ⁇ l each.
  • [0182] [30] 16 ng of DNA obtained from [29], M13FII (SEQ ID NO: 58) (1.6 pmol / ⁇ 1) 21 or M13RII (SEQ ID NO: 59) (1.6 pmol / ⁇ ⁇ ) 2 ⁇ 6 ⁇ l of DTCS kit Premix (Beckman coulter) was mixed, and RNase-Free water was added to make a total volume of 10;
  • PCR was performed at 96 ° C for 5 minutes, followed by 30 cycles of 96 ° C, 20 seconds, 58 ° C, 20 seconds, 60 ° C, and 4 minutes, followed by reaction at 72 ° C for 5 minutes. .
  • [0183] Transfer the PCR reaction product obtained in [30] to a 1.5 ml tube, and add 1 ⁇ l of 3 M NaOAc, 0.1 ⁇ EDTA 11 and 20 ⁇ g / ml glycogen solution (Nacalai Tesque, Inc.) 1 ⁇ l 1 was mixed well, and 60 ⁇ l of cold 100% ethanol was added and mixed. After centrifugation at 4 ° C and 14000 rpm for 15 minutes to remove the supernatant, washing the pellet with 70% ethanol, centrifugation again at 14000 rpm and 2 minutes to remove the supernatant was performed twice. Then, after centrifugal drying for 30 to 40 minutes, deionized formamide (Beckman coulter) 401 was added and mixed well. Sequence analysis was performed using the CEQ 2000 DNA Analysis System (Beckman coultei).
  • MHO reacted the library obtained in [11] with the library obtained in [11]
  • MHO reacted the library obtained in [11] with [18] at 50 ° C for 30 minutes, then at 99 ° C for 5 minutes, and reacted with angio.
  • the library, MI2 which selected tensin II as an antigen and recovered in [25]
  • 100 X expressed in C (%) (the number of arrays having converged arrays in A in Figs. 11 and 12) / In frame was 33% for Mil, 57% for MI2, and 87% for MI3 according to the selection.
  • 100 X in C (%) (the number of arrays with converged arrays in B in Figs. 11 and 12) / In frame shows a high value by turning the selection to 50% for MK1 and 40% for MK2.
  • the selection using Lewis X as the antigen showed that the sequence converged to the sequence shown in B.
  • 100 X (the number of arrays with converged arrays in A in Figs. 11 and 12) / In frame in C (%) is 0% for MM1 and 13% for MM2, and selection is performed once. Only by turning, the sequence shown in A was not found, and only by turning the selection twice, was it possible to find the sequence shown in A for one clone.
  • 50 ° C in [18] for MM1 and MM2 Mil, MI2, and MI3 perform the reaction at 50 ° C for 30 minutes at [18], followed by the reaction at 99 ° C for 5 minutes, while only reacting for 30 minutes.
  • Fig. 11 and Fig. 12 show the strains generated by TreeViewPPC after sequence alignment by clustalx based on the amino acid sequence, as a result of the sequence analysis and analysis performed in [31].
  • a surrounded by a line indicates the one converged by the selection of Angiotensin II
  • B surrounded by a line indicates the one converged by the selection of Lewis X.
  • the number of the clone is shown after the library abbreviation.
  • a surrounded by a line shows more than 90% homology as a result of analysis by GENETYX-MAC at the protein level compared to MI3-55, and B surrounded by a line is analyzed by GENETYX-MAC at the protein level compared to MK1-17 As a result, 90% or more homology was shown.
  • the amino acid sequence is shown as an odd SEQ ID NO: 61-117.
  • the sequence was determined as follows according to CDR sequence number i3 ⁇ 4, Martin, A. R. Accessing the Kabat Antibody Sequence Dataoase by Computer PROTEINS: Structure, Function and Genetics, 25 (1996), 130-133.
  • Each CDR of MI3-8, MK1-15, MK1-17, MK1-24, MK2-19, and MK2-8 has the following amino acid numbers.
  • PCR was performed at 94 ° C for 5 minutes, followed by 25-30 cycles of 94 ° C, 30 seconds, 58 ° C, 30 seconds, 68 ° C, 2 minutes, and then reaction at 68 ° C for 5 minutes. went.
  • the amplified genes were confirmed to have 900-1000 bp bands by 1% agarose gel electrophoresis.
  • FIG. 13 shows the results of Western blotting of [37] of ⁇ 3-55 (right two). The left three are Western blot controls. Carboxy-terminal from lane left
  • FLAG-BAP fosion protein 100 ng ⁇ 50 ng ⁇ 20 ng ⁇ Biotynylated SDS—PAGE Standards low range (Bio-rad) 21 1, MI3-55 [37] from 3rd to 6th The collected 15 1 and 51 were subjected to 15% polyacrylamide electrophoresis and transferred to a PBDF membrane.
  • Anti FLAG M2-Mouse (Sigma) was used as the primary antibody, Goat anti-mouse IgG (H + L) -HRP conjugate (Sigma) and Avidin- HRP conjugate (Bio- rad) was used as a two-following ⁇ I this ECL Western blotting detection detected in Reagents (Amersham biosciences) 7 this 1 / es Tan blot.
  • MI3-55 a band was detected at a molecular weight of about 31,000 daltons, which was consistent with the calculated value of the single-chain antibody.
  • Biacore used the Biacore 3000 system. Immobilization was performed on the sensor chip Bl (CM4) by the amine coupling method. Flow cells 1 and 2 contain 0.2 M N-ethyl- ⁇ '-dimethylaminopropylcarbodiimide (EDC) and 50 mM N-hydroxysuccinimide (NHS) Activation was performed with the solution for 10 minutes. Next, 50 M streptavidin (Sigma) (0.02 M potassium phosphate buffer, pH 6.5) was mixed with 201 and 10 mM acetate buffer, pH 5.0 980 ⁇ l in flow cell 2 for 10 min. mM acetate buffer pH 5.0 was allowed to flow for 10 minutes.
  • EDC N-ethyl- ⁇ '-dimethylaminopropylcarbodiimide
  • NHS N-hydroxysuccinimide
  • flow cells 1 and 2 were blocked with 1 M ethanolamine pH 8.5 for 10 minutes.
  • Flow cell 2 has a 2024 or 2305 response unit attached to the sensor chip surface.
  • 50 mM NaCl and 1 M NaCl 10 were flown into the flow cells 1 and 2 five times, and then 10 1 of angiotensin ⁇ -pyotin (0.1 ⁇ ) was flown into the flow cell 2 in which the 2024 response unit was bonded to the sensor chip surface.
  • 3 ⁇ l of Lewis-X-sp-Piotin (0.1 ⁇ ) was flowed into flow cell 2 in which the 2305 response unit was connected to the sensor chip surface.
  • Biacore was analyzed using a PP6T buffer having a composition of 50 mM potassium phosphate, 150 mM NaCl, pH 6.0, 0.1% Tween 20 at a flow rate of 201 / min.
  • the samples obtained in [35] or [37] were injected with KINJECT. Regeneration was performed with Glycine 1.5 at 201, 50 mM NaCl, 1 M NaCl 101.
  • the response curve was calculated by combining the flow cell 2 and the flow cell 1 with the bow I to calculate the combined response unit.
  • FIG. 14 shows a selection using angiotensin II as an antigen.
  • MI3-55, MI3-42, MI3-28, MI3-41 This is a bar graph showing KD calculated by translating MI3-34, MI3-5, MI3-15, and MI3-26 using [34] and then analyzing with Biacore. It showed very high affinity values of 0.4-1.5 nM, demonstrating that the single chain antibodies selected by the method of the present invention have very high affinity.
  • FIG. 15 shows that in the selection using Lewis X as an antigen, MK1-1, MK1-24, MK2-19, and MK1-17 among the sequences converging to the sequences shown in FIG. 11 and FIG.
  • This is a bar graph showing the KD calculated by translating and analyzing with Biacore. It showed a value of 20-43 nM, indicating that the single-chain antibody selected by the method of the present invention was a high affinity antibody.
  • Fig. 16 shows that after translating MI3-55 at [34], processing it at 4 ° C, 60 ° C, or 99 ° C for 5 minutes, and then performing the processing of [35], the Biacore of [39] The results of the analysis are shown in a bar graph.
  • Blocking one (Nakarai Tester Co., Ltd.): The blocking agent diluted with Milli Q (1: 4) was caloried, and blocking was performed overnight at 4 ° C. or 1 to several hours at 25 ° C. Next, the plate was washed four times with PBS (Nacalai Tester Co., Ltd.) 200 1 and the sample obtained in [35] or [37] was added to 100 1 plate. At this time, in order to examine antagonistic inhibition, a mixture of angiotensin II or Free Lewis X and a single-chain antibody sample in advance was added.
  • PBS Nacalai Tester Co., Ltd.
  • Multiskan JX (Dainippon Pharmaceutical Co., Ltd.) 450 nm and 630 nm were measured with a 96-well microplate reader, and the value of 630 nm was subtracted from the value of 450 nm.
  • Figure 17 shows that MI3-55 was translated at [34] and then treated at 4 ° C or 60 ° C or 99 ° C for 5 minutes.
  • the undiluted solution treated at 99 ° C for 5 minutes had a 0.5 dilution to 99% of 21% of the value treated at 4 ° C for 5 minutes.
  • C, treated for 5 minutes had a 44% value compared to that treated at 4 ° C, 5 minutes, but the same value was obtained by diluting the treated at 4 ° C, 5 minutes to 0.125, respectively.
  • MI3-55 treated at 99 ° C. for 5 minutes and treated in [35] has a binding activity of about 12.5% or more, and the single-chain antibody selected by the method of the present invention is extremely low. It was shown to be heat resistant.
  • the present invention is a technique for applying protein functional maturation to an evolutionary molecular engineering technique (IW method) to rapidly and inexpensively select a protein in a test tube.
  • a desired protein can be obtained by constructing a protein cDNA library, converting to an assigning molecule (IW) library, selecting, collecting genes, and repeating this cycle several times.
  • IW assigning molecule
  • a library obtained by adding a mutation (point mutation, DNA shuffling) to a known protein gene it is possible to select a protein with higher functionality, higher stability, and higher expression. is there.
  • Preliminary heat treatment before binding the library to the antigen denatures the unstable protein group to ensure that the three-dimensional structure is maintained stably or that the protein is quickly unwound. Only high protein groups can be selected.
  • the obtained protein can be expressed in a cell-free translation system in the presence of a reducing agent such as DTT, or expressed in large quantities in the cytoplasm of Escherichia coli in a reductive environment, and is stable to reducing agents. is there.

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Abstract

A method of quickly and efficiently selecting a high-functioning protein or a nucleic acid encoding the same by selecting a protein interacting with a target molecule or a nucleic acid encoding the same through the following steps (a) to (d). (a) The step of constructing a protein-encoding DNA library. (b) The step of transcribing DNAs in the library constructed in the above (a), attaching a spacer carrying puromycin to the 3’-end of the transcribed RNAs, and then constructing a library of molecules assigned to genotype and phenotype in a cell-free translation system. (c) The step of heating the library of the assigned molecules. (d) The step of bonding each assigned molecule to the target molecule, thoroughly washing, eluting and then amplifying the nucleic acid moiety by reverse transcription -PCR or PCR.

Description

明 細 書  Specification
高機能性タンパク質の迅速、高効率な選択法、それによつて得られる高 機能性タンパク質、およびその製造方法と利用方法  Method for selecting high-functional protein quickly and efficiently, high-functional protein obtained thereby, and method for producing and using the same
技術分野  Technical field
[0001] 本発明は、高機能性タンパク質の選択法、それによつて得られる高機能性タンパク 質、およびその製造方法と利用方法に関する。  The present invention relates to a method for selecting a highly functional protein, a highly functional protein obtained thereby, and a method for producing and using the same.
背景技術  Background art
[0002] 標的分子と相互作用するタンパク質の相互作用は、タンパク質の機能に深く関与し ており、より優れた機能を有するタンパク質を得る試みがなされている。例えば、タン パク質の一種の抗体分子のもつ、抗原に対する高い特異性と親和性は、幅広い分 野での利用価値が高ぐ抗体を自在にまた効果的に創製できる系の構築研究が盛 んに行われている。本来、抗体は B細胞内でドメインシャッフリングと体細胞変異を繰 り返し (親和性成熟: affinity maturation)、ある抗原に対してより特異性と親和性が高 められたものだけが免疫系で作用することができる。  [0002] The interaction of a protein that interacts with a target molecule is deeply involved in the function of the protein, and attempts have been made to obtain a protein having a better function. For example, the high specificity and affinity for antigens of a kind of protein antibody molecule has been actively studied for the construction of a system that can freely and effectively create antibodies that have high utility in a wide range of fields. Has been done. Originally, antibodies repeat domain shuffling and somatic mutation in B cells (affinity maturation), and only those with higher specificity and affinity for a certain antigen act on the immune system can do.
[0003] 抗体の親和性の研究においては、その均一性の観点からモノクローナル抗体が一 般に用いられている力 現在、モノクローナル抗体の調製法は細胞融合法 (ハイプリ ドーマ技術) (非特許文献 1)が主流である。しかし、この細胞融合法は非常に手間が かかる上に、高価な試薬を必要とし、また時間が力かる(約 1年間)。さらに、動物の免 疫操作が必要であるため、自己抗原や毒物に対する抗体を選択することが難しいと いった種々の問題を有している。最近、これらの問題を解決する方法としてファージ ディスプレイ (非特許文献 2)やリボソームディスプレイ (非特許文献 3) t ヽつた進化分 子工学的手法 (核酸 [遺伝情報]とタンパク質 [機能情報]を直接対応付ける技術)がモ ノクローナル抗体の選択に利用されている。すなわち、大きな多様性を有する抗体ラ イブラリーを構築し、目的とする抗体 (対応付け分子)の選択、濃縮を繰り返すこと〖こ よって、比較的容易に目的抗体を得ることができる。特に、非常に柔軟性の高いぺプ チドリンカーで片方の鎖の C末端ともう片方の N末端を結合させた一本鎖抗体 (非特 許文献 4)を対象とする方法が知られて!/ヽる。 [0004] しかしながら、これらの技術においても、まだいくつかの欠点がある。ファージデイス プレイは、現実的な DNAライブラリーの上限は 109 (抗体の場合)程度で、ライブラリー サイズが小さいこと、生物学的なシステム (大腸菌への感染、増幅など)を利用するた めに、予期し得ないバイアス (変異等)がかかってしまう。さらに、大腸菌に対して毒性 を持つ抗原は使用できな 、。リボソームディスプレイは無細胞翻訳系を用いた完全な in vitroの実験系であるためこれらのバイアスは力からず、また、ライブラリーの上限は 101Q-10Uが可能とされている。しかし、 mRNA-タンパク質-リボソーム三者複合体の形 成効率が 0.2%と非常に低く (非特許文献 3)、 in vitro viruS(IVV)法 (非特許文献 5))の数 百分の 1である。また、その複合体が非常に不安定であるため、バイオバニング操作 中により強い選択圧をかけられないという問題がある。ここで、バニングにおける選択 圧は抗体濃縮における最も重要な因子であり、強 、選択圧かけられな 、と 、うことは より多くの選択サイクルを繰り返す必要性が生じる。また、一種類の抗原に対して、安 定性、親和性の異なる多種多様な抗体が得られるため、より高機能なものを得るため には、多くの検体のクロー-ング、発現、分析を必要とする。このことは、たいへんな 重労働であるだけでなく時間もコストも力かる。 [0003] In the study of antibody affinity, the power of monoclonal antibodies being generally used from the viewpoint of homogeneity At present, monoclonal antibodies are prepared by cell fusion (Hyperidoma technology) (Non-patent Document 1). ) Is the mainstream. However, this cell fusion method is very complicated, requires expensive reagents, and is time consuming (about one year). In addition, there are various problems that it is difficult to select antibodies against self-antigens and toxic substances due to the necessity of immunizing animals. Recently, phage display (Non-patent document 2) and ribosome display (Non-patent document 3) have been developed to solve these problems. Associated technology) is used for selection of monoclonal antibodies. That is, by constructing an antibody library having great diversity and repeating the selection and enrichment of the target antibody (assigning molecule), the target antibody can be obtained relatively easily. In particular, a method is known for single-chain antibodies (Non-Patent Document 4) in which the C-terminal of one chain is linked to the N-terminal of the other with a very flexible peptide linker! / ヽYou. [0004] However, these techniques still have some disadvantages. The phage display uses a realistic DNA library with an upper limit of about 10 9 (for antibodies), small library size, and the use of biological systems (e.g., E. coli infection, amplification, etc.). Will have an unexpected bias (mutation, etc.). Furthermore, antigens that are toxic to E. coli cannot be used. Since ribosome display is a complete in vitro experimental system using a cell-free translation system, these biases are ineffective, and the upper limit of the library can be 10 1 Q -10 U. However, the formation efficiency of the mRNA-protein-ribosome ternary complex is extremely low at 0.2% (Non-patent document 3), and is several hundredths of that of the in vitro viru S (IVV) method (Non-patent document 5). It is. In addition, since the complex is very unstable, there is a problem that a stronger selection pressure cannot be applied during the biobanning operation. Here, the selection pressure in the banner is the most important factor in the antibody concentration, and if the selection pressure is not strongly applied, it becomes necessary to repeat more selection cycles. In addition, since a wide variety of antibodies with different stability and affinity can be obtained for one type of antigen, it is necessary to clone, express, and analyze many samples in order to obtain more functional ones. And This is not only a lot of hard work, but also time and money.
[0005] これまで、タンパク質を試験管内で合成する無細胞翻訳系として、小麦胚芽の系( 非特許文献 6)及び大腸菌の系 (非特許文献 7)において、タンパク質の大量発現が研 究されてきている。それに伴い、タンパク質の大量発現が可能な安定した翻訳テンプ レートの開発として、 mRNAの安定性向上と翻訳効率向上のために、一般的には 3'UTRが使われるが (非特許文献 8)、 mRNAの化学構造の置換や修飾などの方法 (非 特許文献 9)を用いる。  [0005] As cell-free translation systems for synthesizing proteins in vitro, large-scale expression of proteins has been studied in a wheat germ system (Non-Patent Document 6) and an E. coli system (Non-Patent Document 7). ing. Along with this, 3'UTR is generally used to improve the stability and translation efficiency of mRNA as a stable translation template capable of expressing a large amount of protein (Non-Patent Document 8). A method such as substitution or modification of the chemical structure of mRNA (Non-Patent Document 9) is used.
[0006] 我々は、 1997年に、進化分子工学のスクリーニングツールである〃 in vitro virus [0006] We established a screening tool for evolutionary molecular engineering in 1997, 〃 in vitro virus
(IW)〃の構築に世界に先駆けて成功した (非特許文献 10)。 IVV法では、 3'末端にピ ユーロマイシンを結合した mRNAを铸型として無細胞翻訳反応を行うことにより、タン パク質と mRNAとがピューロマイシンを介して共有結合した RNA-タンパク質連結分子 (IW)のライブラリーを構築することができる。この対応づけ分子のライブラリーの中 力 標的分子に結合するタンパク質を in vitroでスクリーニングした後、逆転写 PCRに より遺伝子を増幅し解読することができる。その後我々は、 IW法をプロテオーム解析 に応用するための基礎研究を開始し、ヒトゃマウス由来の cDNAライブラリーから IW ライブラリーを構築し、その中から、タンパク質や核酸、薬剤などの標的分子と相互作 用する未知のタンパク質を in vitroで迅速かつ高感度にスクリーニングする技術を確 立してきた。最近、我々は小麦胚芽抽出液の無細胞翻訳系で、 IVVと C末端ラベル 化タンパク質を高効率に発現できる铸型 DNAの開発に成功した (非特許文献 5)。本 発明者等は、 mRNA (遺伝子型)とタンパク質 (表現型)を無細胞翻訳系にお 、てピュ 一口マイシンを介して連結させ、対応付け分子である IWを効率よく構築する方法を 先に提案している (特許文献 1;特許文献 2;特許文献 3;特許文献 4)。 (IW) 〃 was successfully pioneered in the world (Non-Patent Document 10). In the IVV method, an RNA-protein linked molecule (IW) in which protein and mRNA are covalently linked via puromycin by performing a cell-free translation reaction using type III mRNA containing a piuromycin bound to the 3 'end. ) Library can be constructed. After screening in vitro proteins that bind to the target molecules of this library of mapping molecules, the genes can be amplified and decoded by reverse transcription PCR. After that, we performed a proteome analysis of the IW method. Started basic research to apply it to humans and mice, constructed an IW library from a cDNA library derived from human and mouse, and extracted unknown proteins that interact with target molecules such as proteins, nucleic acids, and drugs from them. A technique for rapid and sensitive screening in vitro has been established. Recently, we have successfully developed a type II DNA that can express IVV and C-terminally labeled proteins with high efficiency using a cell-free translation system of wheat germ extract (Non-Patent Document 5). The present inventors have previously described a method for efficiently constructing IW, an assigning molecule, by linking mRNA (genotype) and protein (phenotype) to a cell-free translation system via pure bitemycin. Patent Document 1; Patent Document 2; Patent Document 3; Patent Document 4).
[0007] さらに我々は、 IW法の構築過程で、低濃度のピューロマイシン誘導体を無細胞翻 訳系に投入すると、合成されたタンパク質の C末端に結合することを見出した。この原 理を応用して、例えば、蛍光色素をピューロマイシンに連結させたィ匕合物を用いれば 、タンパク質の C末端を蛍光色素で標識することが可能となった (非特許文献 11;非 特許文献 12)。本発明者等は、タンパク質の C末端を無細胞翻訳系において効率よ くラベル化する方法先に提案している (特許文献 5;特許文献 6;特許文献 7)。  [0007] Furthermore, during the construction process of the IW method, we found that when a low-concentration puromycin derivative was introduced into a cell-free translation system, it bound to the C-terminus of the synthesized protein. Applying this principle, for example, by using a conjugate having a fluorescent dye linked to puromycin, it became possible to label the C-terminal of the protein with the fluorescent dye (Non-Patent Document 11; Patent Document 12). The present inventors have previously proposed a method for efficiently labeling the C-terminus of a protein in a cell-free translation system (Patent Document 5; Patent Document 6; Patent Document 7).
[0008] タンパク質の詳細な機能や立体構造の解析、抗体の作製のためには大量のタンパ ク質を必要とする。そのため内在性の目的タンパク質を精製し、必要量得ることは困 難であるため、 目的タンパク質の cDNAを適した宿主に入れ発現させた組み換えタン ノ ク質を用いることになる。一本鎖抗体を発現させる宿主としては、大腸菌、酵母、昆 虫細胞や動物由来の培養細胞などさまざまなものが考えられる。その中でも大腸菌 は、短期間で安価に大量のタンパク質が得られるので便利である。また、組み換えバ キュロウィルスを昆虫細胞に感染させる系を使うと、大腸菌の系では難し力つたタン パク質の発現が可能になる。  [0008] A large amount of protein is required for detailed function and three-dimensional structure analysis of proteins and production of antibodies. For this reason, it is difficult to purify the endogenous target protein and obtain a required amount thereof. Therefore, a recombinant protein in which cDNA of the target protein is expressed in a suitable host and used is used. Various hosts for expressing a single-chain antibody can be considered, such as Escherichia coli, yeast, insect cells, and animal-derived cultured cells. Among them, E. coli is convenient because a large amount of protein can be obtained in a short period of time at low cost. In addition, the use of a system that infects insect cells with a recombinant baculovirus allows expression of proteins that is difficult with the E. coli system.
[0009] また、ポストゲノム機能解析研究のために開発されてきた!/、ろ!/、ろな解析ツールに も抗体が重要な役割を占めている。その一つとしてタンパク質の検出、定量が挙げら れる。そのためには、特異性の高い、高親和性の抗体の利用が不可欠である。抗体 を用いたタンパク質の検出方法として、 in vitroの方法では、ウェスタンブロット法、免 疫染色法、蛍光抗体染色法、抗体チップ法、また in vivoの方法では免疫沈降法など がある。このような手法を用いてタンパク質を検出するためには、抗体に予め蛍光を 発するタンパク質、例えば GFPを融合させておくか、抗体を酵素タンパク質、例えば 西洋ヮサビのペルォキシダーゼやアルカリ性ホスファターゼを融合させて、その酵素 活性を指標にする。 [0009] It has also been developed for post-genome functional analysis research! /, Ro! Antibodies play an important role in various analytical tools. One of them is the detection and quantification of proteins. For that purpose, the use of highly specific and high affinity antibodies is essential. Methods for detecting proteins using antibodies include in vitro methods such as Western blotting, immunostaining, fluorescent antibody staining, and antibody chip methods, and in vivo methods such as immunoprecipitation. In order to detect proteins using such a technique, the antibody must be pre- An emitted protein, for example, GFP is fused, or an antibody is fused to an enzyme protein, for example, horseradish peroxidase or alkaline phosphatase, and the enzyme activity is used as an index.
[0010] 抗体を用いたタンパク質間相互作用の検出法には、表面プラズモン共鳴法、蛍光 共鳴エネルギー移動法、蛍光偏光解消法、エバネッセント場イメージング法、蛍光相 関分光法、蛍光イメージング法、固相酵素免疫検定法などがある。とりわけ、蛍光相 関分光法(Fluorescence Correlation Spectroscopy : FCS)は、測定に必要な試料量 が少なく(およそフェムトリットル)、測定時間が短く(およそ 10秒)、 HTSのための自動 化が容易である(実際に EVOTEC社では 1日で 10万検体以上のスクリーニングを行う ウルトラ HTSを目指した装置の開発を行なっている)等の長所があり、検出系として優 れている (非特許文献 13)。さらに 2種類の蛍光色素を用いる蛍光相互相関分光法( Fluorescence し ross—し orrelation Spectroscopy : Fしじ¾ノで【¾、 1種類の 光色 を用 いる FCSでは困難であった同程度の大きさをもつ分子間の相互作用も検出が可能で あり、タンパク質相互作用の HTSへの応用が期待されている。一般に、タンパク質相 互作用の検出系では、固相化のためのタグや蛍光色素等のプローブでタンパク質を 修飾する必要がある。本発明者等は、ピューロマイシン等の核酸誘導体を用いて無 細胞翻訳系中でタンパク質の C末端を修飾する方法を先に提案して ヽる(特許文献 5 、特許文献 6、特許文献 3)。この方法は、従来の化学修飾法や蛍光タンパク質融合 法に比べて、タンパク質の機能を損ないにくい等の利点がある。  [0010] Methods for detecting protein-protein interaction using antibodies include surface plasmon resonance, fluorescence resonance energy transfer, fluorescence depolarization, evanescent field imaging, fluorescence correlation spectroscopy, fluorescence imaging, and solid-phase imaging. There are enzyme immunoassays and the like. In particular, Fluorescence Correlation Spectroscopy (FCS) requires a small amount of sample (approximately femtoliter), requires a short measurement time (approximately 10 seconds), and is easy to automate for HTS. (Actually, EVOTEC is developing a device aiming for Ultra HTS, which screens more than 100,000 samples per day), and is superior as a detection system (Non-Patent Document 13). In addition, fluorescence cross-correlation spectroscopy using two types of fluorescent dyes (fluorescence and ross-relation spectroscopy): [で, comparable size that was difficult with FCS using one type of light color It is also possible to detect the interaction between molecules having a protein, and it is expected that the protein interaction will be applied to HTS.In general, in a protein interaction detection system, a tag for immobilization, a fluorescent dye, etc. The present inventors have previously proposed a method of modifying the C-terminus of a protein in a cell-free translation system using a nucleic acid derivative such as puromycin (patent). Literature 5, Patent Literature 6, Patent Literature 3) This method has the advantage that the function of the protein is less likely to be impaired than the conventional chemical modification method and fluorescent protein fusion method.
[0011] IW法は、 mRNAを無細胞翻訳系等において発現させる際、リボソーム上で、 mRNA とタンパク質がピューロマイシンを介して化学的に結合した mRNA-タンパク質連結分 子を構築する方法である (非特許文献 10;非特許文献 12;非特許文献 5)。さらに、そ の mRNA-タンパク質連結分子を試験管内淘汰法により陶太し、選択された対応付け 分子の mRNA部分を逆転写 PCRにより増幅することにより取得することができる。  [0011] The IW method is a method for constructing an mRNA-protein linking molecule in which mRNA and a protein are chemically linked via puromycin on the ribosome when mRNA is expressed in a cell-free translation system or the like ( Non-patent document 10; Non-patent document 12; Non-patent document 5). Furthermore, the mRNA-protein-linked molecule can be obtained by in vitro selection, and the mRNA portion of the selected assigning molecule can be amplified by reverse transcription PCR.
[0012] 市販の各種無細胞翻訳系は還元剤である DTT (ジチオスレィトール)が添加されて いるため、抗体を代表とする S-S結合を有するタンパク質の発現には不向きである。 特に、小麦胚芽無細胞翻訳系はその発現に DTTが必須であるため、これまで一本鎖 抗体の cDNAライブラリーから高い結合活性をもった抗体のスクリーニングと発現例は ない。それ故、小麦胚芽無細胞翻訳系で DTT存在下でも、 cDNAライブラリ一力ゝら高 い結合活性をもった所望の抗体がスクリーニングできる系の確立が望まれている。 非特許文献 l : Kohler, G. and Milstein, C. (1975) Nature 256, 495 [0012] Since various commercially available cell-free translation systems contain DTT (dithiothreitol) as a reducing agent, they are not suitable for expression of a protein having an SS bond represented by an antibody. In particular, since DTT is essential for the expression of the wheat germ cell-free translation system, screening and expression examples of antibodies with high binding activity from single-chain antibody cDNA libraries have been Absent. Therefore, it is desired to establish a wheat embryo cell-free translation system capable of screening for a desired antibody having a very high binding activity even in the presence of DTT. Non-patent document l: Kohler, G. and Milstein, C. (1975) Nature 256, 495
非特許文献 2 : Smith, G.P. (1985) Science 228, 1315-1317 Non-Patent Document 2: Smith, G.P. (1985) Science 228, 1315-1317
非特許文献 3 : Hanes, J. and Pluckthun, A. (1997) Proc. Natl. Acad. Sci. USA 94,Non-Patent Document 3: Hanes, J. and Pluckthun, A. (1997) Proc. Natl. Acad. Sci. USA 94,
4937-4942 4937-4942
非特許文献 4 : Huston, J. S., Margolies, M. N., Haber, E. (1996) Adv. Protein Chem., 49, 329 Non-Patent Document 4: Huston, J.S., Margolies, M.N., Haber, E. (1996) Adv. Protein Chem., 49, 329.
非特許文献 5 : Miyamoto- Sato, E., et al., (2003) Nucleic Acids Res., 31, e78 非特許文献 6 : Madin K, et al. (2000) Proc. Natl. Acad. Sci. USA., 97, 559-564 非特許文献 7 : Shimizu, Y. et al. (2001) Nat. BiotechnoL, 19, 751-755 Non-patent document 5: Miyamoto-Sato, E., et al., (2003) Nucleic Acids Res., 31, e78 Non-patent document 6: Madin K, et al. (2000) Proc. Natl. Acad. Sci. USA ., 97, 559-564 Non-Patent Document 7: Shimizu, Y. et al. (2001) Nat.BiotechnoL, 19, 751-755.
非特許文献 8 : Sachs. A.B., et al. (1997) Cell 89, 831-838 Non-Patent Document 8: Sachs.A.B., Et al. (1997) Cell 89, 831-838
非特許文献 9 : Ueda. T" et al. (1991) Nucleic Acids Symp Ser. 25, 151—152 非特許文献 10 : Nemoto, N., et al., (1997) FEBS Lett., 414, 405-408 Non-Patent Document 9: Ueda. T "et al. (1991) Nucleic Acids Symp Ser. 25, 151-152 Non-Patent Document 10: Nemoto, N., et al., (1997) FEBS Lett., 414, 405- 408
特許文献 1:特開平 10-816636号公報 Patent Document 1: JP-A-10-816636
特許文献 2:国際公開第 W098/16636号パンフレット Patent Document 2: International Publication No. W098 / 16636 pamphlet
特許文献 3:特開 2002-176987号公報 Patent Document 3: Japanese Patent Application Laid-Open No. 2002-176987
特許文献 4:国際公開第 WO02/48347号パンフレット Patent Document 4: International Publication No. WO02 / 48347 pamphlet
非特許文献 l l : Nemoto, N., et al. (1999) FEBS Lett. 462, 43-46 Non-patent literature l l: Nemoto, N., et al. (1999) FEBS Lett. 462, 43-46
非特許文献 12 : Miyamoto- Sato, E" et al. (2000) Nucleic Acids Res. 28, 1176-1182 特許文献 5 :特開平 11-322781号公報 Non-patent Document 12: Miyamoto-Sato, E "et al. (2000) Nucleic Acids Res. 28, 1176-1182 Patent Document 5: JP-A-11-322781
特許文献 6:特開 2000-139468号公報 Patent Document 6: JP-A-2000-139468
特許文献 7:国際公開第 WO02/46395号パンフレット Patent Document 7: International Publication WO02 / 46395 pamphlet
非特許文献 13 :金城政孝 (1999)蛋白質核酸酵素 44 : 1431-1438 Non-Patent Document 13: Masataka Kaneshiro (1999) Protein nucleic acid enzyme 44: 1431-1438
発明の開示 Disclosure of the invention
本発明の課題は、高機能性タンパク質またはそれをコードする核酸を迅速かつ高 効率に選択する方法を提供することである。また、本発明の他の課題は、高機能性タ ンパク質またはそれをコードする核酸、並びに、その製造方法及び利用方法を提供 することである。 An object of the present invention is to provide a method for rapidly and efficiently selecting a highly functional protein or a nucleic acid encoding the same. Another object of the present invention is to provide a highly functional protein or a nucleic acid encoding the same, and a method for producing and using the same. It is to be.
[0014] 本発明者らは、タンパク質をコードする核酸のライブラリーを、 IW法を用いて作製 すると、タンパク質の選択において、従来は、タンパク質の失活により目的のものが得 られな 、と考えられて 、たような高温での加熱処理により高!、淘汰圧をかけることが でき、その結果、高機能性タンパク質を迅速かつ高効率に選択することができること を見出した。また、 S-S結合を有するタンパク質を、還元剤を必要とする無細胞翻訳 系で翻訳しても高機能性タンパク質を選択することが可能であることを見出した。以 上の知見に基づき、本発明は完成された。  [0014] The present inventors believe that, when a library of nucleic acids encoding a protein is prepared by the IW method, the target is not obtained conventionally by inactivation of the protein in the selection of the protein. As a result, they have found that heat treatment at such a high temperature can apply a high selection pressure, and as a result, a high-functional protein can be selected quickly and efficiently. In addition, they have found that a high-functional protein can be selected even when a protein having an SS bond is translated by a cell-free translation system that requires a reducing agent. The present invention has been completed based on the above findings.
[0015] 従って、本発明では、以下のものを提供する。  Therefore, the present invention provides the following.
(1)標的分子と相互作用するタンパク質またはそれをコードする核酸の選択法であつ て、以下の工程 (a)— (d)を含むことを特徴とする選択法。  (1) A method for selecting a protein that interacts with a target molecule or a nucleic acid encoding the same, comprising the following steps (a) to (d).
(a)タンパク質をコードする DNAのライブラリーを調製する工程。  (a) a step of preparing a library of DNAs encoding the protein;
(b) (a)で調製されたライブラリーの DNAを転写し、転写された RNAの 3'末端にピュー口 マイシンを含むスぺーサーを連結した後、無細胞翻訳系にお ヽて遺伝子型と表現型 の対応付け分子を調製することにより対応付け分子のライブラリーを構築する工程。 (b) After transcribing the DNA of the library prepared in (a) and ligating a spacer containing pewmic mycin to the 3 'end of the transcribed RNA, the cells are genotyped in a cell-free translation system. Constructing a library of mapping molecules by preparing mapping molecules of phenotype and phenotype.
(c)対応付け分子のライブラリーを加熱処理する工程。 (c) heating the library of assigning molecules;
(d)対応付け分子を標的分子に対して結合させ、十分洗浄した後、溶出し、対応付け 分子の核酸部を逆転写- PCRまたは PCRによって増幅させる工程。  (d) a step of binding the assigning molecule to the target molecule, washing sufficiently, eluting, and amplifying the nucleic acid portion of the assigning molecule by reverse transcription-PCR or PCR.
(2)標的分子が抗原であり、タンパク質が一本鎖抗体である(1)の選択法。  (2) The method according to (1), wherein the target molecule is an antigen and the protein is a single-chain antibody.
(3)スぺーサ一がポリエチレングリコールを含む(1)の選択法。  (3) The method according to (1), wherein the spacer contains polyethylene glycol.
(4) (d)で増幅した DNAを (a)のライブラリ一として用いて、(a)— (d)の操作を繰り返すェ 程をさらに含む(1)の選択法。  (4) The selection method of (1), further comprising the step of repeating the steps (a) to (d) using the DNA amplified in (d) as a library in (a).
(5)加熱処理する際の条件が、 50-100°C、 1-30分の範囲力 選択される(1)の選択 法。  (5) The selection method of (1), in which the conditions for the heat treatment are selected in the range of 50-100 ° C for 1-30 minutes.
(6) (d)の工程の前に対応付け分子の RNA部を逆転写により RNA-DNAノヽイブリツドに する工程をさらに含む(1)の選択法。  (6) The method of (1), further comprising a step of reverse transcription of the RNA portion of the assigning molecule into an RNA-DNA hybrid prior to the step (d).
(7)逆転写の前に、逆転写反応を阻害する無細胞翻訳系由来の物質を除去する(6 )の選択法。 (8)無細胞翻訳系が、チオール化合物を含む無細胞翻訳系である(1)の選択法。(7) The selection method according to (6), wherein prior to reverse transcription, a substance derived from a cell-free translation system that inhibits the reverse transcription reaction is removed. (8) The method according to (1), wherein the cell-free translation system is a cell-free translation system containing a thiol compound.
(9)無細胞翻訳系が、小麦胚芽抽出液、ゥサギ網状赤血球抽出液、又は、大腸菌 S-30抽出液の無細胞翻訳系である(1)の選択法。 (9) The method according to (1), wherein the cell-free translation system is a wheat germ extract, a heron reticulocyte extract, or an Escherichia coli S-30 extract.
(10)標的分子と相互作用するタンパク質の製造法であって、(1)一(9)のいずれか の選択法により標的分子と相互作用するタンパク質をコードする核酸を選択するェ 程、および、選択された核酸を翻訳してタンパク質を製造する工程を含む製造法。 (10) A method for producing a protein that interacts with a target molecule, the method comprising: (1) selecting a nucleic acid encoding a protein that interacts with the target molecule by any one of the selection methods (1) and (9); A production method comprising a step of producing a protein by translating a selected nucleic acid.
(11)標的分子が抗原であり、タンパク質が一本鎖抗体である(10)の製造法。 (11) The method according to (10), wherein the target molecule is an antigen and the protein is a single-chain antibody.
(12)抗原がアンジォテンシン IIである(11)の製造法。  (12) The method according to (11), wherein the antigen is angiotensin II.
(13)抗原が Lewis Xである(11)の製造法。  (13) The method according to (11), wherein the antigen is Lewis X.
(14)一本鎖抗体を製造する工程が、選択された核酸を、チオール化合物を含む無 細胞翻訳系で翻訳することを含む(11)の製造法。  (14) The method according to (11), wherein the step of producing a single-chain antibody comprises translating the selected nucleic acid with a cell-free translation system containing a thiol compound.
(15)無細胞翻訳系が、小麦胚芽抽出液、ゥサギ網状赤血球抽出液、または、大腸 菌 S-30抽出液である(14)の製造法。  (15) The method according to (14), wherein the cell-free translation system is a wheat germ extract, a heron reticulocyte extract, or an E. coli S-30 extract.
(16)—本鎖抗体を製造する工程が、選択された核酸で生細胞を形質転換させ、そ の生細胞内で一本鎖抗体を発現させることを含む(11)の製造法。  (16) The method according to (11), wherein the step of producing a single-chain antibody comprises transforming a living cell with the selected nucleic acid and expressing the single-chain antibody in the living cell.
(17)一本鎖抗体を製造する工程が、選択された核酸によりコードされる一本鎖抗体 と、酵素又は緑色蛍光タンパク質 (Green Fluorescent Protein: GFP)との融合タンパク 質として製造することを含む(11)の製造法。  (17) The step of producing a single-chain antibody includes producing as a fusion protein a single-chain antibody encoded by the selected nucleic acid and an enzyme or green fluorescent protein (GFP). (11) Manufacturing method.
(18) (1)一(9)のいずれかの選択法により選択された核酸を、 C末端ラベル化剤の 存在下で無細胞翻訳系で翻訳することによりタンパク質の C末端をラベルイ匕する方 法。  (18) (1) A method for labeling the C-terminus of a protein by translating the nucleic acid selected by any one of the selection methods of (9) in a cell-free translation system in the presence of a C-terminal labeling agent. Law.
(19)アンジォテンシン IIに対する結合活性を有する一本鎖抗体であって、下記 (A)又 は (B)に示すアミノ酸配列を有する一本鎖抗体。  (19) A single-chain antibody having angiotensin II-binding activity, which has the amino acid sequence shown in the following (A) or (B):
(A)配列番号 61、 63, 65, 67, 69, 71、 73, 75, 77, 79, 81、 83, 85, 87, 89, 9 1、 93、 95、 97、 99、 101、 103また ίま 105に示すアミノ酸酉己歹 lj。  (A) SEQ ID NO: 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103 or Pamino 105 is an amino acid rooster.
(B) (A)のアミノ酸配列と相同性が 90%以上のアミノ酸配列を有するアミノ酸配列。 (B) An amino acid sequence having an amino acid sequence having 90% or more homology with the amino acid sequence of (A).
(20) (19)の一本鎖抗体をコードする核酸。 (20) The nucleic acid encoding the single-chain antibody according to (19).
(21) Lewis Xに対する結合活性を有する一本鎖抗体であって、下記 (A)又は (B)に示 すアミノ酸配列を有する一本鎖抗体。 (21) A single-chain antibody having binding activity to Lewis X, which is represented by the following (A) or (B): A single-chain antibody having an amino acid sequence.
(A)配列番号 107、 109、 111、 113、 115または 117に示すアミノ酸配列。  (A) the amino acid sequence of SEQ ID NO: 107, 109, 111, 113, 115 or 117;
(B) (A)のアミノ酸配列と相同性が 90%以上のアミノ酸配列を有するアミノ酸配列。 (B) An amino acid sequence having an amino acid sequence having 90% or more homology with the amino acid sequence of (A).
(22) (21)の一本鎖抗体をコードする核酸。 (22) A nucleic acid encoding the single-chain antibody of (21).
(23) (11)一(17)のいずれかの製造法によって得られた抗体を用いてタンパク質を 免疫学的に検出する方法。  (23) A method for immunologically detecting a protein using the antibody obtained by the production method according to any one of (11) to (17).
(24)ウェスタンプロット法、免疫染色法、蛍光抗体染色法、抗体チップ法、免疫沈降 法である(23)の検出方法。  (24) The detection method of (23), which is a Western plot method, an immunostaining method, a fluorescent antibody staining method, an antibody chip method, and an immunoprecipitation method.
(25) (11)一(17)のいずれかの製造法によって得られたタンパク質と、標的分子とを 接触させ、タンパク質と標的分子との相互作用を検出することを含む、分子間相互作 用を検出する方法。  (25) (11) Intermolecular interaction including contacting a protein obtained by the production method according to any one of (17) with a target molecule and detecting an interaction between the protein and the target molecule. How to detect.
(26)蛍光相関分光法、蛍光イメージングアナライズ法、蛍光共鳴エネルギー移動法 、エバネッセント場分子イメージング法、蛍光偏光解消法、表面プラズモン共鳴法、 又は、固相酵素免疫検定法である(25)の検出方法。  (26) Fluorescence correlation spectroscopy, fluorescence imaging analysis, fluorescence resonance energy transfer, evanescent field molecular imaging, fluorescence depolarization, surface plasmon resonance, or enzyme-linked immunosorbent assay (25) Method.
(27)ヒト又はその他の動物由来の DNAライブラリーから(2)の選択法により選択され た核酸の配列に基づいて、その核酸にコードされる可変領域をヒトの IgGの可変領域 と置換することによって構築されるヒトまたはヒト型抗体。  (27) Substituting a variable region encoded by a human IgG variable region from a human or other animal-derived DNA library based on the nucleic acid sequence selected by the selection method in (2), based on the nucleic acid sequence Human or humanized antibodies constructed by
(28) (27)の抗体を有効成分とする治療剤。  (28) A therapeutic agent comprising the antibody of (27) as an active ingredient.
図面の簡単な説明 Brief Description of Drawings
[図 1]本発明で使用される一本鎖抗体 cDNAライブラリーの構築。 [FIG. 1] Construction of a single-chain antibody cDNA library used in the present invention.
[図 2]IW法による IVVライブラリーの選択サイクル。 [Figure 2] IVV library selection cycle by IW method.
[図 3]アンジォテンシン II-ピオチンの化学構造式。 [FIG. 3] Chemical structural formula of angiotensin II-pyotin.
[図 4]Lewis X-sp-ビォチンの化学構造式。 [Figure 4] Chemical structural formula of Lewis X-sp-biotin.
[図 5] [15]で得られた翻訳溶液を 8M尿素存在下 5%ポリアクリルアミド電気泳動で対応 付け分子を確認したもの。上は電気泳動ゲル (電気泳動写真)。下の棒グラフは上の 電気泳動ゲルの FITCの蛍光を MOLECULAR IMAGER FX (Bio-rad)で測定し定量 したもの。 MHO : [11]で得られたライブラリー; Mil : [11]で得られたライブラリーを [18] において 50°C、 30分間次いで 99°C、 5分間反応させアンジォテンシン IIを抗原として セレクションし [25]で回収したライブラリー; MI2 :MI1を [18]において 50°C、 30分間次 いで 99°C、 5分間反応させアンジォテンシン IIを抗原としてセレクションし [25]で回収 したライブラリー; MI3 :MI2を [18]において 50°C、 30分間次いで 99°C、 5分間反応させ アンジォテンシン IIを抗原としてセレクションし [25]で回収したライブラリー。 [Figure 5] The translation solution obtained in [15] was identified by 5% polyacrylamide electrophoresis in the presence of 8M urea to identify the corresponding molecules. The top is an electrophoresis gel (electrophoresis photograph). The lower bar graph shows the FITC fluorescence of the upper electrophoresis gel measured and quantified using MOLECULAR IMAGER FX (Bio-rad). MHO: The library obtained in [11]; Mil: The library obtained in [11] was reacted in [18] at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes, using angiotensin II as an antigen. The library selected and collected in [25]; MI2: MI1 was reacted in [18] at 50 ° C for 30 minutes, then at 99 ° C for 5 minutes, and angiotensin II was selected as an antigen and collected in [25]. Library; MI3: A library collected by reacting MI2 in [18] at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes, selecting angiotensin II as an antigen, and collecting in [25].
[図 6] [15]で得られた翻訳溶液を 8M尿素存在下 5%ポリアクリルアミド電気泳動で対応 付け分子を確認したもの (電気泳動写真)。 MHO : [11]で得られたライブラリー; MK1 : [FIG. 6] The translation solution obtained in [15], in which the corresponding molecules were confirmed by 5% polyacrylamide electrophoresis in the presence of 8M urea (electrophoresis photograph). MHO: Library obtained in [11]; MK1:
[11]で得られたライブラリーを [18]において 50°C、 30分間次いで 99°C、 5分間反応さ せ Lewis Xを抗原としてセレクションし [25]で回収したライブラリー; MK2 :MK1を [18] において 50°C、 30分間次いで 99°C、 5分間反応させ Lewis Xを抗原としてセレクション し [25]で回収したライブラリー。 The library obtained in [11] was reacted in [18] at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes, and Lewis X was selected as an antigen, and the library recovered in [25]; MK2: MK1 In [18], the library was reacted at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes, and Lewis X was selected as an antigen, and the library recovered in [25].
[図 7] [15]で得られた翻訳溶液を 8M尿素存在下 5%ポリアクリルアミド電気泳動で対応 付け分子を確認したもの (電気泳動写真)。 MM1 : [11]で得られたライブラリーを [18] において 50°C、 30分間反応させアンジォテンシン IIを抗原としてセレクションし [25]で 回収したライブラリー; MM2 :MM1を [18]において 50°C、 30分間反応させアンジォテ ンシン IIを抗原としてセレクションし [25]で回収したライブラリー; MP1: [11]で得られ たライブラリーを [15]において 99°C、 5分間反応させその後 [20] [21]においてアンジ ォテンシン IIを抗原としセレクションを行ったあとに [18]において 50°C、 30分間次いで 99°C、 5分間反応させて [22]以降の実験を行!、 [25]で回収したライブラリー。  [FIG. 7] The translation solution obtained in [15], in which the corresponding molecules were confirmed by 5% polyacrylamide electrophoresis in the presence of 8M urea (electrophoresis photograph). MM1: The library obtained in [11] was reacted at 50 ° C for 30 minutes in [18], and angiotensin II was selected as an antigen, and the library was recovered in [25]; MM2: MM1 was recovered in [18]. The library collected by [25] by reacting at 50 ° C for 30 minutes and selecting angiotensin II as an antigen; MP1: The library obtained by [11] was reacted at [15] at 99 ° C for 5 minutes, and then [20] After selection using angiotensin II as an antigen in [21], the reaction was carried out in [18] at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes, and the experiments following [22] were performed! 25].
[図 8] [21]で得られた溶出液を [22]で PCRし 1%ァガロースゲル電気泳動に付したもの 。上は電気泳動ゲル (電気泳動写真)。下の棒グラフは上の電気泳動ゲルのェチジ ゥムブロマイドの吸収を MOLECULAR IMAGER FX (Bio- rad)で測定し定量したもの [Fig. 8] The eluate obtained in [21] was subjected to PCR in [22] and subjected to 1% agarose gel electrophoresis. The top is an electrophoresis gel (electrophoresis photograph). The lower bar graph shows the absorption of ethidium bromide in the upper electrophoresis gel measured and quantified using MOLECULAR IMAGER FX (Bio-rad).
[図 9] [20]で得られた Flowを 1000倍希釈したものと [20]で得られた Washおよび [21] で得られた溶出液 (Elute)を 1倍と 10倍希釈したものを [22]で PCRし 1%ァガロースゲル 電気泳動に付したもの (電気泳動写真)。 [Figure 9] The flow obtained in [20] was diluted 1000-fold, and the Wash obtained in [20] and the eluate (Elute) obtained in [21] were diluted 1- and 10-fold. PCR performed in [22] and subjected to 1% agarose gel electrophoresis (electrophoresis photograph).
[図 10] [20]で得られた Flowを 1000倍希釈したものと [20]で得られた Washおよび [21] で得られた溶出液を 1倍と 10倍希釈したものを [22]で PCRし 1%ァガロースゲル電気泳 動に付したもの。上は電気泳動ゲル (電気泳動写真)。下の棒グラフは上の電気泳動 ゲルのェチジゥムブロマイドの吸収を MOLECULAR IMAGER FX (Bio- rad)で測定し 定量したもの。 Fは Flow、 Wは Wash、 Eは溶出液を 1倍、 E0.1は溶出液を 10倍希釈の 略。 [Figure 10] The flow obtained in [20] was diluted 1000-fold, and the Wash obtained in [20] and the eluate obtained in [21] were diluted 1- and 10-fold [22] PCR performed with 1% agarose gel and electrophoresis. The top is an electrophoresis gel (electrophoresis photograph). Lower bar graph shows upper electrophoresis Absorption of ethidium bromide in gel is measured and quantified using MOLECULAR IMAGER FX (Bio-rad). F stands for Flow, W stands for Wash, E stands for 1-fold dilution, and E0.1 stands for 10-fold dilution.
[図 11] [31]で配列分析を行い解析した結果インフレームのものについてアミノ酸配列 による clustakによる配列アラインメント後 TreeViewPPCにより作成した系統樹。線で 囲った Aはアンジォテンシン IIのセレクションで収束したもの、線で囲った Bは Lewis X のセレクションで収束したものを示す。ライブラリーの略号の後にクローンの番号を示 した。  [Fig. 11] A phylogenetic tree created by TreeViewPPC after sequence alignment by clustak based on amino acid sequences for in-frame results obtained by performing sequence analysis in [31]. A surrounded by a line indicates the one converged by the selection of Angiotensin II, and B surrounded by a line indicates the one converged by the selection of Lewis X. The number of the clone was shown after the library abbreviation.
[図 12]図 11の線で囲った Aおよび Bの拡大図。 Aはアンジォテンシン IIのセレクション で収束したもの、 Bは Lewis Xのセレクションで収束したもの。  FIG. 12 is an enlarged view of A and B surrounded by the line in FIG. A: Converged by Angiotensin II selection, B: Converged by Lewis X selection.
[図 13]ウェスタンブロットの結果(電気泳動写真)。右 2本は、 MI3-55の [37]について ウェスタンブロットしたもの。左 3本はウェスタンブロットのコントロール。  [FIG. 13] Western blot results (electrophoresis photograph). The right two are Western blots of MI3-55 [37]. Left three are Western blot controls.
[図 14]アンジォテンシン IIを抗原としたセレクションで得られた配列を [34]で翻訳を行 つた後 [39]のビアコアで分析した結果を棒グラフで示したもの。  [FIG. 14] A bar graph showing the results obtained by translating the sequence obtained by selection using angiotensin II as an antigen in [34] and then analyzing it in Biacore in [39].
[図 15]Lewis Xを抗原としたセレクションで得られた配列を [34]で翻訳を行った後 [39] のビアコアで分析した結果を棒グラフで示したもの。  [FIG. 15] A bar graph showing the result obtained by translating the sequence obtained by selection using Lewis X as an antigen with [34] and then analyzing with Biacore [39].
[図 16]MI3-55を [34]で翻訳を行った後 4°Cまたは 60°Cまたは 99°C、 5分間処理し [39 ]のビアコアで分析した結果を棒グラフで示したもの。  [FIG. 16] A bar graph showing the results of translating MI3-55 at [34], treating it at 4 ° C. or 60 ° C. or 99 ° C. for 5 minutes, and analyzing with Biacore of [39].
[図 17]MI3-55を [34]で翻訳を行った後 4°Cまたは 60°Cまたは 99°C、 5分間処理し [40 ]の ELISAで分析した結果を棒グラフで示したもの。  [FIG. 17] A bar graph showing the result of translating MI3-55 at [34], treating it at 4 ° C. or 60 ° C. or 99 ° C. for 5 minutes, and analyzing the result by ELISA [40].
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0017] 本発明の、標的分子と相互作用するタンパク質またはそれをコードする核酸の選択 法は、ライブラリーの対応付け分子の核酸部が、標的分子と相互作用するタンパク質 をコードすること、対応付け分子のライブラリーを加熱処理することの他は、通常の対 応付け分子を用いる核酸の選択法 (以下「IW法」とも 、う)に従って行うことができる [0017] The method for selecting a protein that interacts with a target molecule or a nucleic acid that encodes the protein according to the present invention is characterized in that the nucleic acid portion of the assigning molecule in the library encodes a protein that interacts with the target molecule; Except for subjecting the library of molecules to heat treatment, the method can be performed according to a conventional nucleic acid selection method using a corresponding molecule (hereinafter, also referred to as "IW method").
[0018] 「標的分子」とは、選択対象タンパク質と相互作用する分子を意味し、具体的にはタ ンパク質、核酸、糖鎖、低分子化合物などが挙げられる。タンパク質としては、選択対 象タンパク質と相互作用する能力を有する限り特に制限はなぐタンパク質の全長で あっても結合活性部位を含む部分ペプチドでもよい。またアミノ酸配列、およびその 機能が既知のタンパク質でも、未知のタンパク質でもよい。これらは、合成されたぺプ チド鎖、生体より精製されたタンパク質、あるいは cDNAライブラリ一等カゝら適当な翻 訳系を用いて翻訳し、精製したタンパク質等でも標的分子として用いることができる。 合成されたペプチド鎖はこれに糖鎖が結合した糖タンパク質であってもよ ヽ。これら のうち好ましくはアミノ酸配列が既知の精製されたタンパク質力 あるいは cDNAライ ブラリー等力も適当な方法を用いて翻訳および精製されたタンパク質を用いることが できる。 [0018] "Target molecule" means a molecule that interacts with a protein to be selected, and specifically includes proteins, nucleic acids, sugar chains, low molecular weight compounds, and the like. As a protein, selection vs. As long as it has the ability to interact with the elephant protein, there is no particular limitation on the full length of the protein or a partial peptide containing a binding active site. The protein may be a protein whose amino acid sequence and function are known or unknown. These can be used as a target molecule even with a synthesized peptide chain, a protein purified from a living body, or a protein translated using an appropriate translation system such as a cDNA library, and purified. The synthesized peptide chain may be a glycoprotein having a sugar chain bonded thereto. Among these, preferably, a protein which is translated and purified using a suitable method for a purified protein having a known amino acid sequence or a cDNA library can be used.
[0019] 核酸としては、選択対象タンパク質と相互作用する能力を有する限り、特に制限は なぐ DNAまたは RNAも用いることができる。また、塩基配列または機能が既知の核 酸でも、未知の核酸でもよい。好ましくは、タンパク質に結合能力を有する核酸として の機能、および塩基配列が既知のもの力、あるいはゲノムライブラリ一等力 制限酵 素等を用いて切断単離してきたものを用いることができる。糖鎖としては、選択対象タ ンパク質と相互作用する能力を有する限り、特に制限はなぐその糖配列あるいは機 能が、既知の糖鎖でも未知の糖鎖でもよい。好ましくは、既に分離解析され、糖配列 あるいは機能が既知の糖鎖が用いられる。低分子化合物としては、選択対象タンパ ク質と相互作用する能力を有する限り、特に制限はない。機能が未知のものでも、あ るいはタンパク質に結合する能力が既に知られて 、るものでも用いることができる。  [0019] As the nucleic acid, any DNA or RNA can be used as long as it has an ability to interact with the protein to be selected. Further, the nucleic acid may have a known nucleotide sequence or function, or may have an unknown nucleic acid. Preferably, those having a function as a nucleic acid capable of binding to a protein and having a known base sequence, or those that have been cut and isolated using a genomic library first-class restriction enzyme or the like can be used. The sugar chain is not particularly limited as long as it has the ability to interact with the protein to be selected, and the sugar sequence or function may be a known sugar chain or an unknown sugar chain. Preferably, a sugar chain which has already been separated and analyzed and whose sugar sequence or function is known is used. The low molecular weight compound is not particularly limited as long as it has the ability to interact with the protein to be selected. Either those whose functions are unknown or those whose ability to bind to a protein is already known can be used.
[0020] これら標的分子が本発明の選択対象タンパク質と行う「相互作用」又は標的分子間 の「相互作用」とは、通常は、二つの分子間の共有結合、疎水結合、水素結合、ファ ンデルワールス結合、および静電力による結合のうち少なくとも 1つ力 生じる分子間 に働く力による作用を示す力 この用語は最も広義に解釈すべきであり、いかなる意 味においても限定的に解釈してはならない。共有結合としては、配位結合、双極子 結合を包含する。また静電力による結合とは、静電結合の他、電気的反発も包含す る。また、上記作用の結果生じる結合反応、合成反応、分解反応も相互作用に包含 される。  [0020] The term "interaction" between these target molecules and the target protein of the present invention or "interaction" between the target molecules usually means a covalent bond, a hydrophobic bond, a hydrogen bond, a Van der Waals between two molecules. At least one of a bond and a bond by electrostatic force. A force indicating an action by a force acting between molecules. This term should be interpreted in the broadest sense, and should not be interpreted restrictively in any sense. The covalent bond includes a coordinate bond and a dipole bond. The coupling by electrostatic force includes not only electrostatic coupling but also electric repulsion. In addition, a binding reaction, a synthesis reaction, and a decomposition reaction resulting from the above-described actions are also included in the interaction.
[0021] 相互作用の具体例としては、抗原と抗体間の結合および解離、タンパク質レセプタ 一とリガンドの間の結合および解離、接着分子と相手方分子の間の結合および解離 、酵素と基質の間の結合および解離、核酸とそれに結合するタンパク質の間の結合 および解離、情報伝達系におけるタンパク質同士の間の結合と解離、糖タンパク質と タンパク質との間の結合および解離、糖鎖とタンパク質との間の結合および解離、ま たは、低分子化合物とタンパク質との間の結合および解離が挙げられる。 [0021] Specific examples of the interaction include binding and dissociation between an antigen and an antibody, and a protein receptor. Binding and dissociation between a molecule and a ligand, binding and dissociation between an adhesion molecule and a partner molecule, binding and dissociation between an enzyme and a substrate, binding and dissociation between a nucleic acid and a protein bound thereto, proteins in a signal transduction system Binding and dissociation between glycoproteins and proteins, binding and dissociation between sugar chains and proteins, or binding and dissociation between low molecular weight compounds and proteins. Can be
[0022] IW法は mRNAがタンパク質合成阻害剤であるピューロマイシンまたはその誘導体 を介してタンパク質と共有結合するため、リボソームディスプレイやファージディスプレ ィよりもはるかに安定性が高い。従って、リボソームディスプレイやファージディスプレ ィの 2つの技術よりもより強い選択圧でのバニングが可能であり、これによつて特異性 だけでなくより安定な標的分子と相互作用するタンパク質を選択することができ、加え て、高 、濃縮効果が得られるためより少な 、選択サイクルで希望のタンパク質を得る ことができる。またさらに、ほとんど単一にまで濃縮されるため、多くのサンプルを解析 する必要がない。これらにともない操作が簡便、大幅な時間の短縮とコストの削減が 可會 になる。  [0022] The IW method is much more stable than ribosome display or phage display because the mRNA is covalently bound to the protein via the protein synthesis inhibitor puromycin or a derivative thereof. Therefore, it is possible to perform a higher selective pressure baning than the two techniques of ribosome display and phage display, thereby selecting not only specificity but also a protein that interacts with a more stable target molecule. In addition, the desired protein can be obtained in a smaller number of selection cycles because of the higher and higher concentration effects. Furthermore, it is not necessary to analyze many samples because it is concentrated to almost single. With these, the operation is simple, and the time and cost can be greatly reduced.
[0023] また、ライブラリー内の高機能性抗体 (特異性、親和性、安定性等)の存在確立はラ イブラリーの大きさ(多様性)に依存する。ここで、 IW法の選択可能なライブラリーの 上限は、条件が整えば 1013以上が可能であり、他のどの方法よりも大きなライブラリー を対象にできる。また、リボソームディスプレイと同様に完全な in vitroの実験系である ため、不必要なバイアスが力からない。また、得られたタンパク質は DTT等の還元剤 を含有する無細胞翻訳系で機能性タンパク質として発現できるため、多検体の大量 調製が可能であり、それにともないハイスループットィ匕が容易となる。 [0023] The establishment of highly functional antibodies (specificity, affinity, stability, etc.) in the library depends on the size (diversity) of the library. Here, the upper limit of the selectable library of the IW method can be 10 13 or more if conditions are satisfied, and a library larger than any other method can be targeted. In addition, since it is a complete in vitro experimental system like ribosome display, unnecessary bias is not applied. In addition, since the obtained protein can be expressed as a functional protein in a cell-free translation system containing a reducing agent such as DTT, it is possible to prepare a large number of samples, thereby facilitating high-throughput filtration.
[0024] このようにして選択されたタンパク質は、その機能に応じて各種の分野で利用できる 。例えば、タンパク質が一本鎖抗体である場合には、このようにして選択された高親 和性一本鎖抗体は、細胞外や細胞内でのタンパク質の検出や相互作用検出に利用 できる。また、マウス抗体 cDNAライブラリ一力も選択された高親和性一本鎖抗体は、 ヒト IgG抗体の可変領域や CDR領域 (complementarity determining region)と入れ換え ることによって、キメラ型 IgG抗体やヒト型化したマウス IgG抗体を作れる。これらの抗体 は、ヒトに投与したときに惹起されるヒト抗マウス抗体の産生が少ないか、ほとんどない 。さらに、ヒト抗体 cDNAライブラリ一力も選択された高親和性一本鎖抗体は、ヒ HgG 抗体の可変領域と入れ換えることによって、完全なヒトモノクローナル IgG抗体を作れ る。これは、アナフィラキシー症状を引き起こさない抗体医薬として利用することが可 能である。 [0024] The protein thus selected can be used in various fields depending on its function. For example, when the protein is a single-chain antibody, the high affinity single-chain antibody thus selected can be used for extracellular or intracellular protein detection and interaction detection. The high-affinity single-chain antibody, which has been selected for the mouse antibody cDNA library, is replaced with a human IgG antibody variable region and CDR region (complementarity determining region) to produce a chimeric IgG antibody or a humanized mouse antibody. Make IgG antibodies. These antibodies produce little or no human anti-mouse antibody when elicited when administered to humans . Furthermore, a high-affinity single-chain antibody, which has been selected as a human antibody cDNA library, can produce a fully human monoclonal IgG antibody by replacing the variable region of the human HgG antibody. This can be used as an antibody drug that does not cause anaphylactic symptoms.
[0025] 本発明の選択対象タンパク質として使用される一本鎖抗体は、 V鎖と V鎖がリンカ  [0025] The single-chain antibody used as the protein to be selected in the present invention has a V chain and a V chain
H L  H L
一ペプチドを介して結合した通常の一本鎖抗体 (単鎖 Fvフラグメント (scFv)とも呼ば れる)でよ 、。本発明にお 、て用いられる一本鎖抗体をコードする DNAライブラリーと しては、多数の一本鎖抗体をコードする DNAを含むライブラリーであれば特に制限さ れるものではないが、理論的にはあらゆる抗原に対応しうる、 109以上の多種多様な 抗体をコードする DNAを含むものを用いることが好ま U、。力かる抗体 DNAライブラリ 一としては、市販のものを含め、通常の試験管内抗体選択系において用いられる高 等脊椎動物、好ましくはマウス、ヒト、 -ヮトリ、ャギ、ラクダの脾臓および B細胞由来の 天然型抗体 DNAライブラリーであればどのようなものでもよい。例えば、マウス天然型 抗体 cDNAライブラリー(Krebber, A. et al. (1997)) J. Immun. Methods. 201, 35-55: Engberg, J. (1996) Molecular Biotech. 6, 287- 310)はマウスの脾臓から mRNAを抽出 し、抗体の H鎖及び L鎖の可変領域 (V , V )をコードする遺伝子断片を RT-PCRによ Ordinary single-chain antibodies (also called single-chain Fv fragments (scFv)) linked via one peptide. In the present invention, the DNA library encoding a single-chain antibody used in the present invention is not particularly limited as long as it is a library containing DNAs encoding a large number of single-chain antibodies. In particular, it is preferable to use one containing DNA encoding more than 10 9 different antibodies, which can correspond to any antigen. Powerful antibody DNA libraries include, but are not limited to, those derived from higher vertebrates, preferably mouse, human, -petri, goat, camel spleen and B cells, used in conventional in vitro antibody selection systems, including commercially available ones. Any natural antibody DNA library may be used. For example, a mouse native antibody cDNA library (Krebber, A. et al. (1997)) J. Immun. Methods. 201, 35-55: Engberg, J. (1996) Molecular Biotech. 6, 287-310) MRNA was extracted from the spleen of the mouse, and the gene fragments encoding the variable regions (V, V) of the antibody H and L chains were analyzed by RT-PCR.
H L  H L
つて cDNAとし、それらを PCRによって増幅する。さらに得られた断片力も Vの C末端  And amplify them by PCR. Furthermore, the obtained fragment force is also the C-terminal of V
H  H
と Vの N末端を (Gly Ser)リンカ一で繋ぎ合わせるように DNA断片を合成することでマ し 4 4  By synthesizing a DNA fragment such that the N-terminals of V and V are connected by a (Gly Ser) linker.
ウス一本鎖抗体 cDNAライブラリーが完成となる。ここで、繋ぎ合わせるリンカ一配列 は比較的自由度の高いものであればどのようなものでも力まわないが、一般的に (Gly Ser)配列 (配列番号 120)を用いることが好ましい。また、繋ぎ合わせの順番は V The mouse single-chain antibody cDNA library is completed. Here, any linker sequence can be used as long as it has a relatively high degree of freedom, but it is generally preferable to use a (Gly Ser) sequence (SEQ ID NO: 120). The order of joining is V
4 4 し4 4
-(Gly Ser) - Vでもよい。 -(Gly Ser)-V is fine.
4 4 H  4 4 H
[0026] また、あらゆる変異型の抗体 DNAライブラリーも有利に用いることができる。例えば、  [0026] In addition, any mutant type antibody DNA library can be advantageously used. For example,
(l) n- CoDeR:抗体 cDNAから CDR(H鎖 3つ、 L鎖 3つ)のみを PCRによって取り出し、 それらを大腸菌およびファージで発現し易い抗体フレームに組み込んだライブラリー (n- CoDeR:多様性は 2 X 109) (Jirholt, P. et al. (1998) GENE 215, 471-476: (l) n-CoDeR: A library that extracts only CDRs (three H chains and three L chains) from antibody cDNA by PCR and incorporates them into an antibody frame that is easy to express in E. coli and phage (n-CoDeR: diverse Sex is 2 X 10 9 ) (Jirholt, P. et al. (1998) GENE 215, 471-476:
Soderlind, E. et al. (2000) Nature Biotechnol 18. 852-856)。(2)抗体の超可変部位 (CDR)にランダム配列 (NNK, NNB)を導入し、その多様性を増加させたライブラリー( Hayashi, N. et al. (1994) Biotechniqes 17, 310-345)。(3) HuCAL :ヒト抗体は構造的 な多様性が 7種類の H鎖 (subclass: VH1A, VH1B, VH2- 6)と 7種類の L鎖 (subclass:V κ 1-V K 4, V 1- V 3)を組み合わせたもの(計 49種類)によって約 95%以上がカバ 一されている。これら 14種類の抗体の CDR3(H鎖 L鎖両方)にランダム配列を導入し たライブラリー。 (Knappik, A. et al. (2000) J. Mol. Biol. 296, 57-86: Hanes, J et al.Soderlind, E. et al. (2000) Nature Biotechnol 18.852-856). (2) A library that introduces random sequences (NNK, NNB) into the hypervariable region (CDR) of an antibody to increase its diversity ( Hayashi, N. et al. (1994) Biotechniqes 17, 310-345). (3) HuCAL: Human antibodies have 7 types of H chains (subclass: VH1A, VH1B, VH2-6) and 7 types of L chains (subclass: Vκ1-VK4, V1-V) About 95% or more is covered by the combination of 3) (49 types in total). A library in which random sequences have been introduced into CDR3 (both H and L chains) of these 14 types of antibodies. (Knappik, A. et al. (2000) J. Mol. Biol. 296, 57-86: Hanes, J et al.
(2000) Nature Biotech. 18, 1287-1292)。(4) point mutationと DNA shufflingを組み合 わせて、単一の抗体遺伝子にランダムな変異を導入したライブラリー。(Jermutus, L.(2000) Nature Biotech. 18, 1287-1292). (4) A library that combines point mutation and DNA shuffling to introduce random mutations into a single antibody gene. (Jermutus, L.
(2001) Proc. Natl. Acad. Sci. USA 98, 75-80)などが挙げられる。これらのライブラリ 一は、バニングのスピード、抗体の安定性、発現、機能性等を高めるように設計され ており、本発明への適応はより効果的である。 (2001) Proc. Natl. Acad. Sci. USA 98, 75-80). These libraries have been designed to enhance the speed of Banning, antibody stability, expression, functionality, and the like, and adaptation to the present invention is more effective.
[0027] 以下、本発明の選択法の各工程について説明する。  Hereinafter, each step of the selection method of the present invention will be described.
(a)工程は、標的分子と相互作用するタンパク質をコードする DNAのライブラリーを 調製する工程である。このタンパク質をコードする DNAのライブラリ一は、通常の方法 に従って調製することができる。  Step (a) is a step of preparing a library of DNAs encoding proteins that interact with the target molecule. A library of DNAs encoding this protein can be prepared according to a conventional method.
[0028] (b)工程は、(a)で調製されたライブラリーの DNAを転写し、転写された RNAの 3'末端 にピューロマイシンを含むスぺーサーを連結した (以下、ここで調製されるものを「翻 訳テンプレート」と称することがある)後、無細胞翻訳系にお 、て遺伝子型と表現型の 対応付け分子を調製することにより対応付け分子のライブラリーを構築する工程であ る。この工程は通常の IW法におけるものと同様に行うことできる。詳細については後 記で説明する力 スぺーサ一はポリエチレングリコールを含むものであることが好まし い。  [0028] In the step (b), the DNA of the library prepared in (a) is transcribed, and a spacer containing puromycin is ligated to the 3 'end of the transcribed RNA (hereinafter, prepared here). Is sometimes referred to as a “translation template”), and then a library of mapping molecules is prepared in a cell-free translation system by preparing mapping molecules for genotypes and phenotypes. You. This step can be performed in the same manner as in the ordinary IW method. The force spacer, which will be described in more detail below, preferably contains polyethylene glycol.
[0029] (c)工程は、対応付け分子のライブラリーを加熱処理する工程である。ここでの加熱 処理とは、通常の IW法における処理では負荷されないような加熱条件に暴露するこ とを意味する。たとえば、対応付け分子を翻訳後そのまま標的分子と結合させる場合 には、翻訳の間にはない加熱条件に暴露することであり、対応付け分子の RNA部分 を逆転写により RNA-DNAノヽイブリツドにして力 標的分子と結合させる場合には、翻 訳及び逆転写の間にはない加熱条件に暴露することである。この温度は、翻訳され て力 標的分子に結合させるまでの工程に応じて、通常には、 50-100°C、 1-30分の 範囲から選択される。強い選択圧という点では、翻訳だけがある場合並びに翻訳及 び逆転写がある場合のいずれでも、好ましくは 80°C以上、より好ましくは 90°C以上の 温度で加熱処理をする。 The step (c) is a step of subjecting the library of assigning molecules to heat treatment. Here, the heat treatment means exposure to heating conditions that would not be imposed by the treatment in the normal IW method. For example, in the case where the assigning molecule is directly bound to the target molecule after translation, it is necessary to expose the subject to a heating condition that does not exist during translation, and the RNA portion of the assigning molecule is converted into an RNA-DNA hybrid by reverse transcription. When binding to a target molecule, exposure is to heating conditions that are not between translation and reverse transcription. This temperature is typically 50-100 ° C for 1-30 minutes, depending on the process of translation and force binding to the target molecule. Selected from a range. In terms of strong selection pressure, heat treatment is preferably performed at a temperature of 80 ° C or higher, more preferably 90 ° C or higher, regardless of whether there is only translation or translation and reverse transcription.
[0030] ( の工程の前に対応付け分子の RNA部を逆転写により RNA-DNAノヽイブリッドにす ることが好ま U、。これにより RNAの担体および標的分子への非特異的な結合を阻止 することができる。また、逆転写を行う場合には、逆転写の前に、逆転写反応を阻害 する無細胞翻訳系由来の物質を除去することが好ましい。除去の方法としては、ゲル ろ過カラム、スピンカラム、ニッケルカラム等を用いる分画が挙げられる。  [0030] It is preferable that the RNA portion of the assigning molecule be converted into an RNA-DNA hybrid by reverse transcription before the step of (U. This prevents nonspecific binding of the RNA to the carrier and the target molecule. When performing reverse transcription, it is preferable to remove a substance derived from a cell-free translation system that inhibits the reverse transcription reaction before reverse transcription. , A spin column, a nickel column and the like.
[0031] 加熱処理、逆転写、及び、分画の順序は特に限定されないが、通常には、加熱処 理、分画、逆転写の順、または、分画、逆転写、加熱処理の順が好ましい。  [0031] The order of the heat treatment, the reverse transfer, and the fractionation is not particularly limited, but usually, the order of the heat treatment, the fractionation, and the reverse transfer, or the order of the fractionation, the reverse transfer, and the heat treatment is generally. preferable.
(d)工程は、対応付け分子を標的分子に対して結合させ、十分洗浄した後、溶出し 、対応付け分子の核酸部を逆転写- PCRまたは PCRによって増幅させる工程である。 この工程は通常の IW法におけるものと同様に行うことできる。溶出は、通常には遊 離の標的分子を含む溶液で溶出することにより行うことができる。  Step (d) is a step in which the assigning molecule is bound to the target molecule, washed sufficiently, then eluted, and the nucleic acid portion of the assigning molecule is amplified by reverse transcription-PCR or PCR. This step can be performed in the same manner as in the ordinary IW method. Elution can usually be performed by elution with a solution containing a free target molecule.
[0032] 本発明の選択法においては、(d)で増幅した DNAを (a)のライブラリ一として用いて、 (a)— (d)の操作を繰り返すことが好ましい。繰り返す場合には、(d)で増幅した DNAを (a)のライブラリ一として用いるための処理をする。例えば、各サイクルごとに 5'UTR配 列(増幅プライマー配列- SP6プロモーター配列- Ω 29ェンハンサー配列)を N末端に 付加する。しカゝしながら、抗体の N末端はそれぞれでその配列が異なるため、 N末端 に共通配列を導入する必要がある。後記実施例では T7タグ (MASMTGGQQMG (配 列番号 118》を用いた力 このタグは一般的に使用されているものであればどのよう なものでもかまわな ヽ。 C末端側には FLAGタグ (DYKDDDDK (配列番号 119))を導入 した。一般的にライブラリーを構築する上で、 PCRでの増幅、化学合成等によるある 程度の好まれない変異 (STOPコドン、挿入、欠失)の導入は避けられない。そこで、 C 末端のタグによってインフレーム (in-frame)のものだけを選び出すことが可能になる。 このタグもまた一般的に使用されているものであればどのようなものでも力まわない。  [0032] In the selection method of the present invention, it is preferable to repeat the operations (a) to (d) using the DNA amplified in (d) as a library of (a). In the case of repetition, a process for using the DNA amplified in (d) as a library in (a) is performed. For example, a 5 'UTR sequence (amplification primer sequence-SP6 promoter sequence-Ω 29 enhancer sequence) is added to the N-terminus in each cycle. However, since the N-terminal of the antibody has a different sequence, it is necessary to introduce a consensus sequence at the N-terminal. In the examples described later, a T7 tag (MASMTGGQQMG (SEQ ID NO: 118)) This tag may be any commonly used tag. FLAG tag (DYKDDDDK (SEQ ID NO: 119)) Generally, in constructing a library, avoid introducing some undesired mutations (STOP codon, insertion, deletion) by PCR amplification, chemical synthesis, etc. Therefore, the C-terminal tag allows you to select only in-frame tags, which can also be used with any commonly used tag. Absent.
[0033] 本発明の選択法で使用される IWライブラリーの一例を、図 1を参照して説明する。  An example of the IW library used in the selection method of the present invention will be described with reference to FIG.
タンパク質は一本鎖抗体である。マウス脾臓由来抗体 (IgG) mRNAの H鎖 L鎖それぞ れの可変部を RT-PCRで抽出し、 H鎖の C末端と L鎖の N末端を (Gly Ser)のリンカ The protein is a single chain antibody. H chain and L chain of mouse spleen-derived antibody (IgG) mRNA These variable regions were extracted by RT-PCR, and the C-terminus of the H chain and the N-terminus of the L chain were
4 4 一 でつなげて一本鎖抗体 cDNAライブラリーとする。これらを試験管内転写によって mRNAとし、この mRNAの 3'末端にピューロマイシンを有する PEGスぺーサーを結合さ せる。これらを無細胞翻訳系で翻訳すると mRNAとその配列に対応する抗体がピュー ロマイシンを介して共有結合した IWライブラリーが構築される。このライブラリ一は 10 13以上の多様性を有しており、既知の抗体選択法の中で最も大きなライブラリーであ る。 4 4 Connect them to form a single-chain antibody cDNA library. These are converted into mRNA by in vitro transcription, and a PEG spacer having puromycin is bound to the 3 ′ end of the mRNA. When these are translated by a cell-free translation system, an IW library is constructed in which mRNA and an antibody corresponding to the sequence are covalently linked via puromycin. This library one has a 10 1 3 or more diversity, Ru largest libraries der among known antibody selection methods.
[0034] 一本鎖抗体 DNAライブラリーを用いた場合の本発明の選択法の工程の概要を図 2 に示す。このような選択サイクルによって、すなわち、選択、増幅、ライブラリーの再構 築、翻訳を繰り返すことで、希望とする抗体を濃縮する。ここで、選択時にある種の選 択圧を力 4ナることで、得られる抗体の性質と濃縮効率が決まる。すなわち、不安定な 抗体を除去するタンパク質レベルでの選択圧と、非特異的に結合するものを除去す るための選択圧である。本発明では、 IW法を採用することにより、従来の方法では 目的の抗体が得られないような高い選択圧をかけても目的の抗体が選択されること が半 lj明した。  FIG. 2 shows an outline of the steps of the selection method of the present invention when a single-chain antibody DNA library is used. The desired antibody is enriched by such a selection cycle, ie, by repeating selection, amplification, library restructuring, and translation. Here, by applying a certain selection pressure at the time of selection, the properties of the obtained antibody and the concentration efficiency are determined. The selection pressure at the protein level to remove unstable antibodies and the selection pressure to remove non-specifically bound ones. In the present invention, it has been clarified that, by employing the IW method, the target antibody can be selected even when a high selection pressure is applied so that the target antibody cannot be obtained by the conventional method.
[0035] 以下、具体的に本発明の翻訳テンプレート、 C末端修飾タンパク質、翻訳テンプレ ートによる C末端ラベルイ匕法および対応付け方法についての実施例を記述するが、 下記の実施例は本発明につ 、ての具体的認識を得る一助とみなすべきものであり、 本発明の範囲は下記の実施例により何ら限定されるものでない。  Hereinafter, specific examples of the translation template, C-terminal modified protein, and C-terminal labeling method and association method using the translation template of the present invention will be described. This is to be regarded as helping to obtain all the specific recognitions, and the scope of the present invention is not limited by the following examples.
[0036] 選択対象タンパク質のコード部は、 5'末端領域、 ORF領域、 3'末端領域カゝらなり、 5' 末端に Cap構造があってもなくてもよい。また、コード部の 3'末端領域は、 A配列として ポリ Ax8配列、あるいは X配列として Xhol配列や 4塩基以上で (C又は G)NN(C又は G) の配列を持つもの、および A配列と X配列の組み合わせとしての XA配列がある。 A配 列、 X配列、あるいは XA配列の上流に親和性タグ配列として Flag-tag配列、からなる 構成が考えられる。ここで、親和性タグ配列としては HA-tagや IgGのプロテイン A(zドメ イン)などの抗原抗体反応を利用したものや His-tagなど、タンパク質を検出あるいは 精製できるいかなる手段を用いるための配列でも力まわない。ここで、翻訳効率に影 響する範囲としては、 XA配列の組み合わせが重要であり、 X配列のなかで、最初の 4 塩基が重要であり、(C又は G)NN(C又は G)の配列を持つものが好ましい。また、 5'末 端領域は、転写プロモーターと翻訳ェンハンサ一からなり、転写プロモーターは T7/T3ある 、は SP6などが利用でき、特に制限はな!/、が、小麦の無細胞翻訳系では、 翻訳のェンノ、ンサ一配列としてオメガ配列やオメガ配列の一部を含む配列を利用す ることが好ましぐプロモーターとしては、 SP6を用いることが好ましい。翻訳ェンハンサ 一のオメガ配列の一部 (029)は、 TMVのオメガ配列 (Gallie D.R., Walbot V. (1992) Nucleic Acids Res., 20, 4631- 4638)の一部を含んだものである。 [0036] The coding part of the protein to be selected comprises a 5 'terminal region, an ORF region, and a 3' terminal region, and may or may not have a Cap structure at the 5 'terminal. In addition, the 3 'terminal region of the coding portion is a poly Ax8 sequence as an A sequence, an Xhol sequence as an X sequence, a sequence having (C or G) NN (C or G) with 4 or more bases, and an A sequence. There is an XA array as a combination of X arrays. A configuration comprising an A sequence, an X sequence, or a Flag-tag sequence as an affinity tag sequence upstream of the XA sequence can be considered. Here, the affinity tag sequence is a sequence using any means capable of detecting or purifying a protein, such as one utilizing an antigen-antibody reaction such as HA-tag or IgG protein A (z domain) or His-tag. But I can't help. Here, as the range that affects translation efficiency, the combination of XA sequences is important. The base is important, and one having a (C or G) NN (C or G) sequence is preferred. In addition, the 5 'terminal region is composed of a transcription promoter and a translation enhancer.Transcription promoters are T7 / T3, SP6, etc. can be used, and there is no particular limitation! /, But in a wheat cell-free translation system, As a promoter which preferably uses an omega sequence or a sequence containing a part of an omega sequence as the translation sequence, it is preferable to use SP6. A part of the omega sequence (029) of the translation enhancer includes a part of the omega sequence of TMV (Gallie DR, Walbot V. (1992) Nucleic Acids Res., 20, 4631-4638).
[0037] ポリエチレングリコール (PEG)部(スぺーサ一)は、 CCA領域、 PEG領域、ドナー領域 力 なる。最低限必要な構成は、ドナー領域である。翻訳効率の点では、ドナー領域 のみならず PEG部を持つものが好ましぐさらにアミノ酸との結合能力のないピュー口 マイシンを持つことが好まし!/、。 PEG領域のポリエチレングリコールの分子量の範囲 は、 400— 30,000で、好ましくは 1,000— 10,000、より好ましくは 2,000— 6,000である。ま た、 CCA領域にはピューロマイシン(Puromycin)を含む構成と含まな!/、構成が可能で ある。 [0037] The polyethylene glycol (PEG) portion (spacer) is a CCA region, a PEG region, and a donor region. The minimum required configuration is the donor region. From the viewpoint of translation efficiency, those having a PEG moiety as well as the donor region are more preferable, and those having a pure mouth mycin which has no binding ability to amino acids are preferable! The molecular weight range of the polyethylene glycol in the PEG region is from 400 to 30,000, preferably from 1,000 to 10,000, and more preferably from 2,000 to 6,000. Also, the CCA domain can be configured with or without Puromycin!
[0038] 対応付け分子の構築に用いる場合には、無細胞翻訳系にお 、て、そこで翻訳され たタンパク質の C末端にスぺーサ一が結合できるようにピューロマイシンを含む構成 が用いられる。ピューロマイシンを含むとはその誘導体を含むことも包含する。誘導体 の例としては以下のものが挙げられる。 3し N-アミノアシルピューロマイシンアミノヌク レオシド (3 '—Ν— Aminoacylpuromycin aminonucleoside, PANS—アミノ酸)、例えばァミノ 酸部がグリシンの PANS- Gly、 ノ《リンの PANS- Val、ァラニンの PANS- Ala、その他、全 アミノ酸に対応する PANS-全アミノ酸が利用できる。また、化学結合として 3'-アミノア デノシンのァミノ基とアミノ酸のカルボキシル基が脱水縮合した結果形成されたアミド 結合でつながった 3'-N-アミノアシルアデノシンアミノヌクレオシド(  [0038] When used for constructing the assigning molecule, a configuration containing puromycin is used in a cell-free translation system so that a spacer can bind to the C-terminus of the protein translated there. Including puromycin includes including derivatives thereof. Examples of derivatives include the following. 3 N-aminoacylpuromycin aminonucleosides (3'-Ν—Aminoacylpuromycin aminonucleoside, PANS-amino acids), such as PANS-Gly for glycine in the amino acid portion, PANS-Val for phosphorus, PANS-Ala for alanine, etc. PANS-all amino acids corresponding to all amino acids can be used. In addition, 3'-N-aminoacyl adenosine aminonucleoside (an amide bond formed as a result of dehydration condensation of the amino group of 3'-aminoadenosine and the carboxyl group of amino acid as a chemical bond)
3'- Aminoacyladenosine aminonucleoside, AANS-アミノ酸)、例えばアミノ酸部がグリ シンの AANS-Gly、 ノ リンの AANS-Val、ァラニンの AANS-Ala、その他、全アミノ酸に 対応する AANS-全アミノ酸が利用できる。また、ヌクレオシドあるいはヌクレオシドとァ ミノ酸のエステル結合したものなども利用できる。その他、ヌクレオシドあるいはヌクレ オシドに類似した化学構造骨格を有する物質と、アミノ酸あるいはアミノ酸に類似した 化学構造骨格を有する物質を化学的に結合可能な結合様式のものなら全て利用す ることができる。 CCA領域 (CCA)の 5'側に 1残基以上の DNAおよび/または RNAからな る塩基配列を持つことが好ましい。塩基の種類としては、 C〉(U又は T)〉G〉Aの順で好 ましい。配列としては、 dC- Puromycin, rC- Puromycinなど、より好ましくは 3'-Aminoacyladenosine aminonucleoside (AANS-amino acid), for example, AANS-Gly of glycine, AANS-Val of norin, AANS-Ala of alanine, and other AANS-amino acids corresponding to all amino acids can be used. In addition, nucleosides or those in which a nucleoside and an amino acid are ester-linked can also be used. Other substances that have a chemical structural skeleton similar to nucleosides or nucleosides, Any substance can be used as long as the substance has a chemical structure and can be chemically bonded. It is preferable to have a nucleotide sequence comprising one or more residues of DNA and / or RNA on the 5 'side of the CCA region (CCA). As the type of base, C> (U or T)>G> A is preferable. As the sequence, dC-Puromycin, rC-Puromycin and the like, more preferably
dCdC-Puromycin, rCrC— Puromycin, rCdC— Puromycin, dCrC— Puromycinなどの酉己歹 [J で、アミノアシル- tRNAの 3'末端を模倣した CCA配列 (Philipps G.R. (1969) Nature 223, 374-377)が適当である。  dCdC-Puromycin, rCrC—Puromycin, rCdC—Puromycin, dCrC—Puromycin, etc. [J, a CCA sequence that mimics the 3 'end of aminoacyl-tRNA (Philipps GR (1969) Nature 223, 374-377) Appropriate.
[0039] C末端ラベルイ匕の場合など翻訳のみに用いる翻訳テンプレートでは、ピュー口マイ シンを含まない構成、または、アミノ酸との結合能力のないピューロマイシンを含む構 成が好ま 、。上記のピューロマイシン誘導体のァミノ基がアミノ酸と結合する能力を 欠いたあらゆる物質、およびピューロマイシンを欠いた CCA領域も考えられる力 リボ ソーム上でタンパク質と結合不能なピューロマイシンを含むことで、より翻訳効率を高 められる。その理由は定かではないが、タンパク質と結合不能なピューロマイシンがリ ポソームを刺激することでターンオーバーが促進される可能性がある。結合不能なピ ユーロマイシンでは、上記のピューロマイシンを適切な方法でアミノ酸と結合不能とす る。 [0039] In a translation template used only for translation, such as in the case of C-terminal labeling, a structure containing no puromycin or a structure containing puromycin having no binding ability to amino acids is preferable. Any substance in which the amino group of the above puromycin derivative lacks the ability to bind to amino acids, and the CCA region lacking puromycin may also be a potential force.Includes puromycin, which cannot bind proteins on the ribosome. Efficiency can be increased. The reason is unclear, but puromycin, which cannot bind to proteins, may stimulate turnover by stimulating liposomes. In the case of uncombined piuromycin, the above puromycin is made uncombinable with amino acids in an appropriate manner.
[0040] PEG部は修飾物質を有する構成が可能である。このことによって、翻訳テンプレート を回収、精製による再利用、あるいは固定ィ匕などのためのタグとして利用することが 出来る。少なくとも 1残基の DNAおよび/または RNAの塩基に修飾物質として、蛍光物 質、ピオチン、あるいは His-tagなど各種分離タグなどを導入したものが可能である。 また、コード部の 5'末端領域を SP6+029とし、 3'末端領域を、たとえば、 Flag+Xhol+A (n=8)とすることで、各長さは、 5'末端領域で約 60bp、 3'末端領域で約 40bpであり、 PCRのプライマーにアダプター領域として設計可能な長さである。これによつて新た な効果が生み出された。すなわち、あらゆるベクターやプラスミドや cDNAライブラリー カゝら PCRによって、 5'末端領域と 3'末端領域をもったコード部を簡単に作成可能となり 、このコード部に、 3'UTRの代わりとして PEG部をライゲーシヨンすることで、翻訳効率 の高 、翻訳テンプレートを得られる。  [0040] The PEG portion can be configured to have a modifying substance. As a result, the translation template can be recovered, reused by purification, or used as a tag for fixing. As a modifying substance, a fluorescent substance, biotin, or various separation tags such as His-tag may be introduced into at least one base of DNA and / or RNA. By setting the 5 'end region of the coding part to SP6 + 029 and the 3' end region to, for example, Flag + Xhol + A (n = 8), each length is about 60 bp in the 5 'end region. It is about 40 bp in the 3 'end region, and has a length that can be designed as an adapter region in a PCR primer. This has created a new effect. That is, any vector, plasmid or cDNA library PCR can easily create a coding part having a 5'-terminal region and a 3'-terminal region. By ligating the translation template, a translation template with high translation efficiency can be obtained.
[0041] PEG部とコード部のライゲーシヨンは、その方法については、一般的な DNAリガーゼ を用いるものや光反応による連結など何でもよぐ特に限定されるものではない。 RNA リガーゼを用いるライゲーシヨンでは、コード部でライゲーシヨン効率に影響を与える 範囲としては 3'末端領域の A配列が重要であり、少なくとも 2残基以上の dAおよび/ま たは rAの混合あるいは単一のポリ A連続鎖であり、好ましくは、 3残基以上、より好まし くは 6から 8残基以上のポリ A連続鎖である。 PEG部のドナー領域の 5'末端の DNAお よび/または RNA配列は、ライゲーシヨン効率を左右する。コード部と PEG部を、 RNAリ ガーゼでライゲーシヨンするためには、少なくとも 1残基以上を含むことが必要であり、 ポリ A配列をもつァクセプターに対しては、少なくとも 1残基の dC (デォキシシチジル酸 )あるいは 2残基の dCdC (ジデォキシシチジル酸)が好ましい。塩基の種類としては、 C 〉(U又は T)〉G〉Aの順で好ましい。さらに、ライゲーシヨン反応時に、 PEG領域と同じ分 子量のポリエチレングリコールを添加することが好ましい。 [0041] The ligation of the PEG part and the code part is performed by a general DNA ligase method. The method is not particularly limited, and any method such as a method using a compound or a photoreaction can be used. In the ligation using RNA ligase, the A sequence in the 3'-terminal region is important as the range that affects the ligation efficiency in the coding region, and at least 2 residues of dA and / or rA mixed or single It is a poly A continuous chain, preferably a poly A continuous chain of 3 or more residues, more preferably 6 to 8 residues or more. The DNA and / or RNA sequence at the 5 'end of the donor region of the PEG part affects ligation efficiency. In order to ligate the coding region and the PEG region with RNA ligase, it is necessary to include at least one or more residues.For an receptor having a poly A sequence, at least one residue of dC (doxycytidylic acid) is required. ) Or dCdC (dideoxycytidylic acid) having two residues. The type of the base is preferably in the order of C> (U or T)>G> A. Further, it is preferable to add polyethylene glycol having the same molecular weight as the PEG region during the ligation reaction.
[0042] 次に、選択対象タンパク質の C末端修飾 (ラベル化)につ!/、て述べる。修飾剤の存 在下で、選択対象タンパク質の翻訳テンプレートを用いた翻訳によって合成された、 修飾剤で C末端修飾された選択対象タンパク質であり、翻訳テンプレートと、修飾剤 力もなる。ここでの特徴は、特に翻訳テンプレートのコード部の構成にある。以下詳細 に記述する。  Next, the C-terminal modification (labeling) of the protein to be selected will be described. The protein to be selected, which is synthesized by the translation of the protein to be selected with the translation template in the presence of the modifying agent, and is C-terminally modified with the modifying agent, and also serves as the translation template and the modifying agent. The feature here lies particularly in the configuration of the code part of the translation template. The details are described below.
[0043] 翻訳テンプレートの PEG部は、ピューロマイシンがアミノ酸と連結出来ないことを特 徴とする。また、コード部も C末端ラベルイ匕に適した構成としては、 3'末端領域が、 XA 配列であることが重要であり、 X配列のなかで、最初の 4塩基が重要で、(C又は G)NN(C又は G)の配列を持つものが好ましい。ここでも、コード部の 5'末端領域を SP6+029とし、 3'末端領域を、たとえば、 Flag+Xhol+A (n=8)とすることで、各長さは、 5'末端領域で約 60bp、 3'末端領域で約 40bpであり、 PCRのプライマーにアダプター 領域として設計できる長さである。これによつて、 5'末端領域と 3'末端領域をもったコ 一ド部を PCRによって簡単に作成可能となり、このコード部に 3'UTRの代わりとして PEG部をライゲーシヨンすることで、 C末端ラベル化に適した翻訳効率の高 、翻訳テ ンプレートを得られる。  [0043] The PEG portion of the translation template is characterized in that puromycin cannot be linked to amino acids. In addition, as a structure suitable for C-terminal labeling, it is important that the 3 ′ terminal region is an XA sequence, and the first 4 bases are important in the X sequence, and (C or G ) Those having the sequence of NN (C or G) are preferred. Again, the length of the 5 'end region is about 5% in the 5' end region by setting the 5 'end region of the coding region to SP6 + 029 and the 3' end region to, for example, Flag + Xhol + A (n = 8). The length is about 60 bp and about 40 bp in the 3'-terminal region, and it can be designed as an adapter region for PCR primers. This makes it possible to easily create a code portion having a 5'-terminal region and a 3'-terminal region by PCR, and ligating a PEG portion instead of the 3'UTR to this code portion to obtain a C-terminal portion. A translation template with high translation efficiency suitable for labeling can be obtained.
[0044] 修飾剤は、タンパク質の翻訳系でのペプチド転移反応、すなわち、リボソーム上で のペプチド転移反応によってタンパク質と結合し得る基 (残基を含む)をもつァクセプ ター部が、ヌクレオチドリンカ一を介して修飾部と結合した構成をもつ。この修飾剤の 存在下でタンパク質合成を行い、得られる C末端修飾タンパク質を精製し、分子間相 互作用の検出系を用いることによって、タンパク質の検出や相互作用の検出が可能 となる。修飾部には、 PEG部と同様に修飾物質が含まれる。修飾物質として、非放射 性修飾物質の具体例としては、蛍光性、非蛍光性修飾物質等が挙げられる。蛍光性 物質としては、フルォレセイン系列、ローダミン系列、 Cy3、 Cy5、ェォシン系列、 NBD 系列等の蛍光色素や、緑色蛍光タンパク質 (GFP)等の蛍光性タンパク質がある。ま た、非蛍光性物質としては、ピオチンのような補酵素、タンパク質、ペプチド、糖類、 脂質類、色素、ポリエチレングリコール等、何らかの目印となり得る化合物であればい かなるものでもよい。修飾剤においては、修飾部が蛍光基、タンパク質と結合する基 、または、その両方をもつことが好ましい。ァクセプター部は、タンパク質の翻訳系で 、ペプチド転移反応によってタンパク質と結合し得る基をもち、好ましくはピュー口マイ シン又はその誘導体の残基をもつ。ピューロマイシンはアミノアシル tRNAと類似した 構造をもち、タンパク質合成を阻害する抗生物質として知られているが、低濃度では タンパク質の C末端に結合することが知られている(Miyamoto-Sato, E. et al. (2000) Nucleic Acids Res. 28: 1176-1182)。用いることができるピューロマイシン誘導体は、 ピューロマイシンと類似した構造を有し、タンパク質の C末端に結合することができる 物質であればいかなるものでもよい。具体例としては、 3'-N-アミノアシルピュー口マイ シンアミノヌクレオシド、 3'-N-アミノアシルアデノシンアミノヌクレオシド等が挙げられる 。修飾部とァクセプター部との間をつなぐヌクレオチドリンカ一とは、具体的には、リボ ヌクレオチドまたはデォキシリボヌクレオチドが 1個ないし複数個つながった核酸また は核酸誘導体であり、特に好ましい例として、シトシン塩基を含むリボヌクレオチド( -rC-)またはデォキシリボヌクレオチド (_dC-)が 1個な 、し複数個つながったィ匕合物 が挙げられる。その他、修飾部とァクセプター部との間に挿入することによって修飾タ ンパク質の収量を上げることができる物質であれば 、かなるものでもよ、。本発明修 飾剤においては、ヌクレオチドリンカ一力 デォキシシチジル酸、 2'-デォキシシチ ジル -(3',5')-2'-デォキシチジル酸、リボシチジル酸、又は、リボシチジル -(3',5')-リボ シチジル酸であることが好まし!/、。 [0045] 修飾剤は、上記修飾部とァクセプター部とを所望のヌクレオチドリンカ一を介して、 それ自体既知の化学結合方法によって結合させることにより製造することができる。 具体的には、例えば、適当な保護基で保護された上記ァクセプター部を固相担体上 に結合させ、核酸合成機を用いてヌクレオチドリンカ一としてヌクレオチドホスホアミダ イト、およびデォキシヌクレオチドホスホアミダイト、機能性修飾物質として蛍光物質や ピオチンなどを結合したホスホアミダイトを順次結合させた後、脱保護を行うことによつ て作製することができる。上記各部の種類、あるいは結合の種類によっては液相合成 法で結合させるかあるいは両者を併用することもできる。また、機能性修飾物質として ニッケル等の金属イオンを用いる場合には、金属イオンが配位しうる-トリ口トリ酢酸 やイミノジ酢酸等のキレート性の試薬を結合させ、次 、で金属イオンを配位させること ができる。 [0044] The modifying agent is an axepept having a group (including a residue) capable of binding to a protein by a transpeptidation reaction in a protein translation system, ie, a transpeptidation reaction on a ribosome. The linker has a configuration in which the linker is linked to the modifier via a nucleotide linker. By performing protein synthesis in the presence of this modifying agent, purifying the resulting C-terminal modified protein, and using a system for detecting an intermolecular interaction, it becomes possible to detect the protein and the interaction. The modifying part contains a modifying substance as in the case of the PEG part. Specific examples of the non-radioactive modifying substance as the modifying substance include fluorescent and non-fluorescent modifying substances. Examples of the fluorescent substance include fluorescent dyes such as fluorescein series, rhodamine series, Cy3, Cy5, eosin series, and NBD series, and fluorescent proteins such as green fluorescent protein (GFP). In addition, as the non-fluorescent substance, any compound may be used as a marker, such as a coenzyme such as biotin, a protein, a peptide, a saccharide, a lipid, a dye, and polyethylene glycol. In the modifying agent, the modifying portion preferably has a fluorescent group, a group that binds to a protein, or both. The receptor section has a group capable of binding to a protein by a transpeptidation reaction in a protein translation system, and preferably has a residue of pure mouth mycin or a derivative thereof. Puromycin has a structure similar to aminoacyl-tRNA and is known as an antibiotic that inhibits protein synthesis, but is known to bind to the C-terminus of proteins at low concentrations (Miyamoto-Sato, E. et. al. (2000) Nucleic Acids Res. 28: 1176-1182). The puromycin derivative that can be used is any substance having a structure similar to puromycin and capable of binding to the C-terminus of the protein. Specific examples include 3′-N-aminoacyl pure mouth aminoaminonucleoside, 3′-N-aminoacyladenosine aminonucleoside and the like. The nucleotide linker that connects between the modification part and the receptor part is, specifically, a nucleic acid or a nucleic acid derivative in which one or more ribonucleotides or deoxyribonucleotides are connected, and particularly preferred examples include: An example is a conjugate having one ribonucleotide (-rC-) or one deoxyribonucleotide (_dC-) containing a cytosine base. In addition, any substance that can increase the yield of the modified protein by inserting it between the modified part and the receptor part may be used. In the modifying agent of the present invention, the nucleotide linker is deoxycytidylic acid, 2'-deoxycitidyl- (3 ', 5')-2'-deoxytidylic acid, ribocytidylic acid, or ribocytidyl- (3 ', 5')- Preferably it is ribocytidylic acid! / ,. [0045] The modifying agent can be produced by binding the above-mentioned modified portion and the acceptor portion via a desired nucleotide linker by a chemical bonding method known per se. Specifically, for example, the above-mentioned receptor portion protected with an appropriate protecting group is bound to a solid-phase carrier, and a nucleotide phosphoramidite and a deoxynucleotide phosphoramidite are used as a nucleotide linker using a nucleic acid synthesizer. It can be produced by sequentially binding a phosphoramidite to which a fluorescent substance or biotin is bound as a functional modifier, and then performing deprotection. Depending on the type of the above components or the type of bonding, they can be combined by a liquid phase synthesis method, or both can be used in combination. When a metal ion such as nickel is used as the functional modifier, the metal ion can be coordinated with a chelating reagent such as triacetate triacetic acid or iminodiacetic acid, and then the metal ion is distributed in the next step. Can be placed.
[0046] 翻訳テンプレートの PEG部は、ピューロマイシンがアミノ酸と連結できることを特徴と する以外は前記したものとと同様である。また、コード部も前記したものと同様である 力 特に、対応付けに適した構成としては、 3'末端領域を A配列にすることが重要で あり、トータル蛋白の対応付けの効率が著しく向上してフリータンパク質の量が激減 することが確認された。ここでも、コード部の 5'末端領域を SP6+029とし、 3'末端領域 を、たとえば、 Flag+Xhol+A (n=8)とすることで、各長さは、 5'末端領域で約 60bp、 3'末 端領域で約 40bpであり、 PCRのプライマーにアダプター領域として設計できる長さで ある。これによつて、あらゆるベクターやプラスミドや cDNAライブラリーから PCRによつ て、 5'末端領域と 3'末端領域をもったタンパク質のコード部を簡単に作成可能となり、 PEG部をライゲーシヨンすることで、対応付け効率の高!、翻訳テンプレートが得られる 。また、 PEGによって C末端修飾されたタンパク質は、タンパク質の検出や相互作用 検出などにおいて、コード部を利用しない場合、たとえば、 FCCS測定、蛍光リーダー 、プロテインチップなどに応用する場合は、 RNase Aなどで意図的に切断することが 好ましい。切断することによって、コード部の妨害によるタンパク質間相互作用の検出 の困難性が解消出来る。  [0046] The PEG portion of the translation template is the same as described above, except that puromycin can be linked to amino acids. In addition, the coding portion is the same as that described above. In particular, as a configuration suitable for association, it is important that the 3 ′ end region is an A sequence, and the efficiency of association of total proteins is significantly improved. It was confirmed that the amount of free protein was drastically reduced. Again, the length of the 5'-end region is about 5% in the 5'-end region by setting the 5'-end region of the coding region to SP6 + 029 and the 3'-end region to, for example, Flag + Xhol + A (n = 8). It is 60 bp, about 40 bp in the 3 'terminal region, and has a length that can be designed as an adapter region for PCR primers. This makes it possible to easily create a coding region for a protein having a 5'-terminal region and a 3'-terminal region from any vector, plasmid or cDNA library by PCR, and to ligate the PEG region. , High association efficiency !, and translation templates. In addition, PEG-C-terminally modified proteins can be used with RNase A when the coding part is not used for protein detection or interaction detection, for example, when applied to FCCS measurement, fluorescent readers, protein chips, etc. It is preferable to cut intentionally. Cleavage can eliminate the difficulty of detecting protein-protein interactions due to interference with the coding region.
[0047] タンパク質 cDNAライブラリーから IWを形成させるには、無細胞翻訳系を用いる。具 体的には、ジチオスレィトール (DTT)や β -メルカプトエタノールのようなチオール化 合物を含む、又は含まない小麦胚芽抽出液、ゥサギ網状赤血球抽出液、大腸菌 S-30抽出液を用いる。特に、これまで DTTを含む無細胞翻訳系では、活性のある抗 体をタンパク質として発現させることが困難であつたが、本発明では DTT存在下でも IWを用いることにより、活性のある抗体を選択することが可能であることがわ力つた。 これらの無細胞タンパク質合成系の中に、上記タンパク質の翻訳テンプレートをカロえ 、 25— 37°Cで 1一数時間保温することによって IWを形成させる。 C末端ラベル化の 場合は、同時に 1一 100 Mの修飾剤をカロえると、 C末端修飾タンパク質が合成される 。合成された IVVは、そのまま次の、加熱処理工程、又は逆転写の工程に供する。 [0047] In order to form IW from the protein cDNA library, a cell-free translation system is used. Specifically, thiolation such as dithiothreitol (DTT) and β-mercaptoethanol A wheat germ extract containing or not containing the compound, a heron reticulocyte extract, and an Escherichia coli S-30 extract are used. In particular, it has been difficult to express an active antibody as a protein in the cell-free translation system containing DTT, but in the present invention, it is possible to select an active antibody by using IW even in the presence of DTT. I was able to do it. In these cell-free protein synthesis systems, the translation template of the above protein is caloried, and incubated at 25-37 ° C for several hours to form IW. In the case of C-terminal labeling, when one hundred and 100 M modifiers are simultaneously consumed, a C-terminal modified protein is synthesized. The synthesized IVV is directly used for the next heat treatment step or reverse transcription step.
[0048] 対応付け分子 (「IW」ともいう)を翻訳後直接加熱する場合は、通常には 50-100°C で 1-30分の範囲力 選択される条件で処理する。予め IWの mRNA部を逆転写酵素 によって RNA-DNAハイブリッドにする場合は、無細胞翻訳系に逆転写反応を阻害す る物質が含まれているので、除去することが好ましい。除去操作には、ゲルろ過、好 ましくは Sephadex G200(Amersham Bioscience社製)、又はスピンカラム、好ましくは Ultrafree MC, 100,000 NMWL (ミリポア社製)、又は大腸菌の無細胞翻訳系の PURESYSTEM (ポストゲノム研究所社製)の場合は、ニッケル榭脂を用いる。  In the case where the assigning molecule (also referred to as “IW”) is directly heated after translation, it is usually processed at 50-100 ° C. under a condition that a force in a range of 1-30 minutes is selected. When the mRNA portion of IW is converted into an RNA-DNA hybrid with a reverse transcriptase in advance, the cell-free translation system contains a substance that inhibits the reverse transcription reaction. For the removal operation, gel filtration, preferably Sephadex G200 (manufactured by Amersham Bioscience), or spin column, preferably Ultrafree MC, 100,000 NMWL (manufactured by Millipore), or PURESYSTEM (post-genome) of E. coli cell-free translation system (Manufactured by Research Institute), use nickel resin.
[0049] 逆転写反応 (RT)により、 IWの mRNA部を RNA- DNAハイブリッドにすることにより、 mRNA部の担体および抗原への非特異的吸着が阻止できる。逆転写反応は、 mRNA を 65°Cで加熱変性させた後、 4°Cに冷却してァニールさせ、 ReverTra Ace(TOYOBO 社製)を加えて、 50°Cで 30分反応させる。逆転写酵素は、逆転写反応を行うものであ れば、いかなる酵素、いかなる条件であってもよい。上記の条件に限定されるもので はない。 mRNA部を RNA-DNAノヽイブリツドにした IVVの加熱処理条件も、通常には 50-100°Cで 1-30分の範囲力 選択される条件である力 逆転写の最高温度より高い 温度とする。  [0049] By making the mRNA portion of the IW into an RNA-DNA hybrid by the reverse transcription reaction (RT), nonspecific adsorption of the mRNA portion to the carrier and the antigen can be prevented. In the reverse transcription reaction, mRNA is denatured by heating at 65 ° C, then cooled to 4 ° C to anneal, ReverTra Ace (TOYOBO) is added, and the reaction is performed at 50 ° C for 30 minutes. The reverse transcriptase may be any enzyme under any conditions as long as it performs a reverse transcription reaction. It is not limited to the above conditions. The heat treatment conditions of IVV with mRNA part as RNA-DNA hybrid are also usually in the range of 1 to 30 minutes at 50-100 ° C. Force that is the condition selected is higher than the maximum temperature of reverse transcription. .
[0050] RT-PCRおよび PCRに使用される DNAポリメラーゼは、 PCR反応に用いられるもので あれば特に制限されるものではないが、高い増幅効率と、 PCRの忠実度 (fidelity)が高 すぎないものがより好ましい。本発明で使用した KOD Dash(TOYOBO製品)は極めて 微量の铸型 DNAからも効率よく増幅が可能であり、また適度な変異が導入されるため 、より高機能なものに進化させることができる。 [0051] 標的分子はペプチド (化学合成された、又は天然から単離された、又はタンパク質 の部分消化物であってもよい)、タンパク質、核酸 (DNA、又は RNA)、糖類、種々の低 分子化合物、金属、金属化合物など、あらゆる化合物や物質が該当する。 [0050] The DNA polymerase used for RT-PCR and PCR is not particularly limited as long as it is used for the PCR reaction, but the high amplification efficiency and the fidelity of the PCR are not too high. Are more preferred. The KOD Dash (TOYOBO product) used in the present invention can be efficiently amplified even from a very small amount of type I DNA, and can be evolved into a more highly functional one because appropriate mutations are introduced. [0051] The target molecule is a peptide (which may be chemically synthesized or isolated from nature or may be a partial digest of a protein), a protein, a nucleic acid (DNA or RNA), a saccharide, various small molecules. All compounds and substances, such as compounds, metals and metal compounds, are applicable.
[0052] 本発明に用いられる標的分子は態様に応じて、榭脂、ビーズ等の固相に結合させ る力 固相に結合させる方法としては、修飾物質を介して結合させるものと、それ以 外の部分により結合させるものが挙げられる。  [0052] Depending on the embodiment, the target molecule used in the present invention may be a force capable of binding to a solid phase such as a resin or a bead. One that is bound by an outer portion is included.
[0053] 修飾物質を介して結合させる場合に用いられる修飾物質は、通常には、特定のポリ ペプチドに特異的に結合する分子 (以下、「リガンド」と称することがある。)であり、固 相表面には該リガンドと結合する特定のポリペプチド (以下、「アダプタータンパク質」 と称することがある)を結合させる。アダプタータンパク質には、結合タンパク質、受容 体を構成する受容体タンパク質、抗体なども含まれる。アダプタータンパク質 Zリガン ドの組み合わせとしては、例えば、アビジンおよびストレプトアビジン等のピオチン結 合タンパク質 Zピオチン、マルトース結合タンパク質 Zマルトース、 Gタンパク質 Zグ ァニンヌクレオチド、ポリヒスチジンペプチド Zニッケルある 、はコバルト等の金属ィォ ン、グルタチオン S—トランスフェラーゼ Zグルタチオン、 DNA結合タンパク質 ZDN A、抗体 Z抗原分子 (ェピトープ)、カルモジュリン Zカルモジュリン結合ペプチド、 A TP結合タンパク質 ZATP、あるいはエストラジオール受容体タンパク質 Zエストラジ オールなどの各種受容体タンパク質 Zそのリガンドなどが挙げられる。  [0053] The modifying substance used in the case of binding via the modifying substance is usually a molecule that specifically binds to a specific polypeptide (hereinafter, may be referred to as "ligand"). A specific polypeptide (hereinafter, sometimes referred to as “adaptor protein”) that binds to the ligand is bound to the phase surface. The adapter protein also includes a binding protein, a receptor protein constituting the receptor, an antibody, and the like. Examples of the combination of adapter protein Z ligands include biotin-binding protein Z-biotin such as avidin and streptavidin, maltose-binding protein Z-maltose, G-protein Z guanine nucleotide, polyhistidine peptide Z-nickel, and cobalt. Various receptors such as metal ion, glutathione S-transferase Z glutathione, DNA binding protein ZDNA, antibody Z antigen molecule (epitope), calmodulin Z calmodulin binding peptide, ATP binding protein ZATP, or estradiol receptor protein Z estradiol Body protein Z and its ligands.
[0054] これらの中で、アダプタータンパク質 Zリガンドの組み合わせとしては、アビジンおよ びストレプトアビジンなどのピオチン結合タンパク質、マルトース結合タンパク質 Zマ ルトース、ポリヒスチジンペプチド Zニッケルあるいはコバルト等の金属イオン、グルタ チオン s—トランスフェラーゼ Zダルタチオン、などが好ましぐ特にストレプトァビジ ン zピオチンの組み合わせが最も好ましい。これらの結合タンパク質は、それ自体既 知のものであり、該タンパク質をコードする DNAは既にクローユングされている。  [0054] Among these, combinations of adapter protein Z ligands include a biotin binding protein such as avidin and streptavidin, a maltose binding protein Z maltose, a polyhistidine peptide Z a metal ion such as nickel or cobalt, and glutathione. Particularly preferred is a combination of streptavidin z-biotin, in which s-transferase Z daltathione is preferred. These binding proteins are known per se, and the DNA encoding the protein has already been closed.
[0055] アダプタータンパク質の固相表面への結合は、それ自体既知の方法を用いることが できるが、具体的には、例えば、タンニン酸、ホルマリン、ダルタルアルデヒド、ピルビ ックアルデヒド、ビス ジァゾ化べンジゾン、トルエン- 2,4-ジイソシァネート、アミノ基、 活性エステルに変換可能なカルボキシル基、又はホスホアミダイドに変換可能な水 酸基もしくはアミノ基などを利用する方法を用いることができる。 [0055] The binding of the adapter protein to the surface of the solid phase can be carried out by a method known per se, and specifically, for example, tannic acid, formalin, dartalaldehyde, pyrvicaldehyde, benzodiazobenzidine , Toluene-2,4-diisocyanate, amino group, carboxyl group convertible to active ester, or water convertible to phosphoramidite A method using an acid group or an amino group can be used.
[0056] 修飾物質以外の部分により固相に結合させる場合は、通常タンパク質、核酸、糖鎖 、低分子化合物を固相に結合させるのに用いられる既知の方法、具体的には例えば 、タンニン酸、ホルマリン、グルタルアルデヒド、ピルビックアルデヒド、ビスージァゾィ匕 ベンジゾン、トルエン- 2, 4-ジイソシァネート、アミノ基、活性エステルに変換可能な力 ルポキシル基、又はホスホアミダイドに変換可能な水酸基もしくはァミノ基などを利用 する方法を用いることができる。  When binding to the solid phase by a moiety other than the modifying substance, a known method usually used to bind proteins, nucleic acids, sugar chains, and low molecular weight compounds to the solid phase, specifically, for example, tannic acid , Formalin, glutaraldehyde, pyrvicaldehyde, bis-diazodyl benzidine, toluene-2,4-diisocyanate, amino group, hydroxyl group which can be converted to active ester, or hydroxyl or amino group which can be converted to phosphoamidide Can be used.
[0057] 固相担体は、好ましくはァガロースビーズ、磁性体が包埋されたァガロースビーズ が好ましい。固相表面と抗原分子との距離は、立体的な観点から 30オングストローム 以上離れて!/、ることが好ま U、。  [0057] The solid phase carrier is preferably agarose beads or agarose beads in which a magnetic substance is embedded. The distance between the solid surface and the antigen molecule should be more than 30 angstroms from a steric point of view!
[0058] 高機能性タンパク質を取得する方法として一般的には 2つの方法が挙げられる。以 下、高親和性抗体を例として説明する。 1つは選択実験に使用する抗原濃度を低く 設定することで、その濃度の抗体のみを選択する方法である。この方法は回収される 抗体の量も当然少なくなるため効率が悪ぐそれにともなって、設定できる抗原濃度 も回収可能な濃度に制限される。 2つめは off- rate selection (Hawkins, R. (1992) J. Mol. Biol. 226, 889—896; Boder, E. (1997) Nat. Biotechnol. 15, 553—557; Jermutus, L. (2001) Proc. Natl. Acad. Sci. USA 98, 75- 80)と呼ばれるもので、抗原と抗体の解 離速度を利用した方法である。解離定数 (Kd)は結合速度定数 (on-rate)と解離速度 定数 (off-rate)の比で表されるが、生体高分子の結合定数はほぼ一定の範囲内 (104 - 106M— ^ にあるため実質の Kdは解離速度定数によって決まる。従って、解離速度 が遅い抗体ほどその親和性は高いため、抗原での溶出時間を長くすればするほど、 より親和性の高い抗体を濃縮することができる。し力しながら、この方法は 1サイクル の選択実験に有する時間が長いといった問題がある。この問題を解決する方法とし て、抗原で溶出するのではなぐ抗原または抗体を配列特異的にプロテア一ゼで消 化させることによって溶出する方法がある。このプロテアーゼは TMVプロテアーゼゃ 因子 (factor) Xaを代表とする極めて基質特異性の高いものから、トリプシンやプロティ ナーゼ Kと 、つた配列特異性の低 、ものまでを含むあらゆるプロテアーゼによって制 限されない。以上の方法は本発明においても適応可能であり、また組み合わせること で極めて有効な方法となる。 [0058] There are generally two methods for obtaining a highly functional protein. Hereinafter, a high affinity antibody will be described as an example. One is to set the antigen concentration to be low in the selection experiment and select only the antibody at that concentration. In this method, the amount of antibody to be recovered is naturally small, and the efficiency is low. Accordingly, the antigen concentration that can be set is limited to the concentration that can be recovered. The second is off-rate selection (Hawkins, R. (1992) J. Mol. Biol. 226, 889-896; Boder, E. (1997) Nat. Biotechnol. 15, 553-557; Jermutus, L. (2001 Natl. Acad. Sci. USA 98, 75-80), which uses the dissociation rate of antigen and antibody. Although the dissociation constant (Kd) is represented by the ratio of the association rate constant (on-rate) and dissociation rate constant (off-rate), association constant of biopolymers substantially within a certain range (10 4 - 10 6 M — Because of the ^, the real Kd is determined by the dissociation rate constant, so antibodies with a slower dissociation rate have a higher affinity, so the longer the elution time with the antigen, the more concentrated the antibody with higher affinity However, this method has the disadvantage that it takes longer to perform one cycle of a selection experiment, and a solution to this problem is to use an antigen or antibody sequence-specific instead of eluting with the antigen. The protease is eluted by quenching with proteinase, which has a very high substrate specificity, such as TMV protease factor Xa, from trypsin, proteinase K, and the like. Specificity The above methods are also applicable to the present invention, and are not limited by any proteases including, but not limited to, Is an extremely effective method.
[0059] 本発明の選択法により選択された核酸を用いてタンパク質を製造する方法、すなわ ち、本発明の選択法により標的分子と相互作用するタンパク質をコードする核酸を選 択する工程、および、選択された核酸を翻訳してタンパク質を製造する工程を含む製 造法も提供される。  [0059] A method for producing a protein using the nucleic acid selected by the selection method of the present invention, that is, a step of selecting a nucleic acid encoding a protein that interacts with a target molecule by the selection method of the present invention, and A method for producing a protein by translating a selected nucleic acid is also provided.
[0060] 核酸を選択する工程は、本発明の選択法に関して説明したとおりである。  [0060] The step of selecting a nucleic acid is as described for the selection method of the present invention.
核酸を用いたタンパク質の製造は、通常の方法に従って行うことできる。例えば、核 酸を無細胞翻訳系で翻訳してもよ 、し、核酸を導入したプラスミドを用いることなどに より核酸で大腸菌等の生細胞を形質転換して、その生細胞内でタンパク質を発現さ せてもよい。無細胞翻訳系としては、ジチオスレィトールや j8 -メルカプトエタノールの ようなチオール化合物を含む無細胞翻訳系、好ましくは、小麦胚芽抽出液、ゥサギ網 状赤血球抽出液、または、大腸菌 S-30抽出液が挙げられる。  Production of a protein using a nucleic acid can be performed according to a usual method. For example, the nucleic acid may be translated by a cell-free translation system, or a living cell such as Escherichia coli may be transformed with the nucleic acid by using a plasmid into which the nucleic acid has been introduced, and the protein may be expressed in the living cell. You may let them. As the cell-free translation system, a cell-free translation system containing a thiol compound such as dithiothreitol or j8-mercaptoethanol, preferably, a wheat germ extract, a rabbit egret reticulocyte extract, or an Escherichia coli S-30 extract Liquid.
[0061] タンパク質を発現させる際には、選択された核酸によりコードされるタンパク質と、酵 素又は緑色蛍光タンパク質 (Green Fluorescent Protein: GFP)との融合タンパク質とし て発現させてもよい。  [0061] When expressing the protein, the protein may be expressed as a fusion protein of a protein encoded by the selected nucleic acid and an enzyme or a green fluorescent protein (GFP).
[0062] 本発明の製造法により得られるタンパク質の例としては一本鎖抗体が挙げられる。  [0062] Examples of the protein obtained by the production method of the present invention include a single-chain antibody.
一本鎖抗体の例としては、抗原がアンジォテンシン IIの場合には、後記実施例に示 す Mil- 16 (61)、 Mil- 4 (63)、 MI2- 10 (65)、 MI2- 34 (67)、 MI2- 41 (69)、 MI2- 48 (7 1)、 MI3- 15 (73)、 MI3- 26 (75)、 MI3- 28 (77)、 MI3- 34 (79)、 MI3- 41 (81)、 MI3- 42 (83)、 MI3- 47 (85)、 MI3- 5 (87)、 MI3- 55 (89)、 MI3- 62 (91)、 MI3- 64 (93)、 MI3- 66 (95)、 MI3- 74 (97)、 MK2- 3 (99)、 MM2- 11 (101)、 MP1- 36 (103)、 MP1-41 (105)が挙げられる (括弧内の数字はアミノ酸配列の配列番号を示す)。ま た、抗原力 ewis Xである場合には、後記実施例に示す MI3-8 (107)、 MK1-15 (109 )、 MK1- 17 (111)、 MK1- 24 (113)、 MK2- 8 (115)、 MK2- 19 (117)が挙げられる(括 弧内の数字はアミノ酸配列の配列番号を示す)。これらの抗体のアミノ酸配列に相同 性の高いアミノ酸配列(例えば、 90%以上 (相同性計算法: GENETYX- MAC Version 7.3 (SOFTWARE DEVELOPMENT CO., LTD)の Maximum matching))または、これ らの抗体のアミノ酸配列において 1または数個(通常には 30個以下)の残基の置換、 欠失または挿入を有するアミノ酸を有するものは、同様な結合活性を有するものが多 いと予想される。したがって、本発明は、以下の抗体およびこれらをコードする核酸も 提供する。 As an example of a single-chain antibody, when the antigen is angiotensin II, Mil-16 (61), Mil-4 (63), MI2-10 (65), MI2-34 (67), MI2-41 (69), MI2-48 (71), MI3- 15 (73), MI3-26 (75), MI3-28 (77), MI3-34 (79), MI3-41 (81), MI3-42 (83), MI3-47 (85), MI3--5 (87), MI3-55 (89), MI3-62 (91), MI3-64 (93), MI3-66 ( 95), MI3-74 (97), MK2-3 (99), MM2-11 (101), MP1-36 (103), MP1-41 (105) (the numbers in parentheses indicate the amino acid sequence Number). In the case of antigenicity ewis X, MI3-8 (107), MK1-15 (109), MK1-17 (111), MK1-24 (113), MK2-8 ( 115) and MK2-19 (117) (the number in parentheses indicates the amino acid sequence SEQ ID NO). Amino acid sequences with high homology to the amino acid sequences of these antibodies (for example, 90% or more (Homology calculation: Maximum matching of GENETYX-MAC Version 7.3 (SOFTWARE DEVELOPMENT CO., LTD))) or the amino acid sequences of these antibodies Substitution of one or several (usually 30 or less) residues in the amino acid sequence, It is expected that many amino acids having a deletion or insertion have similar binding activities. Therefore, the present invention also provides the following antibodies and nucleic acids encoding them.
[0063] アンジォテンシン IIに対する結合活性を有する一本鎖抗体であって、下記 (A)又は (B)に示すアミノ酸配列を有する一本鎖抗体。  [0063] A single-chain antibody having angiotensin II binding activity, which has the amino acid sequence shown in the following (A) or (B).
(A)配列番号 61、 63, 65, 67, 69, 71、 73, 75, 77, 79, 81、 83, 85, 87, 89, 9 1、 93、 95、 97、 99、 101、 103また ίま 105に示すアミノ酸酉己歹 lj。  (A) SEQ ID NO: 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103 or Pamino 105 is an amino acid rooster.
(B) (A)のアミノ酸配列と相同性が 90%以上のアミノ酸配列を有するアミノ酸配列。  (B) An amino acid sequence having an amino acid sequence having 90% or more homology with the amino acid sequence of (A).
[0064] Lewis Xに対する結合活性を有する一本鎖抗体であって、下記 (A)又は (B)に示すァ ミノ酸配列を有する一本鎖抗体。 [0064] A single-chain antibody having binding activity to Lewis X, having the amino acid sequence shown in the following (A) or (B).
(A)配列番号 107、 109、 111、 113、 115または 117に示すアミノ酸配列。  (A) the amino acid sequence of SEQ ID NO: 107, 109, 111, 113, 115 or 117;
(B) (A)のアミノ酸配列と相同性が 90%以上のアミノ酸配列を有するアミノ酸配列。  (B) An amino acid sequence having an amino acid sequence having 90% or more homology with the amino acid sequence of (A).
[0065] また、本発明の選択法により選択された核酸を、 C末端ラベル化剤の存在下で無細 胞翻訳系で翻訳することによりタンパク質の C末端をラベル化する方法も提供される 。ラベル化は、特開平 11-322781、特開 2000-139468、 WO 02/46395に記載されたよ うにして行うことができる。無細胞翻訳系としては、小麦胚芽抽出液、ゥサギ網状赤血 球抽出液、大腸菌 S-30抽出液が挙げられる。 [0065] Also provided is a method for labeling the C-terminus of a protein by translating the nucleic acid selected by the selection method of the present invention with a cell-free translation system in the presence of a C-terminal labeling agent. Labeling can be performed as described in JP-A-11-322781, JP-A-2000-139468, and WO 02/46395. Cell-free translation systems include wheat germ extracts, egret reticulocyte extracts, and E. coli S-30 extracts.
[0066] 生細胞を用いてタンパク質を大量に発現させる具体例としては、大腸菌、枯草菌、 好熱菌、酵母等の細菌から、昆虫細胞、哺乳類等の培養細胞、さらに線虫、ショウジ ヨウバエ、ゼブラフィッシュ、マウス等に至るまでいかなる細胞でもよい。これらの細胞 の中に、上記 C末端ラベルイ匕ある ヽは対応付けされた両修飾タンパク質を直接導入 することもできるし、あるいは、上記翻訳テンプレートを導入し、 C末端ラベル化の場 合は、同時に 1一 100 /z Mの修飾剤を電気穿孔法、マイクロインジェクション法等により 細胞の中に導入し、細胞の至適生育温度で数時間保温することによって修飾タンパ ク質が合成される。対応付けの場合は、上記翻訳テンプレートを導入し、細胞の至適 生育温度で数時間保温することによって対応付け分子が合成される。合成された両 修飾タンパク質は、細胞を破砕することによって回収し次の精製プロセスまたは検出 プロセスに供することができる。また、そのまま細胞の中で検出プロセスに供すること も可能である。翻訳テンプレートは、用いる翻訳系に合わせて適切なものを選択する [0066] Specific examples of expressing proteins in large amounts using living cells include bacteria such as Escherichia coli, Bacillus subtilis, thermophiles, and yeast, cultured cells such as insect cells and mammals, as well as nematodes, Drosophila, Any cells, such as zebrafish and mice, may be used. In these cells, the above-mentioned C-terminal labeled 匕 can directly introduce both of the associated modified proteins, or, if the above-described translation template is introduced and the C-terminal labeling is carried out, simultaneously. 11. A modified protein is synthesized by introducing a 100 / zM modifier into cells by electroporation, microinjection, or the like, and keeping the cells at the optimal growth temperature for several hours. In the case of mapping, the mapping molecule is synthesized by introducing the above translation template and keeping the cells at the optimal growth temperature for several hours. Both synthesized modified proteins can be recovered by disrupting the cells and subjected to subsequent purification or detection processes. In addition, subject to the detection process in the cell as it is Is also possible. Select an appropriate translation template according to the translation system used
[0067] 大腸菌でタンパク質を大量発現させるには、発現ベクターに目的の cDNAを導入す ることが必要となる。この時発現ベクターに含まれるもので最も重要なのは、 cDNAを[0067] In order to express a large amount of a protein in Escherichia coli, it is necessary to introduce a target cDNA into an expression vector. At this time, the most important thing contained in the expression vector is cDNA
RNAに転写させるためのプロモーターで多様なものが用いられている。発現しようと するタンパク質は、宿主である大腸菌に対して毒性をもつ場合もあるため、ほとんど のベクターでは何らかの形で発現を制御するようになって 、る。この制御は発現タン ノ^質をコードする遺伝子を組込んだベクターで大腸菌を形質転換してカゝら発現誘 導をかけるまでの間、大腸菌の増殖を妨げないためにも重要である。 Various promoters are used for transcription into RNA. Since the protein to be expressed may be toxic to the host Escherichia coli, most vectors have some form of expression control. This control is also important so that the growth of E. coli is not hindered until E. coli is transformed with a vector containing the gene encoding the expression protein and expression is induced.
[0068] プロモーターのすぐ下流、クローユングサイトのすぐ上流にはリボソーム結合部位、 [0068] Immediately downstream of the promoter and immediately upstream of the claw site, a ribosome binding site,
Shine-Dalgarno配列と呼ばれる塩基配列が含まれて!/、る。一般には使用するプロモ 一ターの下流域に付随してくるものをそのまま利用することが多 、。この部分は mRNAに転写されたあと、開始コドン AUGとともにリボソーム RNAに結合する。 Contains a base sequence called Shine-Dalgarno sequence! In general, it is often the case that what is attached to the downstream area of the promoter used is used as it is. After this part is transcribed into mRNA, it binds to ribosomal RNA along with the start codon AUG.
Shine-Dalgarno配列と開始コドンの間が 5— 10塩基の時に最も翻訳効率がよいといわ れている。現在では融合タンパク質として発現させることがほとんどであるため、巿販 のベクターに合わせて最適化されたプロモーターとそのすぐ下流に存在する融合タ ンパク質の開始コドンをそのまま利用することになる。  It is said that the translation efficiency is highest when there is 5-10 bases between the Shine-Dalgarno sequence and the start codon. At present, most of them are expressed as fusion proteins. Therefore, a promoter optimized for a commercially available vector and the start codon of the fusion protein immediately downstream of the promoter are used as they are.
[0069] 翻訳の効率ィ匕の観点からは、翻訳開始点付近の mRNAの高次構造が重要である。 From the viewpoint of translation efficiency, the higher-order structure of mRNA near the translation start point is important.
このため目的遺伝子の N末端付近をコードする塩基配列を、アミノ酸の置換なしに極 力アデ-ンゃチミンに富んだものに変換することが必要なこともある。さらに、高効率 の発現を期待するならば、大腸菌におけるアミノ酸のコドンの使用頻度を考慮し、大 腸菌に最適なコドンに置換することも重要である。  For this reason, it may be necessary to convert the nucleotide sequence encoding the vicinity of the N-terminus of the target gene into a product that is as rich as possible in adenyl- thymine without amino acid substitution. Furthermore, if high-efficiency expression is expected, it is important to consider the frequency of amino acid codon usage in Escherichia coli and to substitute codons optimal for E. coli.
[0070] また、通常大腸菌での発現系は細胞質内で行われるが、細胞質は還元的な環境 下にあるため、抗体のような S-S結合を有するタンパク質は封入体となってしまう。そこ で、抗体の場合には、大腸菌発現は酸ィ匕的な環境下にあるペリブラズム内で行うの が一般的である。この場合、抗体の N末端にシグナル配列(例えば pel Bリーダーや ompT)を導入することで達成される。し力しながら、ペリブラズムでの発現系は細胞質 に比べてその発現量が極めて低い。ここで、本発明で得られる抗体は DTTが存在す る還元的な環境下で選択されたものであるため、大腸菌の細胞質内で発現させた場 合にぉ 、ても、水溶性の機能性抗体として発現させることができる。 [0070] In general, the expression system in Escherichia coli is performed in the cytoplasm. However, since the cytoplasm is in a reducing environment, proteins having an SS bond, such as antibodies, become inclusion bodies. Therefore, in the case of an antibody, E. coli expression is generally performed in periplasm under an irritating environment. In this case, it is achieved by introducing a signal sequence (for example, pel B leader or ompT) into the N-terminus of the antibody. However, the expression level in periplasm is extremely low compared to the cytoplasm. Here, DTT exists in the antibody obtained in the present invention. Since it has been selected under a reducing environment, it can be expressed as a water-soluble functional antibody even when expressed in the cytoplasm of Escherichia coli.
[0071] 本発明の製造法によって得られた一本鎖抗体は、タンパク質の免疫学的検出に使 用できる。免疫学的検出の方法の例としては、ウェスタンプロット法、免疫染色法、蛍 光抗体染色法、抗体チップ法、免疫沈降法が挙げられる。  [0071] The single-chain antibody obtained by the production method of the present invention can be used for immunological detection of a protein. Examples of the immunological detection method include a Western blot method, an immunostaining method, a fluorescent antibody staining method, an antibody chip method, and an immunoprecipitation method.
[0072] また、本発明の製造法によって得られた一本鎖抗体と、タンパク質とを接触させ、一 本鎖抗体とタンパク質との相互作用を検出することにより、タンパク質相互作用を検 出することができる。タンパク質相互作用の検出の方法としては、蛍光相関分光法、 蛍光イメージングアナライズ法、蛍光共鳴エネルギー移動法、エバネッセント場分子 イメージング法、蛍光偏光解消法、表面プラズモン共鳴法、固相酵素免疫検定法が 挙げられる。  In addition, the protein interaction is detected by bringing the single-chain antibody obtained by the production method of the present invention into contact with a protein and detecting the interaction between the single-chain antibody and the protein. Can be. Methods for detecting protein interactions include fluorescence correlation spectroscopy, fluorescence imaging analysis, fluorescence resonance energy transfer, evanescent field molecular imaging, fluorescence depolarization, surface plasmon resonance, and enzyme-linked immunosorbent assay. Can be
[0073] ペプチドやタンパク質を、一本鎖抗体を用いて検出するためには、一本鎖抗体とぺ プチド、タンパク質を何らかの修飾剤、例えば蛍光色素で修飾したり、酵素や蛍光タ ンパク質 (GFP)との融合タンパク質を構築したりして、検出する必要がある。一本鎖抗 体とペプチド、タンパク質を蛍光色素で修飾する方法については前記した。融合タン パク質の構築は、 GFP又は酵素、好ましくはアルカリ性ホスファターゼゃ西洋ヮサビの ペルォキシダーゼを一本鎖抗体の C末端又は N末端に融合させる。このような一本 鎖抗体の融合タンパク質を無細胞翻訳系又は大腸菌やバキュロウィルス、動物細胞 の系で発現、精製して得る。 GFPの場合は蛍光で、アルカリ性ホスファターゼの場合 は可視光で、西洋ヮサビのペルォキシダーゼの場合は、化学発光をルミノメーターで 検出する。また、タンパク質の検出には、一本鎖抗体のタグ (Flag-tag、 T7-tag、 HA-tag等)の二次抗体と、 GFP又はアルカリ性ホスファターゼゃ西洋ヮサビのペルォ キシダーゼとの融合タンパク質を用いてもよい。以下に、一本鎖抗体を用いたぺプチ
Figure imgf000030_0001
、て述べる。
[0073] In order to detect a peptide or protein using a single-chain antibody, the single-chain antibody, the peptide, and the protein are modified with some modifying agent, for example, a fluorescent dye, or an enzyme or a fluorescent protein ( It is necessary to construct a fusion protein with GFP) and detect it. The method for modifying single-chain antibodies, peptides and proteins with fluorescent dyes has been described above. Construction of the fusion protein involves fusing GFP or an enzyme, preferably alkaline phosphatase {horseradish rust) peroxidase, to the C-terminus or N-terminus of the single-chain antibody. Such a single-chain antibody fusion protein is obtained by expression and purification in a cell-free translation system or a system of Escherichia coli, baculovirus, or animal cells. Fluorescence is detected for GFP, visible light for alkaline phosphatase, and chemiluminescence for horseradish peroxidase with a luminometer. For protein detection, a fusion protein of a secondary antibody of a single-chain antibody tag (Flag-tag, T7-tag, HA-tag, etc.) and GFP or alkaline phosphatase ゃ horseradish peroxidase is used. May be. The following describes a peptide using a single-chain antibody.
Figure imgf000030_0001
, To be described.
[0074] (1)抗体による免疫染色  (1) Immunostaining with antibody
研究対象となる分子の解析を始める際に、その機能を推測する手段の一つにその 分子の局在性の検討が挙げられる。 目的分子の発現を検討するためにさまざまな方 法が開発されて 、るが、その!/、ずれもが実験材料の準備や実験系の確立に多くの時 間と手間を要する。それらの方法と比較して抗体を用いた組織染色は操作が比較的 簡便であり、準備するものは対象分子を認識する抗体のみである。ある分子の解析を 始めた時点でその分子を認識する抗体はなんらかの方法で手に入つている場合が 多いので、初めての人でもすぐに実験を開始できる。また遺伝子の発現という形では なぐタンパク質の存在を直接確認できるというのも本方法の優れた点である。しかし ながらすベての抗体が組織染色に用いることができるわけではない。その分子が生 体内でとる構造を認識できな 、抗体では良好な結果は望めな 、。組織染色に適した 抗体を準備できるかが勝負の分かれ目になる。 When starting to analyze a molecule to be studied, one of the means to estimate its function is to examine the localization of that molecule. Various methods have been developed to study the expression of the target molecule, but this is often the case in preparing experimental materials and establishing experimental systems. It takes time and effort. Compared with these methods, tissue staining using antibodies is relatively simple in operation, and only an antibody that recognizes the target molecule is prepared. When you start analyzing a molecule, antibodies that recognize that molecule are often obtained in some way, so even the first person can start the experiment immediately. Another advantage of the present method is that the presence of a protein can be directly confirmed in the form of gene expression. However, not all antibodies can be used for tissue staining. Good results cannot be expected with antibodies because they cannot recognize the structure of the molecule in vivo. The determination of the ability to prepare antibodies suitable for tissue staining is a key game.
[0075] 基本的な原理はウェスタンプロットと変わらな 、が、組織染色の場合には組織中に タンパク質をホルムアルデヒドなどでィ匕学的に固定しその局在を検出する。固定され た標的タンパク質を一次抗体および二次抗体を用いて検出するわけだ力 そのシグ ナルが微弱で検出が困難である場合には、目的のシグナルのみを特異的に増幅す る方法も開発されている。  [0075] The basic principle is the same as that of the Western plot, but in the case of tissue staining, the protein is fixed in the tissue with formaldehyde or the like, and its localization is detected. Ability to detect immobilized target protein using primary and secondary antibodies If the signal is weak and difficult to detect, a method has been developed to specifically amplify only the target signal. ing.
[0076] (2)蛍光抗体染色による内在性タンパク質の細胞内局在の観察  (2) Observation of intracellular localization of endogenous protein by fluorescent antibody staining
機能が不明であるタンパク質を解析するうえで、その分子を特異的に認識する抗体 を用いて細胞内における内在性タンパク質の局在を観察することは、非常に有用な 情報をもたらしてくれることが多 、。 GFP融合タンパク質として発現した場合などとは 異なり、生細胞のままその分子の動きを観察するというわけにはいかないが、刺激や 薬剤の処理に対する応答や細胞運動といったさまざまな状態における目的タンパク 質の局在の変化を観察することができる。 目的とするタンパク質に対する抗体がない 場合でもそのタンパク質にタグをつけた状態で細胞内に一過的に発現させるなどの 方法を用いることにより、タグに対する抗体を用いて同様に目的の分子の局在を観察 することはできる。し力しながらこの場合に観察されたタグつきタンパク質の局在は、 必ずしもそのタンパク質の実際の局在と一致するとは限らず、過剰に発現させたこと による結果であることも少なくない。実験の操作は比較的に容易であるが、抗体や目 的タンパク質の局在によっては固定方法の条件検討が必要になる点、そして何よりも 注目しているタンパク質に対する特異的な抗体を用意することが重要なポイントであ る。 [0077] 適切な固定法により細胞を固定した後、界面活性剤などを用いて膜を透過化( permealize;浸透性を高くする)し、 目的の分子に対する特異的な抗体 (一次抗体)を 用いて処理することにより、細胞内において目的タンパク質 抗体の複合体を形成さ せる。細胞内には非特異的に結合した抗体が多く残っているのでこれを洗浄して除 き、次に一次抗体を認識しさらに蛍光物質で標識してある抗体(二次抗体)を用いて 目的タンパク質 -一次抗体 -二次抗体という複合体を形成させる。一次抗体と同様に 非特異的に結合している二次抗体を洗浄して除き、封入し、顕微鏡で観察する。 In analyzing proteins of unknown function, observing the localization of endogenous proteins in cells using an antibody that specifically recognizes that molecule can provide very useful information. Many,. Unlike when expressed as a GFP fusion protein, etc., it is not possible to observe the movement of the molecule as it is in a living cell, but the localization of the target protein in various states such as response to stimulation and treatment of drugs and cell motility. You can observe the change in location. Even when there is no antibody against the protein of interest, localization of the molecule of interest using the antibody against the tag can be achieved by using a method such as transiently expressing the protein in a cell with the tag attached. Can be observed. However, the localization of the tagged protein observed in this case does not always coincide with the actual localization of the protein, and is often the result of overexpression. Although the operation of the experiment is relatively easy, it is necessary to examine the conditions of the immobilization method depending on the localization of the antibody and the target protein, and above all, prepare a specific antibody against the protein of interest Is an important point. After fixing the cells by an appropriate fixing method, the membrane is permealized (permealized; increased permeability) using a surfactant or the like, and a specific antibody (primary antibody) against the target molecule is used. Thus, a complex of the target protein and antibody is formed in the cells. Since many non-specifically bound antibodies remain in the cells, they are removed by washing, and then an antibody that recognizes the primary antibody and is labeled with a fluorescent substance (secondary antibody) A complex of protein-primary antibody-secondary antibody is formed. Like the primary antibody, the non-specifically bound secondary antibody is washed away, encapsulated, and observed under a microscope.
[0078] (3)ウェスタンブロット法  [0078] (3) Western blotting
ウェスタンプロットとは、電気泳動後のタンパク質を電気的にメンブレンに転写する 作業、あるいはそのような作業を含む一連の実験を言う。  The Western plot refers to an operation of electrically transferring a protein after electrophoresis to a membrane, or a series of experiments including such an operation.
[0079] タンパク質の電気泳動で一般的によく用いられる方法が SDS-PAGEである。試料に 、還元剤である SDSと負の電荷をもつ 2-メルカプトエタノールなどをカ卩えて熱処理する ことにより、タンパク質の高次構造がほぼ完全に破壊される。また、 SDSがタンパク質 の分子量比(1 : 1.4)でタンパク質に結合するため、均一な荷電状態となる。これをポリ アクリルアミドゲル中で泳動すると、タンパク質の分子量にしたがって分離することが できる。 [0079] A commonly used method for protein electrophoresis is SDS-PAGE. When the sample is treated with SDS as a reducing agent and 2-mercaptoethanol having a negative charge and heat-treated, the higher order structure of the protein is almost completely destroyed. In addition, since SDS binds to the protein at a protein molecular weight ratio (1: 1.4), the charge state becomes uniform. When this is electrophoresed in a polyacrylamide gel, it can be separated according to the molecular weight of the protein.
[0080] SDS-PAGE後、ゲル内に展開したタンパク質を電気泳動的にメンブレンに転写し、 試料中の目的タンパク質に対して特異的な抗体とメンブレン上で反応させて、 目的の タンパク質を免疫的に検出する。  [0080] After SDS-PAGE, the protein developed in the gel is electrophoretically transferred to a membrane, and reacted with an antibody specific to the target protein in the sample on the membrane to immunologically target the target protein. To be detected.
[0081] ウェスタンブロットは、ゲル内に展開されたタンパク質バンドをほとんど拡散させるこ となくメンブレン上に転写できる。また、抗原抗体反応の特異性が高ぐ感度が高いこ とも特徴である。したがって、 目的のタンパク質に対する抗体が入手できれば、特定 のタンパク質を高感度で検出できる、手軽でよい方法と言える。  [0081] Western blots can be transferred onto a membrane with little diffusion of the protein bands developed in the gel. It is also characterized by high sensitivity and high specificity of the antigen-antibody reaction. Therefore, if an antibody against the target protein is available, it can be said that it is a simple and convenient method that can detect a specific protein with high sensitivity.
[0082] ここでは、 SDS-PAGEおよび酵素活性を用いたウェスタンブロットの手法について 記述するが、特にこのプロトコルに限定されるわけではない。ウェスタンブロットの操 作の流れを以下に示す。 (1) SDSなどを含むサンプルバッファーでタンパク質試料を 変性させた後、 SDS-PAGEによりタンパク質を分子量に従って分離する。(2)泳動後 、ゲルをメンブレンと重ねて転写装置にセットし、ゲル内に展開したタンパク質バンド を電気的にメンブレン上に転写 (プロッティング)する。 (3)タンパク質への非特異的 吸着を防ぐためのブロッキングを行った後、 目的タンパク質を特異的に認識する抗体 と一次抗体反応させる。(4)一次抗体分子を特異的に認識する二次抗体 (発色酵素 つき)と二次抗体反応させる。(5)二次抗体につけた発色酵素の酵素活性、例えば ペルォキシダーゼ活性やアルカリ性ホスファターセ活性を利用して発色反応させ、 目 的タンパク質を検出する。 [0082] Here, the method of Western blot using SDS-PAGE and enzyme activity is described, but it is not particularly limited to this protocol. The flow of the Western blot operation is shown below. (1) After denaturing the protein sample with a sample buffer containing SDS, etc., separate proteins according to molecular weight by SDS-PAGE. (2) After electrophoresis, the gel is placed on the transfer device with the gel layered on it, and the protein band developed in the gel Is electrically transferred (plotted) onto the membrane. (3) After blocking to prevent nonspecific adsorption to the protein, a primary antibody reaction is performed with an antibody that specifically recognizes the target protein. (4) The secondary antibody reacts with a secondary antibody (with a chromogenic enzyme) that specifically recognizes the primary antibody molecule. (5) A color reaction is performed using the enzyme activity of the chromogenic enzyme attached to the secondary antibody, for example, a peroxidase activity or an alkaline phosphatase activity, and the target protein is detected.
[0083] (4)免疫沈降法  [0083] (4) Immunoprecipitation method
免疫沈降法(免沈、 Immunoprecipitation, IP)とは、特異的な抗原 抗体反応および 、一本鎖抗体の C末端部をピオチンで修飾したり、種々のタグ、例えば Flag-tag、 T7-tag、 HA-tag、 His-tagなど(これはァガロースビーズに共有結合しているストレプト アビジンやタグ抗体と結合するので、遠心により簡単に溶液と分けられる)を用いるこ とにより、使用した抗体に対応する抗原タンパク質だけを分離、精製する方法である 。ただ現在は、単に抗原タンパク質だけを分離、精製するのみならず、むしろ「洗い」 の強度や用いるライセートの界面活性剤を適度に弱めることで、抗体に直接結合す る抗原タンパク質と一緒に複合体を作っているタンパク質を検出し、 in vivoでのタン ノ ク質-タンパク質相互作用を立証する際に最もよく使われる。そのため、 in vitroで の結合しか立証できな!/、プル ダウンアツセィに比べ、検出できた際の意味合!ヽは大 きい。ただ免疫沈降法を行うにあたっては、特異性と親和性が高い抗体が必要であ る。この点、本発明で選択される高親和性抗体は目的に叶っている。一般に用いる 抗体はウェスタンブロットよりも高いタイター(107— IOVOI 1)を必要とし、かつ特異性 もある程度以上が要求される。 Immunoprecipitation (immunoprecipitation, IP) refers to a specific antigen-antibody reaction and modification of the C-terminal portion of a single-chain antibody with biotin, various tags such as Flag-tag, T7-tag, By using HA-tag, His-tag, etc. (this binds to streptavidin or tag antibody covalently bound to agarose beads, it can be easily separated from the solution by centrifugation), and the antigen corresponding to the used antibody can be obtained. It is a method to separate and purify only proteins. However, at present, not only is the antigen protein separated and purified, but rather the complex with the antigen protein that directly binds to the antibody by appropriately weakening the washing strength and the lysate detergent used. It is most often used to detect proteins that make proteins and to demonstrate protein-protein interactions in vivo. Therefore, binding can only be demonstrated in vitro! /, Compared to pull-down assays, the significance of detection is greater! However, when performing immunoprecipitation, an antibody with high specificity and affinity is required. In this regard, the high-affinity antibodies selected in the present invention serve the purpose. Commonly used antibodies require higher titers (10 7 -IOVOI 1 ) than Western blots, and require a certain degree of specificity.
[0084] また、上述したように洗浄に用いるバッファー、洗浄の回数、可溶化バッファーの組 成になどに応じて分離できるタンパク質複合体の様相が変化する。以下にプロトコル を示すが、特に限定されるものでない。(1)ライセートの回収:免疫沈降を行う準備段 階としてまず細胞な 、し組織力ゝらタンパク質溶液を得る必要がある。ここでどのような 濃度、組成の溶液を作るかで後の免疫沈降の結果も変わってくる。ライセートにはい ろ!、ろなタンパク質が含まれて 、る。 (2)抗原 抗体反応:ライセートに目的とするタン ノ ク質を認識する一本鎖抗体を加えることで抗原 抗体反応を起こさせる。 (3)ビー ズと抗体との結合:このままでは溶液内の抗体を分離できないため、それを可能とす るよう、ストレプトアビジンやタグ抗体を共有結合させたァガロースビーズを加える。ス トレブトアビジンやタグ抗体は一本鎖抗体の C末端の修飾されたピオチンやタグを特 異的に認識して結合するため、この操作によって一本鎖抗体がビーズに結合した形 となり、分離ができるようになる。(4)分離、精製:遠心することによりビーズを下に落と し、上清を除くことで分離する。さらに洗浄バッファーを加え、混和することで洗浄し、 非特異的に結合しているタンパク質をある程度取り除く。(5)精製したタンパク質複合 体の解析:タンパク質複合体に含まれて ヽるものをビーズカゝらタンパク質を溶出させ、 SDS— PAGEで分離することによって解析する。検出の方法としては、ウェスタンブロッ ティング (既知の場合で抗体をもって 、る時)や CBB染色などが用いられる。 CBB染 色して、未知のバンドが検出できた場合、 MSで固定することもできる。 As described above, the aspect of the protein complex that can be separated changes depending on the buffer used for washing, the number of washings, the composition of the solubilization buffer, and the like. The protocol is shown below, but is not particularly limited. (1) Recovery of lysate: As a preparation step for performing immunoprecipitation, it is necessary to first obtain a cell-free protein solution from cells. Depending on the concentration and composition of the solution, the results of subsequent immunoprecipitation will also vary. The lysate contains various proteins and various proteins. (2) Antigen-antibody reaction: An antigen-antibody reaction is caused by adding a single-chain antibody that recognizes the target protein to the lysate. (3) Bee Binding of antibody to antibody: Since the antibody in the solution cannot be separated as it is, add agarose beads to which streptavidin or a tag antibody is covalently bonded to enable this. Streptavidin and tag antibodies specifically recognize and bind to the C-terminal modified biotin and tag of the single-chain antibody, and this operation results in the single-chain antibody bound to the beads and separated. Will be able to (4) Separation and purification: The beads are dropped down by centrifugation and separated by removing the supernatant. Add wash buffer and mix by washing to remove non-specifically bound proteins to some extent. (5) Analysis of the purified protein complex: Analyze what is contained in the protein complex by eluting the beaded protein and separating it by SDS-PAGE. As a detection method, Western blotting (when an antibody is used when known) and CBB staining are used. If unknown bands can be detected by staining with CBB, they can be fixed with MS.
[0085] 本発明により得られる標的分子と相互作用するタンパク質 (以下、「機能性タンパク 質」ともいう)を用いた分子間相互作用の解析方法においては、通常には、上記で得 られた修飾機能性タンパク質と標的分子を、修飾物質の種類や反応系の種類などに より適宜組み合わせて接触せしめ、修飾機能性タンパク質又は標的分子が発する信 号にお 、て両分子間の相互作用に基づ!/、て発生される上記信号の変化を測定する ことにより相互作用を解析する。また、相互作用の解析は、例えば、蛍光相関分光法 、蛍光イメージングアナライズ法、蛍光共鳴エネルギー移動法、エバネッセント場分 子イメージング法、蛍光偏光解消法、表面プラズモン共鳴法、又は、固相酵素免疫 検定法により行われる。また、上記で得られた 2組の修飾機能性タンパク質と標的分 子を、修飾物質の種類や反応系の種類などにより適宜組み合わせて接触せしめ、修 飾機能性タンパク質が発する信号にぉ 、て両抗原分子間の相互作用に基づ 、て発 生される上記信号の変化を測定することにより相互作用を解析する。相互作用の解 析は、例えば、蛍光相関分光法、蛍光イメージングアナライズ法、蛍光共鳴エネルギ 一移動法、エバネッセント場分子イメージング法、蛍光偏光解消法、表面プラズモン 共鳴法、又は、固相酵素免疫検定法により行われる。以下これらの方法の詳細につ いては記述する。標的分子及び相互作用は、上記に説明した通りである。 [0085] In the method for analyzing an intermolecular interaction using a protein (hereinafter, also referred to as "functional protein") that interacts with a target molecule obtained by the present invention, the modification obtained above is usually used. The functional protein and the target molecule are brought into contact with each other by appropriately combining them according to the type of the modifying substance, the type of the reaction system, etc., and the signal generated by the modified functional protein or the target molecule is based on the interaction between the two molecules. ! / Analyze the interaction by measuring the change in the signal generated. The analysis of the interaction may be performed, for example, by fluorescence correlation spectroscopy, fluorescence imaging analysis, fluorescence resonance energy transfer, evanescent field molecular imaging, fluorescence depolarization, surface plasmon resonance, or solid-phase enzyme immunoassay. It is done by law. Further, the two sets of the modified functional protein obtained above and the target molecule are brought into contact with each other by appropriately combining them according to the type of the modifying substance, the type of the reaction system, and the like. The interaction is analyzed by measuring a change in the signal generated based on the interaction between the antigen molecules. Interaction analysis can be performed, for example, by fluorescence correlation spectroscopy, fluorescence imaging analysis, fluorescence resonance energy transfer, evanescent field molecular imaging, fluorescence depolarization, surface plasmon resonance, or enzyme-linked immunosorbent assay. Is performed by The details of these methods are described below. The target molecules and interactions are as described above.
[0086] 用いられる機能性タンパク質は、態様に応じて修飾物質により修飾して用いること ができる。修飾物質は、通常、蛍光性物質などの非放射性修飾物質から選択される 。蛍光物質としては、フリーの官能基 (例えばカルボキシル基、水酸基、アミノ基など) を持ち、タンパク質、核酸等の上記一本鎖抗体と連結可能な種々の蛍光色素、例え ばフルォレセイン系列、ローダミン系列、 Cy3、 Cy5、ェォシン系列、 NBD系列などの いかなるものであってもよい。その他、色素など修飾可能な化合物であれば、その化 合物の種類、大きさは問わない。 [0086] The functional protein to be used may be used after being modified with a modifying substance according to the embodiment. Can do. The modifier is usually selected from non-radioactive modifiers such as fluorescent substances. As the fluorescent substance, various fluorescent dyes having free functional groups (for example, carboxyl group, hydroxyl group, amino group, etc.) and capable of being linked to the above-mentioned single-chain antibodies such as proteins and nucleic acids, for example, fluorescein series, rhodamine series, Any of Cy3, Cy5, eosin series, NBD series and the like may be used. In addition, the type and size of the compound are not limited as long as the compound can be modified such as a dye.
[0087] これらの修飾物質は、標的分子と修飾機能性タンパク質との間の相互作用に基づ いて発生される信号の変化の測定又は解析方法に適したものが適宜用いられる。 上記修飾物質の機能性タンパク質への結合は、それ自体既知の適当な方法を用 いて行うことができる。具体的には、例えば、上に記載した C末端を修飾する方法等 を用いることができる。また、修飾機能性タンパク質または本発明に用いられる標的 分子は態様に応じて、固相に結合させる場合があるが、固相に結合させる方法として は、修飾物質を介して結合させるものと、それ以外の部分により結合させるものが挙 げられる。 [0087] As these modifying substances, those suitable for a method of measuring or analyzing a change in a signal generated based on an interaction between a target molecule and a modified functional protein are appropriately used. The above-mentioned modifying substance can be bound to the functional protein using an appropriate method known per se. Specifically, for example, the above-described method of modifying the C-terminus can be used. In addition, the modified functional protein or the target molecule used in the present invention may be bound to a solid phase depending on the embodiment. Those that are combined by other parts are listed.
[0088] 修飾物質を介して結合させる場合に用いられる修飾物質は、通常には、特定のポリ ペプチドに特異的に結合する分子 (以下、「リガンド」と称することがある。)であり、固 相表面には該リガンドと結合する特定のポリペプチド (以下、「アダプタータンパク質」 と称することがある)を結合させる。アダプタータンパク質には、結合タンパク質、受容 体を構成する受容体タンパク質、抗体なども含まれる。アダプタータンパク質 Zリガン ドの組み合わせとしては、例えば、アビジンおよびストレプトアビジン等のピオチン結 合タンパク質 Zピオチン、マルトース結合タンパク質 Zマルトース、 Gタンパク質 Zグ ァニンヌクレオチド、ポリヒスチジンペプチド Zニッケルある 、はコバルト等の金属ィォ ン、グルタチオン S—トランスフェラーゼ Zグルタチオン、 DNA結合タンパク質 ZDN A、抗体 Z抗原分子 (ェピトープ)、カルモジュリン Zカルモジュリン結合ペプチド、 A TP結合タンパク質 ZATP、あるいはエストラジオール受容体タンパク質 Zエストラジ オールなどの各種受容体タンパク質 Zそのリガンドなどが挙げられる。  [0088] The modifying substance used in the case of binding via a modifying substance is usually a molecule that specifically binds to a specific polypeptide (hereinafter, may be referred to as "ligand"). A specific polypeptide (hereinafter, sometimes referred to as “adaptor protein”) that binds to the ligand is bound to the phase surface. The adapter protein also includes a binding protein, a receptor protein constituting the receptor, an antibody, and the like. Examples of the combination of adapter protein Z ligands include biotin-binding protein Z-biotin such as avidin and streptavidin, maltose-binding protein Z-maltose, G-protein Z guanine nucleotide, polyhistidine peptide Z-nickel, and cobalt. Various receptors such as metal ion, glutathione S-transferase Z glutathione, DNA binding protein ZDNA, antibody Z antigen molecule (epitope), calmodulin Z calmodulin binding peptide, ATP binding protein ZATP, or estradiol receptor protein Z estradiol Body protein Z and its ligands.
[0089] これらの中で、アダプタータンパク質 Zリガンドの組み合わせとしては、アビジンおよ びストレプトアビジンなどのピオチン結合タンパク質、マルトース結合タンパク質 Zマ ルトース、ポリヒスチジンペプチド Zニッケルあるいはコバルト等の金属イオン、グルタ チオン s—トランスフェラーゼ Zダルタチオン、などが好ましぐ特にストレプトァビジ ン Zピオチンの組み合わせが最も好ましい。これらの結合タンパク質は、それ自体既 知のものであり、該タンパク質をコードする DNAは既にクローユングされている。 [0089] Among these, combinations of adapter protein Z ligands include biotin-binding proteins such as avidin and streptavidin, and maltose-binding protein Z-matrix. Particularly preferred is a combination of streptavidin Z-biotin, which is preferably a metal ion such as lactose, polyhistidine peptide Z nickel or cobalt, glutathione s-transferase Z daltathione, and the like. These binding proteins are known per se, and the DNA encoding the protein has already been closed.
[0090] アダプタータンパク質の固相表面への結合は、それ自体既知の方法を用いることが できるが、具体的には、例えば、タンニン酸、ホルマリン、ダルタルアルデヒド、ピルビ ックアルデヒド、ビス ジァゾ化べンジゾン、トルエン- 2,4-ジイソシァネート、アミノ基、 活性エステルに変換可能なカルボキシル基、又はホスホアミダイドに変換可能な水 酸基あるいはアミノ基などを利用する方法を用いることができる。 [0090] The binding of the adapter protein to the solid phase surface can be carried out by a method known per se, and specifically, for example, tannic acid, formalin, dartalaldehyde, pyrvicaldehyde, benzodiazobenzidine A method utilizing a toluene-2,4-diisocyanate, an amino group, a carboxyl group convertible to an active ester, or a hydroxyl group or amino group convertible to a phosphoramidide can be used.
[0091] 修飾物質以外の部分により固相に結合させる場合は、通常タンパク質、核酸、糖鎖 、低分子化合物を固相に結合させるのに用いられる既知の方法、具体的には例えば 、タンニン酸、ホルマリン、グルタルアルデヒド、ピルビックアルデヒド、ビスージァゾィ匕 ベンジゾン、トルエン- 2, 4-ジイソシァネート、アミノ基、活性エステルに変換可能な力 ルポキシル基、又はホスホアミダイドに変換可能な水酸基あるいはアミノ基などを利 用する方法を用いることができる。 When binding to a solid phase by a moiety other than a modifying substance, a known method usually used for binding proteins, nucleic acids, sugar chains, and low molecular weight compounds to a solid phase, specifically, for example, tannic acid , Formalin, glutaraldehyde, pyrvicaldehyde, bis-diazoi di-benzizone, toluene-2,4-diisocyanate, amino group, hydroxyl group that can be converted to active ester, or hydroxyl group or amino group that can be converted to phosphoamidide A method can be used.
[0092] 「測定」とは解析のために用いられる信号の変化を収集するための手段であり、い 力なる意味においても限定的に解釈してはならない。用いられる測定法としては、例 えば、蛍光相関分光法、蛍光共鳴エネルギー移動法、エバネッセント場分子イメージ ング法、蛍光偏光解消法、蛍光イメージングアナライズ法、表面プラズモン共鳴法、 固相酵素免疫検定法など、分子間相互作用を検出できるあらゆる系が利用可能であ る。 [0092] "Measurement" is a means for collecting changes in signals used for analysis, and should not be construed as limiting in any sense. Examples of the measuring method used include fluorescence correlation spectroscopy, fluorescence resonance energy transfer, evanescent field molecular imaging, fluorescence depolarization, fluorescence imaging analysis, surface plasmon resonance, and enzyme-linked immunosorbent assay. Any system that can detect intermolecular interactions is available.
[0093] 光ネ目関分光法 (Fluorescence し orrelation Spectroscopy (Fし S): Eigen, M., et al.,  [0093] Fluorescence and orrelation Spectroscopy (F and S): Eigen, M., et al.,
Proc. Natl. Acad. Sci. USA, 91, 5740-5747(1994))は、共焦点レーザー顕微鏡等の 下で、粒子の流動速度、あるいは拡散率、容積収縮等を測定する方法であり、本発 明においては、修飾機能性タンパク質 (C末端修飾機能性タンパク質)と抗原分子間 の相互作用により元の修飾分子 1分子の並進ブラウン運動の変化を測定することに より、相互作用する分子を測定することができる。具体的には試料粒子が励起光によ り励起されて、試料液容積の一部において蛍光を放射し、この放射光を測定し光子 割合を得る。この値は、特定の時間に観測されている空間容積中に存在する粒子の 数と共に変化する。上述した種々のパラメータ一は自己相関関数を使用してこの信 号の変動から算出され得る。この FCSを行う為の装置もカールツァイス (Zeiss)社等か ら市販されており、本方法においてもこれらの装置を用いて解析を行うことができる。 この方法を用いて機能性タンパク質 標的分子間および標的分子間相互作用の測 定又は解析を行う場合、 C末端修飾機能性タンパク質または標的分子の ヽずれも溶 液として供することが必要である (液相法)。標的分子は修飾の必要はない。また相 互作用を調べようとする C末端修飾機能性タンパク質より非常に分子量の小さい分子 は、 C末端修飾機能性タンパク質のブラウン運動に影響を及ぼさないため本方法に おいてはふさわしくない。 Proc. Natl. Acad. Sci. USA, 91, 5740-5747 (1994)) is a method for measuring the flow velocity, diffusivity, volume contraction, etc. of particles under a confocal laser microscope or the like. In the present invention, the interaction between the modified functional protein (C-terminal modified functional protein) and the antigen molecule is measured by measuring the change in the translational Brownian motion of one original modified molecule, thereby measuring the interacting molecules. can do. Specifically, the sample particles are excited by the excitation light, emit fluorescence in a part of the sample solution volume, and the emitted light is measured and Get the percentage. This value varies with the number of particles present in the volume of space observed at a particular time. The various parameters mentioned above can be calculated from the variation of this signal using an autocorrelation function. Instruments for performing this FCS are also commercially available from Carl Zeiss and the like, and analysis can be performed using these instruments also in this method. When measuring or analyzing the interaction between functional proteins and target molecules and the interaction between target molecules using this method, it is necessary to provide the solution of the C-terminal modified functional protein or target molecule as a solution (liquid Phase method). The target molecule does not need to be modified. Molecules whose molecular weight is much smaller than that of the C-terminally modified functional protein whose interaction is to be examined are not suitable for this method because they do not affect the Brownian motion of the C-terminally modified functional protein.
[0094] しかし、 2種類の蛍光色素を用いる蛍光相互相関分光法 (FCCS)は、 1種類の蛍光 色素を用いる FCSでは困難であった同じくらいの分子量をもつタンパク質間の相互作 用も検出できる。 2種類の蛍光色素を用いる他の方法としては蛍光共鳴エネルギー 移動(FRET)法が知られて!/、るが、 FRETが生じるためには 2つの蛍光色素が 40— 50 A以内に近接する必要があり、タンパク質の大きさや蛍光色素の付 ヽて 、る位置に よっては、相互作用していても FRETが観測されない危険性がある。 FCCS法では相 互相関の検出は蛍光色素間の距離に依存しないので、そのような問題がない。一方 、他の検出系である蛍光偏向解消法と比較すると、 FCCS法は必要なサンプル量が 少なぐ検出時間が短ぐ HTSのための自動化が容易等の長所がある。さらに FCCS 法では蛍光標識された分子の大きさや数というきわめて基本的な情報が得られるの で、表面プラズモン共鳴法のように汎用的な用途に利用できる可能性がある。両者の 違いは、表面プラズモン共鳴法ではタンパク質が固定化された状態で相互作用を検 出するのに対して、 FCCS法ではより天然の状態に近い溶液中の相互作用を見ること ができる点にある。 FCCS法では、タンパク質の固定ィ匕が必要ないかわりに、タンパク 質を蛍光色素で標識する必要があるが、本発明により、この課題を克服することが可 能となった。 [0094] However, fluorescence cross-correlation spectroscopy (FCCS) using two types of fluorescent dyes can also detect interactions between proteins with similar molecular weights, which was difficult with FCS using one type of fluorescent dye. . Another method using two types of fluorescent dyes is known as fluorescence resonance energy transfer (FRET)! / However, two fluorescent dyes must be within 40-50 A for FRET to occur Depending on the size of the protein and the location of the fluorescent dye, there is a risk that FRET may not be observed even when the protein interacts. In the FCCS method, the cross-correlation detection does not depend on the distance between the fluorescent dyes, so there is no such problem. On the other hand, when compared with other detection systems such as the fluorescence depolarization method, the FCCS method has advantages such as a small sample amount required, a short detection time, and easy automation for HTS. Furthermore, the FCCS method can provide very basic information such as the size and number of fluorescently labeled molecules, and may be used for general-purpose applications such as surface plasmon resonance. The difference between the two is that the surface plasmon resonance method detects the interaction in a state where the protein is immobilized, whereas the FCCS method allows the interaction in a solution that is closer to the natural state to be observed. is there. In the FCCS method, instead of the necessity of immobilizing proteins, it is necessary to label proteins with fluorescent dyes. The present invention has made it possible to overcome this problem.
[0095] 本方法にお!、て 2種類の蛍光色素で修飾された 2組のタンパク質 標的分子の系 において、接触せしめる方法としては、両標的分子が相互作用するに十分な程度に 接触する方法であれば如何なるものであってもよ 、が、好ましくは市販の FCCS用装 置の測定用ゥエルに通常生化学的に用いられる緩衝液等に適当な濃度でそれぞれ 発光波長が重ならない蛍光色素で標識された 2種の C末端修飾機能性タンパク質を 溶解した溶液を投入し、さらに同緩衝液に適当な濃度で 2種の未標識の標的分子を 溶解した溶液を投入する方法によって行われる。 [0095] In this method, in a system of two sets of protein target molecules modified with two types of fluorescent dyes, a method for bringing the two into contact with each other is sufficient to allow the two target molecules to interact with each other. Any method may be used as long as it comes into contact with the substrate, but preferably, the emission wavelengths do not overlap at a concentration appropriate for a buffer or the like normally used for biochemistry in a measuring well of a commercially available FCCS device. A solution in which two types of C-terminal modified functional proteins labeled with a fluorescent dye are dissolved, and a solution in which two unlabeled target molecules are dissolved in the same buffer at an appropriate concentration are added. Be done.
[0096] この方法において、同時に多数の解析を行う方法としては、例えば上記 FCCS用測 定装置の各測定用ゥエルにそれぞれ異なる複数の C末端修飾機能性タンパク質を投 入し、これにそれぞれの機能性タンパク質に結合し得る標的分子溶液を投入するか 、あるいは特定の C末端修飾機能性タンパク質を投入し、各ゥエルに互いに異なる複 数種の標的分子溶液を投入する方法が用いられる。  [0096] In this method, as a method of performing a large number of analyzes at the same time, for example, a plurality of different C-terminal modified functional proteins are respectively injected into each measurement well of the above-described measurement device for FCCS, and the function of each protein is added to each. A method is used in which a target molecule solution capable of binding to a functional protein is charged, or a specific C-terminal modified functional protein is charged, and a plurality of different target molecule solutions are charged into each well.
[0097] 蛍光イメージングアナライズ法 (抗体チップ法)は、固相化された分子に、修飾分子 を接触せしめ、両分子の相互作用により、固相化された分子上にとどまった修飾分子 から発せられる蛍光を、市販の蛍光イメージングアナライザーを用いて測定又は解析 する方法である。  [0097] In the fluorescent imaging analysis method (antibody chip method), a modified molecule is brought into contact with a solid-phased molecule, and emitted from the modified molecule remaining on the solid-phased molecule due to the interaction between both molecules. This is a method of measuring or analyzing fluorescence using a commercially available fluorescence imaging analyzer.
[0098] この方法を用いて機能性タンパク質 標的分子間、標的 標的分子間相互作用の 測定又は解析を行う場合、 C末端修飾機能性タンパク質または標的分子の ヽずれか 一方は上記した方法により固相化されていることが必要である。標的分子は固相化し て用いる場合には修飾されて 、るものと、されて 、な 、もののどちらも利用可能であ る。また、固相化しないで用いる場合には上記した修飾物質により修飾されているこ とが必要である。 C末端修飾機能性タンパク質は、修飾部を介して固定化されている ものも、修飾部以外の部分、例えば GSTのような融合タンパク質や Flag-tag、 His-tag 等で固定ィ匕されて 、るものも用いることができる。  [0098] When measuring or analyzing the interaction between a functional protein and a target molecule using this method, one of the C-terminal modified functional protein and the target molecule may be immobilized by the method described above. Needs to be When the target molecule is used in the form of a solid phase, it can be modified, and can be used. In addition, when used without being immobilized, it is necessary to be modified with the above-mentioned modifying substance. The C-terminal modified functional protein, even if it is immobilized via the modified part, is immobilized with a part other than the modified part, for example, a fusion protein such as GST, Flag-tag, His-tag, etc. Can also be used.
[0099] C末端修飾機能性タンパク質、または標的分子を固相化するための基板としては、 通常タンパク質や核酸等を固定ィ匕するのに用いられる-トロセルロースメンブレンや ナイロンメンブレン、あるいはスライドガラスやプラスチック製のマイクロプレート等も用 いることがでさる。  [0099] A substrate for immobilizing a C-terminal modified functional protein or a target molecule is usually used for immobilizing proteins, nucleic acids, or the like-trocellulose membrane, nylon membrane, slide glass, or the like. Plastic microplates can also be used.
[0100] 本方法にお!、て修飾標的分子ある!、は機能性タンパク質を固相化分子へ接触せ しめる方法としては、両分子が相互作用するに十分な程度に接触する方法であれば 、かなるものであってもよ 、が、好ましくは修飾標的分子ある!/、は C末端修飾機能性 タンパク質を生化学的に通常使用される緩衝液に適当な濃度で溶解した溶液を作 成し、これを固相表面に接触させる方法が好ましい。 [0100] In the present method, there is a modified target molecule !, a method for bringing a functional protein into contact with a solid-phased molecule as long as the two molecules can be brought into contact with each other to an extent sufficient for interaction. It is possible to use a modified target molecule! /, To prepare a solution in which a C-terminal modified functional protein is dissolved at an appropriate concentration in a buffer commonly used in biochemistry. However, a method of bringing this into contact with the surface of the solid phase is preferred.
[0101] 両分子を接触せしめた後、好ましくは過剰に存在する修飾標的分子あるいは C末 端修飾機能性タンパク質を同緩衝液等により洗浄する工程を行い、固相上にとどま つた標的分子あるいは c末端修飾機能性タンパク質の修飾物質力も発せられる蛍光 信号、又は固相化されている修飾分子力 発せられる蛍光と固相上にとどまった修 飾分子から発せられる蛍光が混ざり合った信号を、市販のイメージングアナライザー を用いて測定あるいは解析することにより、固相化された分子と相互作用する分子を 同定することができる。  [0101] After the two molecules are brought into contact with each other, preferably, a step of washing the excessively present modified target molecule or C-terminal modified functional protein with the same buffer or the like is carried out, and the target molecule or c-terminal remaining on the solid phase is removed. A fluorescent signal that also emits the power of the modifying substance of the terminally modified functional protein, or a signal in which the fluorescent light emitted by the modified molecule that is immobilized on the solid phase and the fluorescent light that is emitted from the modifying molecule that remains on the solid phase are used as a commercially available signal. Molecules that interact with immobilized molecules can be identified by measurement or analysis using an imaging analyzer.
[0102] この方法において、同時に多数の解析を行う方法としては、例えば上記固相表面 に、複数の c末端修飾機能性タンパク質あるいは修飾又は非修飾標的分子を番地 付けして固相化する方法、あるいは 1種類の C末端修飾機能性タンパク質または修 飾もしくは非修飾標的分子に固相化されて 、な 、複数種の c末端修飾機能性タンパ ク質または修飾標的分子を接触させる方法等が用いられる。複数種の C末端修飾機 能性タンパク質または修飾標的分子を接触させる場合には、固相にとどまった該分 子を緩衝液の濃度の差等により解離させて取得し、これを既知の方法により分析する ことにより同定できる。  [0102] In this method, as a method for performing a large number of analyzes at the same time, for example, a method in which a plurality of c-terminal modified functional proteins or modified or unmodified target molecules are addressed and immobilized on the solid phase surface, Alternatively, a method of immobilizing one kind of C-terminal modified functional protein or a modified or unmodified target molecule on a solid phase and contacting a plurality of kinds of c-terminal modified functional proteins or modified target molecules is used. . When a plurality of types of C-terminal modified functional proteins or modified target molecules are brought into contact with each other, the molecules remaining on the solid phase are obtained by dissociation due to a difference in buffer concentration or the like, and this is obtained by a known method. It can be identified by analysis.
[0103] 蛍光共鳴エネルギー移動(FRET)法は、 2種類の蛍光色素を用いる他の分子間相 互作用検出法としてよく知られている。 FRETとは、 2種類の蛍光色素の一方(ェネル ギー供与体)の蛍光スペクトルと、もう一方(エネルギー受容体)の吸収スペクトルに 重なりがあるとき、 2つの蛍光色素間の距離が十分小さいと、供与体力 の発光が起 こらないうちに、その励起エネルギーが受容体を励起してしまう確率が高くなる現象 をいう。したがって、相互作用を検出したい 2つのタンパク質を、それぞれ供与体およ び受容体となる蛍光色素で標識しておき、供与体を励起すれば、 2つのタンパク質が 相互作用しない場合は、蛍光色素間の距離が大きいため FRETは起こらず、供与体 の蛍光スペクトルが観察される力 2つのタンパク質が相互作用して蛍光色素間の距 離が小さくなると、 FRETにより受容体の蛍光スペクトルが観察されるので、蛍光スぺク トルの波長の違 、からタンパク質間相互作用の有無を判別することができる。蛍光色 素としては、供与体がフルォレセイン、受容体がローダミンという組み合わせがよく用 いられている。また最近では、蛍光緑色タンパク質 (GFP)の波長の異なる変異体の 組み合わせにより、細胞の中で FRETを観察し相互作用を検出する試みがなされて いる。この方法の欠点としては、 FRETが生じるために 2つの蛍光色素が 40— 50 A以 内に近接する必要があるため、タンパク質の大きさや蛍光色素の付いている位置に よっては、相互作用していても FRETが観測されない危険性があるという点が挙げら れる。 [0103] The fluorescence resonance energy transfer (FRET) method is well known as another intermolecular interaction detection method using two types of fluorescent dyes. FRET means that when the fluorescence spectrum of one of two types of fluorescent dyes (energy donor) and the absorption spectrum of the other (energy acceptor) overlap, if the distance between the two fluorescent dyes is small enough, A phenomenon in which the probability that the excitation energy excites the acceptor increases before emission of the donor force occurs. Therefore, two proteins whose interaction is to be detected are labeled with a fluorescent dye that serves as a donor and an acceptor, respectively, and when the donor is excited, the two proteins do not interact with each other. FRET does not occur because the distance is large, and the force of observing the fluorescence spectrum of the donor When the distance between the fluorescent dyes decreases due to the interaction of the two proteins, the fluorescence spectrum of the acceptor is observed by FRET. , Fluorescent spark The presence or absence of protein-protein interaction can be determined from the difference in the wavelength of the torr. As a fluorescent dye, a combination of fluorescein as a donor and rhodamine as an acceptor is often used. Recently, attempts have been made to detect the interaction by observing FRET in cells using a combination of mutants of fluorescent green protein (GFP) having different wavelengths. The disadvantage of this method is that, depending on the size of the protein and the location of the fluorescent dye, the two fluorescent dyes must be close to each other within 40-50 A for FRET to occur. However, there is a risk that FRET may not be observed.
[0104] エバネッセント場分子イメージング法とは、 Funatsu, T., et al., Nature, 374,  [0104] The evanescent field molecular imaging method is described in Funatsu, T., et al., Nature, 374,
555-559 (1995)等に記載されている方法で、ガラス等の透明体に固相化した分子に 溶液として第 2の分子を接触せしめ、これにエバネッセント場が発生する角度でレー ザ一光等の光源を照射し、発生したエバネッセント光を検出器によって測定又は解 析する方法である。これらの操作は、それ自体既知のエバネッセント場蛍光顕微鏡 装置を用いて行うことができる。  555-559 (1995), etc., contact a second molecule as a solution with a molecule immobilized on a transparent body such as glass, and contact the laser at an angle where an evanescent field is generated. And the like, and the generated evanescent light is measured or analyzed by a detector. These operations can be performed using an evanescent field fluorescence microscope apparatus known per se.
[0105] この方法を用いてタンパク質間相互作用の測定又は解析を行う場合、 C末端修飾 機能性タンパク質または標的分子の 、ずれか一方は上記した方法により固相化され ていることが必要である。標的分子は固相化する場合は修飾の必要はないが、固相 化しないで用いる場合には上記した修飾物質により修飾されていることが必要である [0105] When measuring or analyzing protein-protein interaction using this method, it is necessary that either the C-terminal modified functional protein or the target molecule is immobilized by the above-described method. . The target molecule does not need to be modified when immobilized, but when used without immobilization, it must be modified with the above-mentioned modifier.
[0106] C末端修飾機能性タンパク質または標的分子を固相化するための基板としては、ガ ラス等の材質の基板が用いられ、好ましくは石英ガラスが用いられる。また、レーザー 光の散乱等を防ぐために表面を超音波洗浄したものが好まし 、。 As a substrate for immobilizing the C-terminal-modified functional protein or the target molecule, a substrate made of a material such as glass is used, and quartz glass is preferably used. It is also preferable that the surface is ultrasonically cleaned to prevent scattering of laser light.
[0107] 本方法にお!、て固相化して 、な 、C末端修飾機能性タンパク質または修飾標的分 子を固相化分子へ接触せしめる方法としては、両分子が相互作用するに十分な程 度に接触する方法であればいかなるものであってもよいが、好ましくは固相化してい ない C末端修飾機能性タンパク質または修飾標的分子を生化学的に通常使用され る緩衝液に適当な濃度で溶解した溶液を作成し、これを固相表面に滴下する方法が 好ましい。 [0108] 両分子を接触せしめた後、エバネッセント場照明により励起された蛍光を CCDカメ ラ等の検出器を用いて測定することにより、固相化された分子と相互作用する分子を 同定することができる。 [0107] In the present method, a method for bringing a C-terminal modified functional protein or a modified target molecule into contact with an immobilized molecule by immobilizing the molecule to the immobilized molecule is sufficient to allow both molecules to interact with each other. Any method may be used as long as it can be contacted with each other, but it is preferable that the non-immobilized C-terminal modified functional protein or the modified target molecule is added at a concentration appropriate for a buffer commonly used in biochemistry. A preferred method is to prepare a dissolved solution and drop it on the surface of the solid phase. [0108] After the two molecules are brought into contact, the fluorescence excited by the evanescent field illumination is measured using a detector such as a CCD camera to identify the molecules that interact with the immobilized molecules. Can be.
[0109] この方法において、同時に多数の解析を行う方法としては、例えば上記基板に、複 数の C末端修飾機能性タンパク質または修飾標的分子を番地付けして固相化する 方法等が用いられる。  In this method, as a method of performing a large number of analyzes at the same time, for example, a method of addressing a plurality of C-terminal modified functional proteins or modified target molecules on the substrate and immobilizing the same on the substrate is used.
[0110] 蛍光偏光法(Perran, J., et al., J. Phys. Rad., 1, 390-401(1926))は、蛍光偏光で励 起された蛍光分子が、励起状態の間、定常状態を保っている場合には同一の偏光 平面で蛍光を放射するが、励起された分子が励起状態中に回転ブラウン運動等を 行った場合に、放射された蛍光は励起光とは異なった平面になることを利用する方 法である。分子の運動はその大きさに影響を受け、蛍光分子が高分子である場合に は、励起状態の間の分子の運動はほとんどなぐ放射光は偏光を保ったままになって いるのに対して、低分子の蛍光分子の場合は、運動速度が速いために放射光の偏 光が解消される。そこで、平面偏光で励起された蛍光分子から放射される蛍光の強 度を、元の平面とそれに垂直な平面とで測定し、両平面の蛍光強度の割合力 この 分子の運動性およびその存在状態に関する情報が得られるものである。この方法に よれば、夾雑物があってもこれに影響されることなぐ蛍光修飾された分子と相互作 用する標的分子の挙動を追跡できる。これは蛍光修飾された分子と標的分子が相互 作用するときにのみ、偏光度の変化として測定される力もである。  [0110] The fluorescence polarization method (Perran, J., et al., J. Phys. Rad., 1, 390-401 (1926)) uses a method in which a fluorescent molecule excited by fluorescence polarization emits light during the excited state. When the steady state is maintained, fluorescence is emitted in the same plane of polarization.However, when the excited molecule performs a rotating Brownian motion or the like during the excited state, the emitted fluorescence is different from the excitation light. This is a method that utilizes the fact that it becomes a plane. The motion of a molecule is affected by its size, and when the fluorescent molecule is a macromolecule, the motion of the molecule during the excited state is almost negligible, whereas the emitted light remains polarized. In the case of a low-molecular fluorescent molecule, the polarization of the emitted light is eliminated because the moving speed is high. Therefore, the intensity of the fluorescence emitted from the fluorescent molecule excited by the plane-polarized light is measured on the original plane and a plane perpendicular to the original plane. The information about is obtained. According to this method, the behavior of a target molecule that interacts with a fluorescence-modified molecule without being affected by contaminants can be tracked. This is also a force that is measured as a change in the degree of polarization only when the target molecule interacts with the fluorescently modified molecule.
[0111] この方法を行うための装置としては例えば BECON (Panyera社製)等が巿販されて おり、本方法もこれらの装置を用いることにより行うことができる。  [0111] As an apparatus for performing this method, for example, BECON (manufactured by Panyera) or the like is commercially available, and this method can also be performed by using these apparatuses.
この方法を用いてタンパク質間相互作用の測定又は解析を行う場合、 C末端修飾 機能性タンパク質または標的分子の 、ずれも溶液として供する必要である。標的分 子は修飾の必要はない。また相互作用を調べようとする C末端修飾機能性タンパク 質より非常に分子量の小さい分子は、 C末端修飾機能性タンパク質のブラウン運動 に影響を及ぼさな 、ため本方法にぉ 、てはふさわしくな 、。  When measuring or analyzing protein-protein interaction using this method, it is necessary to provide the solution of the C-terminal modified functional protein or target molecule as a solution. The target molecule does not need to be modified. In addition, a molecule having a molecular weight much smaller than that of the C-terminal modified functional protein whose interaction is to be examined does not affect the Brownian motion of the C-terminal modified functional protein, and thus is not suitable for the present method. .
[0112] 本方法において 2組の C末端修飾機能性タンパク質 標的分子を接触せしめる方 法としては、両分子が相互作用するに十分な程度に接触する方法であれば如何なる ものであってもよいが、好ましくは市販の蛍光偏光解消装置の測定用ゥエルに通常 生化学的に用いられる緩衝液等に適当な濃度で c末端修飾機能性タンパク質を溶 解した溶液を投入し、さらに同緩衝液に適当な濃度で標的分子を溶解した溶液を投 入する方法によって行われる。 [0112] In the present method, any method of contacting the two sets of C-terminal modified functional protein target molecules can be used as long as the two molecules can be brought into contact enough to interact with each other. However, preferably, a solution prepared by dissolving the c-terminal modified functional protein at an appropriate concentration in a buffer or the like normally used in biochemistry is added to a measuring well of a commercially available fluorescence depolarizer. Further, the method is carried out by injecting a solution in which the target molecule is dissolved at an appropriate concentration in the same buffer.
[0113] 本方法において測定する 2つの標的間の相互作用は、最適の組み合わせを検出 するためには、相互作用の程度を数値ィ匕することが有効である。相互作用の程度を 示す指標としては、例えば一定濃度の C末端修飾機能性タンパク質に対して、極大 蛍光偏光度を与える最小標的物濃度の値等を用いることができる。  [0113] In order to detect the optimal combination of the interaction between two targets measured in the present method, it is effective to numerically indicate the degree of the interaction. As an index indicating the degree of interaction, for example, a value of a minimum target substance concentration that gives a maximum fluorescence polarization degree to a certain concentration of a C-terminal modified functional protein can be used.
[0114] 表面プラズモン共鳴法とは、金属 Z液体界面で相互作用する分子によって表面プ ラズモンが励起され、これを反射光の強度変化で測定する方法である(Cullen, D.C., et al., Biosensors, 3(4), 211-225(1987-88))。この方法を用いてタンパク質 標的分 子間相互作用の測定又は解析を行う場合、 C末端修飾タンパク質は上記した方法に より固相化されていることが必要であるが、標的分子の修飾は必要ない。  [0114] The surface plasmon resonance method is a method in which surface plasmons are excited by molecules interacting at the metal-Z liquid interface, and are measured by changes in the intensity of reflected light (Cullen, DC, et al., Biosensors). , 3 (4), 211-225 (1987-88)). When measuring or analyzing the interaction between protein and target molecules using this method, the C-terminal modified protein must be immobilized by the method described above, but the target molecule does not need to be modified. .
[0115] C末端修飾機能性タンパク質を固相化するための基板としては、ガラスの等の透明 基板上に金、銀、白金等の金属薄膜が構成されたものが用いられる。透明基板とし ては、通常表面プラズモン共鳴装置用に用いられるものであればいかなるものであつ てもよぐレーザー光に対して透明な材料力もなるものとして一般的にはガラス等から なるものであり、その厚さは 0. 1— 5mm程度のものが用いられる。また金属薄膜の膜 厚は 100— 2000 A程度が適当である。このような表面プラズモン共鳴装置用固基 板として市販されているものも用いることができる。 C末端修飾機能性タンパク質の上 記基板への固相化は前述した方法により行うことができる。  [0115] As a substrate for immobilizing the C-terminal-modified functional protein, a transparent substrate such as glass on which a metal thin film of gold, silver, platinum or the like is formed is used. The transparent substrate is generally made of glass, etc., as long as it has a material power that is transparent to laser light, regardless of what is usually used for a surface plasmon resonance device. Its thickness is about 0.1-5 mm. The appropriate thickness of the metal thin film is about 100-2000A. A commercially available solid substrate for such a surface plasmon resonance device can also be used. The C-terminal modified functional protein can be immobilized on the substrate by the above-described method.
[0116] 本方法において標的分子を C末端修飾機能性タンパク質へ接触せしめる方法とし ては、両分子が相互作用するに十分な程度に接触する方法であれば 、かなるもので あってもよいが、好ましくは標的分子を生化学的に通常使用される緩衝液に適当な 濃度で溶解した溶液に固相化された C末端修飾機能性タンパク質を接触させる方法 を用いることができる。  [0116] In the present method, the method of bringing the target molecule into contact with the C-terminal-modified functional protein may be any method as long as the two molecules come into contact with each other to an extent sufficient for interaction. Preferably, a method can be used in which the C-terminal-modified functional protein immobilized on a solid phase is brought into contact with a solution in which a target molecule is dissolved in a buffer commonly used in biochemistry at an appropriate concentration.
[0117] これらの行程は市販の表面プラズモン共鳴装置、例えば BIAcore2000 (Pharmacia Biosensor社製)によってもよい。両分子を接触せしめた後、それ自体既知の表面ブラ ズモン共鳴装置を用いて、それぞれの反射光の相対強度の時間的変化を測定する こと〖こより、固相化された修飾抗原分子と一本鎖抗体との相互作用が解析できる。 [0117] These steps may be performed using a commercially available surface plasmon resonance device, for example, BIAcore2000 (Pharmacia Biosensor). After contacting both molecules, a surface brush known per se The interaction between the immobilized modified antigen molecule and the single-chain antibody can be analyzed by measuring the temporal change in the relative intensity of each reflected light using a Summon resonance apparatus.
[0118] この方法において、同時に多数の解析を行う方法としては、例えば上記表面プラズ モン共鳴装置に用いられる基板に、複数の標的分子を番地付けして固相化し、 1種 類の機能性タンパク質を接触させるか、あるいは 1種類の固相化された C末端修飾機 能性タンパク質に複数種の標的分子を接触させる方法等が用いられる。  [0118] In this method, as a method of performing a large number of analyzes at the same time, for example, a plurality of target molecules are addressed and immobilized on a substrate used for the surface plasmon resonance apparatus, and one type of functional protein is used. Or a method of contacting a plurality of types of target molecules with one type of immobilized C-terminal functional protein.
[0119] 固相酵素免疫検定法(Enzyme Linked Immunosorbent Assay (ELISA): Crowther, J.R., Methods in Molecular Biology, 42 (1995))は、固相上に固定化した抗原に対し 、抗体を含む溶液を接触せしめ、両分子の相互作用(抗原抗体反応)により、固相化 された抗原上にとどまった抗体をこれと特異的に結合する修飾分子 (IgG等)力 発 せられる蛍光、あるいは修飾分子を基質とする色素から発せられる信号を、市販の検 出器 (ELISAリーダー)を用いて測定又は解析する方法である。  [0119] A solid-phase enzyme immunoassay (Enzyme Linked Immunosorbent Assay (ELISA): Crowther, JR, Methods in Molecular Biology, 42 (1995)) uses a solution containing an antibody to an antigen immobilized on a solid phase. The molecules are brought into contact with each other, and the interaction (antigen-antibody reaction) between the two molecules causes the antibody remaining on the immobilized antigen to bind to the modified molecule (such as IgG) that specifically binds to the fluorescent or modified molecule. This is a method of measuring or analyzing a signal emitted from a dye as a substrate using a commercially available detector (ELISA reader).
[0120] この方法を用いてタンパク質 抗原分子間相互作用の測定又は解析を行う場合、 抗原となる C末端修飾機能性タンパク質を上記した方法により固相化されて 、ること が必要である。また抗体となる標的分子は上記した修飾物質により修飾されて 、るこ とが必要である。  [0120] When measuring or analyzing protein-antigen molecule interaction using this method, it is necessary that the C-terminal modified functional protein serving as an antigen be immobilized by the above-described method. Also, the target molecule to be an antibody needs to be modified with the above-mentioned modifying substance.
[0121] 抗原となる C末端修飾機能性タンパク質を固相化するための基板としては、通常 E LISAに用いられるプラスチック製のマイクロプレート等も用いることができる。  [0121] As a substrate for immobilizing a C-terminal modified functional protein serving as an antigen, a plastic microplate or the like usually used for ELISA can also be used.
本方法において抗体となる修飾標的分子を固相分子へ接触せしめる方法としては 、両分子が相互作用するに十分な程度に接触する方法であればいかなるものであつ てもよいが、好ましくは修飾標的分子を生化学的に通常使用される緩衝液に適当な 濃度で溶解した溶液を作成し、これをマイクロプレートに注入する方法が好まし 、。  The method for bringing the modified target molecule to be an antibody into contact with the solid phase molecule in the present method may be any method as long as the two molecules are brought into contact with each other to an extent sufficient for interaction. A method is preferred in which a solution is prepared by dissolving a molecule in an appropriate concentration in a buffer commonly used in biochemistry, and the solution is injected into a microplate.
[0122] 両分子を接触せしめた後、好ましくは過剰に存在する固相化分子に結合していな い修飾分子を同緩衝液等により洗浄する工程を行い、固相上にとどまった修飾分子 から発せられる蛍光を、市販の ELISAリーダー等を用いて測定あるいは解析するこ とにより、固相化された抗原分子と相互作用する分子を同定することができる。  [0122] After the two molecules are brought into contact with each other, preferably, a step of washing the excessively present modified molecules that are not bound to the immobilized molecules with the same buffer or the like is performed to remove the modified molecules remaining on the solid phase. By measuring or analyzing the emitted fluorescence using a commercially available ELISA reader or the like, a molecule that interacts with the immobilized antigen molecule can be identified.
[0123] この方法において、同時に多数の解析を行う方法としては、例えば上記マイクロプ レートの各穴にそれぞれ異なる複数の修飾標的分子を固相化する方法が用いられる 相互作用する分子の同定方法としては、上記のそれぞれの方法により測定され、相 互作用が認められた標的分子は、該分子の一次構造が未知の場合、それ自体既知 の適当な方法により、その一次構造を解析することができる。具体的には、相互作用 を認められた標的分子がタンパク質の場合、アミノ酸分析装置等によりアミノ酸配列 を解析し、一次構造を特定することができる。また、標的分子が核酸の場合には、塩 基配列決定方法により、オート DNAシーケンサーなどを用いれば塩基配列を決定 することができる。 [0123] In this method, as a method for performing a large number of analyzes at the same time, for example, a method of immobilizing a plurality of different modified target molecules on each well of the microplate is used. As a method for identifying interacting molecules, the target molecule, which has been measured by each of the above methods and has been found to have an interaction, is identified by an appropriate method known per se when the primary structure of the molecule is unknown. The primary structure can be analyzed. Specifically, when the target molecule for which interaction has been recognized is a protein, the primary structure can be identified by analyzing the amino acid sequence using an amino acid analyzer or the like. Further, when the target molecule is a nucleic acid, the base sequence can be determined by a base sequence determination method using an auto DNA sequencer or the like.
[0124] C末端修飾機能性タンパク質や標的分子の固相化のための装置としては、上記に 記載した C末端修飾機能性タンパク質や標的分子の修飾部を介した固相への固定 化方法を行うために、既知の適切な手段を組み合わせて装置を構築することもできる 。本装置における各手段自体はそれぞれ既知のものであり、これらの手段における、 基板の保持、 C末端修飾タンパク質溶液の添加、洗浄等の各操作は、それ自体既知 の方法により行えばよい。これらの操作を組み合わせ、全自動又は半自動の、 C末端 修飾タンパク質の固相化のための装置を構築することができる。  [0124] As an apparatus for immobilizing a C-terminal modified functional protein or target molecule on the solid phase, the above-described method for immobilizing a C-terminal modified functional protein or target molecule on a solid phase via a modified portion is used. To do so, the device can be constructed by combining known suitable means. Each means in the apparatus is known, and operations such as holding a substrate, adding a C-terminal modified protein solution, and washing may be performed by a method known per se. By combining these operations, a fully automatic or semi-automatic device for immobilizing a C-terminal modified protein can be constructed.
[0125] タンパク質間相互作用測定のための装置としては、上記に記載したタンパク質-標 的分子間相互作用測定を行うために、既知の適切な手段を組み合わせて装置を構 築することもできる。本装置における各手段自体はそれぞれ既知のものであり、これら の手段における、基板の保持、抗原分子の添加、洗浄、信号検出等の各操作は、そ れ自体既知の方法により行えばよい。これらの操作を組み合わせ、全自動又は半自 動の、タンパク質間相互作用測定のための装置を構築することができる。  [0125] As an apparatus for measuring protein-protein interaction, an apparatus can be constructed by combining known appropriate means for performing the above-described protein-target molecular interaction measurement. The respective means in the present apparatus are known, and the respective operations such as holding of the substrate, addition of antigen molecules, washing, and signal detection in these means may be performed by methods known per se. By combining these operations, a fully-automatic or semi-automatic device for protein-protein interaction measurement can be constructed.
[0126] マウス抗体 cDNAライブラリ一力 本発明の方法によって選択された高親和性一本 鎖抗体は、ヒト IgG抗体の可変領¾½ 'や CDR¾¾½\complementarity determining region) と入れ換えることによって、キメラ型 IgG抗体やヒト型化したマウス IgG抗体を作れる。こ れらの抗体は、ヒトに投与したときに惹起されるヒト抗マウス抗体の産生が少ないか、 ほとんどない。さらに、ヒト抗体 cDNAライブラリ一力も本発明の方法に選択された高親 和性一本鎖抗体は、ヒ HgG抗体の可変領域と入れ換えることによって、完全なヒトモ ノクローナル IgG抗体を作れる。これは、アナフィラキシー症状を引き起こさない抗体 医薬として利用することが可能である。 [0126] High-affinity single-chain antibody selected by the method of the present invention can be used as a chimeric IgG antibody by replacing the variable region of human IgG antibody or CDR (complementarity determining region). And humanized mouse IgG antibodies. These antibodies produce little or no human anti-mouse antibody when elicited when administered to humans. Furthermore, a highly human single-chain antibody selected by the method of the present invention, which is a human antibody cDNA library, can produce a fully human monoclonal antibody by replacing the variable region of the human HgG antibody. This is an antibody that does not cause anaphylactic symptoms It can be used as a medicine.
[0127] したがって、ヒト又はその他の動物由来の DNAライブラリ一力も本発明の選択法に より選択された核酸の配列に基づいて、その核酸にコードされる可変領域をヒトの IgG の可変領域と置換することによって構築されるヒトまたはヒト型抗体が提供される。ま た、この抗体を有効成分とする治療剤が提供される。アンジォテンシン IIに対して結 合する抗体は、高血圧の治療に中和抗体として使用できると考えられる。すなわち、 アンジォテンシン IIの生理作用は血圧上昇作用なので、抗体はアンジォテンシン IIと 結合して血圧上昇作用を抑えると考えられる。また、 Lewis Xに対して結合する抗体 は、癌の治療に使用できると考えられる。細胞が癌化すると細胞表面に Lewis Xが発 現するようになるので、それを標的として抗体を用いることができる。例えば、癌細胞 を殺す抗癌剤やリシンなどの毒素を結合させて、全身または局所的に投与し、抗体 力 Sミサイルのように癌細胞を集中的に攻撃し、癌に特異的かつ効果的なミサイル治療 を行うことができる。また、ミサイル治療に用いる製剤の一種として、脂質二重膜小胞 に薬剤を入れ、その表面に抗体を結合させたィムノリボソーム製剤としてもょ 、。  [0127] Therefore, based on the sequence of a nucleic acid selected by the selection method of the present invention, a human or other animal-derived DNA library is substituted with a variable region encoded by the nucleic acid and a human IgG variable region. A human or humanized antibody constructed thereby. Also provided is a therapeutic agent comprising the antibody as an active ingredient. Antibodies that bind to angiotensin II could be used as neutralizing antibodies in the treatment of hypertension. That is, since the physiological action of angiotensin II is a blood pressure increasing action, it is considered that the antibody binds to angiotensin II and suppresses the blood pressure increasing action. Antibodies that bind to Lewis X could also be used to treat cancer. When cells become cancerous, Lewis X is expressed on the cell surface, and antibodies can be used to target it. For example, an anticancer drug that kills cancer cells or a toxin such as ricin is bound and administered systemically or locally, and the cancer cells are intensively attacked like antibody-powered S missiles, resulting in cancer-specific and effective missiles Treatment can be provided. Also, as a kind of preparation used for missile treatment, an immunoribosome preparation in which a drug is put in a lipid bilayer vesicle and an antibody is bound to the surface thereof is used.
[0128] 以下、具体的に本発明の実施例を記述するが、下記の実施例は本発明について の具体的認識を得る一助とみなすべきものであり、本発明の範囲は下記の実施例に より何ら限定されるものでない。  [0128] Hereinafter, specific examples of the present invention will be described. However, the following examples should be regarded as helping to obtain a specific understanding of the present invention, and the scope of the present invention is limited to the following examples. It is not limited at all.
実施例 1  Example 1
[0129] 一本鎖抗体をコードする核酸の選択  [0129] Selection of nucleic acid encoding single-chain antibody
(I) ライブラリーの作成  (I) Creating a library
[1]一本鎖抗体 cDNAライブラリーの製造には新たに作成したライブラリーを用いた  [1] Single-chain antibody cDNA library was prepared using a newly created library
[0130] 始めに H鎖 DNA溶液の調製を行った。マウス脾臓 Poly A+ RNA (5 μ §/ μ \) ( [0130] First, an H-chain DNA solution was prepared. Mouse spleen Poly A + RNA (5 μ § / μ \) (
DEPC-処理水)(CLONTECH社)を RNase-Free水で 100倍に希釈したものを 11 1、 5 X RT緩衝液 (ΤΟΥΟΒΟ) 22 μ 1、 10 mM dNTPs(TOYOBO) 11 1、 MulgGl/2 forward プライマー (配列番号 48) (lpmol/ μ 1) 27.5 μ 1、 MulgG3 forwardプライマー (配列番号 47)(lpmol/ μ 1) 27.5 μ 1を混和させ、 65°Cで 9分間反応後、直ちに 4°Cに冷却し、 4°C で 2分間放置した後、 ReverTra Ace(TOYOBO) 5.5 μ 1、 RNase inhibitor(TOYOBO) 5.5 /z lをカ卩え、 50°Cで 30分間、次いで 99°Cで 5分間反応させ cDNA-Hを合成した。 DEPC-treated water) (CLONTECH) diluted 100-fold with RNase-Free water 11 1, 5 X RT buffer (ΤΟΥΟΒΟ) 22 μl, 10 mM dNTPs (TOYOBO) 111, MulgGl / 2 forward Primer (SEQ ID NO: 48) (lpmol / μ1) 27.5 μ1, MulgG3 forward primer (SEQ ID NO: 47) (lpmol / μ1) 27.5 μl, mix, react at 65 ° C for 9 minutes, then immediately 4 ° C And leave it at 4 ° C for 2 minutes.ReverTra Ace (TOYOBO) 5.5 μ1, RNase inhibitor (TOYOBO) 5.5 / zl was prepared and reacted at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes to synthesize cDNA-H.
[0131] [2] [1]で合成した cDNA-H溶液 5 1、下記の HBプライマーリストに示した各 HBプ ライマー(lpmol/ μ 1) 2.5 1、 10 X PCR緩衝液 (ΤΟΥΟΒΟ) 2.5 μ 1、 MulgGl/2 forward プライマー (配列番号 48)(lpmol/ μ 1) 1.25 μ 1、 MulgG3 forwardプライマー (配列番号 47)(lpmol/ μ 1) 1.25 μ 1、 KOD DASHポリメラーゼ (TOYOBO) 0.25 μ 1を混和させ、 RNase-Free水を添加し全体量を 25 μ 1としてそれぞれ PCR反応させた。 [0131] [2] cDNA-H solution 51 synthesized in [1] 51, each HB primer (lpmol / μ1) 2.5 1 shown in the following HB primer list, 10X PCR buffer (ΤΟΥΟΒΟ) 2.5 μl 1, MulgGl / 2 forward primer (SEQ ID NO: 48) (lpmol / μ1) 1.25 μ1, MulgG3 forward primer (SEQ ID NO: 47) (lpmol / μ1) 1.25 μ1, KOD DASH polymerase (TOYOBO) 0.25 μ1 After mixing, RNase-Free water was added to make a total volume of 25 μl, and each was subjected to PCR reaction.
[0132] [表 1] 表 1 HBプライマーリスト [0132] [Table 1] Table 1 HB primer list
配列 名称 配列番号 Sequence name Sequence number
GACTGGTGGACAGCAAATGGGT GAKGTRMAGCTTCAGGAGTC HB1 1GACTGGTGGACAGCAAATGGGT GAKGTRMAGCTTCAGGAGTC HB1 1
GACTGGTGGACAGCAAATGGGT GAGGTBCAGCTBCAGCAGTC HB2 2GACTGGTGGACAGCAAATGGGT GAGGTBCAGCTBCAGCAGTC HB2 2
GACTGGTGGACAGCAAATGGGT CAGGTGCAGCTGAAGSASTC HB3 3GACTGGTGGACAGCAAATGGGT CAGGTGCAGCTGAAGSASTC HB3 3
GACTGGTGGACAGCAAATGGGT GAGGTCCARCTGCAACARTC HB4 4GACTGGTGGACAGCAAATGGGT GAGGTCCARCTGCAACARTC HB4 4
GACTGGTGGACAGCAAATGGGT CAGGTYCAGCTBCAGCARTC HB5 5GACTGGTGGACAGCAAATGGGT CAGGTYCAGCTBCAGCARTC HB5 5
GACTGGTGGACAGCAAATGGGT CAGGTYCARCTGCAGCAGTC HB6 6GACTGGTGGACAGCAAATGGGT CAGGTYCARCTGCAGCAGTC HB6 6
GACTGGTGGACAGCAAATGGGT CAGGTCCACGTGAAGCAGTC HB7 7GACTGGTGGACAGCAAATGGGT CAGGTCCACGTGAAGCAGTC HB7 7
GACTGGTGGACAGCAAATGGGT GAGGTGAASSTGGTGGAATC HB8 8GACTGGTGGACAGCAAATGGGT GAGGTGAASSTGGTGGAATC HB8 8
GACTGGTGGACAGCAAATGGGT GAVGTGAWGYTGGTGGAGTC HB9 9GACTGGTGGACAGCAAATGGGT GAVGTGAWGYTGGTGGAGTC HB9 9
GACTGGTGGACAGCAAATGGGT GAGGTGCAGSKGGTGGAGTC HB10 10GACTGGTGGACAGCAAATGGGT GAGGTGCAGSKGGTGGAGTC HB10 10
GACTGGTGGACAGCAAATGGGT GAKGTGCAMCTGGTGGAGTC HB11 11GACTGGTGGACAGCAAATGGGT GAKGTGCAMCTGGTGGAGTC HB11 11
GACTGGTGGACAGCAAATGGGT GAGGTGAAGCTGATGGARTC HB12 12GACTGGTGGACAGCAAATGGGT GAGGTGAAGCTGATGGARTC HB12 12
GACTGGTGGACAGCAAATGGGT GAGGTGCARCTTGTTGAGTC HB13 13GACTGGTGGACAGCAAATGGGT GAGGTGCARCTTGTTGAGTC HB13 13
GACTGGTGGACAGCAAATGGGT GARGTRAAGCTTCTCGAGTC HB14 14GACTGGTGGACAGCAAATGGGT GARGTRAAGCTTCTCGAGTC HB14 14
GACTGGTGGACAGCAAATGGGT GAAGTGAARSTTGAGGAGTC HB15 15GACTGGTGGACAGCAAATGGGT GAAGTGAARSTTGAGGAGTC HB15 15
GACTGGTGGACAGCAAATGGGT CAGGTTACTCTRAAAGWGTSTG HB16 16GACTGGTGGACAGCAAATGGGT CAGGTTACTCTRAAAGWGTSTG HB16 16
GACTGGTGGACAGCAAATGGGT CAGGTCCAACTVCAGCARCC HB17 17GACTGGTGGACAGCAAATGGGT CAGGTCCAACTVCAGCARCC HB17 17
GACTGGTGGACAGCAAATGGGT GATGTGAACTTGGAAGTGTC HB18 18GACTGGTGGACAGCAAATGGGT GATGTGAACTTGGAAGTGTC HB18 18
GACTGGTGGACAGCAAATGGGT GAGGTGAAGGTCATCGAGTC HB19 19 GACTGGTGGACAGCAAATGGGT GAGGTGAAGGTCATCGAGTC HB19 19
PCRは、 96°C、 5分間反応後、 96°C、 30秒間、 50°C、 30秒間、 72°C、 1分間を 25サイ クル行った後 72°C、 5分間反応を行った。増幅した遺伝子は 2%ァガロースゲル電気泳 動によりそれぞれ 500-900bPのパンドを確認し、フエノール/クロ口ホルム処理を行つ た。すなわち同量のフエノール:クロ口ホルム:イソアミルアルコール(25:24:1)を加えよ く混和させ 4°Cで 13,200rpm、 5分間遠心し、水層部のみを新しいチューブに移しもう 一度同量のフエノール:クロ口ホルム:イソアミルアルコール(25:24:1)をカ卩えよく混和さ せ 4°Cで 13,200rpm、 5分間遠心し、水層部のみを新しいチューブに移した。得られた 溶液についてエタノール沈殿を行った。すなわち 20 mg/mlグリコーゲン溶液 (ナカラ ィテスタ株式会社) 1 1、 3 M酢酸ナトリウム(pH 5.2) 0.1倍量、 100%エタノール 3倍 量を添加し氷上で 15分間放置後、 13,200rpm、 20分間遠心して上清を除去して、 1ml の冷却した 70%エタノールにてペレットを洗浄後、再び 13,200rpm、 2分間遠心して上 清を除去した。その後、約 15分間遠心乾燥した後、各 DNA(19種)を RNase-free水 20 1に溶解した。 PCR was performed at 96 ° C for 5 minutes, followed by 25 cycles of 96 ° C, 30 seconds, 50 ° C, 30 seconds, 72 ° C, and 1 minute, followed by reaction at 72 ° C for 5 minutes. Amplified gene respectively check the Pando of 500-900B P by 2% Agarosugeru electrophoresis movement, Gyotsu a phenol / black port Holm process It was. That is, add the same volume of phenol: cloth form: isoamyl alcohol (25: 24: 1), mix well, centrifuge at 4 ° C for 13,200 rpm for 5 minutes, transfer only the aqueous layer to a new tube, and re-equivalent volume. Phenol: cloth form: isoamyl alcohol (25: 24: 1) was mixed well and centrifuged at 13,200 rpm for 5 minutes at 4 ° C, and only the aqueous layer was transferred to a new tube. The resulting solution was subjected to ethanol precipitation. That is, add a 20 mg / ml glycogen solution (Nacala Tester Co., Ltd.) 0.1 volume of 1,3 M sodium acetate (pH 5.2) and 3 volumes of 100% ethanol, leave on ice for 15 minutes, and continue at 13,200 rpm for 20 minutes. The supernatant was removed by taking care, and the pellet was washed with 1 ml of cooled 70% ethanol, and then centrifuged again at 13,200 rpm for 2 minutes to remove the supernatant. Then, after centrifugal drying for about 15 minutes, each DNA (19 species) was dissolved in RNase-free water 201.
[0134] [3] [2]で合成した各 DNA溶液 (19種) 1 μ 1、 ΗΒプライマーリストに示した対応する各 ΗΒプライマー(lOpmol/ 1) 2 1、 10 X PC R緩衝液 (TOYOBO) 10 μ 1、 (2 mM) dNTPs(TOYOBO) 10 1、下記の HFプライマーリストに示した VH forwardプライマー HF1:HF2:HF3:HF4(1:1:1:1)混合液 (lOpmol/ μ \) 2 μ KOD DASHポリメラーゼ (TOYOBO) 0.5 μ 1を混和させ、 RNase- Free水を添カ卩し全体量を 100 μ 1としてそれぞ れ PCR反応させた。  [3] [3] 1 μl of each DNA solution (19 types) synthesized in [2], 各 each corresponding 対 応 primer (lOpmol / 1) 21 shown in the primer list, 10 X PCR buffer (TOYOBO) ) 10 μ1, (2 mM) dNTPs (TOYOBO) 101, VH forward primer HF1: HF2: HF3: HF4 (1: 1: 1: 1) mixture shown in the HF primer list below (lOpmol / μ \ ) 2 μKOD DASH polymerase (TOYOBO) 0.5 μl was mixed, RNase-Free water was added to make a total volume of 100 μl, and each was subjected to PCR reaction.
[0135] [表 2] 表 2 HFプライマ一リスト  [Table 2] Table 2 List of HF primers
配列 名称 配列番号 Sequence name Sequence number
GATGGCCGAGACGTTTTGGCCGAGGAAACGGTGACCGTGGT HF1 20GATGGCCGAGACGTTTTGGCCGAGGAAACGGTGACCGTGGT HF1 20
GATGGCCGAGACGTTTTGGCCGAGGAGACTGTGAGAGTGGT HF2 21GATGGCCGAGACGTTTTGGCCGAGGAGACTGTGAGAGTGGT HF2 21
GATGGCCGAGACGTTTTGGCCGCAGAGACAGTGACCAGAGT HF3 22GATGGCCGAGACGTTTTGGCCGCAGAGACAGTGACCAGAGT HF3 22
GATGGCCGAGACGTTTTGGCCGAGGAGACGGTGACTGAGGT HF4 23 GATGGCCGAGACGTTTTGGCCGAGGAGACGGTGACTGAGGT HF4 23
[0136] PCRは、 96°C、 5分間反応後、 96°C、 30秒間、 50°C、 30秒間、 72°C、 1分間を 20サイ クル行った後、 72°C、 5分間反応を行った。増幅した遺伝子は 2%ァガロースゲル電気 泳動によりそれぞれ 500-900bpのバンドを確認し、フエノール/クロ口ホルム処理及び エタノール沈殿を行った。その後、約 15分間遠心乾燥した後、各 DNA(19種)を RNase— free水 10 μ 1に溶解した。 [0137] [4] [3]で得られた 19種の DNAを 2%低融点ァガロースゲル (Sigma)電気泳動に付し それぞれのバンドを切り出した。チューブに、切り出したァガロースゲルの断片と RNase-free水 100 μ 1をカ卩ぇ 70°Cで 15分間加熱後、 TE飽和フエノール処理を行った。 すなわち同量の TE飽和フエノール(8-キノリノール含有)を加えよく混和させ 4°Cで 13,200rpm、 5分間遠心し、水層部のみを新しいチューブに移しもう一度同量の TE飽 和フエノール (8-キノリノール含有)を加えよく混和し 4°Cで 13,200rpm、 5分間遠心し、 水層部のみを新し 、チューブに移した。得られた水層部をフエノール/クロ口ホルム処 理し、続いて得られた溶液についてエタノール沈殿を行った。得られたペレットは約 15分間遠心乾燥した後、各 DNA(19種)を RNase-free水 10 1に溶解した。各 DNA溶 液にっ 、て 260nmの吸収を測定し [0136] PCR was performed at 96 ° C for 5 minutes, followed by 20 cycles of 96 ° C, 30 seconds, 50 ° C, 30 seconds, 72 ° C, and 1 minute, followed by a reaction at 72 ° C for 5 minutes. Was done. The amplified gene was confirmed to have a band of 500-900 bp by 2% agarose gel electrophoresis, and was subjected to phenol / cloth form treatment and ethanol precipitation. Then, after centrifuging and drying for about 15 minutes, each DNA (19 species) was dissolved in 10 μl of RNase-free water. [4] The 19 types of DNA obtained in [4] and [3] were subjected to 2% low melting point agarose gel (Sigma) electrophoresis to cut out each band. The cut agarose gel fragment and 100 μl of RNase-free water were heated in a tube at 70 ° C. for 15 minutes, and then treated with TE-saturated phenol. That is, add the same amount of TE-saturated phenol (containing 8-quinolinol), mix well, centrifuge at 13,200 rpm for 5 minutes at 4 ° C, transfer only the aqueous layer to a new tube, and again use the same amount of TE-saturated phenol (8-quinolinol). (Containing quinolinol), mixed well, and centrifuged at 13,200 rpm for 5 minutes at 4 ° C. Only the aqueous layer was renewed and transferred to a tube. The obtained aqueous layer was subjected to phenol / chloroform-form treatment, and then the obtained solution was subjected to ethanol precipitation. The obtained pellet was centrifugally dried for about 15 minutes, and each DNA (19 species) was dissolved in RNase-free water 101. Measure the absorbance at 260 nm for each DNA solution.
HB1:HB2:HB3:HB4:HB5:HB6:HB7:HB8:HB9:HB10:HB11:HB12:HB13:HB14:HB15 : HB16:HB17:HB18:HB19 (8:9:4:4:12:4:1:4:12:4:4:2:2:4:4:8:6:1:1)の比率で混和さ せ合計 0.5pmolの H鎖 DNA溶液とした。  HB1: HB2: HB3: HB4: HB5: HB6: HB7: HB8: HB9: HB10: HB11: HB12: HB13: HB14: HB15: HB16: HB17: HB18: HB19 (8: 9: 4: 4: 12: 4: 1: 4: 12: 4: 4: 2: 2: 4: 4: 8: 6: 1: 1) to give a total 0.5 pmol H chain DNA solution.
[0138] [5]次に L鎖 DNA溶液の調製を行った。マウス脾臓 Poly A+ RNA (5 μ §/ μ \) ([5] Next, an L-chain DNA solution was prepared. Mouse spleen Poly A + RNA (5 μ § / μ \) (
DEPC-処理水)(CLONTECH社)を RNase-Free水で 100倍に希釈したものを 10 1、 5 X RT緩衝液 (TOYOBO) 20 1、 10 mM dNTPs(TOYOBO) 10 1、 MuCK forwardプラ イマ一 (配列番号 49)(1ρπιο1/ /ζ 1) 50 1を混和させ、 65°Cで 9分間反応後直ちに 4°Cに 冷却し、 4°Cで 2分間放置した後、 ReverTra Ace(TOYOBO) 5 1、 RNase DEPC-treated water) (CLONTECH) diluted 100-fold with RNase-Free water was used for 101, 5X RT buffer (TOYOBO) 201, 10 mM dNTPs (TOYOBO) 101, MuCK forward primer (SEQ ID NO: 49) (1ρπιο1 / / ζ 1) 50 1 is mixed, cooled to 4 ° C immediately after reacting at 65 ° C for 9 minutes, and left at 4 ° C for 2 minutes.ReverTra Ace (TOYOBO) 5 1, RNase
inhibitor(TOYOBO) 5 μ 1を加え、 50°Cで 30分間、次いで 99°Cで 5分間反応させ cDNA-Lを合成した。  5 μl of inhibitor (TOYOBO) was added, and reacted at 50 ° C. for 30 minutes and then at 99 ° C. for 5 minutes to synthesize cDNA-L.
[0139] [6] [5]で合成した cDNA- L溶液 5 μ 1に、下記の LBプライマーリストに示した各 LBプ ライマー(lpmol/ μ 1) 2.5 1、 10 X PCR緩衝液 (TOYOBO) 2.5 μ 1、 MuCK forwardプラ イマ一 (配列番号 49) (lpmol/ μ 1) 2.5 μ 1、 KOD DASHポリメラーゼ (TOYOBO) 0.25 μ 1を加え、 RNase- Free水を添カ卩し全体量を 25 μ 1としてそれぞれ PCR反応させた。  [6] [5] 5 μl of the cDNA-L solution synthesized in [5] was added to each of the LB primers (lpmol / μ1) 2.5 1, 10X PCR buffer (TOYOBO) shown in the LB primer list below. 2.5 μ1, MuCK forward primer (SEQ ID No. 49) (lpmol / μ1) Add 2.5 μ1, KOD DASH polymerase (TOYOBO) 0.25 μ1, add RNase-Free water, add 25 μl, and reduce the total volume to 25 μ The PCR reaction was performed as 1 for each.
[0140] [表 3] 表 3 LBプライマーリスト [0140] [Table 3] Table 3 LB primer list
配列 名称 配列番号 Sequence name Sequence number
GTGGCAGCATTGAGGGTCGCGAYATCCAGCTGACTCAGCC LB1 24GTGGCAGCATTGAGGGTCGCGAYATCCAGCTGACTCAGCC LB1 24
GTGGCAGCATTGAGGGTCGCGAYATTGTTCTCWCCCAGTC LB2 25GTGGCAGCATTGAGGGTCGCGAYATTGTTCTCWCCCAGTC LB2 25
GTGGCAGCATTGAGGGTCGCGAYATTGTGMTMACTCAGTC LB3 26GTGGCAGCATTGAGGGTCGCGAYATTGTGMTMACTCAGTC LB3 26
GTGGCAGCATTGAGGGTCGCGAYATTGTGYTRACACAGTC LB4 27GTGGCAGCATTGAGGGTCGCGAYATTGTGYTRACACAGTC LB4 27
GTGGCAGCATTGAGGGTCGCGAYATTGTRATGACMCAGTC LB5 28GTGGCAGCATTGAGGGTCGCGAYATTGTRATGACMCAGTC LB5 28
GTGGCAGCATTGAGGGTCGCGAYATTMAGATRAMCCAGTC LB6 29GTGGCAGCATTGAGGGTCGCGAYATTMAGATRAMCCAGTC LB6 29
GTGGCAGCATTGAGGGTCGCGAYATTCAGATGAYDCAGTC LB7 30GTGGCAGCATTGAGGGTCGCGAYATTCAGATGAYDCAGTC LB7 30
GTGGCAGCATTGAGGGTCGCGAYATYCAGATGACACAGAC LB8 31GTGGCAGCATTGAGGGTCGCGAYATYCAGATGACACAGAC LB8 31
GTGGCAGCATTGAGGGTCGCGAYATTGTTCTCAWCCAGTC LB9 32GTGGCAGCATTGAGGGTCGCGAYATTGTTCTCAWCCAGTC LB9 32
GTGGCAGCATTGAGGGTCGCGAYATTGWGCTSACCCAATC LB10 33GTGGCAGCATTGAGGGTCGCGAYATTGWGCTSACCCAATC LB10 33
GTGGCAGCATTGAGGGTCGCGAYATTSTRATGACCCARTC LB11 34GTGGCAGCATTGAGGGTCGCGAYATTSTRATGACCCARTC LB11 34
GTGGCAGCATTGAGGGTCGCGAYRTTKTGATGACCCARAC LB12 35GTGGCAGCATTGAGGGTCGCGAYRTTKTGATGACCCARAC LB12 35
GTGGCAGCATTGAGGGTCGCGAYATTGTGATGACBCAGKC LB13 36GTGGCAGCATTGAGGGTCGCGAYATTGTGATGACBCAGKC LB13 36
GTGGCAGCATTGAGGGTCGCGAYATTGTGATAACYCAGGA LB14 37GTGGCAGCATTGAGGGTCGCGAYATTGTGATAACYCAGGA LB14 37
GTGGCAGCATTGAGGGTCGCGAYATTGTGATGACCCAGWT LB15 38GTGGCAGCATTGAGGGTCGCGAYATTGTGATGACCCAGWT LB15 38
GTGGCAGCATTGAGGGTCGCGAYATTGTGATGACACAACC LB16 39GTGGCAGCATTGAGGGTCGCGAYATTGTGATGACACAACC LB16 39
GTGGCAGCATTGAGGGTCGCGAYATTTTGCTGACTCAGTC LB17 40GTGGCAGCATTGAGGGTCGCGAYATTTTGCTGACTCAGTC LB17 40
GTGGCAGCATTGAGGGTCGCGATGCTGTTGTGACTCAGCAATC LB λ 41 GTGGCAGCATTGAGGGTCGCGATGCTGTTGTGACTCAGCAATC LB λ 41
[0141] PCRは、 96°C、 5分間反応後、 96°C、 30秒間、 48°C、 30秒間、 72°C、 1分間を 25サイ クル行った後 72°C、 5分間反応を行った。増幅した遺伝子は 2%ァガロースゲル電気泳 動によりそれぞれ 500-900bpのバンドを確認し、フエノール/クロ口ホルム処理を行つ た。得られた溶液についてエタノール沈殿を行った。その後、約 15分間遠心乾燥した 後、各 DNA(18種)を RNase-free水 20 μ 1に溶解した。 [0141] PCR was performed at 96 ° C for 5 minutes, followed by 25 cycles of 96 ° C, 30 seconds, 48 ° C, 30 seconds, 72 ° C, and 1 minute, followed by reaction at 72 ° C for 5 minutes. went. The amplified gene was confirmed to have a band of 500-900 bp by 2% agarose gel electrophoresis, and was subjected to phenol / clonal form treatment. The obtained solution was subjected to ethanol precipitation. Then, after centrifugal drying for about 15 minutes, each DNA (18 species) was dissolved in 20 μl of RNase-free water.
[0142] [7] [6]で合成した各 DNA溶液 (18種) 1 μ 1、 LBプライマーリストに示した対応する各 LBプライマー (lOpmol/ μ ΐ) 2 μ 10 X PCR緩衝液 (TOYOBO) 10 1、 2 mM dNTPs(TOYOBO) 10 μ 1、下記の LFプライマーリストに示した LH forwardプライマー LF1:LF2:LF3:LF4:LF (1:1:1:1)混合液 (lOpmol/ 1) 2 1、 KOD DASHポリメラーゼ (TOYOBO) 0.5 μ 1を混和させ、 RNase-Free水を添加し全体量を 100 /ζ 1としてそれぞ れ PCR反応させた。 [0142] [7] 1 μl of each DNA solution synthesized in [6], 1 μl of each corresponding LB primer shown in the LB primer list (lOpmol / μΐ) 2 μ10 X PCR buffer (TOYOBO) 10 1, 2 mM dNTPs (TOYOBO) 10 μ1, LH forward primer LF1: LF2: LF3: LF4: LF (1: 1: 1: 1) mixture shown in the LF primer list below (lOpmol / 1) 2 1.Mix 0.5 μl of KOD DASH polymerase (TOYOBO) and add RNase-Free water to make the total volume 100 / ζ1. The PCR reaction was performed.
[0143] [表 4] 表 4 LFプライマーリスト [Table 4] Table 4 LF primer list
配列 名称 配列 Array Name Array
CTTGTCGTCGTCGTCCTTGTAGTCACGTTTGATTTCCAGCTTGG LF1 42CTTGTCGTCGTCGTCCTTGTAGTCACGTTTGATTTCCAGCTTGG LF1 42
CTTGTCGTCGTCGTCCTTGTAGTCACGTTTTATTTCCAGCTTGG LF2 43CTTGTCGTCGTCGTCCTTGTAGTCACGTTTTATTTCCAGCTTGG LF2 43
CTTGTCGTCGTCGTCCTTGTAGTCACGTTTTATTTCCAACTTTG LF3 44CTTGTCGTCGTCGTCCTTGTAGTCACGTTTTATTTCCAACTTTG LF3 44
CTTGTCGTCGTCGTCCTTGTAGTCACGTTTCAGCTCCAGCTTGG LF4 45CTTGTCGTCGTCGTCCTTGTAGTCACGTTTCAGCTCCAGCTTGG LF4 45
CTTGTCGTCGTCGTCCTTGTAGTCACCTAGGACAGTCAGTTTGG LF ^ 46 CTTGTCGTCGTCGTCCTTGTAGTCACCTAGGACAGTCAGTTTGG LF ^ 46
[0144] PCRは、 96°C、 5分間反応後、 96°C、 30秒間、 48°C、 30秒間、 72°C、 1分間を 20サイ クル行った後、 72°C、 5分間反応を行った。増幅した遺伝子は 2%ァガロースゲル電気 泳動によりそれぞれ 500-900bpのバンドを確認し、フエノール/クロ口ホルム処理及び エタノール沈殿を行った。その後、約 15分間遠心乾燥した後、各 DNA(18種)を RNase— free水 10 μ 1に溶解した。 [0144] PCR was performed at 96 ° C for 5 minutes, followed by 20 cycles of 96 ° C, 30 seconds, 48 ° C, 30 seconds, 72 ° C, and 1 minute, followed by a reaction at 72 ° C for 5 minutes. Was done. The amplified gene was confirmed to have a band of 500-900 bp by 2% agarose gel electrophoresis, and was subjected to phenol / cloth form treatment and ethanol precipitation. Then, after centrifugal drying for about 15 minutes, each DNA (18 kinds) was dissolved in 10 μl of RNase-free water.
[0145] [8] [7]で得られた 18種の DNAを 2%低融点ァガロースゲル (Sigma)電気泳動に付し それぞれのバンドを切り出した。チューブに、切り出したァガロースゲルの断片と RNase-free水 100 1をカ卩え、 70°Cで 15分間加熱後、 TE飽和フエノール処理を行った 。得られた水層部をフエノール/クロ口ホルム処理し続 ヽて得られた溶液にっ 、てエタ ノール沈殿を行った。得られたペレットは約 15分間遠心乾燥した後、各 DNA(18種)を RNase-free水 10 μ 1〖こ溶解した。各 DNA溶液について 260nmの吸収を測定し し B1:し B2:し B3:し B4:し B5:し B6:し B7:し B8:し B9:し B10:し B11:し B12:し B13:し B14:し B15:し B1 6:LB17:LB (2:4:8:8:8:16:12:4:4:8:16:16:12:4:4:2:2:1)の比率で混和させ合計 0.5pmolの L鎖 DNA溶液とした。  [0145] The eighteen kinds of DNAs obtained in [8] and [7] were subjected to 2% low melting point agarose gel (Sigma) electrophoresis, and each band was cut out. The cut agarose gel fragment and RNase-free water 1001 were added to a tube, heated at 70 ° C for 15 minutes, and then treated with TE-saturated phenol. The obtained aqueous layer was treated with phenol / chloroform, followed by ethanol precipitation using the resulting solution. The obtained pellet was centrifugally dried for about 15 minutes, and each DNA (18 species) was dissolved in 10 μl of RNase-free water. Measure the absorbance of each DNA solution at 260 nm. B14: then B15: then B1 6: LB17: LB (2: 4: 8: 8: 8: 16: 12: 4: 4: 8: 16: 16: 12: 4: 4: 2: 2: 1) The mixture was mixed at a ratio to obtain a total 0.5 pmol L chain DNA solution.
[0146] [9] H鎖と L鎖を一本ィ匕するためにさらに PCRを行った。 [4]で合成した H鎖 DNA溶 液 0.5pmol、 [8]で合成した L鎖 DNA溶液 0.5pmol、 5'UTR (配列番号 57)(lpmol/ 1) 0.5 μ 1、 McD— Linker+ (配列番号 56)(lpmol/ μ 1) 0.5 μ 1、 McD 3'UTR (His Tag) (配列 番号 55)(lpmol/ μ 1) 0.5 1、 10 X PCR緩衝液 (TOYOBO) 5 1、 2 mM  [9] PCR was further performed to ligate one H chain and one L chain. 0.5 pmol of H chain DNA solution synthesized in [4], 0.5 pmol of L chain DNA solution synthesized in [8], 5'UTR (SEQ ID NO: 57) (lpmol / 1) 0.5 μ1, McD—Linker + (SEQ ID NO: 56) (lpmol / μ1) 0.5 μ1, McD 3'UTR (His Tag) (SEQ ID NO: 55) (lpmol / μ1) 0.5 1, 10 X PCR buffer (TOYOBO) 51, 2 mM
dNTPs(TOYOBO) 5 1、 KOD DASHポリメラーゼ (TOYOBO) 0.25 μ 1を混和させ、 1^&36 6水を添カ卩し全体量を45.75 μ 1として PCR反応させた。 Mix dNTPs (TOYOBO) 51 and KOD DASH polymerase (TOYOBO) 0.25 μl, 1 ^ & 366 water was added to the mixture to make a total volume of 45.75 μl, and PCR was performed.
[0147] PCRは、 96°C、 5分間反応後、 96°C、 30秒間、続!、て 5分間のスロープで 58°Cとし、 58°C、 30秒間、 72°C、 1分間を 10サイクル行った後、 72°C、 5分間反応を行った。  [0147] PCR was performed at 96 ° C for 5 minutes, followed by 96 ° C for 30 seconds, followed by ramping to 58 ° C for 5 minutes, and 58 ° C for 30 seconds, 72 ° C for 1 minute. After 10 cycles, the reaction was performed at 72 ° C for 5 minutes.
[10]続いて、 [9]の PCR反応溶液 45.75 μ 1に、 McD- F (配列番号 52)(lpmol/ μ \) 2 μ 1、 McD— R (His Tag) (配列番号 50)(lpmol/ 1) 2 1、 KOD DASHポリメラーゼ (TOYOBO) 0.25 μ 1をカ卩えさらに PCR反応させた。  [10] Subsequently, to 45.75 μl of the PCR reaction solution of [9], 2 μl of McD-F (SEQ ID NO: 52) (lpmol / μ \), McD-R (His Tag) (SEQ ID NO: 50) (lpmol / 1) 21, 0.25 μl of KOD DASH polymerase (TOYOBO) was added and PCR was performed.
[0148] PCRは、 96°C、 5分間反応後、 96°C、 30秒間、 58°C、 30秒間、 72°C、 1分間を 15サイ クル行った後、 72°C、 5分間反応を行った。  [0148] PCR was performed at 96 ° C for 5 minutes, followed by 15 cycles of 96 ° C, 30 seconds, 58 ° C, 30 seconds, 72 ° C, and 1 minute, followed by reaction at 72 ° C for 5 minutes. Was done.
[11] [10]で得られた DNAを 1%低融点ァガロースゲル (Sigma)電気泳動に付しそれ ぞれのバンドを切り出した。チューブに、切り出したァガロースゲルの断片と  [11] The DNA obtained in [10] was subjected to 1% low melting point agarose gel (Sigma) electrophoresis to cut out each band. Place the cut agarose gel fragment in a tube
RNase-free水 100 1をカ卩え、 70°Cで 15分間加熱後、 TE飽和フエノール処理を行った 。得られた水層部をフエノール/クロ口ホルム処理し続 ヽて得られた溶液にっ 、てエタ ノール沈殿を行った。得られたペレットは約 15分間遠心乾燥した後、 DNAを  RNase-free water 100 1 was prepared, heated at 70 ° C. for 15 minutes, and then treated with TE-saturated phenol. The obtained aqueous layer was treated with phenol / chloroform, followed by ethanol precipitation using the resulting solution. The obtained pellet is centrifuged and dried for about 15 minutes,
RNase— free水 10 μ 1に溶解した。  RNase—dissolved in 10 μl of free water.
[0149] (II) ライブラリーの転写  [II] (II) Transcription of library
[12] [11]で得られた DNAまたは [25]で得られた DNA lpmol、 5 X SP6緩衝液 4 1、 ATP (lOOmM) 1 μ 1、 CTP (lOOmM) 1 μ 1、 UTP (lOOmM) 1 μ 1、 GTP (lOmM) 2 1、キ ヤップアナログ (m7G(5')PPP(5')G) (Invitrogen) (40mM) 2.5 μ 1、ェンザィムミックス SP6RNAポリメラーゼ(Promega) 2 1を混和させ、 RNase- Free水を添カ卩し全体量を 20 μ 1とし、 37°Cで 2時間 30分間反応後、 RQ1 RNase- Free DNase (Promega) 5 1を添加 しさらに 37°Cで 1時間反応させた。  [12] DNA obtained in [11] or DNA obtained in [25] lpmol, 5 X SP6 buffer 41, ATP (lOOmM) 1 μ1, CTP (lOOmM) 1 μ1, UTP (lOOmM) 1 μl, GTP (lOmM) 21, cap analog (m7G (5 ′) PPP (5 ′) G) (Invitrogen) (40 mM) 2.5 μl, Enzymemix SP6 RNA polymerase (Promega) 21 Mix, add RNase-Free water to make a total volume of 20 μl, react at 37 ° C for 2 hours 30 minutes, add RQ1 RNase-Free DNase (Promega) 51, and add 1 at 37 ° C. Allowed to react for hours.
[0150] [13] [12]で得られた RNAを RNeasy Mini kit (Qiagen)により精製した。すなわち転写 反応液に RNase- Free水を添カ卩し全体量を 100 μ 1とし、 RLT緩衝液 (Qiagen) 350 μ 1、 2-メルカプトエタノール 5 1、 100%エタノール 250 μ 1を加え、 RNeasyミニスピンカラ ムに供し、 4°C、 12,000 rpm、 16秒間遠心後排出された溶液を除去し、 RPE緩衝液 (Qiagen^OO /z lを同カラムに加え、 4°C、 12,000 rpm、 16秒間遠心後排出された溶液 を除去し、さらに RPE緩衝液 (Qiagen^OO /z 1を同カラムに加え、 4°C、 12,000 rpm、 2分 間遠心後排出された溶液を除去し、同カラムを新しいチューブに差し替え、 4°C、 12,000 rpm、 1分間遠心し、再び同カラムを新しいチューブに差し替え同カラムに RNase-Free水を 30.5 1添カ卩し、 10分間氷上で放置後、 4°C、 14000 rpm、 1分間遠心 し RNA溶液として回収した。 [0150] The RNA obtained in [13] and [12] was purified using the RNeasy Mini kit (Qiagen). That is, RNase-Free water was added to the transcription reaction solution to make the total volume 100 μl, and 350 μl of RLT buffer (Qiagen), 51 of 2-mercaptoethanol 51, and 250 μl of 100% ethanol were added, and the RNeasy mini Spin the column, centrifuge at 4 ° C, 12,000 rpm for 16 seconds, remove the discharged solution, add RPE buffer (Qiagen ^ OO / zl to the column, centrifuge at 4 ° C, 12,000 rpm, 16 seconds) After that, remove the discharged solution, add RPE buffer (Qiagen ^ OO / z 1) to the column, centrifuge at 4 ° C, 12,000 rpm for 2 minutes, remove the discharged solution, and replace the column with a new one. Replace with tube, 4 ° C, Centrifuge at 12,000 rpm for 1 minute, replace the same column with a new tube again, add RNase-Free water to the column, add 30.10 ml of RNase-free water, leave on ice for 10 minutes, and centrifuge at 4 ° C, 14000 rpm for 1 minute to RNA. Collected as a solution.
[0151] PEGスぺーサ一とのライゲーシヨン [0151] Ligation with PEG spacer
[14] [13]で得られた RNA溶液 29.5 μ 1、 Τ4 ligation 10 X緩衝液 5 1、 0.1 M DTT 1 1、 40 mM ATP 0.5 μ 1、 100% DMSO 5 1、 0.1% BSA 1 μ 1、 RNase  [14] RNA solution obtained in [13] 29.5 μ1, Τ4 ligation 10X buffer 51, 0.1 M DTT 11, 1, 40 mM ATP 0.5 μ1, 100% DMSO 51, 0.1% BSA 1 μ1 , RNase
inhibitor(TOYOBO) 1 μ 1、ポリエチレングリコール(PEG)を含むスぺーサ一分子(特 開 2002-176987 ; (dC) T(F1) PEG(2000) (dC ) Puro (記号の意味は特開 2002-276987  inhibitor (TOYOBO) 1 μl, a spacer molecule containing polyethylene glycol (PEG) (Japanese Patent 2002-176987; (dC) T (F1) PEG (2000) (dC) Puro -276987
P 2 p P P 2  P 2 p P P 2
に記載されている通りである) (10 nmol) 1 μ 1、ポリエチレングリコール(PEG) 2000 (日 本油脂) (30 nmol) 1 1、 T4 RNAリガーゼ (Takara)(250 υ/ μ \) 5 μ 1を混和させ、遮 光条件下 15°C、 12-15時間または遮光条件下 4°C、約 40時間反応させた。得られたス ぺーサ一分子が結合した RNAは RNeasy Mini kit (Qiagen)により精製した。  (10 nmol) 1 μl, polyethylene glycol (PEG) 2000 (Japanese fat) (30 nmol) 11 1, T4 RNA ligase (Takara) (250 υ / μ \) 5 μl 1 was mixed and reacted at 15 ° C for 12-15 hours under light-shielding conditions or at 4 ° C for approximately 40 hours under light-shielding conditions. The resulting RNA to which one spacer molecule was bound was purified using the RNeasy Mini kit (Qiagen).
[0152] (III) 翻訳 [0152] (III) Translation
[15] [14]で得られたスぺーサ一分子が結合した RNA 2.5 pmol、小麦胚芽抽出液( 5mM DTTを含む) (Promega) 12.5 μ 1、 Amino Acid mixture, Complete (ImM) ( Promega) 2 1、 RNase inhibitor(TOYOBO) 2 1、 CH COOK (1M) (Promega) 2 1を  [15] 2.5 pmol of RNA bound to one spacer molecule obtained in [14], wheat germ extract (containing 5 mM DTT) (Promega) 12.5 μ1, Amino Acid mixture, Complete (ImM) (Promega) 2 1, RNase inhibitor (TOYOBO) 2 1, CH COOK (1M) (Promega) 2 1
3  Three
混和させ、 RNase-Free水を添加し全体量を とし、遮光条件下 25°Cで 1時間反応 させ翻訳を行った。  After mixing, RNase-Free water was added to make the total volume of the mixture, and the reaction was carried out at 25 ° C for 1 hour under light-shielded conditions to perform translation.
[0153] (IV)ライブラリーの逆転写 [0153] (IV) Reverse transcription of library
[16]翻訳溶液の逆転写反応の前処理を行った。 RNase-Free水で調製し組成が 50 mMリン酸カリウム、 150 mM NaCl、 pH 6.0、 0.1% Tween 20である PP6T緩衝液にて膨 潤および平衡化させた Sephadex G200 (Amersham Biosciences)ゲル 1 mlをカラム(バ ィォラッド)に充填したものに [15]で得られた翻訳溶液 25 μ 1を供し、 2滴ずつチュー ブに集め 5本目から 8本目までを回収した。  [16] Pretreatment of the reverse transcription reaction of the translation solution was performed. 1 ml of Sephadex G200 (Amersham Biosciences) gel prepared with RNase-Free water, swollen and equilibrated with PP6T buffer having a composition of 50 mM potassium phosphate, 150 mM NaCl, pH 6.0, 0.1% Tween 20 25 μl of the translation solution obtained in [15] was applied to the one packed in a column (Viorad), and two drops were collected in a tube to collect the fifth to eighth tubes.
[0154] [17]または UltrafreeMC 100,000NWL (ミリポア)に [15]で得られた翻訳溶液 25 μ 1 を供し 4°C、 13,200 rpm、 20分間遠心し下層の溶液を回収した。 [0154] 25 µl of the translation solution obtained in [15] was applied to [17] or UltrafreeMC 100,000 NWL (Millipore), and the mixture was centrifuged at 13,200 rpm for 20 minutes at 4 ° C to recover the lower layer solution.
[18] [16]または [17]のいずれかの前処理溶液、 5 X RT緩衝液 (TOYOBO) 80 1、 [18] Pretreatment solution of either [16] or [17], 5X RT buffer (TOYOBO) 80 1,
(10 mM) dNTPs(TOYOBO) 40 μ 1、 McD-R (His Tag) (配列番号 5O)(10pmol/ μ 1) 20 μ 1を混和させ、 RNase- Free水を添カ卩し全体量を 360 μ 1とし、 65°Cで 9分間反応後直 ちに 4°Cに冷却し 4°Cで 2分間放置した後、 ReverTra Ace(TOYOBO) 20 μ 1、 RNase inhibitor(TOYOBO) 20 1を加えた。 50°Cで 30分間次いで 99°Cで 5分間反応、または 、 50°Cで 30分間反応させた。 (10 mM) dNTPs (TOYOBO) 40 μ1, McD-R (His Tag) (SEQ ID NO: 5O) (10 pmol / μ1) 20 After mixing 1 μl and adding RNase-Free water to make the total volume 360 μl, cool to 4 ° C immediately after reacting at 65 ° C for 9 minutes and leave at 4 ° C for 2 minutes. 20 μl of ReverTra Ace (TOYOBO) and RNase inhibitor (TOYOBO) 201 were added. The reaction was carried out at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes, or at 50 ° C for 30 minutes.
[0155] (V) 標的抗原の固定ィ匕  (V) Immobilization of target antigen
[19]榭脂(UltraLink Immobilized Neutr Avidin Plus) (Pierce) 50 を 1.5 mlチュ ーブに取り、 RNase-Free水 500 μ 1でけん濁させ、 4°C、 3,000 rpm、 1分間遠心し上清 を除去し再び RNase- Free水 500 μ 1でけん濁させ、 4°C、 3,000 rpm、 1分間遠心し上 清を除去した。組成が 100 mM Tris- HC1、 150 mM NaCl、 pH 7.5、 0.1% Tween 20で ある ELISA緩衝液に溶解した (0.4 M)アンジォテンシン II-ピオチンまたは (0.4 M) Lewis X- sp-ビォチンを、 500 1カ卩ぇ 25°Cにて 1時間ロータリーミキサー(Nissin)で回 転攪拌した。図 3にアンジォテンシン II-ピオチンの化学構造式および図 4に Lewis X-sp-ピオチンの化学構造式をそれぞれ示した。 ELISA緩衝液に溶解した 5 mMピオ チン 7 1をカ卩えさらに 25°Cにて 30分間ロータリーミキサー (Nissin)で回転攪拌した。 ELISA緩衝液 500 μ 1でけん濁させ、 4°C、 3,000 rpm、 1分間遠心し上清を除去し再び ELISA緩衝液 500 μ 1でけん濁させ、 4°C、 3,000 rpm、 1分間遠心し上清を除去した。  [19] Transfer 1.5 ml of UltraLink Immobilized Neutr Avidin Plus (Pierce) 50 to a 1.5 ml tube, suspend with 500 μl of RNase-Free water, centrifuge at 3,000 rpm for 1 minute at 4 ° C, and supernatant. Was removed and suspended again with 500 μl of RNase-Free water, and centrifuged at 3,000 rpm for 1 minute at 4 ° C. to remove the supernatant. (0.4 M) angiotensin II-biotin or (0.4 M) Lewis X-sp-biotin dissolved in an ELISA buffer having a composition of 100 mM Tris-HC1, 150 mM NaCl, pH 7.5, 0.1% Tween 20 The mixture was rotated and stirred with a rotary mixer (Nissin) at 500 ° C for 1 hour at 25 ° C. FIG. 3 shows the chemical structural formula of angiotensin II-biotin, and FIG. 4 shows the chemical structural formula of Lewis X-sp-biotin. 5 mM biotin 71 dissolved in the ELISA buffer was added and further stirred at 25 ° C. for 30 minutes with a rotary mixer (Nissin). Centrifuge with 500 μl of ELISA buffer, centrifuge at 3,000 rpm for 1 minute at 4 ° C, remove the supernatant, resuspend with 500 μl of ELISA buffer, and centrifuge for 1 minute at 3,000 rpm at 4 ° C. The supernatant was removed.
[0156] 10 Xマレイン酸緩衝液 4 ml (Dig wash and block buffer set)(Roche)とブロッキング 10  [0156] 10 X maleic acid buffer 4 ml (Dig wash and block buffer set) (Roche) and blocking 10
Xブロッキング溶液 4.45ml (Dig wash and block buffer set)(Roche)、および  X blocking solution 4.45 ml (Dig wash and block buffer set) (Roche), and
RNase-Free水 36 mlを混和させたブロッキング剤 500 μ 1でけん濁させ、 4°C、 3,000 rpm、 1分間遠心し上清を除去し、再び、同様のブロッキング剤 600 μ 1を加え 2 mlチュ 一ブに榭脂を移した。  Suspended with 500 μl of blocking agent mixed with 36 ml of RNase-Free water, centrifuged at 3,000 rpm for 1 minute at 4 ° C, removed the supernatant, added 600 μl of the same blocking agent again, and added 2 ml. The resin was transferred to a tube.
[0157] (VI) 標的抗原を認識する IVV抗体の選択  (VI) Selection of IVV Antibody Recognizing Target Antigen
[20]ブロッキング剤 600 μ 1にけん濁した榭脂に [18]で得られた逆転写反応したラ イブラリーの 400 /z lを加え、 4°Cにて 15分間ミニディスクローター(Bio craft)で回転 攪拌した。  [20] Blocking agent Add 400 / zl of the reverse-transcribed library obtained in [18] to the suspended resin in 600 µl of the suspended resin, and use a mini disc rotor (Biocraft) at 4 ° C for 15 minutes. The rotation was stirred.
[0158] シリンジ 5mL(Terumo)と Wizard Minicolumn (Promega)と注射針 18G X  [0158] Syringe 5mL (Terumo), Wizard Minicolumn (Promega) and injection needle 18G X
l/2"(Terumo)を直列につなぎ、上記のけん濁液 lmlを供し注射筒で押し出した。その 溶液を Flowとして一部 4°Cで保存した。 PP6T緩衝液に注射針を差し込み注射筒で 5 ml吸い上げ、再び押し出した。この操作を 6回行った。 l / 2 "(Terumo) were connected in series, lml of the above suspension was provided and extruded with a syringe. The solution was partially stored as a flow at 4 ° C. Insert the syringe needle into PP6T buffer and insert the syringe In 5 ml and pumped out again. This operation was performed six times.
[0159] さらにアンジォテンシン II (最終濃度 1 nM)または Lewis X(最終濃度 1 nM)を含む [0159] Further contains angiotensin II (final concentration 1 nM) or Lewis X (final concentration 1 nM)
PP6T緩衝液にて上記の操作を 2回行った。この操作の最後の溶出液を Washとして一 部 4°Cで保存した。アンジォテンシン 11(100 mM) 20 μ 1、 Ribonucleic acid-core from torula Yeast RNA Type Π— C (5 ^ g/ ^ 1) 2 μ \, PP6T緩衝液 178 μ 1または Lewis X 0.5 mgを93.6 μ 1を ΡΡ6Τ緩衝液に溶解させたものと、 Ribonucleic acid-core from torula Yeast RNA Type II- C (5 μ g/ μ 1) 0.95 μ 1を混和させ約 94.6 μ 1としテルモシ リンジとテルモ注射針をはずし Wizard Minicolumnにけん濁させ榭脂を回収した。この 榭脂けん濁液を 4°Cにて 1時間ミニディスクローター(Bio craft)で回転攪拌した。 The above operation was performed twice with PP6T buffer. The eluate at the end of this operation was partially stored at 4 ° C as Wash. Angiotensin 11 (100 mM) 20 μ 1, Ribonucleic acid-core from torula Yeast RNA Type Π-- C (5 ^ g / ^ 1) 2 μ \, PP6T buffer 178 μ 1 or Lewis X 0.5 mg 93.6 μ 1 dissolved in ΡΡ6Τ buffer and 0.95 μl of Ribonucleic acid-core from torula Yeast RNA Type II-C (5 μg / μ1) to make approximately 94.6 μl, and then add Terumo syringe and Terumo injection needle. The suspension was suspended in a Wizard Minicolumn and the fat was recovered. This fat suspension was rotationally stirred at 4 ° C. for 1 hour with a mini disc rotor (Biocraft).
[0160] [21]Urtra Freeに [20]で得られたけん濁液を供し 10,000 rpm、 3分間遠心し下層に 溶液を回収した。あらかじめ RNase-Free水 100 1をカ卩ぇ 10, 000 rpm、 3分間遠心した Microcon YM- 50に、溶出液 200 μほたは 94.6 μ 1を供し 12,000 rpm、 1.5- 2.5分間遠 心した。さらに RNase- Free水 100 1をカ卩ぇ 12,000 rpm、 1.5- 2.5分間遠心を数回繰り 返し行 、アンジォテンシン Πまたは Lewis Xを除去した。  [21] The suspension obtained in [20] was supplied to [21] Ultra Free, and centrifuged at 10,000 rpm for 3 minutes to recover the solution in the lower layer. RNase-Free water 1001 was previously centrifuged at 10,000 rpm at 10,000 rpm for 3 minutes. Microcon YM-50 was centrifuged at 12,000 rpm for 1.5-2.5 minutes with 200 μl of eluate. Further, RNase-Free water 1001 was repeatedly centrifuged several times at 12,000 rpm for 1.5 to 2.5 minutes to remove angiotensin II or Lewis X.
[0161] (VII) PCRによる cDNAライブラリーの回収  [0161] (VII) Recovery of cDNA library by PCR
[22] [21]で得られた溶出溶液 1 μ 1、 10 X PCR緩衝液 (ΤΟΥΟΒΟ) 10 1、 2 mM dNTPs(TOYOBO) 10 1、 Τ7— tag— F (配列番号 53) (lOpmol/ 1) 2 1、 McD-R his tag (配列番号 50) (lOpmol/ 1) 2 1、 KOD DASHポリメラーゼ (TOYOBO) 1 μ 1を混 和させ、 RNase-Free水を添カ卩し全体量を 100 μ 1としてそれぞれ PCR反応させた。  [22] 1 μl of the elution solution obtained in [21], 10 X PCR buffer (ΤΟΥΟΒΟ) 101, 2 mM dNTPs (TOYOBO) 101, Τ7—tag—F (SEQ ID NO: 53) (lOpmol / 1 ) 21 1, McD-R his tag (SEQ ID NO: 50) (lOpmol / 1) 21 and KOD DASH polymerase (TOYOBO) 1 μl, mix with RNase-Free water, and add 100 μl The PCR reaction was performed as 1 for each.
[0162] PCRは、 96°C、 5分間反応後、 96°C、 30秒間、 58°C、 30秒間、 72°C、 1分間を 25-30 サイクル行った後、 72°C、 5分間反応を行った。増幅した遺伝子は 1%ァガロースゲル 電気泳動によりそれぞれ 900-1000bpのバンドを確認した。  [0162] PCR was performed at 96 ° C for 5 minutes, followed by 25-30 cycles of 96 ° C, 30 seconds, 58 ° C, 30 seconds, 72 ° C, 1 minute, and then 72 ° C, 5 minutes. The reaction was performed. The amplified genes were confirmed to have 900-1000 bp bands by 1% agarose gel electrophoresis.
[0163] [23] [22]で得られた増幅した遺伝子は Wizard Plus Minipreps DNA Purification System (Promega)で精製した。すなわち PCR反応物を 1.5mlチューブに移し Direct purification緩 ¾r^Promega) 100 μ 1、 DNA purification resin (Promega) 1 mlをカ卩ぇ混 和させシリンジ 2.5 mL(Terumo)を使って Wizard Minicolumn (Promega)に供した。シリ ンジで液を押し出して捨て (80%)イソプロパノール 2 mlをカ卩ぇ再び液を押し出して捨て た。 Wizard Minicolumnを新しい 1.5 mlチューブに付け 4°C、 10,000 rpm、 2分間遠心し た後、再び新しい 1.5 mlチューブをつけ、 RNase-Free水 60 μ 1を加え、室温で 10分 間放置した。 4°C、 10,000 rpm、 2分間遠心し、 Wizard Minicolumnを捨て、下層を回収 し、さらに 4°C、 10,000 rpm、 5分間遠心し、生じた沈殿を除き新しい 1.5mlチューブに 上清を回収した。 [0163] [23] The amplified gene obtained in [22] was purified using Wizard Plus Minipreps DNA Purification System (Promega). That is, transfer the PCR reaction product to a 1.5 ml tube, mix 100 μl of Direct purification buffer (¾r ^ Promega) and 1 ml of DNA purification resin (Promega), mix with 1 ml of syrup, and use a 2.5 mL syringe (Terumo) for Wizard Minicolumn (Promega). Was served. The solution was extruded with a syringe and discarded (80%), and 2 ml of isopropanol was extruded again and discarded. Place the Wizard Minicolumn in a new 1.5 ml tube and centrifuge at 4 ° C, 10,000 rpm for 2 minutes. After that, a new 1.5 ml tube was attached again, 60 µl of RNase-Free water was added, and the mixture was left at room temperature for 10 minutes. Centrifuged at 4 ° C, 10,000 rpm for 2 minutes, discarded the Wizard Minicolumn, collected the lower layer, and centrifuged at 4 ° C, 10,000 rpm for 5 minutes to remove the formed precipitate and collected the supernatant in a new 1.5 ml tube. .
[0164] DNA溶液にっ 、て 260nmの吸収を測定し濃度を見積もった。  The concentration of the DNA solution was estimated by measuring the absorption at 260 nm.
(VIII) PCRによるオメガ配列の付カロ  (VIII) Calories of omega sequence by PCR
[24] PCRによるオメガ配列の付カ卩をするために [23]で得られた DNA溶液 0.2 pmol 、 10 X PCRllf Π¾(ΤΟΥΟΒΟ) 10 1、 2 mM dNTPs(TOYOBO) 10 μ 1、 McD 5'UTR ( 配列番号 54)(lpmol/ j 1) 1 j 1、 McD- F (配列番号 52)(10pmol/ u l) 2 u McD- R his tag (配列番号 5O)(10pmol/ 1) 2 1、 KOD DASHポリメラーゼ (TOYOBO) 0.5 μ 1を 混和させ、 RNase-Free水を添カ卩し全体量を 100 μ 1としてそれぞれ PCR反応させた。 [24] For adding omega sequence by PCR, 0.2 pmol of DNA solution obtained in [23], 10 X PCRllf Π¾ (ΤΟΥΟΒΟ) 101, 2 mM dNTPs (TOYOBO) 10 μ1, McD 5 ' UTR (SEQ ID NO: 54) (lpmol / j 1) 1 j1, McD-F (SEQ ID NO: 52) (10 pmol / ul) 2 u McD-R his tag (SEQ ID NO: 50) (10 pmol / 1) 21, KOD 0.5 μl of DASH polymerase (TOYOBO) was mixed, and RNase-Free water was added to the mixture to make a total volume of 100 μl.
[0165] PCRは、 96°C、 5分間反応後、 96°C、 30秒間、 58°C、 30秒間、 72°C、 1分間を 7-10サ イタル行った後、 72°C、 5分間反応を行った。増幅した遺伝子は 1%ァガロースゲル電 気泳動によりそれぞれ約 1000bpのバンドを確認し、フエノール/クロ口ホルム処理及び エタノール沈殿を行った。その後、約 15分間遠心乾燥した後、 DNAを RNase-free水 10 1に溶解した。 [0165] PCR was performed at 96 ° C for 5 minutes, followed by 96 ° C, 30 seconds, 58 ° C, 30 seconds, 72 ° C, 1 minute for 7-10 cycles, and then at 72 ° C, 5 minutes. The reaction was performed for minutes. The amplified gene was confirmed to have a band of about 1000 bp by 1% agarose gel electrophoresis, and was subjected to phenol / cloth-form treatment and ethanol precipitation. After centrifugation and drying for about 15 minutes, the DNA was dissolved in RNase-free water (101).
[0166] [25] [24]で得られた DNAを 1%低融点ァガロースゲル (Sigma)電気泳動に付しそれ ぞれのバンドを切り出した。チューブに、切り出したァガロースゲルの断片と  [0166] The DNA obtained in [25] and [24] was subjected to 1% low melting point agarose gel (Sigma) electrophoresis, and each band was cut out. Place the cut agarose gel fragment in a tube
RNase-free水 100 1をカ卩え、 70°Cで 15分間加熱後、 TE飽和フエノール処理を行った 。得られた水層部をフエノール/クロ口ホルム処理し、続いて得られた溶液についてェ タノール沈殿を行った。得られたペレットは約 15分間遠心乾燥した後、 DNAを RNase— free水 10 μ 1に溶解した。  RNase-free water 100 1 was prepared, heated at 70 ° C. for 15 minutes, and then treated with TE-saturated phenol. The obtained aqueous layer was subjected to phenol / chloroform-form treatment, and then the obtained solution was subjected to ethanol precipitation. The obtained pellet was centrifuged and dried for about 15 minutes, and the DNA was dissolved in 10 μl of RNase-free water.
[0167] 図 5には、上記の工程で得られた各翻訳溶液を 8Μ尿素存在下 5%ポリアクリルアミド 電気泳動に付し対応付け分子を確認した結果を示す。図中、下の棒グラフは上の電 気泳動ゲルの FITCの蛍光を MOLECULAR IMAGER FX (Bio-rad)で測定し定量した 結果を示す。左力も Negaは [11]で得られたライブラリー MH0のクローンのひとつであ る MH0-15、 Posiはライブラリー MI3のクローンのひとつである MI3-55、 MHOは [11]で 得られたライブラリー、 Milは [11]で得られたライブラリーを [18]において 50°C、 30分 間次いで 99°C、 5分間反応させアンジォテンシン IIを抗原としてセレクションし [25]で 回収したライブラリー、 MI2は Milを [18]において 50°C、 30分間次いで 99°C、 5分間反 応させアンジォテンシン IIを抗原としてセレクションし [25]で回収したライブラリー、 MI3は MI2を [18]において 50°C、 30分間次いで 99°C、 5分間反応させアンジォテンシ ン IIを抗原としてセレクションし [25]で回収したライブラリーを示している。 [0167] FIG. 5 shows the results obtained by subjecting each translation solution obtained in the above steps to 5% polyacrylamide electrophoresis in the presence of 8-urea to identify the associating molecules. In the figure, the lower bar graph shows the results of measuring and quantifying the fluorescence of FITC of the upper electrophoresis gel using MOLECULAR IMAGER FX (Bio-rad). Nega is one of the clones of library MH0 obtained in [11], MH0-15, Posi is one of the clones of library MI3, MI3-55, and MHO is the live one obtained in [11]. Larry and Mil use the library obtained in [11] at [18] at 50 ° C for 30 minutes Then, the reaction was performed at 99 ° C for 5 minutes, and angiotensin II was selected as an antigen. The library recovered in [25], MI2 was reacted with [18] at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes. For the library collected by [25], MI2 was reacted with [2] at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes to select angiotensin II as an antigen. And the library recovered in [25].
[0168] MHOではストップコドンまたはミューテーシヨンにより蛋白質に翻訳されないものがか なりの割合で含まれていること力 対応付けの効率は 15%と低いのに対して、 Milでは 25%、 MI2では 37%、 MI3では 41%と対応付けの効率が上昇している。 Negaおよび Posiは インフレームのクローンでありこの場合の対応付け効率はそれぞれ 34%および 40%を 示しているが MI2または MI3においてほぼその対応付け効率と同等の値が得られて いる。従って蛋白レベルでのセレクションが成功しライブラリ一はインフレームの割合 が上昇して 、ることを示して!/、る。  [0168] MHO contains a considerable proportion of proteins that are not translated into proteins due to stop codons or mutations. The efficiency of force association is as low as 15%, whereas that of Mil is 25% and that of MI2 is 25%. The efficiency of association has increased to 37% and 41% for MI3. Nega and Posi are in-frame clones, and the association efficiency in this case is 34% and 40%, respectively. However, MI2 or MI3 has almost the same value as the association efficiency. Therefore, the selection at the protein level was successful, indicating that the library had a higher in-frame rate! /
[0169] 図 6は、図 5と同様に上記の工程で得られた各翻訳溶液を 8M尿素存在下 5%ポリア クリルアミド電気泳動に付し対応付け分子を確認した結果を示す。左から MHOは [11] で得られたライブラリー、 MK1は [11]で得られたライブラリーを [18]において 50°C、 30 分間次いで 99°C、 5分間反応させ Lewis Xを抗原としてセレクションし [25]で回収した ライブラリー、 MK2は MK1を [18]において 50°C、 30分間次いで 99°C、 5分間反応させ Lewis Xを抗原としてセレクションし [25]で回収したライブラリーを示している。 MHOで はストップコドンまたはミューテーシヨンにより蛋白質に翻訳されないものがかなりの割 合で含まれて 、ることから対応付けの効率は 15%と低 、のに対して、 MK1では 41%、 MK2では 36%と対応付けの効率が上昇して!/、る。  [0169] FIG. 6 shows the results obtained by subjecting each translation solution obtained in the above steps to 5% polyacrylamide electrophoresis in the presence of 8M urea in the same manner as in FIG. 5, and confirming the corresponding molecules. From the left, MHO is the library obtained in [11], and MK1 is the library obtained in [11] at [18] at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes, and Lewis X is used as an antigen. The library selected and collected in [25], MK2 was prepared by reacting MK1 in [18] at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes, selecting the library using Lewis X as an antigen and recovering the library collected in [25]. Is shown. In the MHO, a significant percentage of non-translated proteins due to stop codons or mutations contained a low efficiency of 15%, whereas 41% for MK1 and 41% for MK2. Efficiency of association increased with 36%!
[0170] 図 7は、図 5と同様に上記の工程で得られた各翻訳溶液を 8M尿素存在下 5%ポリア クリルアミド電気泳動に付し対応付け分子を確認した結果を示す。左から MM1は [11 ]で得られたライブラリーを [18]において 50°C30分間反応させアンジォテンシン IIを抗 原としてセレクションし [25]で回収したライブラリー、 MM2は MM1を [18]において 50 °C30分間反応させアンジォテンシン IIを抗原としてセレクションし [25]で回収したライ ブラリー、 MP1は [11]で得られたライブラリーを [15]において 99°C、 5分間反応させそ の後 [20] [21]においてアンジォテンシン IIを抗原としセレクションを行ったあとに [18] において 50°C、 30分間次いで 99°C、 5分間反応させて [22]以降の実験を行い [25]で 回収したライブラリーを示している。 MM1では 25%、 MM2では 33%と対応付けの効率が 上昇している。 MM1では [18]において 50°C、 30分間反応させた後 99°C、 5分間反応 させた Milと比較し同様な値であり、また MM2も MI2と比較し遜色な 、対応付け効率 を示すことから蛋白レベルでのセレクションは成功していると考えられる。これに対し て MP1は 8%と対応付けの効率が極端に低く [15]において 99°C、 5分間反応させること により RNAまたは対応付け分子に対し何らかの変性が生じた可能性も考えられた。 FIG. 7 shows the results obtained by subjecting each translation solution obtained in the above steps to 5% polyacrylamide electrophoresis in the presence of 8 M urea and confirming the assigning molecules as in FIG. From the left, MM1 is the library obtained in [11], reacted at 50 ° C for 30 minutes in [18], selected angiotensin II as an antigen, and recovered in [25]. MM2 was MM1 in [18]. The library obtained in [25], MP1 was reacted with the library obtained in [11] at 99 ° C for 5 minutes at [15]. After [20] [21], selection was performed using angiotensin II as an antigen, [18] Shows the library recovered in [25] after performing the reaction from [22] after reacting at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes. The efficiency of association is 25% for MM1 and 33% for MM2. MM1 has the same value as [18] compared to Mil that was reacted at 50 ° C for 30 minutes and then reacted at 99 ° C for 5 minutes at [18], and MM2 also shows an association efficiency that is inferior to MI2 This suggests that the selection at the protein level has been successful. On the other hand, MP1 has an extremely low assignment efficiency of 8%, and it is possible that some denaturation of RNA or the assignment molecule may have occurred by reacting at 99 ° C for 5 minutes in [15].
[0171] 図 8は、 [21]で得られた溶出液を [22]で PCRし 1%ァガロースゲル電気泳動に付した 結果を示し、下の棒グラフは上の電気泳動ゲルのェチジゥムブロマイドの吸収を MOLECULAR IMAGER FX (Bio-rad)で測定し定量したものを示している。左から Negaは [11]で得られたライブラリー MHOのクローンのひとつである MH0-15、 Posiはラ イブラリー MI3のクローンのひとつである MI3-55、 Milは [11]で得られたライブラリーを [18]において 50°C、 30分間次いで 99°C、 5分間反応させアンジォテンシン IIを抗原と してセレクションし [25]で回収したライブラリー、 MI2は Milを [18]において 50°C、 30分 間次いで 99°C、 5分間反応させアンジォテンシン IIを抗原としてセレクションし [25]で 回収したライブラリー、 MI3は MI2を [18]において 50°C、 30分間次いで 99°C、 5分間反 応させアンジォテンシン IIを抗原としてセレクションし [25]で回収したライブラリーを示 している。 Negaでは回収される DNAの量は非常に少なくアンジォテンシン IIに親和性 のある分子のがほとんど含まれて 、な 、ことを示し、 Posiでは回収される DNAの量は 多くアンジォテンシン IIに親和性のある分子が大多数であることを示している。 Mil, MI2ではセレクションで回収される DNAの量はまだ少なくライブラリ一中に含まれるァ ンジォテンシン IIに親和性のある分子の割合が低 、ものと考えられる。これに対して MI3では回収される DNAの量が急激に上昇しライブラリ一中に含まれるアンジォテン シン IIに親和性のある分子の割合が急激に上昇しセレクションを繰り返すことによって 濃縮が起こったことを示すものと考えられる。  [0171] FIG. 8 shows the results of PCR of the eluate obtained in [21] and PCR of the eluate obtained in [22] and 1% agarose gel electrophoresis. The lower bar graph shows ethidium bromide in the upper electrophoresis gel. The figure shows the results obtained by measuring and quantifying the absorption of MOLECULAR IMAGER FX (Bio-rad). From left, Nega is a library obtained in [11], MH0-15, one of the clones of MHO, Posi is a library, MI3-55, one of the clones in library MI3, and Mil is a library obtained in [11]. Was reacted at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes in [18], and the library collected by selecting [25] for angiotensin II as an antigen. C, 30 minutes and then 99 ° C, 5 minutes reaction, library selected from angiotensin II as antigen and collected in [25], MI3 is MI2 in [18] at 50 ° C, 30 minutes and 99 ° C The reaction was performed for 5 minutes, and the library recovered by selecting with angiotensin II as an antigen [25] is shown. In Nega, the amount of DNA recovered was very small, indicating that most molecules with affinity for angiotensin II were contained. This indicates that the affinity molecules are the majority. In Mil and MI2, the amount of DNA recovered by selection is still small, and it is considered that the proportion of molecules having affinity for angiotensin II contained in the library is low. On the other hand, in MI3, the amount of recovered DNA sharply increased, the proportion of molecules with affinity for angiotensin II contained in the library increased sharply, and enrichment occurred due to repeated selection. It is considered to indicate.
[0172] 図 9は、 [20]で得られた Flowを 1000倍希釈したものと [20]で得られた Washおよび [ 21]で得られた溶出液 (Elute)を 1倍と 10倍希釈したものを [22]で PCRし 1%ァガロース ゲル電気泳動に付した結果である。 Eluteに関し、 1は溶出液を1倍希釈、 X 0.1は 溶出液を 10倍希釈の略である。 MK1は [11]で得られたライブラリーを [18]において 50°C、 30分間次いで 99°C、 5分間反応させ Lewis Xを抗原としてセレクションし [25]で 回収したライブラリー、 MK2は MK1を [18]において 50°C、 30分間次いで 99°C、 5分間 反応させ Lewis Xを抗原としてセレクションし [25]で回収したライブラリーを示している 。 Flowに示すように十分大過剰量の分子をセレクションに投入して 、るが Washでは ほとんど DNAのバンドは見えないことからセレクションにおいて Lewis Xに親和性の少 な 、分子を十分洗 、流せて 、ることを示して 、る。また MK1ではセレクションで回収さ れる DNAの量はまだ少なくライブラリ一中に含まれる Lewis Xに親和性のある分子の 割合が低 、ものと考えられる。これに対して MK2では回収される DNAの量が急激に 上昇しライブラリ一中に含まれる Lewis Xに親和性のある分子の割合が急激に上昇し セレクションを繰り返すことによって濃縮が起こったことを示すものと考えられる。 [0172] FIG. 9 shows that the flow obtained in [20] is diluted 1000-fold, the wash obtained in [20] and the eluate (Elute) obtained in [21] are diluted 1- and 10-fold. This is the result of PCR of [22] followed by 1% agarose gel electrophoresis. For Elute, 1 = 1x dilution of eluate, X 0.1 = Abbreviation of 10-fold dilution of eluate. MK1 is a library obtained by reacting the library obtained in [11] at 50 ° C for 30 minutes and then 99 ° C for 5 minutes in [18], selecting Lewis X as an antigen and collecting in [25], and MK2 is MK1. Is reacted at 50 ° C. for 30 minutes and then at 99 ° C. for 5 minutes in [18], and the library recovered using [25] is selected using Lewis X as an antigen. As shown in Flow, a sufficiently large excess amount of the molecule is put into the selection. However, since the DNA band is hardly seen by Wash, the molecule having low affinity for Lewis X in the selection can be sufficiently washed and washed. To show that Also, with MK1, the amount of DNA recovered by selection is still small, and the proportion of molecules with affinity for Lewis X contained in the library is considered to be low. On the other hand, in MK2, the amount of recovered DNA sharply increased, the proportion of molecules with affinity for Lewis X contained in the library increased sharply, indicating that enrichment occurred by repeating selection. It is considered.
[0173] 図 10は、 [20]で得られた Flowを 1000倍希釈したものと [20]で得られた Washおよび  [0173] FIG. 10 shows that the flow obtained in [20] was diluted 1000-fold, and the Wash and [20] obtained in [20] were diluted.
[21]で得られた溶出液を 1倍と 10倍希釈したものを [22]で PCRし 1%ァガロースゲル電 気泳動に付した結果とその電気泳動ゲルのェチジゥムブロマイドの吸収を  The eluate obtained in [21] was diluted 1-fold and 10-fold and subjected to PCR in [22] and subjected to 1% agarose gel electrophoresis, and the absorption of ethidium bromide in the electrophoresis gel was measured.
MOLECULAR IMAGER FX (Bio-rad)で測定し定量した結果を示す。 Fは Flow、 Wは Wash, Eは溶出液を 1倍、 E0.1は溶出液を 10倍希釈の略である。  The results of measurement and quantification with MOLECULAR IMAGER FX (Bio-rad) are shown. F stands for Flow, W stands for Wash, E stands for 1-fold dilution, and E0.1 stands for 10-fold dilution.
[0174] Flowに示すように十分大過剰量の分子をセレクションに投入して 、るが Washでは ほとんど DNAのバンドは見えないことからセレクションにおいてアンジォテンシン IIに 親和性の少な 、分子を十分洗 、流せて 、ることを示して 、る。  [0174] As shown in the flow, a sufficiently large excess amount of the molecule was introduced into the selection. However, since the DNA band was hardly visible in the Wash, the molecule having a low affinity for angiotensin II was sufficiently washed in the selection. , Let it flow, show it.
[0175] Milは [11]で得られたライブラリーを [18]において 50°C、 30分間次いで 99°C、 5分 間反応させアンジォテンシン IIを抗原としてセレクションし [25]で回収したライブラリー 、 MM1は [11]で得られたライブラリーを [18]において 50°C、 30分間させアンジォテン シン IIを抗原としてセレクションし [25]で回収したライブラリーを示している。  [0175] Mil reacted the library obtained in [11] at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes in [18], selected angiotensin II as an antigen, and collected it in [25]. The library, MM1, shows the library obtained in [18] at 50 ° C for 30 minutes in [18], and angiotensin II was selected as an antigen, and recovered in [25].
[0176] Milではセレクションで回収される DNAの量はまだ少なくライブラリ一中に含まれる アンジォテンシン IIに親和性のある分子の割合が低いものと考えられる。これに対し て MM1では回収される DNAの量が非常に多くライブラリ一中に含まれるアンジォテン シン IIに親和性のある分子の割合が多いものと考えられる。すなわち MM1では [18] において 50°C、 30分間反応させただけなのに対して Milでは [18]において 50°C、 30 分間反応させた後 99°C、 5分間反応させていることによって 99°Cに安定性の高い分 子だけがセレクションされ不安定なものは除去される傾向にあることを示して 、る。 実施例 2 [0176] In Mil, the amount of DNA recovered by selection is still small, and it is considered that the proportion of molecules having an affinity for angiotensin II contained in one library is low. On the other hand, in MM1, the amount of recovered DNA is very large, and it is considered that the ratio of molecules having affinity for angiotensin II contained in the library 1 is large. In other words, MM1 was only reacted at 50 ° C for 30 minutes at [18], while Mil was 50 ° C at 30 ° C at [18]. By reacting at 99 ° C for 5 minutes after reacting for 5 minutes, it is shown that only molecules having high stability at 99 ° C tend to be selected and unstable ones tend to be removed. Example 2
[0177] 配列分析 [0177] Sequence analysis
(I) クローニング  (I) Cloning
[26]ライブラリーからクローンを得るために TOPOクロー-ングキット(Invitrogen)を 用いた。 [11]で得られた DNAまたは [25]で得られた DNA 0.05-1 pmol、 taqポリメラー ゼ 10 X緩衝液 (TOYOBO) 2 μ \, dATP (40 mM) 2 μ \, taqポリメラーゼ(Taq Pol) (ΤΟΥΟΒΟ)0.5 μ 1を混和させ、 RNase- Free水を添カ卩し全体量を 20 μ 1として、 72°Cで 15分間、フエノール/クロ口ホルム処理及びエタノール沈殿を行った。その後、約 15分 間遠心乾燥した後、 DNAを RNase- free水 4 μ 1に溶解した。  [26] TOPO Cloning Kit (Invitrogen) was used to obtain clones from the library. DNA obtained in [11] or DNA obtained in [25] 0.05-1 pmol, taq polymerase 10X buffer (TOYOBO) 2 μ \, dATP (40 mM) 2 μ \, taq polymerase (Taq Pol (ΤΟΥΟΒΟ) 0.5 μl was mixed, RNase-free water was added to the mixture, and the total amount was adjusted to 20 μl. The mixture was subjected to phenol / cloth form treatment and ethanol precipitation at 72 ° C. for 15 minutes. After centrifugation and drying for about 15 minutes, the DNA was dissolved in 4 µl of RNase-free water.
[0178] [27] [26]で得られた DNA 4 1、 Topo vector (Invitrogen) 0.5 μ 1、 Salt solution ( [0178] [27] DNA 41 obtained from [26], Topo vector (Invitrogen) 0.5 μ1, Salt solution (
Invitrogen) 1 1を混和させ室温で 20分間放置した。大腸菌にトランスフォームするた めに氷上で溶解したコンビテントセルに上記の混和溶液 5.5 μ 1を入れ氷上で 30分間 放置後 43°C、 45秒間ヒートショックを行った。セルに SOC (Invitrogen)を 250 μ 1入れ 37 °C、 1時間 30分振とう培養器で培養させ 2枚の寒天プレート (500 ml中にトリプトン 5 g 、酵母エキス 2.5 g、 NaCl 5 g、寒天 7.5 g、アンピシリン 25 mg) (シャーレ 9cm)にまい て 37°Cでー晚培養した。 (Invitrogen) 11 was mixed and left at room temperature for 20 minutes. 5.5 μl of the above mixed solution was placed in a competent cell melted on ice in order to transform into E. coli, left on ice for 30 minutes, and then subjected to heat shock at 43 ° C. for 45 seconds. Place 250 μl of SOC (Invitrogen) in the cell and incubate in a shaking incubator at 37 ° C for 1 hour and 30 minutes.Two agar plates (5 g of tryptone, 2.5 g of yeast extract, 5 g of NaCl, 5 g of NaCl, agar in 500 ml) 7.5 g, ampicillin 25 mg) (9 cm petri dish) were cultured at 37 ° C.
[0179] (II) 塩基配列の決定  (II) Determination of base sequence
[28] [27]で培養した寒天プレートに生じたコロニーの一部を爪楊枝でつっつき RNase-Free水 20 1にけん濁させ 99°C、 5分間加熱後急冷し、 - 20°Cでコロニーけん 濁液として保存した。  [28] A portion of the colony formed on the agar plate cultured in [27] is stuck with a toothpick, suspended in RNase-Free water 201, heated at 99 ° C for 5 minutes, quenched, and cooled at -20 ° C. Stored as a suspension.
[0180] [29] [28]で得られたコロニーけん濁液 1 1、 10 X PCR緩衝液 (TOYOBO) 5 1、 2 mM dNTPs(TOYOBO) 5 1、 M13FII (酉己歹 [J番号 58) (10 pmol/ 1) 1 1、 M13RII (酉己 列番号 59) (10 pmol/ 1) 1 1、 KOD DASHポリメラーゼ (TOYOBO) 0.25 μ 1を混和さ せ、 RNase-Free水を添加し全体量を 50 μ 1としてそれぞれ PCR反応させた。  [0180] [29] Colony suspension obtained in [28] 11; 10X PCR buffer (TOYOBO) 51; 2 mM dNTPs (TOYOBO) 51; M13FII (Torigami [J number 58] (10 pmol / 1) 1 1, M13RII (Toroki Column No. 59) (10 pmol / 1) 11 1, KOD DASH polymerase (TOYOBO) 0.25 μl, mix, add RNase-Free water and reduce the total volume PCR reaction was carried out with 50 μl each.
[0181] PCRは、 96°C、 5分間反応後、 96°C、 30秒間、 58°C、 30秒間、 72°C、 1分間を 30サイ クル行った後 72°C、 5分間反応を行った。増幅した遺伝子は 1%ァガロースゲル電気泳 動によりそれぞれ 900-1000bpのバンドを確認した。増幅した遺伝子は Wizard Plus Minipreps DNA Purification System (Promegaで精製し 7こ。 [0181] In the PCR, the reaction was performed at 96 ° C for 5 minutes, followed by 30 cycles of 96 ° C, 30 seconds, 58 ° C, 30 seconds, 72 ° C, and 1 minute, followed by a reaction at 72 ° C for 5 minutes. went. Amplified gene is electrophoresed in 1% agarose gel The band of 900-1000bp was confirmed by the movement. The amplified gene was purified using the Wizard Plus Minipreps DNA Purification System (Promega 7).
[0182] [30] [29]で得られた DNA 16 ng、 M13FII (配列番号 58) (1.6 pmol/ μ 1) 2 1、また は M13RII (配列番号 59) (1.6 pmol/ μ \) 2 μ DTCS kit Premix (Beckman coulter) 6 μ 1を混和させ、 RNase-Free水を添カ卩し全体量を 10 ;ζ 1としてそれぞれ PCR反応させ た。 [0182] [30] 16 ng of DNA obtained from [29], M13FII (SEQ ID NO: 58) (1.6 pmol / μ1) 21 or M13RII (SEQ ID NO: 59) (1.6 pmol / μ \) 2 μ 6 μl of DTCS kit Premix (Beckman coulter) was mixed, and RNase-Free water was added to make a total volume of 10;
PCRは、 96°C、 5分間反応後、 96°C、 20秒間、 58°C、 20秒間、 60°C、 4分間を 30サイク ル行った後、 72°C、 5分間反応を行った。  PCR was performed at 96 ° C for 5 minutes, followed by 30 cycles of 96 ° C, 20 seconds, 58 ° C, 20 seconds, 60 ° C, and 4 minutes, followed by reaction at 72 ° C for 5 minutes. .
[0183] [31] [30]で得られた PCR反応物を 1.5mlチューブに移し、 3 M NaOAc 1 μ 1、 0.1 Μ EDTA 1 1、 20 mg/mlグリコーゲン溶液(ナカライテスク株式会社) 1 μ 1をよく混和させ 、冷 100%エタノール 60 μ 1を加え、混和させた。 4°C、 14000 rpm、 15分間遠心し上清 を除去し 70%エタノールにてペレットを洗浄後、再び 14000rpm、 2分間遠心して上清を 除去しすることを 2回行った。その後、 30-40分間遠心乾燥した後、脱イオン化したホ ルムアミド (Beckman coulter) 40 1を加えよく混和させた。配列分析は CEQ 2000 DNA Analysis System (Beckman coulteiで行つ 7こ。  [0183] [31] Transfer the PCR reaction product obtained in [30] to a 1.5 ml tube, and add 1 μl of 3 M NaOAc, 0.1 μEDTA 11 and 20 μg / ml glycogen solution (Nacalai Tesque, Inc.) 1 μl 1 was mixed well, and 60 μl of cold 100% ethanol was added and mixed. After centrifugation at 4 ° C and 14000 rpm for 15 minutes to remove the supernatant, washing the pellet with 70% ethanol, centrifugation again at 14000 rpm and 2 minutes to remove the supernatant was performed twice. Then, after centrifugal drying for 30 to 40 minutes, deionized formamide (Beckman coulter) 401 was added and mixed well. Sequence analysis was performed using the CEQ 2000 DNA Analysis System (Beckman coultei).
[0184] [表 5]  [0184] [Table 5]
[31] で配列分析を行い解析した結果 Results of sequence analysis performed in [31]
Name In frame Mutation Total In frame (%) C c ( ) IVV (%) Name In frame Mutation Total In frame (%) C c () IVV (%)
MHO 8 6 14 57 0 0 15MHO 8 6 14 57 0 0 15
Mil 6 7 13 46 2 33 25Mil 6 7 13 46 2 33 25
MI2 7 4 11 64 4 57 37MI2 7 4 11 64 4 57 37
MI3 15 6 21 71 13 87 41MI3 15 6 21 71 13 87 41
MK1 6 4 10 60 3 50 41MK1 6 4 10 60 3 50 41
MK2 5 3 8 63 2 40 36MK2 5 3 8 63 2 40 36
MM1 10 12 22 45 0 0 25MM1 10 12 22 45 0 0 25
MM2 8 8 16 50 1 13 33 表 5においては、左のレーンから、 "Name"はセレクションの種類と回数、 "In frame" はインフレームの個数、 "Mutation"はストップコドンまたはミューテーシヨンにより蛋白 質に翻訳されないものの個数、 "Totanま In frameと Mutationの合計、 Ίη frame (%Γは 100 X In frame/Total, "C"は図 11および図 12の Aまたは Bに収束した配列をもつもの の個数、 "C(%)"は 100 X図 11および図 12の Aまたは Bに収束した配列をもつものの個 数/ In frame,〃IW (%Γは、図 5、図 6および図 7より算出された対応付け効率をそれ ぞれ示している。 MM2 8 8 16 50 1 13 33 In Table 5, from the left lane, "Name" is the type and number of selections, "In frame" is the number of in-frames, "Mutation" is protein by stop codon or mutation. Number of untranslated items, "Totan or sum of In frame and Mutation", Ίη frame (% Γ is 100 X In frame / Total, “C” has an array converging to A or B in Figs. 11 and 12) The number of “C (%)” is 100 X The number of those with an array converging to A or B in Figure 11 and Figure 12 / In frame, 〃IW (% Γ is from Figures 5, 6 and 7) Each of the calculated association efficiencies is shown.
[0186] また MHOは [ 11 ]で得られたライブラリ一、 MI 1は [ 11 ]で得られたライブラリーを [ 18] において 50°C、 30分間次いで 99°C、 5分間反応させアンジォテンシン IIを抗原として セレクションし [25]で回収したライブラリー、 MI2は Milを [18]において 50°C、 30分間 次いで 99°C、 5分間反応させアンジォテンシン IIを抗原としてセレクションし [25]で回 収したライブラリー、 MI3は MI2を [18]において 50°C、 30分間次いで 99°C、 5分間反応 させアンジォテンシン IIを抗原としてセレクションし [25]で回収したライブラリー、 MK1 は [11]で得られたライブラリーを [18]において 50°C、 30分間次いで 99°C、 5分間反応 させ Lewis Xを抗原としてセレクションし [25]で回収したライブラリー、 MK2は MK1を [ 18]において 50°C、 30分間次いで 99°C、 5分間反応させ Lewis Xを抗原としてセレクシ ヨンし [25]で回収したライブラリー、 MM1は [11]で得られたライブラリーを [18]におい て 50°C、 30分間反応させアンジォテンシン IIを抗原としてセレクションし [25]で回収し たライブラリー、 MM2は MM1を [18]において 50°C、 30分間反応させアンジォテンシン IIを抗原としてセレクションし [25]で回収したライブラリーを示している。  [0186] In addition, MHO reacted the library obtained in [11] with the library obtained in [11], and reacted the library obtained in [11] with [18] at 50 ° C for 30 minutes, then at 99 ° C for 5 minutes, and reacted with angio. The library, MI2, which selected tensin II as an antigen and recovered in [25], reacted Mil with [18] at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes, and selected angiotensin II as an antigen [25]. For the library collected in [18], MI2 was reacted with [18] at 50 ° C for 30 minutes, then at 99 ° C for 5 minutes, and a library selected as angiotensin II as an antigen and collected in [25], MK1 In the [18], the library obtained in [18] was reacted at 50 ° C for 30 minutes and then at 99 ° C for 5 minutes, and Lewis X was selected as an antigen, and the library collected in [25] was recovered. In [18], react at 50 ° C for 30 minutes, then at 99 ° C for 5 minutes, For the MM1 library, which was selected as a source and collected in [25], the library obtained in [11] was reacted at [18] at 50 ° C for 30 minutes, and angiotensin II was selected as an antigen. MM2 shows the library collected by reacting MM1 at 50 ° C for 30 minutes in [18], selecting angiotensin II as an antigen, and collecting in [25].
[0187] C(%)で示した 100 X (図 11および図 12の Aに収束した配列をもつものの個数)/ In frameは Milで 33%、 MI2で 57%、 MI3で 87%とセレクションに従い上昇しアンジォテン シン IIを抗原としたセレクションでは Aに示した配列に急激に収束することが示された 。 C(%)で示した 100 X (図 11および図 12の Bに収束した配列をもつものの個数)/ In frameは MK1で 50%、 MK2で 40%、とセレクションを回すことによって高い値を示し Lewis Xを抗原としたセレクションでは Bに示した配列に収束することが示された。これ らに対し C(%)で示した 100 X (図 11および図 12の Aに収束した配列をもつものの個数 )/In frameは MM1で 0%、 MM2で 13%でありセレクションを 1回、回しただけでは Aに示 した配列のものはひとつも見られず、セレクションを 2回、回すことではじめて 1つのク ローンが Aに示した配列のものが見いだされた。 MM1、 MM2では [18]において 50°C 30分間反応させただけなのに対して Mil、 MI2、 MI3では [18]において 50°C、 30分間 反応させた後 99°C、 5分間反応させている。従って 99°C、 5分間反応させることで急激 に Aに示した配列に収束したのに対し 99°C、 5分間反応させない MM1、 MM2の場合 では Aに示した配列を見いだすことはできるものの非常に効率が悪いことがわ力つた [0187] 100 X expressed in C (%) (the number of arrays having converged arrays in A in Figs. 11 and 12) / In frame was 33% for Mil, 57% for MI2, and 87% for MI3 according to the selection. In the selection using angiotensin II as an antigen, it was shown to rapidly converge to the sequence shown in A. 100 X in C (%) (the number of arrays with converged arrays in B in Figs. 11 and 12) / In frame shows a high value by turning the selection to 50% for MK1 and 40% for MK2. The selection using Lewis X as the antigen showed that the sequence converged to the sequence shown in B. On the other hand, 100 X (the number of arrays with converged arrays in A in Figs. 11 and 12) / In frame in C (%) is 0% for MM1 and 13% for MM2, and selection is performed once. Only by turning, the sequence shown in A was not found, and only by turning the selection twice, was it possible to find the sequence shown in A for one clone. 50 ° C in [18] for MM1 and MM2 Mil, MI2, and MI3 perform the reaction at 50 ° C for 30 minutes at [18], followed by the reaction at 99 ° C for 5 minutes, while only reacting for 30 minutes. Therefore, the sequence shown in A was suddenly converged by reacting at 99 ° C for 5 minutes, whereas in the case of MM1 and MM2 not reacting at 99 ° C for 5 minutes, the sequence shown in A could be found, but it was very Helped me to be inefficient
[0188] MP1は [11]で得られたライブラリーを [15]において 99°C、 5分間反応させその後 [20 ] [21]においてアンジォテンシン IIを抗原としセレクションを行ったあとに [18]におい て 50°C30分間、 99°C、 5分間反応させて [22]以降の実験を行い [25]で回収したライ ブラリーであるが [31]で配列分析を行い解析した結果インフレームは 2個、ストップコ ドンまたはミューテーシヨンにより蛋白質に翻訳されないものは 11個、合計は 13個、図 11および図 12の Aに収束した配列をもつものは 2個であった。 MP1の場合インフレ一 ムの割合が極端に低くやはり効率が良 、とは 、えな 、が Aに示した配列を見 、だす ことができることがわ力 た。 [0188] MP1 reacted the library obtained in [11] at 99 ° C for 5 minutes in [15], and then performed selection in [20] [21] using angiotensin II as an antigen. After reacting at 50 ° C for 30 minutes and at 99 ° C for 5 minutes, the library obtained in [22] and subsequent experiments performed in [25] was sequenced and analyzed in [31]. Two, eleven were not translated into protein by stop codon or mutation, a total of thirteen, and two had sequences converged to A in FIGS. 11 and 12. In the case of MP1, the ratio of inflation was extremely low and the efficiency was still good, but it was clear that the array shown in A could be found out.
[0189] 図 11および図 12は [31]で配列分析を行い解析した結果インフレームのものにつ いてアミノ酸配列による clustalxによる配列アラインメント後 TreeViewPPCにより作成し た系統榭を示す。線で囲った Aはアンジォテンシン IIのセレクションで収束したもの、 線で囲った Bは Lewis Xのセレクションで収束したものを示す。ライブラリーの略号の 後にクローンの番号を示した。線で囲った Aは MI3-55と比較し蛋白質レベルで GENETYX- MACで解析した結果 90%以上の相同性を示し、線で囲った Bは MK1-17 と比較し蛋白質レベル GENETYX- MACで解析した結果 90%以上の相同性を示した。  [0189] Fig. 11 and Fig. 12 show the strains generated by TreeViewPPC after sequence alignment by clustalx based on the amino acid sequence, as a result of the sequence analysis and analysis performed in [31]. A surrounded by a line indicates the one converged by the selection of Angiotensin II, and B surrounded by a line indicates the one converged by the selection of Lewis X. The number of the clone is shown after the library abbreviation. A surrounded by a line shows more than 90% homology as a result of analysis by GENETYX-MAC at the protein level compared to MI3-55, and B surrounded by a line is analyzed by GENETYX-MAC at the protein level compared to MK1-17 As a result, 90% or more homology was shown.
[0190] 配列決定されたクローンの一部(MI1-16、 MIl-4、 MI2-10、 MI2-34、 MI2-41、  [0190] Some of the sequenced clones (MI1-16, MIl-4, MI2-10, MI2-34, MI2-41,
MI2— 48、 MI3— 15、 MI3— 26、 MI3— 28、 MI3— 34、 MI3— 41、 MI3— 42、 MI3— 47、 MI3— 5、 MI3- 55、 MI3- 62、 MI3- 64、 MI3- 66、 MI3- 74、 MK2- 3、 MM2- 11、 MP1- 36、 MP1-41) について、塩基配列及びそれがコードするアミノ酸配列を 60— 116の偶数の配列番 号に示す。アミノ酸配列を 61— 117の奇数の配列番号に示す。なお、 CDR配列番号 i¾、 Martin, A.し. R. Accessing the Kabat Antibody Sequence Dataoase by Computer PROTEINS: Structure, Function and Genetics, 25 (1996), 130- 133.に従って以下の ように決定した。 [0191] Mil- 16、 Mil- 4、 MI2- 10、 MI2- 34、 MI2- 41、 MI2- 48、 MI3- 15、 MI3- 26、 MI3- 28、 MI3- 34、 MI3- 41、 MI3- 42、 MI3- 47、 MI3- 5、 MI3- 55、 MI3- 62、 MI3- 64、 MI3- 66、 MI3-74, MK2-3, MM2-11、 MPl-36、 MP1-41の各 CDRは以下のアミノ酸番号である MI2—48, MI3—15, MI3—26, MI3—28, MI3—34, MI3—41, MI3—42, MI3—47, MI3—5, MI3-55, MI3-62, MI3-64, MI3- 66, MI3-74, MK2-3, MM2-11, MP1-36, MP1-41), the nucleotide sequence and the amino acid sequence encoded thereby are shown in even-numbered sequence numbers from 60 to 116. The amino acid sequence is shown as an odd SEQ ID NO: 61-117. The sequence was determined as follows according to CDR sequence number i¾, Martin, A. R. Accessing the Kabat Antibody Sequence Dataoase by Computer PROTEINS: Structure, Function and Genetics, 25 (1996), 130-133. [0191] Mil-16, Mil-4, MI2-10, MI2-34, MI2-41, MI2-48, MI3-15, MI3-26, MI3-28, MI3-34, MI3-41, MI3-42 , MI3-47, MI3--5, MI3-55, MI3-62, MI3-64, MI3-66, MI3-74, MK2-3, MM2-11, MPl-36, MP1-41 Is an amino acid number
[0192] [表 6] [0192] [Table 6]
表 6  Table 6
アミノ酸番号  Amino acid number
CDRH1 42-48  CDRH1 42-48
CDRH2 63-78  CDRH2 63-78
CDRH3 111-120  CDRH3 111-120
CDRL1 186-196  CDRL1 186-196
CDRL2 212-218  CDRL2 212-218
CDRL3 251-259  CDRL3 251-259
[0193] MI3-8、 MK1-15, MK1-17、 MK1-24, MK2-19、 MK2-8の各 CDRは以下のアミノ酸 番号である。 [0193] Each CDR of MI3-8, MK1-15, MK1-17, MK1-24, MK2-19, and MK2-8 has the following amino acid numbers.
[0194] [表 7] 表 7 [0194] [Table 7] Table 7
アミノ酸番号  Amino acid number
CDRH1 42-46  CDRH1 42-46
CDRH2 61-77  CDRH2 61-77
CDRH3 110-119  CDRH3 110-119
CDRL1 185-195  CDRL1 185-195
CDRL2 211-217  CDRL2 211-217
CDRL3 250-258 実施例 3  CDRL3 250-258 Example 3
[0195] 抗体の調製 [0195] Preparation of antibody
[32] [28]で得られたコロニーけん濁液 1 μ 1、 10 X PCR緩衝液 (TOYOBO) 10 1、 2 mM dNTPs(TOYOBO) 10 μ 1、 25 mM MgSO 4 1、 McD- F (配列番号 52)(10pmol/ [32] 1 μl of the colony suspension obtained in [28], 10X PCR buffer (TOYOBO) 10 1, 2 mM dNTPs (TOYOBO) 10 μ1, 25 mM MgSO 41, McD-F (SEQ ID NO: 52) (10 pmol /
4  Four
1) 3 1、 McD- R (His Tag)- stop (配列番号 51)(10pmol/ 1) 3 1、 KOD PLUSポリ メラーゼ (TOYOBO) 2 μ 1を混和させ、 RNase- Free水を添カ卩し全体量を 100 μ 1として それぞれ PCR反応させた。  1) 31 1, McD-R (His Tag) -stop (SEQ ID NO: 51) (10 pmol / 1) 3 1, KOD PLUS polymerase (TOYOBO) 2 μl, mix, add RNase-Free water The total volume was 100 µl, and each was subjected to PCR reaction.
[0196] PCRは、 94°C、 5分間反応後、 94°C、 30秒間、 58°C、 30秒間、 68°C、 2分間を 25-30 サイクル行った後、 68°C5分間反応を行った。増幅した遺伝子は 1%ァガロースゲル電 気泳動によりそれぞれ 900-1000bpのバンドを確認した。  [0196] PCR was performed at 94 ° C for 5 minutes, followed by 25-30 cycles of 94 ° C, 30 seconds, 58 ° C, 30 seconds, 68 ° C, 2 minutes, and then reaction at 68 ° C for 5 minutes. went. The amplified genes were confirmed to have 900-1000 bp bands by 1% agarose gel electrophoresis.
[0197] [33]クローン DNAの転写を行った。 [32]で得られた DNA lpmol、 5 X SP6緩衝液 4 μ 1、 ATP (lOOmM) 1 μ 1、 CTP (lOOmM) 1 μ 1、 UTP (lOOmM) 1 μ 1、 GTP (lOmM) 2 μ 1 、キャップアナログ (m7G(5')PPP(5')G) (Invitrogen) (40mM) 2.5 μ 1、ェンザィムミックス SP6RNAポリメラーゼ(Promega) 2 μ 1を混和させ、 RNase-Free水を添カ卩し全体量を 20 μ 1とし、 37°C、 2時間 30分間反応後、 RQ1 RNase-Free DNase (Promega) 5 μ 1を添 加しさらに 37°C、 1時間反応させた。得られた RNAは RNeasy Mini kit (Qiagen)により 精製した。  [33] Transcription of the cloned DNA was performed. DNA lpmol obtained in [32], 5 X SP6 buffer 4 μ1, ATP (lOOmM) 1 μ1, CTP (lOOmM) 1 μ1, UTP (lOOmM) 1 μ1, GTP (lOmM) 2 μ1 , Cap analog (m7G (5 ') PPP (5') G) (Invitrogen) (40 mM) 2.5 μl, Enzymmix SP6 RNA polymerase (Promega) 2 μl, mix with RNase-Free water The whole volume was adjusted to 20 μl, and after reaction at 37 ° C. for 2 hours and 30 minutes, 5 μl of RQ1 RNase-Free DNase (Promega) was added, and the mixture was further reacted at 37 ° C. for 1 hour. The obtained RNA was purified using the RNeasy Mini kit (Qiagen).
[0198] [34] [33]で得られた RNA 20 pmol、小麦胚芽抽出液(5mM DTTを含む) (Promega ) 50 1、 Amino Acid mixture, Complete (ImM) (Promegaノ 8 1、 RNase  [0198] [34] 20 pmol of RNA obtained in [33], wheat germ extract (containing 5 mM DTT) (Promega) 501, Amino Acid mixture, Complete (ImM) (Promega 81, RNase
inhibitor(TOYOBO) 8 1、 CH3COOK (1M) (Promega) 8 μ 1を混和し、 RNase— Free水 を添加し全体量を 100 /z lとし、遮光条件下 25°C、 1-3時間反応させ翻訳を行った。  Mix inhibitor (TOYOBO) 81 and CH3COOK (1M) (Promega) 8 μl, and add RNase-Free water to make the total volume 100 / zl. Was done.
[0199] [35]ミリ Q水で調製し組成が 50mMリン酸カリウム、 150 mM NaCl、 pH 6.0、 0.1% Tween 20である PP6T緩衝液にて膨潤ならびに平衡化させた Sephadex G75  [35] Sephadex G75 swelled and equilibrated with PP6T buffer prepared in Milli-Q water and composed of 50 mM potassium phosphate, 150 mM NaCl, pH 6.0, 0.1% Tween 20
(Amersham Biosciences)ゲル 1 mlをカラム(バイオラッド)に充填したものに [34]で得ら れた翻訳溶液 200 μ 1を供し、 4滴ずつチューブに集め 3本目から 6本目までを回収し た。  (Amersham Biosciences) 200 μl of the translation solution obtained in [34] was supplied to a column (Bio-Rad) packed with 1 ml of gel, and collected in tubes by four drops, and the third to sixth tubes were collected. .
[0200] [36]あるいはミリ Q水で調製し組成が 20 mMリン酸ナトリウム、 500 mM NaCl、 20 mM イミダゾール pH 7.4である His Tag A緩衝液にて膨潤ならびに平衡化させた  [36] [36] Or swelled and equilibrated with His Tag A buffer containing 20 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole pH 7.4 prepared in Milli-Q water
Sephadex G75 (Amersham Biosciences)ゲノレ 1 mlをカラム (バイオラッド)に充填したも のに [34]で得られた翻訳溶液 200 μ 1を供し、 4滴ずつチューブに集め 3本目から 6本 目までを回収した。その溶液に His Tag A緩衝液で平衡化した Ni-NTA agarose (Qiagen)榭脂 100 1を加え、 25°C、 1時間ロータリーミキサー (Nissin)で回転攪拌し た。 His Tag A緩衝液 500 μ 1でけん濁させ、 4°C、 3,000 rpm、 1分間遠心し上清を除 去しする操作を 3回繰り返し、榭脂を回収し、組成が 20 mMリン酸ナトリウム、 500 mM NaCl、 475 mMイミダゾール pH 7.4である His Tag B緩衝液 150 μ 1をカ卩え 25°C、 30分 間- 1時間ロータリーミキサー (Nissin)で回転攪拌した。 Urtra Freeに榭脂けん濁液を 供し 10,000 rpm、 3分間遠心し下層に溶液を回収した。 Sephadex G75 (Amersham Biosciences) 1 ml of Genole was packed in a column (Bio-Rad), and 200 μl of the translation solution obtained in [34] was supplied.Four drops were collected in tubes, and the 3rd to 6th tubes were collected. Collected. Ni-NTA agarose equilibrated with His Tag A buffer (Qiagen) 1001 was added, and the mixture was rotationally stirred at 25 ° C. for 1 hour using a rotary mixer (Nissin). The suspension was suspended three times with 500 μl of His Tag A buffer, centrifuged at 3,000 rpm for 1 minute at 4 ° C, and the supernatant was removed three times. 150 μl of a His Tag B buffer solution of 500 mM NaCl and 475 mM imidazole pH 7.4 was rotated with a rotary mixer (Nissin) at 25 ° C. for 30 minutes to 1 hour. The suspension was supplied to Urtra Free and centrifuged at 10,000 rpm for 3 minutes to recover the solution in the lower layer.
[0201] [37] PP6T緩衝液で平衡化させた Micro Spin G— 25 column(Amersham Biosciences) を 4°C、 735 g、 1分間遠心し、下層に溶出した液を除去し、 [36]で得られた溶液 50 1 をカラムに供した。 4°C、 735g、 2分間遠心し、下層の溶液を回収した。  [0201] [37] Micro Spin G—25 column (Amersham Biosciences) equilibrated with PP6T buffer was centrifuged at 735 g for 1 minute at 4 ° C, and the eluate in the lower layer was removed. The obtained solution 501 was applied to a column. The solution was centrifuged at 4 ° C and 735 g for 2 minutes to recover the lower layer solution.
[0202] あるいはミリ Q水で調製し組成が 50mMリン酸カリウム、 150 mM NaCl、 pH 6.0、 0.1% Tween 20である PP6T緩衝液にて膨潤ならびに平衡化させた Sephadex G75  [0202] Alternatively, Sephadex G75 swelled and equilibrated with a PP6T buffer having a composition of 50 mM potassium phosphate, 150 mM NaCl, pH 6.0, 0.1% Tween 20 prepared in Milli-Q water
(Amersham Biosciences)ゲル 1 mlをカラム(バイオラッド)に充填したものに [36]で得ら れた溶液を 150 μ 1を供し、 4滴ずつチューブに集め 3本目から 6本目までを回収した。  (Amersham Biosciences) 1 ml of the gel was packed in a column (Bio-Rad), 150 μl of the solution obtained in [36] was provided, and the solution was collected in tubes by four drops, and the third to sixth tubes were collected.
[0203] 図 13は、 ΜΙ3-55の [37]についてウェスタンブロットした結果(右 2本)を示す。左 3本 はウェスタンブロットのコントローノレである。レーン左から Carboxy-terminal  [0203] Fig. 13 shows the results of Western blotting of [37] of ΜΙ3-55 (right two). The left three are Western blot controls. Carboxy-terminal from lane left
FLAG-BAP fosion protein (Sigma)を 100 ngゝ 50 ngゝ 20 ngゝ Biotynylated SDS— PAGE Standards low range (Bio- rad) 2 1、 MI3- 55の [37]にお 、て 3本目から 6本目までを 回収した 15 1、 5 1を 15%ポリアクリルアミド電気泳動に付した後 PBDF膜に転写し、 anti FLAG M2- Mouse(Sigma)を一次抗体、 Goat anti-mouse IgG (H+L)- HRP conjugate (Sigma)と Avidin— HRP conjugate (Bio— rad)をニ次饥ィ本とし ECL Western Blotting Detection Reagents (Amersham biosciences)で検出し 7こ1/エスタンブロット。 MI3-55は分子量約 31,000ダルトンにバンドを検出し一本鎖抗体の計算値と一致した 実施例 4 FLAG-BAP fosion protein (Sigma) 100 ng ゝ 50 ng ゝ 20 ng ゝ Biotynylated SDS—PAGE Standards low range (Bio-rad) 21 1, MI3-55 [37] from 3rd to 6th The collected 15 1 and 51 were subjected to 15% polyacrylamide electrophoresis and transferred to a PBDF membrane.Anti FLAG M2-Mouse (Sigma) was used as the primary antibody, Goat anti-mouse IgG (H + L) -HRP conjugate (Sigma) and Avidin- HRP conjugate (Bio- rad) was used as a two-following饥I this ECL Western blotting detection detected in Reagents (Amersham biosciences) 7 this 1 / es Tan blot. For MI3-55, a band was detected at a molecular weight of about 31,000 daltons, which was consistent with the calculated value of the single-chain antibody. Example 4
[0204] ビアコアによる結合の確認 [0204] Confirmation of connection with Biacore
[38]ビアコアはビアコア 3000システムを用いた。センサーチップ Bl (CM4)にァミン カップリング法で固定化を行った。フローセル 1および 2を 0.2 M N-ェチル - Ν'-ジメチ ルァミノプロピルカルボジイミド (EDC)と 50 mM N-ヒドロキシスクシンイミド (NHS)を含む 溶液で 10分間活性化を行った。次にフローセル 2に 50 Mストレプトアビジン (Sigma) (0.02Mリン酸カリウム緩衝液 pH 6.5)を 20 1、 10 mM酢酸緩衝液 pH 5.0 980 μ 1と 混合したものを 10分間、フローセル 1には 10 mM酢酸緩衝液 pH 5.0を 10分間流した 。続いてフローセル 1および 2を 1Mエタノールァミン pH 8.5で 10分間ブロッキングを行 つた。フローセル 2は 2024または 2305レスポンスユニットがセンサーチップ表面に結合 した。さらにフローセル 1および 2に 50 mM NaCl, 1 M NaCl 10 を 5回流した後、 2024レスポンスユニットがセンサーチップ表面に結合したフローセル 2にアンジォテン シン Π-ピオチン (0.1 Μ)を 10 1流した。 2305レスポンスユニットがセンサーチップ表 面に結合したフローセル 2に Lewis- X-sp-ピオチン (0.1 μ Μ)を 3 μ 1流した。最後にフ ローセル 1および 2に Glycine 1.5を 10 1流した結果、アンジォテンシン II-ピオチンの フローセル 2では 35.0レスポンスユニットが、 Lewis-X-sp-ビォチンのフローセル 2では 31.6レスポンスユニットがそれぞれセンサーチップ表面に結合した。理論的 Rmaxはァ ンジォテンシン II-ビォチンのフローセル 2では 801とまたは Lewis-X-sp-ビォチンのフ ローセル 2では 1081と見積もられた。 [38] Biacore used the Biacore 3000 system. Immobilization was performed on the sensor chip Bl (CM4) by the amine coupling method. Flow cells 1 and 2 contain 0.2 M N-ethyl-Ν'-dimethylaminopropylcarbodiimide (EDC) and 50 mM N-hydroxysuccinimide (NHS) Activation was performed with the solution for 10 minutes. Next, 50 M streptavidin (Sigma) (0.02 M potassium phosphate buffer, pH 6.5) was mixed with 201 and 10 mM acetate buffer, pH 5.0 980 μl in flow cell 2 for 10 min. mM acetate buffer pH 5.0 was allowed to flow for 10 minutes. Subsequently, flow cells 1 and 2 were blocked with 1 M ethanolamine pH 8.5 for 10 minutes. Flow cell 2 has a 2024 or 2305 response unit attached to the sensor chip surface. Further, 50 mM NaCl and 1 M NaCl 10 were flown into the flow cells 1 and 2 five times, and then 10 1 of angiotensin Π-pyotin (0.1 Μ) was flown into the flow cell 2 in which the 2024 response unit was bonded to the sensor chip surface. 3 μl of Lewis-X-sp-Piotin (0.1 μΜ) was flowed into flow cell 2 in which the 2305 response unit was connected to the sensor chip surface. Finally, as a result of flowing Glycine 1.5 through flow cells 1 and 2, 35.0 response units were used for flow cell 2 of angiotensin II-biotin, and 31.6 response units were used for flow cell 2 of Lewis-X-sp-biotin. Bound to the surface. The theoretical Rmax was estimated to be 801 for flow cell 2 of angiotensin II-biotin or 1081 for flow cell 2 of Lewis-X-sp-biotin.
[0205] [39]ビアコアの分析は組成が 50 mMリン酸カリウム、 150 mM NaCl, pH 6.0、 0.1% Tween 20である PP6T緩衝液を用い流速は 20 1/分で行った。 [35]または [37]で得 られたサンプルを KINJECTでインジェタトした。再生は Glycine 1.5を20 1、 50mM NaCl, 1 M NaCl 10 1で行った。レスポンスカーブはフローセル 2からフローセル 1を 弓 Iき算し結合のレスポンスユニットを算出した。  [39] Biacore was analyzed using a PP6T buffer having a composition of 50 mM potassium phosphate, 150 mM NaCl, pH 6.0, 0.1% Tween 20 at a flow rate of 201 / min. The samples obtained in [35] or [37] were injected with KINJECT. Regeneration was performed with Glycine 1.5 at 201, 50 mM NaCl, 1 M NaCl 101. The response curve was calculated by combining the flow cell 2 and the flow cell 1 with the bow I to calculate the combined response unit.
[0206] 図 14は、アンジォテンシン IIを抗原としたセレクションで図 11および図 12の Αに示し た配列に収束する配列のうち MI3-55、 MI3-42, MI3-28、 MI3-41、 MI3-34、 MI3-5、 MI3-15、MI3-26を [34]で翻訳を行った後ビアコアで分析し算出した KDを棒グラフで 示したものである。 0.4-1.5 nMの非常に親和性の高い値を示し、本発明の方法で選 択された一本鎖抗体は非常に高親和性であることがわ力つた。  [0206] FIG. 14 shows a selection using angiotensin II as an antigen. Among the sequences converging to the sequences shown in Α in FIGS. 11 and 12, MI3-55, MI3-42, MI3-28, MI3-41, This is a bar graph showing KD calculated by translating MI3-34, MI3-5, MI3-15, and MI3-26 using [34] and then analyzing with Biacore. It showed very high affinity values of 0.4-1.5 nM, demonstrating that the single chain antibodies selected by the method of the present invention have very high affinity.
[0207] 図 15は、 Lewis Xを抗原としたセレクションで図 11および図 12の Bに示した配列に 収束する配列のうち MK1-1、 MK1-24, MK2-19, MK1-17を [34]で翻訳を行った後ビ ァコアで分析し算出した KDを棒グラフで示したものである。 20-43 nMの値を示し、本 発明の方法で選択された一本鎖抗体は高親和性抗体であることがわかった。 [0208] 図 16は、 MI3-55を [34]で翻訳を行った後 4°Cまたは 60°Cまたは 99°C、 5分間処理し [35]の処理を行ったあと [39]のビアコアで分析した結果を棒グラフで示したものであ る。右のレーンから 1はサンプル原液を 0.5は原液の 2分の 1希釈、 0.25は 0.5の 2分の 1 希釈、 0.125は 0.25の 2分の 1希釈を示す。原液を 99°C、 5分間処理したものは 4°C5分 間処理したものに比べて 56%のレスポンスになったものの 4°C5分間処理したものを 0.25に希釈したものと同等のレスポンスを示した。従って MI3-55は 99°C、 5分間処理 し [35]の処理したものでも約 25%の結合活性を有し、本発明の方法で選択された一 本鎖抗体は非常に耐熱性であることが示された。 [0207] FIG. 15 shows that in the selection using Lewis X as an antigen, MK1-1, MK1-24, MK2-19, and MK1-17 among the sequences converging to the sequences shown in FIG. 11 and FIG. This is a bar graph showing the KD calculated by translating and analyzing with Biacore. It showed a value of 20-43 nM, indicating that the single-chain antibody selected by the method of the present invention was a high affinity antibody. [0208] Fig. 16 shows that after translating MI3-55 at [34], processing it at 4 ° C, 60 ° C, or 99 ° C for 5 minutes, and then performing the processing of [35], the Biacore of [39] The results of the analysis are shown in a bar graph. From the right lane, 1 indicates the sample stock solution, 0.5 indicates 1/2 dilution of the stock solution, 0.25 indicates 1/2 dilution of 0.5, and 0.125 indicates 1/2 dilution of 0.25. The stock solution treated at 99 ° C for 5 minutes gave a 56% response compared to that treated at 4 ° C for 5 minutes, but showed a response equivalent to that of the solution treated at 4 ° C for 5 minutes diluted to 0.25. Was. Therefore, MI3-55 treated at 99 ° C for 5 minutes and treated in [35] has about 25% binding activity, and the single-chain antibody selected by the method of the present invention is extremely heat-resistant. It was shown.
実施例 5  Example 5
[0209] ELISAによる結合の確認  [0209] Confirmation of binding by ELISA
[40]Nuncストレプトアビジンコート 96穴マイクロプレートの 1穴あたりにアンジォテン シン II-ビォチンまたは Lewis- X-sp-ビォチン (100 nM)/ELISA緩衝液 200 μ 1をカ卩え 50 °C、 1時間静かに振とうさせ固定ィ匕を行った。プレートの溶液を捨て良く水をきり ELISA緩衝液 200 1をカ卩えるプレートを洗う操作を 3回行った。 ELISA緩衝液 200 μ \ をカロえ 5分間放置した後再び ELISA緩衝液 200 μ 1でプレートを洗う操作を 3回行った 。 Blocking one (ナカライテスタ株式会社):ミリ Q (1:4)で希釈したブロッキング剤をカロ え 4°Cで一晩または 25°Cで 1-数時間ブロッキングを行った。次に PBS (ナカライテスタ 株式会社) 200 1でプレートを洗う操作を 4回行い、 [35]または [37]で得られたサン プノレを 100 1プレートに加えた。このとき拮抗阻害を調べる場合にはアンジォテンシ ン Πまたは Free Lewis Xを一本鎖抗体サンプルとあらかじめ混和させたものを加えた。 25°Cで 0.5-2時間静かに振とうさせ後 PBS (ナカライテスタ株式会社) 200 μ 1でプレー トを洗う操作を 4回行い一次抗体溶液として anti FLAG M2-Mouse (Sigma): Blocking one (ナカライテスタ株式会社): ELISA緩衝液(1:10:190)を 100 1加えた。 25°Cで 0.5- 2時間静かに振とうさせ後 PBS (ナカライテスタ株式会社) 200 μ 1でプレートを洗う 操作を 4回行い二次抗体溶液として Goat anti-mouse IgG (H+L)- HRP conjugate (Sigma): Blocker casein in TBS (PIERCE) (1:400)を 100 μ 1加えた。 25°Cで 0.5- 2時間 静か〖こ振とうさせ後 PBS (ナカライテスタ株式会社) 200 μ 1でプレートを洗う操作を 4回 行い TMB Peroxidase EIA substrate kit (Bio rad) (A:B = 9:1) 100 μ 1カ卩えた。 25°Cで 5-60分間静かに振とうさせ 1 N H SOを 100 1加え反応を停止した。吸光度は [40] Nunc streptavidin-coated 96-well microplate with 200 μl of angiotensin II-biotin or Lewis-X-sp-biotin (100 nM) / ELISA buffer per well at 50 ° C for 1 hour She was shaken gently and fixed. The operation of washing the plate on which the solution of the plate was discarded and draining well and washing the ELISA buffer solution 200 1 was performed three times. After washing 200 μl of the ELISA buffer and leaving it for 5 minutes, the plate was washed three times with 200 μl of the ELISA buffer again. Blocking one (Nakarai Tester Co., Ltd.): The blocking agent diluted with Milli Q (1: 4) was caloried, and blocking was performed overnight at 4 ° C. or 1 to several hours at 25 ° C. Next, the plate was washed four times with PBS (Nacalai Tester Co., Ltd.) 200 1 and the sample obtained in [35] or [37] was added to 100 1 plate. At this time, in order to examine antagonistic inhibition, a mixture of angiotensin II or Free Lewis X and a single-chain antibody sample in advance was added. After gently shaking at 25 ° C for 0.5-2 hours, wash the plate four times with 200 μl of PBS (Nacalai Tester Co., Ltd.), and perform anti-FLAG M2-Mouse (Sigma): Blocking one ( (Nakarai Tester Co., Ltd.): 100 1 of ELISA buffer (1: 10: 190) was added. Shake gently at 25 ° C for 0.5-2 hours, then wash the plate with 200 μl of PBS (Nacalai Tester Co., Ltd.) 4 times, and perform Goat anti-mouse IgG (H + L) -HRP as a secondary antibody solution. 100 μl of conjugate (Sigma): Blocker casein in TBS (PIERCE) (1: 400) was added. After shaking gently at 25 ° C for 0.5-2 hours, wash the plate 4 times with 200 μl of PBS (Nacalai Tester Co., Ltd.), and perform the operation 4 times.TMB Peroxidase EIA substrate kit (Bio rad) (A: B = 9: 1) One hundred microliters were prepared. At 25 ° C The mixture was shaken gently for 5-60 minutes, and the reaction was stopped by adding 100 1 of 1 NH 2 SO 4. Absorbance is
2 4  twenty four
Multiskan JX (大日本製薬株式会社) 96穴マイクロプレートリーダーで 450 nmと 630 nmを測定し 450 nmの値から 630 nmの値を引き算した。  Multiskan JX (Dainippon Pharmaceutical Co., Ltd.) 450 nm and 630 nm were measured with a 96-well microplate reader, and the value of 630 nm was subtracted from the value of 450 nm.
[0210] 図 17は、 MI3-55を [34]で翻訳を行った後 4°Cまたは 60°Cまたは 99°C、 5分間処理し [0210] Figure 17 shows that MI3-55 was translated at [34] and then treated at 4 ° C or 60 ° C or 99 ° C for 5 minutes.
[35]の処理を行ったあと [40]の ELISAで分析した結果を棒グラフで示したものである 。右のレーンから 1はサンプル原液を 0.5は原液の 2分の 1希釈、 0.25は 0.5の 2分の 1 希釈、 0.125は 0.25の 2分の 1希釈、 0.0625は 0.125の 2分の 1希釈を示す。原液を 99°C 、 5分間処理したものは 4°C、 5分間処理したものに比べて 21%の値に、 0.5希釈を 99 。C、 5分間処理したものは 4°C、 5分間処理したものに比べて 44%の値になったものの 、それぞれ 4°C、 5分間処理したものを 0.125に希釈したものと同等の値を示した。従つ て MI3-55は 99°C、 5分間処理し [35]の処理したものでも約 12.5%以上の結合活性を 有し、本発明の方法で選択された一本鎖抗体は、非常に耐熱性であることが示され た。  This is a bar graph showing the result of analysis by ELISA in [40] after performing the treatment in [35]. From the right lane, 1 is the sample stock, 0.5 is the half dilution of the stock, 0.25 is the half dilution of 0.5, 0.125 is the half dilution of 0.25, and 0.0625 is the half dilution of 0.125 . The undiluted solution treated at 99 ° C for 5 minutes had a 0.5 dilution to 99% of 21% of the value treated at 4 ° C for 5 minutes. C, treated for 5 minutes had a 44% value compared to that treated at 4 ° C, 5 minutes, but the same value was obtained by diluting the treated at 4 ° C, 5 minutes to 0.125, respectively. Indicated. Therefore, MI3-55 treated at 99 ° C. for 5 minutes and treated in [35] has a binding activity of about 12.5% or more, and the single-chain antibody selected by the method of the present invention is extremely low. It was shown to be heat resistant.
産業上の利用の可能性  Industrial potential
[0211] 本発明は、タンパク質の機能的成熟を進化分子工学的手法 (IW法)に適用し、試 験管内でのタンパク質選択を安価で迅速に行う技術である。具体的にはタンパク質 cDNAライブラリーの構築、対応付け分子 (IW)ライブラリーへの変換、選択、遺伝子 の回収、このサイクルを数回繰り返す事によって、所望のタンパク質を得ることができ る。さらに、ライブラリーを既知のタンパク質遺伝子に変異(point mutation, DNA shuffling)を加えたものを使用することで、より高機能性、高安定性、高発現のタンパ ク質を選択することも可能である。  [0211] The present invention is a technique for applying protein functional maturation to an evolutionary molecular engineering technique (IW method) to rapidly and inexpensively select a protein in a test tube. Specifically, a desired protein can be obtained by constructing a protein cDNA library, converting to an assigning molecule (IW) library, selecting, collecting genes, and repeating this cycle several times. Furthermore, by using a library obtained by adding a mutation (point mutation, DNA shuffling) to a known protein gene, it is possible to select a protein with higher functionality, higher stability, and higher expression. is there.
[0212] 本発明によれば、強 ヽ選択圧環境下(高温での加熱処理)でタンパク質選択が行 われるため、極めて高い濃縮効果を示し、さらに標的分子に対して強い結合力をも つタンパク質を迅速に選択できる。得られたタンパク質は分子生物学的基礎研究に おける利用は言うまでもなぐ診断薬、治療薬への応用など、その応用範囲は極めて 広い。特に、以下のような優れた効果を奏し得る。  [0212] According to the present invention, since protein selection is performed under a strong selective pressure environment (heat treatment at a high temperature), an extremely high enrichment effect and a protein having a strong binding force to a target molecule are exhibited. Can be quickly selected. The resulting protein has a very wide range of applications, including diagnostic and therapeutic applications, not to mention its use in basic molecular biology research. In particular, the following excellent effects can be obtained.
(1)ライブラリーと抗原を結合させる前に予め加熱処理を行うことで、不安定なタンパ ク質群を変性させ、安定に立体構造が維持されているものか、迅速に巻き戻る安定 性の高 、タンパク質群だけを選択できる。 (1) Preliminary heat treatment before binding the library to the antigen denatures the unstable protein group to ensure that the three-dimensional structure is maintained stably or that the protein is quickly unwound. Only high protein groups can be selected.
(2)同時に逆転写反応を行う場合には、 RNA部分での担体や標的分子への非特異 的な結合が抑えられ、飛躍的に濃縮効率が上昇する。  (2) When performing reverse transcription simultaneously, non-specific binding to the carrier or target molecule in the RNA portion is suppressed, and the enrichment efficiency is dramatically increased.
(3)得られたタンパク質は、 DTT等の還元剤存在下での無細胞翻訳系や還元的環 境の大腸菌の細胞質内での大量発現が可能であり、また還元剤に対しても安定であ る。  (3) The obtained protein can be expressed in a cell-free translation system in the presence of a reducing agent such as DTT, or expressed in large quantities in the cytoplasm of Escherichia coli in a reductive environment, and is stable to reducing agents. is there.

Claims

請求の範囲 The scope of the claims
[1] 標的分子と相互作用するタンパク質またはそれをコードする核酸の選択法であって、 以下の工程 (a)— (d)を含むことを特徴とする選択法。  [1] A method for selecting a protein that interacts with a target molecule or a nucleic acid encoding the same, comprising the following steps (a) to (d).
(a)タンパク質をコードする DNAのライブラリーを調製する工程。  (a) a step of preparing a library of DNAs encoding the protein;
(b) (a)で調製されたライブラリーの DNAを転写し、転写された RNAの 3'末端にピュー口 マイシンを含むスぺーサーを連結した後、無細胞翻訳系にお ヽて遺伝子型と表現型 の対応付け分子のライブラリーを構築する工程。  (b) After transcribing the DNA of the library prepared in (a) and ligating a spacer containing pewmic mycin to the 3 'end of the transcribed RNA, the cells are genotyped in a cell-free translation system. The step of constructing a library of molecules that correspond to phenotypes.
(c)対応付け分子のライブラリーを加熱処理する工程。  (c) heating the library of assigning molecules;
(d)対応付け分子を標的分子に対して結合させ、十分洗浄した後、溶出し、核酸部を 逆転写- PCRまたは PCRによって増幅させる工程。  (d) A step of binding the assigning molecule to the target molecule, washing sufficiently, eluting, and amplifying the nucleic acid portion by reverse transcription-PCR or PCR.
[2] 標的分子が抗原であり、タンパク質が一本鎖抗体である請求項 1記載の選択法。  [2] The selection method according to claim 1, wherein the target molecule is an antigen, and the protein is a single-chain antibody.
[3] スぺーサ一がポリエチレングリコールを含む請求項 1記載の選択法。  [3] The selection method according to claim 1, wherein the spacer contains polyethylene glycol.
[4] (d)で増幅した DNAを (a)のライブラリ一として用いて、上記 (b)— (d)の操作を繰り返す 工程をさらに含む請求項 1記載の選択法。  [4] The selection method according to claim 1, further comprising a step of repeating the above operations (b) to (d) using the DNA amplified in (d) as a library in (a).
[5] 加熱処理する際の条件が、 50-100°C、 1-30分の範囲から選択される請求項 1記載の 選択法。 [5] The selection method according to claim 1, wherein the conditions for the heat treatment are selected from the range of 50-100 ° C and 1-30 minutes.
[6] (d)の工程の前に対応付け分子の RNA部を逆転写して RNA-DNAノヽイブリットとするェ 程をさらに含む請求項 1記載の選択法。  [6] The selection method according to claim 1, further comprising, before the step (d), reverse transcription of the RNA portion of the assigning molecule into an RNA-DNA hybrid.
[7] 逆転写の前に、逆転写反応を阻害する無細胞翻訳系由来の物質を除去する請求項[7] The method of removing a substance derived from a cell-free translation system that inhibits a reverse transcription reaction before reverse transcription.
5記載の選択法。 5. Selection method according to 5.
[8] 無細胞翻訳系がチオール化合物を含む無細胞翻訳系である請求項 1記載の選択法  [8] The selection method according to claim 1, wherein the cell-free translation system is a cell-free translation system containing a thiol compound.
[9] 無細胞翻訳系が、小麦胚芽抽出液、ゥサギ網状赤血球抽出液、又は、大腸菌 S-30 抽出液の無細胞翻訳系である請求項 1記載の選択法。 [9] The selection method according to claim 1, wherein the cell-free translation system is a wheat germ extract, a heron reticulocyte extract, or an Escherichia coli S-30 extract.
[10] 標的分子と相互作用するタンパク質の製造法であって、請求項 1一 9のいずれかに 記載の選択法により標的分子と相互作用するタンパク質をコードする核酸を選択す る工程、および、選択された核酸を翻訳してタンパク質を製造する工程を含む製造法 [10] A method for producing a protein that interacts with a target molecule, the method comprising selecting a nucleic acid encoding a protein that interacts with the target molecule by the selection method according to any one of claims 11 to 19, and Production method including a step of producing a protein by translating a selected nucleic acid
[11] 標的分子が抗原であり、タンパク質が一本鎖抗体である請求項 10記載の製造法。 [11] The production method according to claim 10, wherein the target molecule is an antigen, and the protein is a single-chain antibody.
[12] 抗原がアンジォテンシン IIである請求項 11記載の製造法。 12. The method according to claim 11, wherein the antigen is angiotensin II.
[13] 抗原が Lewis Xである請求項 11の製造法。 [13] The production method according to claim 11, wherein the antigen is Lewis X.
[14] 一本鎖抗体を製造する工程が、選択された核酸を、チオール化合物を含む無細胞 翻訳系で翻訳することを含む請求項 11記載の製造法。  [14] The production method according to claim 11, wherein the step of producing a single-chain antibody comprises translating the selected nucleic acid with a cell-free translation system containing a thiol compound.
[15] 無細胞翻訳系が、小麦胚芽抽出液、ゥサギ網状赤血球抽出液、または、大腸菌 [15] The cell-free translation system is wheat germ extract, egret reticulocyte extract, or Escherichia coli.
S-30抽出液である請求項 14記載の製造法。  15. The production method according to claim 14, which is an S-30 extract.
[16] 一本鎖抗体を製造する工程が、選択された核酸で生細胞を形質転換させ、生細胞 内で一本鎖抗体を発現させることを含む請求項 11記載の製造法。 [16] The production method according to claim 11, wherein the step of producing a single-chain antibody comprises transforming a living cell with the selected nucleic acid and expressing the single-chain antibody in the living cell.
[17] 一本鎖抗体を製造する工程が、選択された核酸によりコードされる一本鎖抗体と酵 素又は緑色蛍光タンパク質 (Green Fluorescent Protein: GFP)との融合タンパク質とし て製造することを含む請求項 11記載の製造法。 [17] The step of producing a single-chain antibody includes producing a single-chain antibody encoded by the selected nucleic acid as a fusion protein of an enzyme or green fluorescent protein (GFP). A method according to claim 11.
[18] 請求項 1一 9のいずれかに記載の選択法により選択された核酸を、 C末端ラベルイ匕 剤の存在下で無細胞翻訳系で翻訳することによりタンパク質の C末端をラベルイ匕する 方法。 [18] A method for labeling the C-terminus of a protein by translating the nucleic acid selected by the selection method according to claim 11 in a cell-free translation system in the presence of a C-terminal labeling agent. .
[19] アンジォテンシン IIに対する結合活性を有する一本鎖抗体であって、下記 (A)又は (B) に示すアミノ酸配列を有する一本鎖抗体。  [19] A single-chain antibody having binding activity to angiotensin II, which has the amino acid sequence shown in the following (A) or (B):
(A)配列番号 61、 63, 65, 67, 69, 71、 73, 75, 77, 79, 81、 83, 85, 87, 89, 9 1、 93、 95、 97、 99、 101、 103また ίま 105に示すアミノ酸酉己歹 lj。  (A) SEQ ID NO: 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103 or Pamino 105 is an amino acid rooster.
(B) (A)のアミノ酸配列と相同性が 90%以上のアミノ酸配列を有するアミノ酸配列。  (B) An amino acid sequence having an amino acid sequence having 90% or more homology with the amino acid sequence of (A).
[20] 請求項 19記載の一本鎖抗体をコードする核酸。 [20] A nucleic acid encoding the single-chain antibody according to claim 19.
[21] Lewis Xに対する一本鎖抗体であって、下記 (A)又は (B)に示すアミノ酸配列を有する 一本鎖抗体。  [21] A single-chain antibody against Lewis X, which has the amino acid sequence shown in the following (A) or (B):
(A)配列番号 107、 109、 111、 113、 115または 117に示すアミノ酸配列。  (A) the amino acid sequence of SEQ ID NO: 107, 109, 111, 113, 115 or 117;
(B) (A)のアミノ酸配列と相同性が 90%以上のアミノ酸配列を有するアミノ酸配列。  (B) An amino acid sequence having an amino acid sequence having 90% or more homology with the amino acid sequence of (A).
[22] 請求項 21記載の一本鎖抗体をコードする核酸。 [22] A nucleic acid encoding the single-chain antibody according to claim 21.
[23] 請求項 11一 17のいずれかに記載の製造法によって得られた一本鎖抗体を用いてタ ンパク質を免疫学的に検出する方法。 [23] A method for immunologically detecting a protein using the single-chain antibody obtained by the production method according to any one of claims 11 to 17.
[24] ウェスタンプロット法、免疫染色法、蛍光抗体染色法、抗体チップ法、免疫沈降法で ある請求項 23に記載の検出方法。 [24] The detection method according to claim 23, which is a Western plot method, an immunostaining method, a fluorescent antibody staining method, an antibody chip method, or an immunoprecipitation method.
[25] 請求項 10— 17のいずれかに記載の製造法によって得られたタンパク質と、標的分 子とを接触させ、タンパク質と標的分子との相互作用を検出することを含む、分子間 相互作用を検出する方法。 [25] An intermolecular interaction comprising contacting a protein obtained by the production method according to any one of claims 10 to 17 with a target molecule and detecting an interaction between the protein and the target molecule. How to detect.
[26] 蛍光相関分光法、蛍光イメージングアナライズ法、蛍光共鳴エネルギー移動法、ェ バネッセント場分子イメージング法、蛍光偏光解消法、表面プラズモン共鳴法、又は[26] fluorescence correlation spectroscopy, fluorescence imaging analysis, fluorescence resonance energy transfer, evanescent field molecular imaging, fluorescence depolarization, surface plasmon resonance, or
、固相酵素免疫検定法である請求項 25に記載の検出方法。 The detection method according to claim 25, which is a solid-phase enzyme immunoassay.
[27] ヒト又はその他の動物由来の DNAライブラリ一力 請求項 2の方法によって選択され た一本鎖抗体をヒトの IgGの可変領域と置換することによって構築されるヒト型および ヒト抗体。 [27] A human and human antibody constructed by substituting a single-chain antibody selected by the method of claim 2 with a human IgG variable region, wherein the human or other animal-derived DNA library is used.
[28] 請求項 27記載の抗体を有効成分とする治療剤。  [28] A therapeutic agent comprising the antibody according to claim 27 as an active ingredient.
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