JP6551923B2 - Test substance measurement FRET molecular sensor - Google Patents

Description

  The present invention relates to a FRET molecular sensor that measures a test substance using fluorescence resonance energy transfer (FRET), that is, a test substance measurement FRET molecular sensor, and more particularly, to a fluorescent target having a fluorescent substance and a test substance binding part. The present invention relates to a test substance sensor, and a test substance measurement FRET molecular sensor provided with a fluorescence competitor that competes with a test substance to bind to or dissociate from a test substance binding part, and utilizes FRET caused by a fluorescent substance and a fluorescence competitor.

  BACKGROUND ART In recent years, development of protein molecule sensors for quantifying test substances has been actively performed in post-genome related technical fields such as drug discovery fields, medical fields, or food fields. Many such molecular sensors using fluorescence resonance energy transfer (FRET) have been developed as protein molecular sensors. In FRET, two fluorescent substances, a donor channel (donor molecule or donor fluorescent molecule) that is a fluorescent energy donor and an acceptor channel (acceptor molecule or acceptor fluorescent molecule) that is a fluorescent energy acceptor exist in close proximity. In some cases, it refers to a nonradiative energy transfer phenomenon in which energy is nonradiatively transferred to the acceptor channel by photoexcitation of the donor channel, and the acceptor channel emits light.

  As a molecular sensor using FRET, conventionally, a bimolecular (intermolecular) molecular sensor in which a donor channel and an acceptor channel are sterically separated is sterically integrated with a donor channel and an acceptor channel. These sensors can be broadly classified into molecular sensors of one molecular type (intramolecular type) (Patent Document 1). The bimolecular (intermolecular) sensor mainly detects intermolecular interactions, and the single molecular (intermolecular) sensor mainly changes molecular structure (structural change that affects FRET) To detect In the case of a single molecule type (intramolecular type) sensor, the amount of the donor channel and the acceptor channel is the same in principle, so that leakage into the acceptor fluorescent protein detection channel, direct excitation of the acceptor fluorescent protein without FRET, etc. Since the problems in the bimolecular (intermolecular) sensor of this are improved, the increase / decrease in the efficiency of FRET can be sufficiently evaluated only by taking the fluorescence amount ratio of the donor channel and the acceptor channel. Many of the current molecular sensors using FRET are single-molecule (intramolecular) sensors.

As a conventional molecular sensor using FRET, for example, a molecular sensor (Patent Document 2) having a donor channel, a plurality of acceptor channels, and a structure change region connected between the donor channel and the acceptor channel, A molecular sensor (Patent Document 3), a donor fluorescent protein, which is a copolymer of a compound to which a donor channel is bound, a compound to which an acceptor channel is bound, and a compound to which neither a donor channel nor an acceptor channel is bound And a acceptor fluorescent molecule, and a molecular sensor (Patent Document 4) that is connected between a donor fluorescent protein and an acceptor fluorescent molecule and includes a specific biomolecule recognition region. In addition, the present inventors previously filed a patent application for a molecular sensor for measuring intracellular inositol 1,4,5 triphosphate (IP ₃ ), which is a single molecule type (intramolecular type) sensor using FRET, It is patented (Patent Document 5).

International Publication No. 2012/043477 brochure JP 2010-233491 A JP 2011-201943 A JP 2014-055577 A Japanese Patent No. 4803976

  However, not only the conventional bimolecular (intermolecular) sensor but also the single molecule (intramolecular) sensor, the test substance binding part existing between the donor channel and the acceptor channel is not present. Depending on the long chain sequence or the degree or mode of structural change caused by the binding of the test substance to the test substance binding site, it leaks into the acceptor fluorescent protein detection channel and does not depend on the FRET of the acceptor fluorescent protein. Problems such as direct excitation or imperceptibility of structural changes that do not affect FRET have occurred, and as a result, there are some that can not play the role of a sensor.

  The present invention has been made to solve such problems, and compared to the conventional single molecule type (intramolecular type) sensor, the length of the arrangement of the test substance binding portion and the test substance The invention provides a FRET molecular sensor that measures analyte using fluorescence resonance energy transfer (FRET), which is extremely sensitive without being affected by the structural change caused by binding to the analyte binding part. The purpose is to

  As a result of intensive studies, the present inventors have found that a fluorescent test substance sensor having a fluorescent substance and a test substance binding part, and a fluorescent competitor substance that competes with the test substance and binds to or dissociates from the test substance binding part. In other words, it has a fluorescent substance which is either a donor channel or an acceptor channel and a test substance binding part, and when the fluorescent substance is a donor channel, it has no acceptor channel and the fluorescent substance has no activity. In the case of a scepter channel, a fluorescent test substance sensor that does not have a donor channel and a fluorescent test substance that competes with the test substance and binds to or dissociates from the test substance binding part, and the fluorescent test substance sensor uses the donor channel. If the fluorescent analyte sensor has an acceptor channel, the fluorescent substance sensor is a donor channel. By using a FRET molecular sensor equipped with a competitive substance, the sensor mode can be included in the bimolecular type (intermolecular type), but when the fluorescent competitor binds to the test substance binding part, the test substance binding The donor channel and the acceptor channel can be close to each other without being affected by the structural length of the portion and the structural change that occurs when the test substance binds to the test substance binding part. As a result, the conventional single molecule type (intramolecular As a result, the inventors have found that the sensitivity as a sensor is greatly improved as compared with a sensor.

(1) A FRET molecular sensor that measures a test substance using fluorescence resonance energy transfer (FRET), which competes with a fluorescent test substance sensor having a fluorescent substance and a test substance binding portion, and a test substance The FRET molecular sensor comprising: a fluorescent competitor that binds to or dissociates from the analyte binding part; and FRET caused by the fluorescent substance and the fluorescent competitor.

(2) The fluorescent test substance sensor is the fluorescent test substance sensor that does not have an acceptor channel when the fluorescent substance is a donor channel and does not have a donor channel when the fluorescent substance is an acceptor channel. And the fluorescent competitor is said fluorescent competitor that is an acceptor channel if the fluorescent analyte sensor has a donor channel and is a donor channel if the fluorescent analyte sensor has an acceptor channel (1 ) FRET molecular sensor according to the above.

(3) A fluorescent test substance sensor is the fluorescent test substance sensor having a fluorescent substance that is a donor channel and a test substance binding part, and the fluorescent competitive substance is a fluorescent competitor that is an acceptor channel. The FRET molecular sensor according to (1) or (2).

(4) The FRET molecular sensor according to any one of (1) to (3), wherein the fluorescent substance is a fluorescent protein or a protein fluorescent labeling substance.

(5) The test substance binding part is a ligand binding part of inositol 1,4,5 triphosphate (IP ₃ ) receptor, and the fluorescent competitor is 9- [5-Deoxy-5- [N-[[1 -[5-[[3 ', 6'-dihydroxy-3-oxospiro (isobenfuran-1,9'-xanthen) -5-yl] amino] thioxoxyl] amino] propyl] -1H-1,2,3-triazol -4-yl]-(3,4-di-O-phosphoryl-α-D-glucopyranosyl) -2-O-phosphoryl-β-D-ribo-pentofuranosyl] adenine (F-ADA) or (2R, 3R, 4S) -4-O-phosphoryl-2- [N-[[1- [5-[[3 ′, 6 ′ dihydroxy-3-oxospiro (isobenzofuran-1,9'-xanthen) -5-yl] amino] thioxomethyl] amino] propyl] -1H-1,2,3-triazol-4-yl] methyl-tetrahydrofuran-tetrahydrofuran The FRET molecular sensor according to any one of (1) to (4), which is −3,4-di-O-phosphoryl-α-D-glucopyranoside (F-LL).

(6) The test substance-binding portion is a paratope-containing polypeptide portion of an antibody, and the fluorescent competitor is a substance specifically bound to or dissociated from the paratope-containing polypeptide portion of the antibody and fluorescently modified ((6) The FRET molecular sensor according to any one of 1) to 4).

(7) A FRET molecular sensor for measuring a test substance using fluorescence resonance energy transfer (FRET), which comprises a fluorescent substance and a test substance binding portion and is immobilized on a carrier And a fluorescence-compatibilizing substance that comprises a fluorescent competitor that competes with the analyte and binds or dissociates with the analyte binding portion, and a carrier-immobilized analyte measuring FRET molecule that utilizes FRET caused by the fluorescent substance and the fluorescent competitor. sensor.

(8) The test substance measurement FRET molecular sensor according to any one of (1) to (6) or the carrier-immobilized test substance measurement FRET molecular sensor according to (7) and a target are brought into contact with each other, excitation A method of measuring a test substance in a subject by using the intensity of fluorescence as an index, comprising the steps of: irradiating the light and measuring the fluorescence generated.

(9) 9- [5-Deoxy-5- [N-[[1- [5-[[3 ′, 6′-dihydroxy-3-oxospiro (isobenzofuran-1,9′-xanthen) -5-yl] amino] thioxomethyl] amino] propyl] -1H-1,2,3-triazol-4-yl]-(3,4-di-O-phosphoryl-α-D-glucopyranosyl) -2-O-phosphoryl-β- D-ribo-pentofuranosyl] adenine (F-ADA) or (2R, 3R, 4S) -4-O-phosphoryl-2- [N-[[1- [5-[[3 ', 6'-dihydroxy-3] -Oxospiro (isobenzofuran-1, 9'-xanthen ) -5-yl] amino] thioxomethyl] amino] propyl] -1H-1,2,3-triazol-4-yl] methyl-tetrahydrofuran-3-yl-3,4-di-O-phosphoryl-α-D -Use of glucopyranoside (F-LL) as a fluorescence competitor in the FRET molecular sensor described in (1) or (2).

  According to the FRET molecular sensor for measuring a test substance according to the present invention, the length of the arrangement of the test substance binding portion and the structural change caused by the test substance binding to the test substance binding portion are not affected. Substances can be quantified with high accuracy, and therefore, even trace amounts of test substances can be quantified, and the determination method with extremely improved sensitivity compared to the conventional determination method using a molecular sensor using FRET It can be established.

It is a figure which shows the preparation flow of the compounds 2-7 which made the compound 1 the starting material. FIG. 6 is a diagram showing a production flow of compound 12 from FITC. FIG. 16 is a drawing showing a production flow of F-ADA (compound 13) from compound 7 and compound 12 and a production flow of F-LL (compound 14) from compound 11 and compound 12. It is a figure which shows the preparation flow of the compounds 8-11 which made the compound 2 the starting material. Diagrams were calculated potency of ^{Ca 2+} release via the IP ₃ receptor _IP 3, F-ADA and F-LL by analyzing the ^{Ca 2+} concentration change in intracellular stores. In the figure, the vertical axis shows the calculated potency of Ca ²⁺ release, and the horizontal axis shows the concentration of IP ₃ , F-ADA or F-LL. It is a diagram showing a configuration of a fluorescence IP ₃ sensor LIBRAvIIS (LvIIS). In the figure, “P” represents a cell membrane localization signal, “ECFP” represents a blue fluorescent protein ECFP, “L1” and “L2” both represent a linker, and “IP ₃ R2 (1-604) with R440Q "Is a peptide consisting of the amino acid sequence 1 to 604 from the N-terminal of the rat type 2 IP ₃ receptor, wherein arginine (R) at position 440 of the sequence is mutated to glutamine (Q), “Venus” indicates the yellow fluorescent protein Venus. Is a diagram of analyzing the specificity of the fluorescent competitor F-ADA and fluorescence IP ₃ sensor LIBRAvIIS. In the figure, the images of Aa and Ab are fluorescence images at the time point before the action of 30 nM F-ADA (time point of arrow B in FIG. 1), and the images of Ac and Ad act on 30 nM F-ADA. Is a diagram showing a fluorescence image at a time point (time point of arrow 3 in B), B is a diagram showing a change in fluorescence due to addition of the fluorescence competitor F-ADA, and C is a fluorescence from a region including cells. It is a figure which shows the fluorescence change at the time of subtracting the fluorescence of BG area | region. The vertical axes of B and C indicate the fluorescence intensity, and the horizontal axis indicates the elapsed time. Moreover, D is a figure which shows fluorescence ratio 480nm / 535nm, the vertical axis | shaft of D shows the value of fluorescence ratio 480nm / 535nm, and a horizontal axis shows elapsed time. Fluorescence IP ₃ sensor LIBRAvI (LvI), LIBRAvII (LvII ), LIBRAvIII (LvIII), LIBRAvN (LvN), is a diagram showing a configuration of LIBRAvIIS (LvIIS) and MP-LIBRAvIIS (MP-LvIIS) . In the figure, “P” indicates cell membrane localization signal, “MP” indicates endoplasmic reticulum localization signal, “ECFP” indicates blue fluorescent protein ECFP, “Cerulean” indicates blue fluorescent protein Cerulean, “L1” “L1H1”, “L2” and “L2-6” all indicate linkers, and “IP ₃ R1 (1-604)” is the 1-604 from the N-terminus of rat type 1 IP ₃ receptor amino acid sequence “IP ₃ R 2 (1-604)” represents a peptide consisting of the amino acid sequence of 1 to 604 from the N-terminus of rat type 2 IP ₃ receptor amino acid sequence, “IP ₃ R 2 (1-604) with R440Q "is a peptide consisting of the amino acid sequence No. 1 to No. 604 from the N-terminus of rat type 2 IP ₃ receptor, and its 4 The peptide which mutated arginine (R) of number 40 to glutamine (Q) is shown, and "IP ₃ R3 (1-604) with K507A" is 1 to 604 from the N-terminus of rat type ₃ IP ₃ receptor amino acid sequence. A peptide consisting of the amino acid sequence of SEQ ID NO: 10, wherein lysine (K) at position 507 of the sequence is mutated to alanine (A), and "Venus" indicates the yellow fluorescent protein Venus. It is a figure which shows the preparation procedure of a LIBRAvIISVm (LvIISVm) gene. In the figure, “→” indicates a part of the vector gene, “MT” indicates a membrane-binding sequence, “ECFP” indicates the sequence of the blue fluorescent protein ECFP, and “LBD” indicates the ligand binding site of the IP ₃ receptor. The “Venus” indicates the yellow fluorescent protein Venus, and the “Venus (m)” indicates the non-chromogenic protein Venus mutant. It is a figure which shows the structure of fluorescence IP ₃ sensor LIBRAVIISVm (LvIISVm). In the figure, “P” indicates a cell membrane localization signal, “ECFP” indicates a blue fluorescent protein ECFP, “L1” and “L2” indicate linkers, and “IP ₃ R2 (1-604) with R440Q” indicates A peptide consisting of an amino acid sequence from the N-terminus to the amino acid number 1 to 604 of the rat type 2 IP ₃ receptor, in which arginine (R) at position 440 of the sequence is mutated to glutamine (Q), "Venus mutants Indicates a non-chromogenic protein Venus mutant. Fluorescent IP ₃ sensors LIBRAvI (LvI), LIBRAvII (LvII), LIBRAvIII (LvIII), LIBRAvN (LvN), LIBRAvIIS (LvIIS) and MP-LIBRAvIIS (MP-LvnmS) when F-ADA is allowed to act And a fluorescence ratio of 535 nm fluorescence. In the figure, the vertical axis indicates the fluorescence ratio of 480 nm / 535 nm, and the horizontal axis indicates the elapsed time. In addition, the values of “10” and “30” and the horizontal bars in the figure indicate the concentration of F-ADA (nM) and the time zone in which F-ADA was applied, respectively. It is a figure which shows the preparation procedure of LIBRAvIISVd (LvIISVd) gene. In the figure, “→” indicates a part of the vector gene, “MT” indicates a membrane binding sequence, “ECFP” indicates a sequence of blue fluorescent protein ECFP, and “LBD” indicates a ligand binding portion of IP ₃ receptor And the "Venus" indicates the yellow fluorescent protein Venus. It is a figure which shows the structure of fluorescence IP ₃ sensor LIBRAVIISVd (LvIISVd). In the figure, “P” indicates a cell membrane localization signal, “ECFP” indicates a blue fluorescent protein ECFP, “L1” indicates a linker, and “IP ₃ R2 (1-604) with R440Q” indicates rat type 2 IP. ₃ shows a peptide consisting of an amino acid sequence of Nos. 1 to 604 from the N-terminal of ₃ receptors, wherein arginine (R) at No. 440 in the sequence is mutated to glutamine (Q). It is a figure which shows the fluorescence intensity (upper stage) of fluorescence IP ₃ sensor LIBRAvIISVd (LvIISVd), and the fluorescence ratio (lower stage) of the fluorescence of 480 nm, and the fluorescence of 535 nm when F-ADA is made to act. In the figure, the vertical axis indicates the fluorescence intensity (upper) or the value of the fluorescence ratio 480 nm / 535 nm (lower), and the horizontal axis indicates the elapsed time (upper and lower). Also, the values “30” and “100” in FIG. A, and the horizontal bars indicate the concentration (nM) of F-ADA that was applied and the time zone in which F-ADA was acting. When allowed to act F-LL, it illustrates fluorescence intensity (top) and 480nm fluorescence and 535nm fluorescence fluorescence ratio of fluorescence IP ₃ sensor LIBRAvIISVd (LvIISVd) a (lower). In the figure, the vertical axis indicates the fluorescence intensity (upper) or the value of the fluorescence ratio 480 nm / 535 nm (lower), and the horizontal axis indicates the elapsed time (upper and lower). Further, the values of “30” and “100” in FIG. A and the horizontal bars indicate the concentration of F-LL (nM) applied and the time zone during which F-LL was applied. When allowed to act IP ₃ after reacted with F-ADA, a diagram illustrating a fluorescence ratio 480 nm / 535 nm fluorescence IP ₃ sensor LIBRAvIISVd (LvIISVd). In the figure, the vertical axis indicates the fluorescence ratio of 480 nm / 535 nm, and the horizontal axis indicates the elapsed time. In addition, the values of “30” and “100” and horizontal bars in the figure indicate the concentration of F-ADA (nM) and the time zone during which F-ADA was applied, the value of “10”, and The horizontal bar indicates the concentration of IP _{3 applied} (μM) and the time zone when IP ₃ was applied. When the IP ₃ was added F-LL after reacted is a diagram showing the fluorescence ratio 480 nm / 535 nm fluorescence IP ₃ sensor LIBRAvIISVd (LvIISVd). In the figure, the vertical axis indicates the fluorescence ratio of 480 nm / 535 nm, and the horizontal axis indicates the elapsed time. Also, the value of "100" in the figure and the horizontal bar indicate the concentration (nM) of F-LL that has been acted and the time zone in which F-LL acted, and "0.1", "1" and " value of 10 "as well as the horizontal bar indicates the time zone in which the concentration ([mu] M) and IP ₃ of IP ₃ which was allowed to act acts. It is a figure which shows the preparation procedure of cyLIBRAvIISVd (cyLvIISVd) gene. In the figure, "ERT" indicates an endoplasmic reticulum binding sequence, "HisTag" indicates a tag peptide for purification consisting of 6 consecutive histidine (His; H) residues, and "Cerulean" indicates a blue fluorescent protein Cerulean. In the figure, “LBD” indicates the sequence of the ligand binding part of IP ₃ receptor, “Venus” indicates the yellow fluorescent protein Venus, and “ECFP” indicates the sequence of blue fluorescent protein ECFP. It is a diagram showing a configuration of a fluorescence IP ₃ sensor cyLIBRAvIISVd (cyLvIISVd). In the figure, “6H” represents a tag peptide for purification consisting of 6 consecutive histidine (His; H) residues, “Cerulean” represents the blue fluorescent protein Cerulean, “L1H1” represents the linker, “IP ₃ R2 (1-604) with R440Q” is a peptide consisting of the amino acid sequence 1 to 604 from the N-terminal of rat type 2 IP ₃ receptor. The peptide mutated to) is shown. Fluorescence IP ₃ sensor cyLIBRAvIISVd (cyLvIISVd) shows a fluorescent image of the binding beads (A), and fluorescence IP ₃ sensor cyLIBRAvIISVd (cyLvIISVd) respectively coupled beads 10 nM, 30 nM, 100 nM or 300nM of F-LL, F-ADA or FITC It is a figure (B, C, and D) which shows each fluorescence change rate at the time of making act. In FIGS. B, C and D, the vertical axis indicates the rate of fluorescence change, and the horizontal axis indicates the concentration (nM). When allowed to act IP ₃ after reacted with F-LL, it illustrates fluorescence ratio 480nm / 535nm (A) and fluorescence rate of change of carrier immobilized fluorescent IP ₃ sensor cyLIBRAvIISVd (cyLvIISVd) a (B). In the figure, the vertical axis indicates the fluorescence ratio of 480 nm / 535 nm, and the horizontal axis indicates the elapsed time. Also, the value of "100" in the figure and the horizontal bar indicate the concentration (nM) of F-LL that has been acted and the time zone in which F-LL acted, and "0.1", "1" and " value of 10 "as well as the horizontal bar indicates the time zone in which the concentration ([mu] M) and IP ₃ of IP ₃ which was allowed to act acts. It is a figure which shows the preparation procedure of OP1-ma (EN) -S gene, OP1-ma (EN) -E gene, and OP1-ma (EN) -ES gene. In the figure, “→” represents a part of the vector gene, “MT” represents the membrane-bound gene sequence, “FLAG” represents the gene sequence of the FLAG tag, and “LT” and “LT” represent the gene sequence of LumioTag "E1" indicates a ScaI cleavage site, "E2" indicates an Eco47III cleavage site, "1-82-E1-LT-E1-83-140-E2-LT-E2-141-604", " 1-82-E1-LT-E1-83-140-E2-141-604 "," 1-82-E1-83-140-E2-LT-E2-141-604 "and" 1-82-E1- “83-140-E2-141-604” indicates the gene sequence of the altered protein of the ligand binding part of the IP ₃ receptor, and “Hx6” is a tag consisting of 6 consecutive histidine (His; H) residues. Shows the gene sequence of the Fluorescence intensity (upper) of fluorescent IP ₃ sensors OP1-ma (EN) -S, OP1-ma (EN) -E, and OP1-ma (EN) -ES when F-ADA is allowed to act; It is a figure which shows the fluorescence ratio (lower stage) of fluorescence of 480 nm and fluorescence of 535 nm. In the figure, the vertical axis indicates the fluorescence intensity (upper) or the value of the fluorescence ratio 480 nm / 535 nm (lower), and the horizontal axis indicates the elapsed time (upper and lower). In addition, the white horizontal bars in the upper graph indicate the time zone in which the DACM has acted, and the black bars indicate the time zone in which the F-ADA has acted. It is a figure which shows the preparation procedure of OP1S-cpC157 gene, OP1S-cpC173 gene, and OP1S-cpC229 gene. In the figure, “→” represents a part of the vector gene, “MT” represents the membrane-bound gene sequence, “FLAG” represents the gene sequence of the FLAG tag, “E1” represents the ScaI cleavage site, “E2” "indicates the Eco47III cutting site," 1-82-E1-83-140-E2-141-604 "represents the gene sequence of the modified protein of the ligand binding portion of the IP ₃ receptor," Hx6 "is six The gene sequence of a tag peptide consisting of consecutive histidine (His; H) residues is shown, wherein “cpSECFP157”, “cpSECFP173” and “cpSECFP229” are the circular permutations of ECFP, respectively, of the fluorescent proteins cpSECFP157, cpSECFP173, and cpSECFP229. The gene sequence is shown as “1-82-cpSECFFP1 57-83-140-E2-141-604 "," 1-82-cpSECFP173-83-140-E2-141-604 ", and" 1-82-cpSECFP229-83-140-E2-141-604 " each shows a gene sequence inserted fluorescent protein cpSECFP157, cpSECFP173, and cpSECFP229 the IP ₃ into a modified protein of the ligand binding portion of the receptor is a circular permutation of ECFP. When allowed to act F-ADA, a diagram showing fluorescence IP ₃ sensors OP1S-cpC157, OP1S-cpC173, and fluorescence intensity of OP1S-cpC229 (top) and 480nm fluorescence and 535nm fluorescence fluorescence ratio of the (bottom) is there. In the figure, the vertical axis indicates the fluorescence intensity (upper) or the value of the fluorescence ratio 480 nm / 535 nm (lower), and the horizontal axis indicates the elapsed time (upper and lower). Moreover, the horizontal bar filled with black in the upper diagram shows the time zone in which F-ADA has acted. Fluorescence ratio of 480 nm fluorescence and 535 nm fluorescence of agarose bead bound fluorescent ABsII-CT2-18 sensor washed with phosphate buffer when fluorescent competitor F-CT2-18 and analyte CT2-18 are allowed to act (A) The fluorescence ratio (B) of the fluorescence at 480 nm and the fluorescence at 535 nm of agarose beads bound fluorescence ABsII-CT2-18 sensor without washing. In the figure, the vertical axis shows the value of the fluorescence ratio 480 nm / 535 nm, and the horizontal axis shows the concentrations of the fluorescence competitor F-CT2-18 and the test substance CT2-18. Fluorescence ratio of 480 nm and 535 nm by excitation of 425 nm of FRET molecular sensor equipped with anti-c-Myc antibody immobilized Protein G beads labeled with DACM and c-Myc peptide labeled with FITC, and labeled with this The change of the fluorescence ratio at the time of making c-Myc which does not coexist is shown. 480 nm and 535 nm fluorescence ratio by excitation of 425 nm of FRET molecular sensor equipped with anti-DYK antibody-immobilized Protein G beads labeled with DACM and DYK peptide labeled with FITC, and unlabeled DYK peptide Shows a change in fluorescence ratio when coexistence of Anti-IP ₃ Receptor Antibody Immobilized Ni-NTA Complexed Protein G Beads Labeled with DACM and His Taged Peptide (DYK Peptide) Labeled with FITC 480 nm by Excitation at 425 nm of FRET Molecular Sensor The fluorescence ratio at 535 nm and the change of the fluorescence ratio when DYK peptide not labeled therewith coexist are shown. A fluorescence ratio of 480 nm and 535 nm by excitation of 425 nm of a FRET molecular sensor including Protein G beads labeled with DACM and an antibody labeled with FITC, and a fluorescence ratio when an unlabeled antibody coexists. Indicates a change in

  Hereinafter, the test substance measurement FRET molecular sensor according to the present invention will be described in detail. A test substance measurement FRET molecular sensor according to the present invention includes a fluorescent test substance sensor having a fluorescent substance and a test substance binding part, and a fluorescence competition that binds to or dissociates from the test substance binding part in competition with the test substance. A molecular sensor comprising a substance and utilizing FRET triggered by a fluorescent substance and a fluorescent competitor.

  The test substance measurement FRET molecular sensor according to the present invention is a bimolecular (intermolecular type) molecular sensor of a fluorescent test substance sensor and a fluorescent competitor, and is a fluorescent substance contained in the fluorescent test substance sensor. And one of the fluorescence competitors may be a donor channel (donor molecule) and the other may be an acceptor channel (acceptor molecule). That is, in this case, when the fluorescent substance is a donor channel, the fluorescent analyte sensor does not have an acceptor channel, and the fluorescent competitor is an acceptor channel, and when the fluorescent substance is an acceptor channel, the fluorescent analyte The sensor does not have a donor channel, and the fluorescence competitor is a donor channel. In this embodiment, the fluorescent substance is a donor channel, the fluorescent analyte sensor does not have an acceptor channel, and the fluorescent competitor is an acceptor channel. Is not limited to this.

  The “fluorescent substance” included in the fluorescent analyte sensor in the present invention is not particularly limited as long as it is a fluorescent substance that can be a donor channel (donor molecule) or an acceptor channel (acceptor molecule) in FRET, and such a fluorescent substance For example, fluorescent proteins and protein fluorescent labeling substances can be mentioned. Examples of such fluorescent proteins include fluorescent proteins such as CFP, YEP, GFP, RFP, BFP, and DsRed, and mutants of these fluorescent proteins may be used. Such variants include ECFP, EYFP, EGFP, ERFP, EBFP and their circular permutations (circular permutations).

  In addition, as the “protein fluorescent labeling substance”, low molecular weight fluorescent substances can be mentioned, for example, naphthalene derivatives such as 1,5-IAEDANS, IAANS, MIANS, etc. which are low molecular weight fluorescent substances as donor channels (donor molecules); Pyrene derivatives such as pyrenemaleimide and pyreneiodoacetamide; coumarin derivatives such as CPM, DCIA, DACIA, DACM, MDCC, IDCC; fluorocein isothiocyanate (FITC), fluorescein maleimide, 5-iodoacetamide fluorescein, 5- Fluorescein derivatives such as bromomethylfluorescein, Oregon green 488 maleimide, Oregon green 488 iodoacetamide; Alexa 488 maleimide, Alexa 532 maleimide, Alexa 546 maleimide, Alexa derivatives such as kusa 568 maleimide, alexa 594 maleimide, alexa 555 maleimide; BODIPY derivatives such as BODIPY493 / 503 bromomethyl, BODIPY499 / 508 maleimide, BODIPY507 / 545 iodoacetamide, BODIPY530 / 550 iodoacetamide; tetramethylrhodamine maleimide, Rhodamine derivatives such as cetamide and rhodamine red maleimide; Texas red derivatives such as Texas red maleimide and Texas red iodoacetamide; and fluoro, which is a low molecular fluorescent substance as an acceptor channel (acceptor molecule) Sein isothiocyanate (FITC), fluorescein maleimide, 5-yo Acetamidofluorescein, 5-bromomethyl fluorescein, Oregon green 488 maleimide, Oregon green 488 iodoacetamide, and other florethein derivatives; Alexa 488 maleimide, Alexa 532 maleimide, Alexa 546 maleimide, Alexa 568 maleimide, Alexa 594 maleimide, Alexa 555 maleimide, Alexa Alexa derivatives such as 633 maleimide, Alexa 647 maleimide, Alexa 660 maleimide, Alexa 680 maleimide, etc .; BODIPY derivatives such as maleimide; Rhodamine derivatives such as tetramethylrhodamine maleimide, tetramethylrhodamine iodoacetamide and rhodamine red maleimide; Texas red derivatives such as Texas red maleimide and Texas red iodoacetamide;

In the present invention, the “test substance binding part” of the fluorescent test substance sensor means a substance capable of forming a specific bond with a test substance or a part thereof, and, for example, the test substance is In the case of inositol 1,4,5 triphosphate (IP ₃ ), the IP ₃ receptor or its ligand binding part, and if the test substance is acetylcholine, the acetylcholine receptor or its ligand binding part, the test substance should be an antigen For example, an antibody against the antigen or a paratope-containing polypeptide portion thereof, a metal-NTA complex for the histidine tag sequence, proteins A, G and L for immunoglobulin, biocin for avidin, an enzyme for a substrate of the enzymatic reaction (eg glutathione-S for glutathione) -Transferase) etc. can be mentioned, respectively.

As the ligand binding part of the IP ₃ receptor, for example, those of a wide range of biological species such as rat, mouse, cow, rabbit, chicken, human, Xenopus, Drosophila, starfish, nematode and the like may be used. For example, the 224-579 th of the amino acid sequence of the IP ₃ receptor isotypes 1 rat (SEQ ID NO: 1), the 224-579 th of the amino acid sequence of the IP ₃ receptor isotypes 2 rat (SEQ ID NO: 2), Of the amino acid sequence of rat IP ₃ receptor isotype 3, positions 225 to 579 (SEQ ID NO: 3), among the amino acid sequence of mouse IP ₃ receptor isotype 1, positions 224 to 579 (SEQ ID NO: 4), The amino acid sequence of IP ₃ receptor isotype 2 at positions 224 to 579 (SEQ ID NO: 5), the amino acid sequence of mouse IP ₃ receptor isotype 3 at positions 225 to 579 (SEQ ID NO: 6), bovine IP ₃ the 224-579-th of the amino acid sequence of the receptor isotypes 1 (SEQ ID NO: 7), bovine IP ₃ receptor isoforms The 224-579-th of the amino acid sequence of type 2 (SEQ ID NO: 8), the 225-579 th of the amino acid sequence of the IP ₃ receptor isotypes 3 of bovine (SEQ ID NO: 9), rabbit IP ₃ receptor isotypes 1 224 to 579th (SEQ ID NO: 10) of the amino acid sequence of SEQ ID NO: 2, 270 to 625th (SEQ ID NO: 11) of the amino acid sequence of rabbit IP ₃ receptor isotype 2, and the amino acid of rabbit IP ₃ receptor isotype 3 378-739 (SEQ ID NO: 12) of the sequence, 224-579th (SEQ ID NO: 13) of the amino acid sequence of chicken IP ₃ receptor isotype 1, and the amino acid sequence of chicken IP ₃ receptor isotype 2 of the 236 to 591-th (SEQ ID NO: 14), amino acids of IP ₃ receptor isotypes 3 chicken The 225-579-th of the column (SEQ ID NO: 15), the IP ₃ receptor isotypes 1 of human 224-579 th of the amino acid sequence (SEQ ID NO: 16), the IP ₃ receptor isotypes 2 human amino acid sequence Of these, the 224th to 579th positions (SEQ ID NO: 17) and the 225th to 579th positions (SEQ ID NO: 18) of the amino acid sequence of human IP ₃ receptor isotype 3 can be used.

The "test substance binding portion" in the present invention, other IP ₃ receptor or ligand binding portion itself that described above, the proteins are also included having their homologs, artificial variants or conservative mutations. In the present specification, a protein having the homologue, the artificial variant, or the conservative mutation is referred to as a conservative variant. Conservative variants can include proteins having a sequence that includes one or a few to several tens of amino acid substitutions, deletions, insertions, additions, and the like.

  Although “one or several to several tens” is different depending on the position of the amino acid residue in the three-dimensional structure of the protein and the kind of amino acid residue, specifically 1 to 50, preferably 1 to 40, More preferably, it means 1 to 30, more preferably 1 to 20, still more preferably 1 to 10, particularly preferably 1 to 5. Also, representative of conservative mutations are conservative substitutions.

  Conservative substitutions are those that can generally be made without altering the physiological activity of the resulting molecule, ie, those that are found in the range of conservative substitutions (Watson et al., Molecular Biology of Gene, etc.), for example If the substitution site is an aromatic amino acid, then Phe, Trp, or Tyr, if the substitution site is a hydrophobic amino acid, if it is a polar amino acid among Leu, Ile, or Val, then Gln. , Asn between basic amino acids, between Lys, Arg and His, between acidic amino acids, between Asp and Glu between amino acids with hydroxyl groups, Ser, Thr It is a mutation which substitutes each other between. Specifically, as a substitution considered as a conservative substitution, a substitution from Ala to Ser or Thr, a substitution from Arg to Gln, His or Lys, a substitution from Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln substitution, Cys to Ser or Ala substitution, Gln to Asn, Glu, Lys, His, Asp or Arg substitution, Glu to Gly, Asn, Gln, Lys or Asp Substitution, substitution of Gly to Pro, substitution of His to Asn, Lys, Gln, Arg or Tyr, substitution of Ile to Leu, Met, Val or Phe, substitution of Leu to Ile, Met, Val or Phe, Substitution of Lys to Asn, Glu, Gln, His or Arg Met to Ile, Leu, Val or Phe substitution, Phe to Trp, Tyr, Met, Ile or Leu substitution, Ser to Thr or Ala substitution, Thr to Ser or Ala substitution, Trp to Phe or A substitution to Tyr, a substitution from Tyr to His, Phe or Trp, and a substitution from Val to Met, Ile or Leu can be mentioned. In addition, amino acid substitutions, deletions, insertions, additions, or inversions as described above include naturally occurring mutations (mutants or variants) such as cases based on individual differences or species differences of the microorganism from which the gene is derived. Also includes those caused by

  Furthermore, the protein having a conservative mutation as described above is, for example, 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, still more preferably, relative to the entire amino acid sequence. It may be a protein having a homology of 98% or more, particularly preferably 99% or more, and having a function equivalent to that of a wild-type protein. In the present specification, “homology” may refer to “identity”.

  "A paratope-containing polypeptide portion of an antibody" refers to an antigen-binding portion of an antibody that binds to an epitope of an antigen (immunogen) that is a test substance, that is, a polypeptide portion containing a paratope; , Fab ′, F (ab ′) 2 and F (v), and the like. Fab and F (ab ') 2 moieties can be made, respectively, by proteolysis of substantially intact antibody molecules with papain and pepsin by methods known in the art. Fab ′ antibody molecule moieties are also known in the art, and the disulfide bond connecting the two heavy chain moieties is reduced with mercaptoethanol and the resulting protein mercaptan is then alkylated with a reagent such as iodoacetamide. Are made from the F (ab ') 2 moiety. Moreover, the presence or absence of immunogenicity with respect to an antigen is not ask | required, and a hapten is contained in an antigen. Furthermore, it may be obvious that an antibody "binds" to a specific antigen is capable of binding to the specific antigen. The term "bind" to the specific antigen also includes cases in which it does not bind to other antigens at all, but also includes cases in which it binds to other antigens and also binds to the specific antigen.

  Also, in the present invention, antibodies can be prepared by any of various methods. For example, in addition to a method for inducing production of serum containing polyclonal antibodies by administering an antigenic fragment as a test substance to an animal, spleen cells are immunized after immunizing an animal (preferably a mouse) with a test substance. A method of preparing a monoclonal antibody (or a test substance-binding fragment thereof) and the like by extracting and fusing with an appropriate myeloma cell line can be mentioned.

  Histidine residues are known to form a complex by chelating with metal ions such as nickel, cobalt, copper, and zinc. By using these metal ions, histidine residues such as histidine tags can be converted. The test substance contained can be detected. These metal ions are introduced into the fluorescent analyte sensor as a complex with NTA, for example, by reacting Isothiocyanobenzyl-NTA with the amino group of the fluorescent analyte sensor and then adding a solution containing the metal ion. Can.

  Proteins A and G are known to specifically bind to the Fc region of immunoglobulins, particularly IgG, and using them as a test substance binding portion, it is possible to detect IgG or its Fc fragment as a test substance it can. Also, protein L is known to specifically bind to the kappa light chain of immunoglobulin, and by using this as a test substance binding part, IgG, IgA, IgM, etc., or their Fab, F (ab (ab) ') 2 or scFv fragments can be detected as a test substance.

  The fluorescent protein which is a fluorescent substance and the test substance binding part may be directly bound and arranged, or may be linked and arranged via a linker. Such a linker is not particularly limited, and peptide sequences used in the art, usually peptide sequences having a length of about 2 to 20 amino acids can be used.

  When the protein fluorescent labeling substance is a fluorescent substance, it may be introduced into the fluorescent test substance sensor by non-peptide bond with the amino acid residue of the fluorescent test substance sensor. For example, when the fluorescent analyte sensor contains a cysteine residue, the protein fluorescent labeling substance is introduced into the fluorescent analyte sensor by reacting the protein fluorescent labeling substance having a maleimide group with the cysteine residue. be able to. The fluorescent test substance sensor into which the protein fluorescent labeling substance has been introduced in this manner causes FRET by binding to the "fluorescent competitor" described later, and functions as a FRET molecular sensor. In addition, introduction of a protein fluorescent labeling substance may be performed on any amino acid residue as long as the binding ability of the test substance binding portion to the ligand is not impaired.

  The fluorescent analyte sensor may optionally have a localization signal. The binding position of the localization signal in the fluorescent test substance sensor may be either N-terminal or C-terminal, and as the localization site, cell membrane, mitochondria, cytoskeleton, nucleus, endoplasmic reticulum, etc. can be mentioned. Furthermore, the fluorescent analyte sensor may have a tag. Such tags include, for example, FLAG tag (FLAG tag), tag tag for purification consisting of histidine (His; H) residue (histidine tag, His-tag), strep-tag, GST (glutathione-S-) Examples include transferase) -tag, HA (hemagglutinin peptide) -tag, Myc-tag and the like.

The fluorescent analyte sensor may be immobilized on an appropriate carrier. As a method of immobilizing a fluorescent test substance sensor on a carrier, in addition to the method of immobilizing via a tag, for example, a carrier on which a molecule (antibody, protein A, protein G) having a binding property to a molecular sensor is immobilized. In addition to the method used, the molecular sensor is biotinylated and bound to a carrier on which avidin or streptavidin is immobilized, and the functional group (NH ₂ group, COOH group, phenol group, etc.) of the fluorescent analyte sensor is reactive. The method etc. which couple | bond with the support | carrier which fixed NH ₂ group, COOH group, an epoxy group, and OH group to have can be mentioned.

  As the carrier for immobilizing the fluorescent test substance sensor in the present invention, for example, polymerizable monomers such as styrene polymerizable monomers, acrylic polymerizable monomers, methacrylic polymerizable monomers and vinyl polymerizable monomers are polymerized. Organic fine particles, magnetic particles, metal oxides, metals, semiconductor compounds, agarose, sepharose, wood boards, glass plates, silicon substrates, natural fibers such as plant fibers, animal fibers, mineral fibers and food fibers; And synthetic fibers, recycled fibers, glass fibers, carbon fibers, and artificial mineral fibers.

In the present invention, the term “fluorescent competitor” refers to a fluorescent substance itself that can be a donor channel (donor molecule) or an acceptor channel (acceptor molecule) in FRET, or a competitor that is modified with such a fluorescent substance. Means a substance that binds to or dissociates from the test substance binding part, for example, a substance corresponding to the ligand itself or an analog thereof, or a substance having a specific binding ability to the test substance binding part . For example, when the test substance binding portion is an IP ₃ receptor, the fluorescent substance-modified IP ₃ , 9- [5-Deoxy-5- [N-[[1- [5-[[3 ', 6' -Dihydroxy-3-oxospiro (isobenzofuran-1,9'-xanthen) -5-yl] amino] thioxomethyl] amino] propyl] -1H-1,2,3-triazol-4-yl]-(3,4-) di-O-phosphoryl-α-D-glucocopyranosyl) -2-O-phosphoryl-β-D-ribo-pentofuranosyl] adenine (F-ADA), (2R, 3R, 4S) -4-O-phosphoryl-2- [N-[[1- [5-[[3 ′, 6′-dihydroxy-3-oxos iro (isobenzofuran-1,9'-xanthen) -5-yl] amino] thioxomethyl] amino] propyl] -1H-1,2,3-triazol-4-yl] methyl-tetrahydrofuran-3-yl-3,4 -Di-O-phosphoryl-α-D-glucocopyranoside (F-LL) or the like can be used as a fluorescence competitor. The structure of F-ADA is shown in the following formula (I), and the structure of F-LL is shown in the following formula (II).

  The method of labeling the competitor for the test substance binding portion with a fluorescent substance can be carried out by methods widely known to those skilled in the art. For example, when the competitor is a protein, the protein fluorescent labeling substance exemplified above may be introduced into the protein by a method known for each. If the competitor is a low molecular weight compound, it may be modified with a fluorescent substance by an organic synthetic chemical method within the range not interfering with the specific binding ability with the test substance binding part.

  In the present invention, “binding” may be used interchangeably with “action”, “interaction”, “specific interaction”, “selective interaction”, “reaction”, or “recognition”. .

  In the present invention, FRET molecular sensors can also be used in cells. For introducing a fluorescent analyte sensor or fluorescent competitor into cells, any known method may be used, and in addition to the method of directly introducing a fluorescent analyte sensor or fluorescent competitor into cells, for example, fluorescence The fluorescent competitor may be introduced after the DNA encoding the fluorescent test substance sensor, which is a fusion protein of the protein and the test substance binding part, is introduced into cells for expression. As a method for introducing such DNA encoding a fluorescent test substance sensor into cells for expression, for example, various DNAs, animals, animals and the like can be obtained by incorporating the DNA coding for such a fluorescent test substance sensor into an appropriate vector. Plants can be transformed. As the vector in that case, for example, plasmid vector, viral vector, Agrobacterium etc. can be mentioned. As a method for introducing a vector into a cell, for example, a lipofection method, a calcium phosphate method, a microinjection method, a proplast fusion method, an electroporation method, a DEAE dextran method, a gene introduction by Gene Gun, or the like can be used.

  Examples of the method for intracellular expression of the fusion protein include a method for producing a transgenic animal or a transgenic plant by introducing a gene in which a fusion protein gene is incorporated into chromosomal DNA into an animal or plant. Production of a transgenic animal or a transgenic plant can also be performed by a known method, and in the production, a fusion gene may be inserted into any site of chromosomal DNA. By using such a transgenic animal or transgenic plant and a fluorescent competitor, it is possible to quantify the test substance in an in vivo system, elucidate the function of the test substance and its receptor, treatment and diagnostic methods. It can be used for the establishment of medicines, screening of drugs, etc. In addition, cells obtained from these transgenic animals and plants can be used for drug screening, etc., and test substance measurement kits and test substance measurement instruments should be created together with fluorescent competitors. You can also.

Next, the method for measuring a test substance in a subject by using the intensity of fluorescence as an indicator in the present invention has the following steps (I) and (II).
(I) measuring an analyte substance FRET molecular sensor or carrier-immobilized analyte substance measuring FRET molecular sensor (target contacting process);
(II) A step of measuring fluorescence generated by irradiating excitation light (irradiation measurement step).

  The target contact step (I) is a step of bringing a fluorescent test substance sensor bound with a fluorescent competitor into contact with a target (subject) that contains the test substance or is assumed to contain the test substance, or A process of introducing a fluorescent competitor after contacting a fluorescent analyte sensor to which a fluorescent competitor is not bound and a target (subject) containing or suspected of containing the analyte. It is either.

  In the irradiation measurement step (II), irradiating excitation light means irradiating light in a range including the excitation maximum wavelength of the donor channel or a wavelength in the vicinity thereof using a desired light source. Besides a light source having a desired wavelength range by using a filter or a spectroscope, it is also possible to use monochromatic light such as a laser or the like. As a laser light source, in addition to a helium cadmium laser (442 nm), a blue diode laser (405 nm), an argon ion laser (457 nm), an LD-pumped solid-state laser (430 nm), and the like can be used. In the case of the two-photon excitation method, a pulsed laser around 800 nm can be used.

  In addition, the measurement of fluorescence is a range including the fluorescence maximum wavelength of the donor channel and / or the acceptor channel or a wavelength in the vicinity thereof, for example, 200 to 800 nm, preferably 300 to 700 nm, more preferably 400 to 600 nm, and even more preferably. Refers to measuring changes in their fluorescence intensity due to FRET in the range of 460-550 nm.

  The quantification of the test substance in the subject can be calculated by preparing a calibration curve in advance using a test substance of known concentration, and can typically be calculated according to the following procedure. First, after contacting a fluorescent analyte sensor with a fluorescent competitor under the absence of a test substance to generate FRET, the fluorescence maximum wavelength of these donor channels and / or acceptor channels or in the vicinity thereof The fluorescence intensity in the wavelength range is measured. Then, a test substance of known concentration is added to this system, and the fluorescence intensity is measured in the state where competition of binding to the fluorescent test substance sensor occurs between the fluorescent competitor and the test substance. The concentration of the test substance is changed to perform the same measurement, and a calibration curve is created by plotting the ratio of the fluorescence intensity of the donor channel to the acceptor channel and the test substance concentration. Thereafter, the above-mentioned target contact step (I) and fluorescence irradiation step (II) are performed using the target, and the mass of the target substance in the target is calculated from the obtained fluorescence intensity ratio.

  One of the preferred embodiments of the measurement method of the present invention is a FRET molecular sensor comprising a fluorescent test substance sensor and a fluorescent competitor substance immobilized on a carrier on a suitable support such as a glass plate for a fluorescence microscope, and a subject. And irradiating the excitation light using a fluorescence microscope to measure the generated fluorescence. Another preferred embodiment of the measurement method of the present invention comprises a fluorescent test substance sensor and a fluorescent competitive substance immobilized on a carrier in a microchannel made of a material capable of measuring fluorescence or a microchip on which the microchannel is mounted. And a step of contacting the target with the FRET molecular sensor, and a step of measuring the fluorescence generated by irradiating the excitation light. In either case, detection sensitivity can be enhanced by condensing the reaction field where FRET occurs on the surface of the carrier and measuring the fluorescence generated by FRET through a short light path.

  Hereinafter, a test substance measurement FRET molecular sensor according to the present invention will be described based on examples. The technical scope of the present invention is not limited to the features shown by these embodiments.

EXAMPLE 1 Preparation of Fluorescent Competitor Binding to Ligand Binding Part of IP ₃ Receptor Binding to Ligand Binding Part of Inositol 1,4,5 Triphosphate (IP ₃ ) Receptor as a Test Substance Binding Part To create a fluorescent competitor.
(1) Preparation of F-ADA 9- [5-Deoxy-5- [N-[[1- [5-[[3 ', 6'-dihydroxy-3-oxospiro (isobenzofuran-1,9'-xanthen)] -5-yl] amino] thioxomethyl] amino] propyl] -1H-1,2,3-triazol-4-yl]-(3,4-di-O-phosphoryl-α-D-glucocopysyl) -2-O -Phosphoryl- [beta] -D-ribo- pentofuranosyl] adenine (F-ADA) was made according to the following procedure.

[1-1] 9- [5-Deoxy-5-ethynyl- (3,4-di-O-phosphoryl-α-D-glucocopyranosyl) -2-O-phosphoryl-β-D-ribo-pentofuranosyl] adenine Preparation First, a precursor of 5'-modified adenophostin derivative synthesis (compound 1, α-disaccharide triflate; T. Mochizuki et al., Org. Lett. 8: 1455-1458, 2006) was used as a starting material 9- [5- Deoxy-5-ethylyl- (3,4-di-O-phosphoryl-α-D-glucopyranyl) -2-O-phosphoryl-β-D-ribo-pentofuranosyl] ad An enine (compound 7) was created.

  Dissolve diisopropylamine (1.88 mL, 13.4 mmol) and HMPA (2.33 mL, 13.4 mmol) in THF (100 mL) and add 1.66 M n-butyllithium in hexane (5.03 mL, at -78.degree. C.). 13.4 mmol) was added. The mixture was warmed to 0 ° C. and stirred for 30 minutes. Trimethylsilylacetylene (1.85 mL, 13.4 mmol) was added again at −78 ° C., and then THF (50 mL) of compound 1 (2.98 g, 3.83 mmol) was added. The solution was added and stirred for another hour. Subsequently, a saturated aqueous solution of ammonium chloride was added and diluted with ethyl acetate, and then separated with a saturated aqueous solution of ammonium chloride and then separated with water. The organic layer was washed with brine and dried over anhydrous sodium sulfate. After filtration, the filtrate was evaporated under reduced pressure, and the residue was dissolved in methanol (150 mL), potassium carbonate (632 mg, 4.60 mmol) was added, and the mixture was stirred for 3 hours. The solvent of the reaction solution was concentrated under reduced pressure, diluted with diethyl ether, and separated from water. The organic layer was washed with saturated brine and dried over anhydrous sodium sulfate. After filtration, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (hexane: AcOEt = 4: 1) to give colorless amorphous solid compound 2 (2.35 g, 3.58 mmol, 94%).

  Subsequently, after 80% aqueous trifluoroacetic acid (50 mL) of Compound 2 (1.00 g, 1.53 mmol) was stirred for 30 minutes, the solvent of the reaction solution was distilled off under reduced pressure, and the residue was purified using toluene. It azeotroped. The residue is dissolved in acetonitrile (33 mL) and dimethylaminopyridine (2.24 g, 18.3 mmol), triethylamine (2.56 mL, 18.3 mmol) and isobutyric anhydride (2.03 mL, 12.2 mmol) are added 2 Stir for hours. Saturated aqueous sodium bicarbonate solution and ethyl acetate were added to the reaction solution to separate the layers, and then the organic layer was washed with water and then with saturated brine. The organic layer was dried over anhydrous sodium sulfate and filtered, and then the solvent of the filtrate was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (hexane: AcOEt = 4: 1) to give compound 3 (1.08 g, 1.38 mmol, 2 steps 90%) as a colorless amorphous solid.

Subsequently, N ⁶ -benzoyladenine (1.44 g, 6.39 mmol) was suspended in a mixed solution (50 mL) of HMDS / pyridine (2/1) and refluxed while heating until the reaction solution became transparent. After distilling off the solvent of the reaction solution, the reaction solution was azeotroped with toluene, and an acetonitrile solution (20 mL) of compound 3 (1.02 g, 1.28 mmol) was added to the residue. After tin tetrachloride (1.81 mL, 7.68 mmol) was added dropwise to the resulting mixed solution under ice-cooling, the mixture was stirred at the same temperature for 1 hour and further at room temperature for 2 hours. Saturated aqueous sodium bicarbonate solution and ethyl acetate were added to the reaction solution to separate the layers, and then the organic layer was washed with water and then with saturated brine. The organic layer was dried using anhydrous sodium sulfate and filtered, and then the filtrate was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane: AcOEt = 4: 1) to give a white amorphous Solid compound 4 (906 mg, 0.971 mmol, 76%) was obtained.

Subsequently, 28% sodium methoxide (21.0 μL, 0.107 mmol) was added to a methanol (2 mL) solution of compound 4 (100 mg, 0.107 mmol), and the mixture was stirred at room temperature for 24 hours. The reaction solution was purified using a WK212 (H ⁺ ) column to obtain Compound 5 (64.9 mg, 0.113 mmol, quant.) As a white amorphous solid.

  Subsequently, imidazolium triflate (58.5 mg, 0.266 mmol) and dibenzyldiisopropylphosphoamidite (80.2 μL, 0.365 mmol) were added to a solution of compound 5 (50.2 mg, 0.0811 mmol) in tetrahydrofuran (4 mL). In addition, the mixture was stirred at room temperature for 24 hours. After a small amount of water was added, mCPBA (62.9 mg, 0.365 mmol) was added at -78 ° C and stirred at room temperature for 30 minutes. Saturated aqueous sodium thiosulfate solution and chloroform were added thereto for liquid separation, and the organic layer was washed with aqueous sodium bicarbonate solution and then with water, and further washed with brine. After drying over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (chloroform: methanol = 99: 1) to give compound 6 (104 mg, 0.0745 mmol) as a white amorphous solid. , 92%).

  Subsequently, Compound 6 (64.2 mg, 0.0458 mmol) was azeotroped three times with toluene, then dissolved in dichloromethane (4 mL), and 1M tribromoborane dichloromethane solution (424 μL, 0.4 mL) at −78 ° C. 424 mmol) was added. After stirring at the same temperature for 10 hours and adding 28% aqueous ammonia, the solvent was distilled off under reduced pressure. The residue was purified by AG resin column (water → 150 mM aqueous TFA) to give compound 7 (25 mg, 0.0369 mmol, 81%) as white isomorphic crystals. The preparation flow of the compounds 2-7 which used the compound 1 as a starting material is shown in FIG. 1, and the NMR spectrum data of the compound 7 are shown below.

NMR spectral data of compound 7 m. p. 158-159 ° C .; ¹ H-NMR (D ₂ O 400 MHz) δ 8.47, 7.38 (each s, each 1 H, H-2, H-8), 6.33 (d, 1 H, H-1 ′ , J = 4.98 Hz), 5.39 (dd, 1H, H-2 ′, J = 4.98 Hz, 4.53 Hz), 5.24 (d, 1H, H−1 ″, J = 3. 62 Hz), 4.70 (t, 1 H, H-4 ', J = 4. 98 Hz), 4.5 1 (dd, 1 H, H-3', J = 9.06 Hz), 4.44 (m, 1 H) , H-3 ''), 4.13 (t, 1 H, H-4 '', J = 9.51 Hz), 3.84-3.73 (m, 4 H, H-6 '' a, 6 ''b,2'',5''), 2.78 (m, 2 H, H-5' a, 5 'b), 2.41 (s, 1 H, alkyne); ¹³ C NMR (D ₂ O 100 MHz) δ 150.49, 148.96 , 145.02, 143.86, 119.44, 98.33, 88.25 (J = 2.40 Hz), 88.02, 80.24, 78.63 (J = 8.40 Hz), 75.80 (J = 3.60 Hz), 75.36, 73.48, 72.87, 72.00, 70.94, 60.65, 45. 25; ³¹ P NMR (D ₂ O 162 MHz) δ 0.81, 0 ESI-LRMS m / z 676 (M + H) ⁺ ; ESI-HRMS calcd for C ₁₈ H ₂₅ N ₅ O ₁₇ P ₃ 676.01176, found 676.01126 (M + H) ⁺ , HPLC purity: RT 7.982, 98.11%

[1-2] N- (3-Azidopropyl) -N- (3 ′, 6′-hydroxy-3-oxospiro [isobenzofuran-1 (3H), 9 ′-[9H] xanthen] -5-yl) -thiourea Next, N- (3-azidopropyl) -N- (3 ', 6'-hydroxy-3-oxospiro [isobenzofuran-1 (3H), 9'-[9H] xanthene from fluorocein isothiocyanate (FITC) ] -5-yl) -thiourea (compound 12).

  Dissolve FITC (20.2 mg, 0.0514 mmol) in saturated aqueous sodium bicarbonate (2 mL), add 3-azidopropylamine (30.8 mg, 0.308 mmol), stir for 3 hours, and remove the solvent under reduced pressure. After evaporation, the residue was purified by C18 reverse phase chromatography (elution solvent; acetonitrile: water = 1: 3, 1: 2, 1: 1) to give compound 12 (24.2 mg, 0.0491 mmol) of red shaped crystals. 96%). The flow of preparation of compound 12 from FITC is shown in FIG. 2 and NMR spectral data is shown below.

NMR spectral data of compound 12 m. p. 185-186 ° C .; ¹ H-NMR (CD ₃ OD, 400 MHz) δ 7.98 (d, 1 H, H-12, J = 1.81 Hz), 7.69 (dd, 1 H, H-10, J = 1 6. 81 Hz, 6.34 Hz), 7.19 (d, 1 H, H-9, J = 8. 16 Hz), 7. 10 (d, 2 H, H-4, 5, J = 9. 06 Hz), 6. 62-6.59 (m, H-1, 2, 7, 8, 4 H), 3.71 (dd, 2 H, J = 6.34 Hz, 6.80 Hz), 3.42 (dd, 2 H, J = 6.80 Hz, 6.34 Hz), 1.92 (m, 2 H); ¹³ C-NMR (CD ₃ OD 125 MHz) δ 182.26, 179.59, 173.67, 159.58, 158.47, 141. 54, 141.19, 132.56, 131.21, 130.83, 125.49, 12 ESI-LRMS m / z 488 (M + H) ⁺ ; ESI-HRMS calcd for 488 5.08, 122. 96, 114. 39, 104. 18, 43. 10341 C ₂₄ H ₁₈ O ₅ N ₅ S, found 488.10429 (M + H) ⁺ , HPLC purity: RT 15.127, 99.053%

[1-3] Preparation of F-ADA Next, compound 7 prepared in Example 1 (1) [1-1] and compound 12 prepared in Example 1 (1) [1-2] ADA (compound 13) was made.

  Compound 12 (7.07 mg, 0.0145 mmol) and copper sulfate (1.65 mg) were added to a solution of compound 7 (7.00 mg, 0.0103 mmol) in a sodium phosphate buffer solution (0.2 M, pH = 7.0, 700 μL). , 0.0103 mmol) and sodium ascorbate (8.19 mg, 0.0412 mmol), and after stirring for 5 hours at room temperature, the reaction solution is subjected to C18 reverse phase column chromatography (elution solvent: acetonitrile: water = 1: 3, Purification by 1: 2, 1: 1) gave F-ADA (compound 13; 10.1 mg, 0.00850 mmol, 83%) as yellow-shaped crystals. The structure of F-ADA is shown in the following formula (I), the production flow of F-ADA (compound 13) from compound 7 and compound 12 is shown in FIG. 3, and the NMR spectrum data is shown below.

NMR spectral data of F-ADA (compound 13) m. p. 294-295 ° C. (dec.); ¹ H-NMR (CDCl ₃ 400 MHz) δ 7.94, 7.89 (each s, each 1H, H-2,8), 7.48 (s, 1H, triazole), 7.35 (s, 1H, fluorescein-H-12), 7.27 (s, 1H, fluorescein-H-10), 7.12 (m, 3H, fluorescein-H-9, 4, 5), 6 .45 (m, 4H, fluorescein-H-1, 2, 7, 8), 6.19 (m, 1H, H-1 ′), 5.15 (m, 2H, H-1 ″, 2 ′ ), 4.49 (m, 1H, H-3 ″), 4.40 (m, 1H, H-3 ′), 4.19 (m, 3H, H-4 ″, triazole-CH ₂ ) , 3.93 (m, 4H, H-4 ', 6''a,6''B,2''), 3.87 (m, H, H-5 '', 3.35,2H, H-5'a, H-5'b), 3.05 (m, CH 2 NH), 1.90 (m, CH 2 CH 2 CH 2 ); ³¹ P-NMR (CDCl ₃ 202 MHz) δ 5.23, 4.67, 4.42; ESI-LRMS m / z 1165 (M + H) ⁺ ; ESI-HRMS calcd for C ₄₂ H ₄₅ O ₂₂ N ₁₀ 165. 15652, found 1165.15385 (M + H) ⁺ , HPLC purity: RT 11.480, 92. 150%

(2) Creation of F-LL (2R, 3R, 4S) -4-O-phosphoryl-2- [N-[[1- [5-[[3 ′, 6′-dihydroxy-3-oxospiro (isobenzofuran- 1,9′-xanthhen) -5-yl] amino] thioxomethyl] amino] propyl] -1H-1,2,3-triazol-4-yl] methyl-tetrahydrofuran-3-yl-3,4-di-O -Phosphoryl-α-D-glucocopyranoside (F-LL) was made according to the following procedure.

Preparation of [2-1] (2R, 3R, 4S) -4-O-phosphoryl-2-ethynylmethyl-tetrahydrofuran-3-yl-3,4-di-O-phosphoryl-α-D-glucopyranoside First, this practice Example 1 (1) Compound 2 (5-Deoxy-5-ethyl-3-O- [2,6-di-O-benzyl-3,4-O- (2) which is a reaction intermediate in [1-1] (2-R, 3R, 4S) -4-O- using, as a starting material, phosphoryl-2-ethynylmethyl-te rahydrofuran-3-yl-3,4-di-O-phosphoryl-α-D-glucopyranoside (Compound 11) was created.

  Compound 2 (50.0 mg, 0.0761 mmol) was dissolved in 90% aqueous trifluoroacetic acid solution (5 mL) and stirred at room temperature for 5 minutes. The reaction mixture was then concentrated under reduced pressure, and the residue was diluted with water (x3) and ethanol. Azeotropically. The residue was dissolved in pyridine (2 mL), acetic anhydride (422 μL, 4.51 mmol) was added, and after stirring for 12 hours at room temperature, the solvent was evaporated under reduced pressure. The residue was partitioned between ethyl acetate and saturated aqueous sodium bicarbonate solution (10 mL × 2), and the organic layer was washed with water (10 mL) and saturated brine (10 mL). After drying over anhydrous sodium sulfate and filtration, the filtrate is concentrated under reduced pressure, and the residue is purified by silica gel chromatography (hexane: AcOEt = 8: 1) to give compound 8 (44 of a white non-crystalline solid) .1 mg, 0.0674 mmol, 2 steps 87%) were obtained.

Subsequently, TMSOTf (10.2 μL, 56.6 μmol) was dropped to a dichloromethane solution (500 μL) of compound 8 (20.0 mg, 28.2 μmol) and triethylsilane (13.0 μL, 56.3 μmol) at 0 ° C. After stirring for 20 minutes at the same temperature, the mixture was warmed to room temperature and stirred for 30 minutes. Saturated aqueous sodium bicarbonate solution and chloroform were added for separation, and then the organic layer was washed with water (4 mL) and then with saturated brine (4 mL). After drying over anhydrous sodium sulfate and filtration, the filtrate was distilled off under reduced pressure to obtain a white solid. The solid was dissolved in MeOH (1 mL) and NaOMe (28% in MeOH, 30 μL) was added and then stirred at room temperature for 12 hours. Purification on a WK 212 (H ⁺ ) column gave compound 9 (8 mg, 0.0171 mmol, quant.) As a white non-crystalline solid.

  Subsequently, according to the procedure for preparing Compound 6, which is a reaction intermediate in Example 1 (1) [1-1], Compound 9 (22.2 mg, 40.2 μmol), 1H-tetrazol (25.3 mg, 360 μmol) ), Dibenzyl diethylphosphoamidite (58.4 mg, 240 μmol) and mCPBA (41.1 mg, 240 μmol) were used to obtain white amorphous solid compound 10 (47.3 mg, 37.5 μmol, 92%).

  Subsequently, Compound 10 (36.0 mg, 0.0281 mmol) was azeotroped with toluene (x3), then dissolved in dichloromethane (2 mL), and 1M tribromoborane dichloromethane solution (450 μL, 0.451 mmol) at −78 ° C. ) Was added and stirred for 7 hours under the same temperature. After adding a small amount of ethanol, the solvent was distilled off under reduced pressure. The residue was purified by C18 reverse phase column using water as an eluting solvent to obtain Compound 11 (13.1 mg, 0.0241 mmol, 86%) of white isomorphic crystals. The preparation flow of compounds 8 to 11 starting from compound 2 is shown in FIG. 4, and NMR spectrum data of compound 11 is shown below.

NMR spectrum data of compound 11 m. p. 143-144 ° C .; ¹ H-NMR (D _20, 500 MHz) δ 5.08 (d, 1 H, H-1 ′, J = 3.43 Hz), 4.76 (m, 1 H, H-2), 4 .36 (dd, 1 H, H-3 ', J = 8.59 Hz, 9.16 Hz), 4.21 (t, 1 H, H-3, J = 5. 73 Hz), 4.05-3.91 ( m, 4H, H-6'a, 6'b, 1a, 1b), 3.71-3. 63 (m, 4H, H-4, 4 ', 5', 2 '), 2.60-2. .46 (m, 2H, H-5a, 5b), 2.30 (s, 1 H, alkyne); ¹³ C-NMR (D ₂ O, 125 MHz) δ 97.59, 80.65, 57.49, 77. 44 (J = 6.00 Hz), 73.38, 72.59, 72.30, 71.96, 71.93 (J = 4.80), 71.05, 70.99 (J = 2. 40), 60.63, 22.29; ³¹ P-NMR (D ₂ O, 202 MHz) δ 0.70, 0.48, 0.13; ESI-LRM m / z 543 (M + H) ⁺ ; ESI-HRMS calcd for C ₁₃ H ₂₂ O ₁₇ P ₃ 543.00753, found 543.00846 (M + H) ⁺

[2-2] Preparation of F-LL Next, F from compound 11 prepared in Example 1 (1) Example 1 (2) [2-1] and compound 12 prepared in [1-2] -LL (compound 14) was made.

Under an argon atmosphere, compound 11 (13.5 mg, 0.0242 mmol), compound 12 (16.0 mg, 0.0331 mmol), dimethyl sulfoxide (0.8 mL) and sodium phosphate buffer (0.2 M, pH = 7. After dissolving in a mixed solution of 0, 0.2 mL), an aqueous solution (500 μL) of copper sulfate (19.2 mg, 0.121 mmol) and sodium ascorbate (95.1 mg, 0.482 mmol) was added and stirred at room temperature for 5 hours. did. The reaction solution is purified by C18 reverse phase column chromatography [eluent: acetonitrile / triethylammonium acetate buffer (5 mM, pH 7.0)], and passed through a KILEX 100 column (K ⁺ form, elution solvent: water) to obtain a yellow definite shape. Crystalline F-LL (compound 14; 5.02 mg, 0.00491 mmol, 20% isolated yield) was obtained. The structure of F-LL is shown in the following formula (II), the production flow of F-LL (compound 14) from compound 11 and compound 12 is shown in FIG. 3, and NMR spectrum data is shown below.

NMR spectral data of F-LL (compound 14) m. p. 298-299 ° C. (dec.); ¹ H-NMR (D ₂ O, 500 MHz) δ 7.80 (m, 1H, fluorescein-H-12), 7.52 (s, 1H, triazole), 7.42 ( m, 1 H, fluorescein-H-10), 7.12 (m, 3 H, fluorescein-H-9, 4, 5), 6, 47 (m, 4 H, fluorescein-H-1, 2, 7, 8) , 5.07 (m, 1H, H -1 '), 4.53 (m, 1H, H-2), 4.38 (m, 3H, H-4, triazole-CH 2), 4.19 ( m, 1 H, H-5 '), 4.09 (m, 1 H, H-3'), 3.92 (1 H, H-3), 3. 80 (m, 3 H, H-1 a, 1 b, 4) '), 3.70 (m, 1H, H-4), 3.62 (m, 1H, H-2'), 3.36 (M, 2H, H-5a , 5b), 3.25 (m, 2H, H-6'a, 6'b), 3.10 (m, 2H, CH 2 N), 2.01 (m, 2H, CH ₂ CH ₂ CH ₂ ) ³¹ P-NMR (D ₂ O, 202 MHz) δ 5.15, 4.51, 4.32; ESI-LRMS m / z 1032 (M + H) ⁺ ; ESI-HRMS calcd for C ₃₇ H ₄₁ O ₂₂ N ₅ P ₃ 1032.11767, found 1032.11795 (M + H) ⁺ , HPLC purity: RT 10.680, 90.679%

(3) Test of Ca ²⁺ Release from Intracellular Ca ²⁺ Storage Site The F-ADA obtained in the present Example 1 (1) and the F-LL obtained in the same (2) are the ligand binding portions of the IP ₃ receptor In order to determine whether it could be a fluorescent competitor that binds to H, the first step was to determine whether Ca ²⁺ is released from the intracellular Ca ²⁺ storage site (Ca ²⁺ store). IP ₃ is an intracellular messenger that induces Ca ²⁺ signal, and when IP ₃ binds to the ligand binding part of IP ₃ receptor which is a Ca ²⁺ channel, IP ₃ receptor is activated and intracellular Ca such as endoplasmic reticulum Ca ²⁺ is released into the cytoplasm from the ²⁺ storage site (Ca ²⁺ store). A confirmation test using this principle was conducted.

[3-1] Preparation of saponin-perforated COS-7 cells into which mag-fura-2 has been incorporated into cells COS-7 cells (RIKEN gene bank) are cultured in an experimental chamber having a volume of about 100 μL. A cell containing 100 μg / mL saponin by incorporating Mag-fura-2 into the cell organelle by allowing a Hanks-Hepes solution containing 10 μM mag-fura-2 to act in the experimental chamber for 30 minutes. The cells in the chamber are perforated with saponin to obtain saponin-perforated cells by exposing the cells to an internal solution-like buffer (ICM; 124 mM KCl, 25 mM NaCl, 1 mM EGTA, 330 μM CaCl ₂ , pH 7.3) for about 3 minutes. after, I a chamber containing 1 mM ATP and 1 mM MgCl ₂ It was replaced with M.

[3-2] Analysis of Ca ²⁺ concentration change in endoplasmic reticulum Ca ²⁺ store by fluorescence imaging method IP of various concentration to saponin perforated cells obtained in Example 1 (3) [3-1] _3. F-ADA and F-LL were allowed to act, and the change in Ca ²⁺ concentration in the endoplasmic reticulum Ca ²⁺ store was analyzed by fluorescence imaging. The fluorescence imaging method is an imaging system (Hamamatsu Photonics) comprising a fluorescence inverted microscope TE2000 (Nikon Instech), EM-CCD camera C9100-13, high-speed wavelength switching device U7773, W-View optical system, and image analysis software AQUACOSMOS. ). Specifically, saponin-perforated cells treated with various concentrations of IP ₃ , F-ADA and F-LL are set in a fluorescent inverted microscope TE2000 (Nicon Instec) and irradiated with light at 345 nm or 380 nm, The fluorescence of 535 ± 12.5 was extracted using a fluorescence filter. The resulting fluorescence was recorded simultaneously with EM-CCD camera, it calculates the fluorescence ratio using an image analysis software, from the change in the fluorescence ratio, by analyzing the Ca ²⁺ concentration change in intracellular stores, IP ₃ The potency of Ca ²⁺ release via IP ₃ receptors of F-ADA and F-LL was calculated. The result is shown in FIG.

As shown in FIG. _5, IP 3, none of the F-ADA and F-LL, concentration dependent fluorescence changes due to ^{Ca 2+} release was confirmed, the EC50 value for 334nM of IP _3, F-ADA Was 2.4 nM and F-LL was 158 nM. From this, it was clarified that Ca ²⁺ can be released from intracellular Ca ²⁺ stores via IP ₃ receptors as well as IP ₃ by F-ADA and F-LL.

(4) Analysis of Specificity of Fluorescent Competitor and Fluorescent IP ₃ Sensor Next, the specificity of F-ADA for fluorescent IP ₃ sensor LIBRAvIIS expressing cells was analyzed according to the following procedure.

[4-1] Preparation of Fluorescent IP ₃ Sensor Expressing Cells The LIBRAvIIS (LvIIS) expression plasmid described in Tanimura et al., J. Biol. Chem., 284: 8910-8917, 2009, was made intracellular It was introduced to prepare a conventional fluorescent IP ₃ sensor LIBRAvIIS (LvIIS) was expressing cells. Specifically, COS-7 cells (RIKEN gene bank) are cultured in an experimental chamber having a volume of about 100 μL, and the LIBRAvIIS (LvIIS) gene (LIVIIS) is used in cells using Lipofectamine 2000 (Life Technologies Corporation). By introducing SEQ ID NO: 19), cells expressing the fluorescent IP ₃ sensor LIBRAvIIS (LvIIS) were obtained. FIG. 6 shows the configuration of the fluorescent IP ₃ sensor LIBRAvIIS (LvIIS). In FIG. 6, “P” indicates a cell membrane localization signal, “ECFP” indicates a blue fluorescent protein ECFP, “L1” and “L2” indicate a linker, and “IP ₃ R2 (1-604) with R440Q” is a peptide consisting of the amino acid mutation R440Q peptide containing the, namely 1-604 number of the amino acid sequence from the N-terminus of the rat type 2 IP ₃ receptors increase the affinity to the ligand binding portion of the rat type 2 IP ₃ receptors A peptide in which arginine (R) at position 440 of the sequence is mutated to glutamine (Q), "Venus" is a mutant of yellow fluorescent protein EYFP and phenylalanine (F) at position 46 of EYFP is leucine (L) ), Phenylalanine (F) at position 64 to leucine (L) and methionine at position 153 (M) to threonine T), a yellow fluorescent protein Venus (T. Nagai et al., Nat. 2) mutated to No. 163 valine (V) to alanine (A) and No. 175 serine (S) to glycine (G), respectively. Biotechnol., 20 (1): 87-90, 2002).

[4-2] Other except that incorporation of mag-fura-2 is the fluorescent IP ₃ sensors expressing saponin perforations cells within organelles creation cells, the first embodiment (3) and [3-1] Based on the same method, fluorescent IP _3- sensor LIBRAVIIS (LvIIS) -expressing saponin-perforated cells were obtained from cells in which the fluorescent IP _3- sensor LIBRAvIIS (LvIIS) obtained in (4) [4-1] was expressed.

[4-3] Analysis by Fluorescence Imaging Method The obtained fluorescence IP _3- sensor LIBRAvIIS (LvIIS) -expressing saponin-perforated cells are reacted with F-ADA to obtain an imaging system (Hamamatsu, Example 1 (3) [3-2]. Fluorescence resonance energy transfer (FRET) was analyzed by fluorescence imaging using Photonics. Specifically, after causing F-ADA to act on the fluorescent IP _3- sensor LIBRAvIIS (LvIIS) -expressing saponin-perforated cells, the sample is set in a fluorescent inverted microscope TE2000 (Nicon Instec Co., Ltd.) and irradiated with 435 nm light. Were separated into two light paths using a W-View optical system 485 nm dichroic mirror, and then fluorescence of 480 ± 15 nm and 535 ± 12.5 nm was extracted using fluorescence filters attached to the respective light paths. Each fluorescence was simultaneously recorded with an EM-CCD camera, and a fluorescence ratio of fluorescence at 480 nm and fluorescence at 535 nm was calculated using image analysis software.

Fluorescence IP ₃ sensor LIBRAvIIS (LvIIS) expressing saponin drilling cells, replacing the ICM containing a fluorescent competitor (F-ADA 30nM), since a slight increase in background fluorescence as well the cells only were observed, by subtracting the background fluorescence from the fluorescence intensity of the cells was analyzed the specificity of the fluorescent competitor F-ADA and fluorescence IP ₃ sensor LIBRAvIIS. The results are shown in FIG.

In FIG. 7, images of Aa and Ab show fluorescence images of time points (time points of arrow 1 of B) before acting with 30 nM F-ADA, and images of Ac and Ad show 30 nM F-ADA. It is a figure which shows the fluorescence image of the time point (point of arrow 1 of B) after making it act, B is a figure which shows the fluorescence change by addition of the fluorescence competition substance F-ADA. In addition, C is a diagram showing a change in fluorescence when the fluorescence of the BG region is subtracted from the fluorescence of the region including the cells for extracting the fluorescence component due to the reaction between the fluorescence competitor F-ADA and the fluorescence IP ₃ sensor LIBRAvIIS. D is a figure which shows the fluorescence ratio 480 nm / 535 nm. As shown in FIG. 7, when the change in fluorescence of the region containing fluorescent IP ₃ sensor expressing cells and the region not containing it (BG) was compared, in the region containing fluorescent IP ₃ sensor expressing cells, the time when F-ADA was added ( Arrow 2) shows that the fluorescence at 480 nm mainly containing CFP fluorescence is reduced, and the fluorescence at 535 nm containing the fluorescence of Venus and the fluorescence competitor F-ADA is increased. In the cell-free BG region, F- It was observed that only the fluorescence at 535 nm of ADA was slightly increased. From these facts, it is clear that the reaction between the fluorescence competitor and the fluorescence IP ₃ sensor lowers the fluorescence ratio (480 nm / 535 nm), and the created fluorescence competitor has the specificity of the fluorescence IP ₃ sensor, That is, it was revealed that CFP and FRET are caused by binding to the ligand binding part of the IP ₃ receptor.

<Example 2> Examination of fluorescent protein in fluorescent IP ₃ sensor The conventional fluorescent IP ₃ sensor has two fluorescent proteins of CFP (ECFP) and YFP (Venus) as fluorescent substances. From the results of 4), if one ligand protein is provided as the ligand binding part of the IP ₃ receptor and the fluorescent substance, it cannot function sufficiently as a fluorescent IP ₃ sensor together with a fluorescent competitor such as F-ADA or F-LL. Since the finding of the availability was obtained, a test was conducted to confirm this.

(1) Preparation of Existing Various Fluorescent IP ₃ Sensor Expressing Cells Based on the same method as in Example 1 (4) [4-1], the previous report (Tanimura et al., J. Biol. Chem., 284: 8910). is a conventional fluorescent IP ₃ sensors listed in -8917,2009) LIBRAvI (LvI) (SEQ ID NO: 20), LIBRAvII (LvII) (SEQ ID NO: 21), LIBRAvIII (LvIII) (SEQ ID NO: 22), LIBRAvN ( LvN) (SEQ ID NO: 23), LIBRAvIIS (LvIIS) (SEQ ID NO: 19) and MP-LIBRAvIIS (MP-LvIIS) (SEQ ID NO: 24) were introduced into the respective cells, and fluorescent IP ₃ sensor LIBRAvI ( LvI), LIBRAvII (LvII), LIBRA III (LvIII), LIBRAvN (LvN), was prepared LIBRAvIIS (LvIIS) and GeneScript Corporation cells MP-LIBRAvIIS you ask synthesized (MP-LvIIS) was expressed in each. The configuration of each fluorescent IP ₃ sensor is shown in FIG. In FIG. 8, “P” indicates a cell membrane localization signal, “MP” indicates an endoplasmic reticulum localization signal, “ECFP” indicates a blue fluorescent protein ECFP, “Cerulean” indicates a blue fluorescent protein Cerulean, “ L1 "," L1H1 "," L2 "and" L2-6 "all indicate linkers, and" IP ₃ R1 (1-604) "is 1 to 604 from the N-terminus of rat type 1 IP ₃ receptor amino acid sequence “IP ₃ R 2 (1-604)” represents a peptide consisting of the amino acid sequence of 1 to 604 from the N terminus of rat type 2 IP ₃ receptor amino acid sequence, “IP ₃ R2 (1-604) with R440Q ”is a peptide consisting of the amino acid sequence 1 to 604 from the N-terminal of rat type 2 IP ₃ receptor, A peptide obtained by mutating arginine (R) of No. 440 to glutamine (Q), wherein “IP ₃ R3 (1-604) with K507A” is Nos. 1 to 604 from the N-terminal of the rat type ₃ IP ₃ receptor amino acid sequence. It is shown that the peptide consisting of the amino acid sequence of SEQ ID NO: 4, and lysine (K) at position 507 of the sequence was mutated to alanine (A), and "Venus" represents the yellow fluorescent protein Venus.

(2) Preparation of fluorescent IP ₃ sensor expression cells in which yellow fluorescent protein does not emit light [2-1] Preparation of fluorescent IP ₃ sensor LIBRAvIISVm (LvIISVm) gene in which yellow fluorescent protein does not emit light Next, fluorescent IP ₃ sensor LIBRAvIIS (LvIIS) A fluorescent IP ₃ sensor LIBRAvIISVm (LvIISVm) in which the Venus of Venus was mutated so as not to cause fluorescence development was prepared according to the following procedure.

First, using the Venus expression plasmid as a template and using the QuikCange II XL site-directed mutagenesis kit (Strategene), and the following PCR primers, Venus mutFP (forward primer) and Venus mutRP (reverse primer), the yellow fluorescent protein Venus The non-chromogenic protein Venus mutant (glycine (G) at position 65 of the Venus sequence to valine (V), tyrosine (Y) at position 66 to asparagine (N), glycine at position 67 G) was mutated to arginine (R) to prepare a mutant) expression plasmid.
Venus mutFP: GTG ACC ACC CTG GTC AAC CGC CTG CAG TG TTC (SEQ ID NO: 25)
Venus mutRP: GAA GCA CTG CAG GCG GTT GAC CAG GGT GGT CAC (SEQ ID NO: 26)

Next, the yellow fluorescent protein Venus gene of the fluorescent IP ₃ sensor LIBRAvIIS (LvIIS) gene and the created non-fluorescent chromogenic protein Venus mutant gene were replaced. Specifically, a LIBRAvIISVm (LvIISVm) gene (SEQ ID NO: 27) was prepared by cutting a LIBRAvIIS (LvIIS) expression plasmid with restriction enzymes EcoRI and NotI and inserting the Venus mutant expression plasmid into the LIBRAvIIS (LvIIS) expression plasmid. . FIG. 9 shows the procedure for preparing the LIBRAvIISVm (LvIISVm) gene. In FIG. 9, “→” represents a part of the vector gene, “MT” represents the membrane-bound gene sequence, “ECFP” represents the gene sequence of the blue fluorescent protein ECFP, and “LBD” represents the IP ₃ receptor. The gene sequence of a ligand binding part is shown, "Venus" shows the gene sequence of yellow fluorescent protein Venus, and "Venus (m)" shows the gene sequence of the non-chromogenic protein Venus mutant.

[2-2] Creation of fluorescent IP ₃ sensor LIBRAvIISVm (LvIISVm) -expressing cells Next, a LIBRAvIISVm (LvIISVm) -expressing plasmid was introduced into cells based on the same method as in Example 1 (4) [4-1]. Thus, cells expressing the fluorescent IP ₃ sensor LIBRAvIISVm (LvIISVm) were prepared. The configuration of the fluorescent IP ₃ sensor LIBRAvIISVm (LvIISVm) is shown in FIG. In FIG. 10, “P” indicates a cell membrane localization signal, “ECFP” indicates a blue fluorescent protein ECFP, “L1” and “L2” indicate linkers, and “IP ₃ R2 (1-604) with R440Q”. Is a peptide consisting of the amino acid sequence 1 to 604 from the N-terminal of the rat type 2 IP ₃ receptor, in which arginine (R) at position 440 of the sequence is mutated to glutamine (Q), and “Venus” “mutant” indicates the chromogenic protein Venus mutant.

(3) Preparation of Fluorescent IP ₃ Sensor Expressing Saponin Perforated Cells and Analysis by Fluorescent Imaging Method Subsequently, based on the same method as in Example 1 (4) [4-2], Example 2 (1) and (2) Each of the fluorescent IP ₃ sensors LIBRAvI (LvI), LIBRAvII (LvII), LIBRAvIII (LvIII), LIBRAvN (LvN), LIBRAvIIS (LvIIS) and MP-LIBRAvIIS (MP-LvIIS) expressed in 2) from respective cells Fluorescent IP ₃ sensor expressing saponin-perforated cells were obtained and treated with F-ADA so that the final concentration was 10 nM or 30 nM, based on the same procedure as in Example 1 (4) [4-3]. , fluorescence of 480nm in each of the fluorescent IP ₃ sensors expressing saponin drilling cells and 5 It was calculated fluorescence fluorescence ratio of 5nm. The results are shown in FIG.

As shown in FIG. 11, in the reaction of fluorescent IP ₃ sensors LIBRAvI (LvI), LIBRAvII (LvII) and LIBRAvIII (LvIII) containing a ligand binding part of type 1 to type 3 IP ₃ receptor with F-ADA, In addition, the fluorescence at 480 nm decreased and the fluorescence at 535 nm increased, and as a result, the fluorescence ratio at 480 nm / 535 nm decreased. A similar decrease in fluorescence ratio was also observed in LIBRAvIIS (LvIIS), which contains an amino acid mutation (R440Q) that increases the affinity to the ligand binding site of the type 2 IP ₃ receptor. Further, the same applies to MP-LIBRAvIIS (MP-LvIIS) in which the cell membrane localization sequence of LIBRAvIIS (LvIIS) is replaced with an endoplasmic reticulum localization sequence and LIBRAvIISVm (LvIISVm) using Venus mutant that does not emit fluorescence due to amino acid substitution of a fluorescent protein. A change in fluorescence ratio was observed. On the other hand, mutation of one of the essential amino acids for IP ₃ binds (K507A) is LIBRAvN (LvN) in fluorescence changes were did not occur at all in a ligand binding portion of the type 3 IP ₃ receptor.

From these facts, it has become clear that these fluorescence changes are caused by the specific reaction of the fluorescent IP ₃ sensor and the fluorescent competitor. Previous studies by the inventors have shown that the binding of LIBRAv I (Lv I), LIBRAv II (LvII), LIBRAvIIS (LvIIS) and LIBRAv III (LvIII) to IP ₃ increases the fluorescence ratio at 480 nm / 535 nm (Tanimura et al., J. Biol. Chem., 284: 8910-8917, 2009). It, these fluorescent changes, conformation of the ligand binding portion of the IP ₃ receptor is changed by the binding of IP _3, CFP linked to the N-terminus and C-terminus of the ligand binding portion of the IP ₃ receptor ( We believe that this is due to the decrease in the efficiency of FRET between ECFP) and YFP (Venus). On the other hand, in the reaction of the fluorescent IP ₃ sensor with the fluorescent competitor in Example 2, the fluorescence ratio was lowered, so it became clear that a completely different principle was working. That is, even if LIBRAvIISVm (LvIISVm) was used in which Venus, which is the FRET acceptor of LIBRAvIIS (LvIIS), was replaced with a non-fluorescent, non-colored Venusant, a large fluorescence change was observed by reaction with the fluorescence competitor F-ADA. Thus, it was shown that the reaction of the fluorescent IP ₃ sensor with the fluorescent competitor does not require the fluorescence of YFP.

<Example 3> Examination of fluorescent IP ₃ sensor provided with one fluorescent protein Next, a fluorescent IP ₃ sensor provided with one fluorescent protein was examined according to the following procedure.

(1) One creation fluorescent IP ₃ sensor with a fluorescent protein fluorescence IP ₃ sensor LIBRAvIIS (LvIIS) fluorescence IP ₃ sensors LIBRAvIISVd removing the Venus gene from the gene (LvIISVd) created the expression vector. Specifically, ECFP and rat type 2 IP purified from LIBRAvIIS (LvIIS) expression plasmid were digested with LIBRAvIIS (LvIIS) expression plasmid and ECFP-C1 expression plasmid (Clontech; SEQ ID NO: 28) with restriction enzymes NheI and EcoRI. _A LIBRAvIISVd (LvIISVd) gene (SEQ ID NO: 29) was prepared by inserting a DNA fragment containing the ₃ receptor ligand binding site gene into the NheI / EcoRI cleavage site of the ECFP-C1 expression plasmid. FIG. 12 shows the procedure for preparing the LIBRAvIISVd (LvIISVd) gene. In FIG. 12, “→” represents a part of the vector gene, “MT” represents the membrane-bound gene sequence, “ECFP” represents the gene sequence of the blue fluorescent protein ECFP, and “LBD” represents the IP ₃ receptor. The gene sequence of the ligand binding part is shown, and “Venus” shows the gene sequence of the yellow fluorescent protein Venus.

(2) Preparation of Fluorescent IP _3- Sensor LIBRAvIISVd (LvIISVd) Expressing Cells Next, LIBRAvIISVd (LvIISVd) expression plasmid is introduced into cells based on the same method as in Example 1 (4) [4-1]. to prepare a cell fluorescence IP ₃ sensor LIBRAvIISVd (LvIISVd) was expressed. The configuration of the fluorescent IP ₃ sensor LIBRAvIISVd (LvIISVd) is shown in FIG. In FIG. 13, “P” indicates a cell membrane localization signal, “ECFP” indicates a blue fluorescent protein ECFP, “L1” indicates a linker, and “IP ₃ R2 (1-604) with R440Q” indicates rat type 2. A peptide consisting of the amino acid sequence 1 to 604 from the N-terminal of the IP ₃ receptor, wherein arginine (R) at position 440 of the sequence is mutated to glutamine (Q).

(3) Following analysis by fluorescence IP ₃ Creating a Sensor expressing saponins drilling cells and fluorescence imaging method, obtained in Example 1 (4) [4-2] on the basis of the same method as the present example 3 (2) Fluorescent IP ₃ sensor-expressing saponin perforated cells are obtained from cells expressing the obtained fluorescent IP ₃ sensor LIBRAvIISVd (LvIISVd), and a fluorescent competitor F-ADA or F-LL is allowed to act so that the final concentration is 30 nM or 100 nM. Otherwise, the fluorescent IP ₃ sensor LIBRAvIISVd (LvIISVd) -expressing saponin perforated cells in which the fluorescent competitor F-ADA or F-LL was acted on the basis of the same method as in Example 1 (4) [4-3] The fluorescence ratio of 480 nm fluorescence and 535 nm fluorescence was calculated. The results are shown in FIG. 14 and FIG.

As shown in FIG. 14, by causing saponin-perforated cells expressing the fluorescent IP ₃ sensor LIBRAvIISVd (LvIISVd) to act with 100 nM F-ADA, 480 nm fluorescence is reduced by about 20% and 535 nm fluorescence is increased by 70% (1 As a result, the 480 nm / 535 nm fluorescence ratio decreased by about 50% (FIG. 14B). Further, as shown in FIG. 15, the same change in the fluorescence ratio was observed with F-LL (FIG. 15A), and the 480 nm / 535 nm fluorescence ratio with 100 nM F-LL decreased by about 20% (FIG. 15B). The change in the fluorescence ratio by the reaction of the fluorescent IP ₃ sensor LIBRAVIIS (LvIIS) with IP ₃ is about 15% (Tanimura et al., J. Biol. Chem., 284: 8910-8917, 2009), the change in fluorescence ratio by reaction with the fluorescent IP ₃ sensor LIBRAvIISVd (LvIISVd) and F-ADA was approximately 150% (1.5-fold) (Figure 14B). From these facts, FRET caused by fluorescent protein (ECFP) and fluorescent competitor having fluorescent binding agent with fluorescent IP ₃ sensor having ligand binding part of one type of fluorescent protein (ECFP) and IP ₃ receptor It has been shown that an IP ₃ measuring FRET molecular sensor can be constructed.

<Example 4> IP fluorescence competition whether consideration fluorescent competitor substance binds to dissociate the ligand-binding portion of the IP ₃ receptors in competition with the analyte IP ₃ is in conflict with the test substance IP ₃ A test for confirming whether or not it binds to or dissociates from the ligand binding part of ₃ receptors was performed according to the following procedure.

Saponin-perforated cells expressing the fluorescent IP ₃ sensor LIBRAvIISVd (LvIISVd) prepared in Example 3 (3) were treated with the fluorescent competitor F-ADA to a final concentration of 30 nM or 100 nM and then 10 μM of IP ₃ Or by applying IP ₃ stepwise so that the final concentration becomes 0.1 μM, 1 μM to 10 μM after the fluorescence competitor F-LL is made to act so that the final concentration becomes 100 nM. Fluorescent IP ₃ sensor LIBRAvIISVd (LvIISVd) in which fluorescent competitors F-ADA and IP ₃ and F-LL and IP ₃ were allowed to act, respectively, based on the same method as in Example 1 (4) [4-3] The fluorescence ratio of fluorescence at 480 nm and fluorescence at 535 nm in the expressed saponin perforated cells was calculated. The results are shown in FIG. 16 and FIG.

As shown in FIG. 16, when a fluorescent competitor F-ADA having an affinity about 150 times that of IP ₃ was allowed to act, F-ADA was bound and the fluorescence ratio of 480 nm / 535 nm decreased. Thereafter, the inside of the experimental chamber was washed, but since the dissociation of F-ADA was extremely slow, the change in the fluorescence ratio of 480 nm / 535 nm was negligible. Subsequently, the addition of IP ₃ promoted the dissociation of F-ADA, and as a result, the fluorescence ratio of 480 nm / 535 nm increased rapidly. On the other hand, as shown in FIG. 17, when a fluorescent competitor F-LL having a low affinity is allowed to act, competition between F-LL and IP ₃ occurs by adding IP _{3 in} the presence of F-LL. , 480 nm / 535 nm fluorescence ratio depending on the concentration of IP ₃ is increased. From these results, it was shown that the fluorescence competitor competes with the test substance IP ₃ and binds to or dissociates from the ligand binding part of the IP ₃ receptor.

<Example 5> Examination of carrier-immobilized IP ₃ measurement FRET molecular sensor In Example 3, a fluorescent IP ₃ sensor having a ligand binding part of one type of fluorescent protein and IP ₃ receptor, and a fluorescent competitor, can be constructed of IP ₃ measurements FRET molecular sensors that utilize FRET fluorescent protein (ECFP) and fluorescent competitor causes showed. Therefore, whether or not a FRET molecular sensor that measures IP ₃ immobilized on a carrier can be constructed by immobilizing the fluorescent IP ₃ sensor on a carrier and combining this with a fluorescent competitor is examined.

(1) Preparation of Carrier Immobilized Fluorescent IP ₃ Sensor [1-1] Preparation of Fluorescent IP ₃ Sensor cyLIBRAvIISVd (cyLvIISVd) Gene First, MP-LIBRAvIIS (MP-LvIIS) (FIG. 8) expression of Example 2 (1) plasmid was digested with restriction enzymes SacI (SEQ ID NO: 24), and recombining after removal of the endoplasmic reticulum localization domain MP, creating a cytoplasmic fluorescent IP ₃ sensor cyLIBRAvIIS (cyLvIIS) expression plasmid (SEQ ID NO: 30) did. Subsequently, the prepared cyLIBRAvIIS (cyLvIIS) expression plasmid was cleaved with restriction enzymes SmaI and HincII, and rejoined to remove the Venus gene, thereby cyLIBRAvIISVd (cyLvIISVd), which is a cytoplasmic fluorescent IP ₃ sensor equipped with one fluorescent protein. ) A gene (SEQ ID NO: 31) was prepared. The procedure for producing the cyLIBRAvIISVd (cyLvIISVd) gene is shown in FIG. In FIG. 18, "ERT" indicates the endoplasmic reticulum binding gene sequence, "HisTag" indicates the gene sequence of a tag peptide consisting of 6 consecutive histidine (His; H) residues, and "Cerulean" indicates a blue fluorescent protein The gene sequence of Cerulean is shown, "LBD" is the gene sequence of the ligand binding part of IP ₃ receptor, "Venus" is the gene sequence of yellow fluorescent protein Venus, and "ECFP" is the gene sequence of blue fluorescent protein ECFP. Indicates.

[1-2] Preparation of bead-immobilized fluorescent IP ₃ sensor cyLIBRAvIISVd (cyLvIISVd) Next, a cyLIBRAvIISVd (cyLvIISVd) expression plasmid was introduced into the cell based on the same method as in Example 1 (4) [4-1]. was introduced to prepare a cell fluorescence IP ₃ sensor cyLIBRAvIISVd (cyLvIISVd) was expressed. The configuration of the fluorescent IP ₃ sensor cyLIBRAvIISVd (cyLvIISVd) is shown in FIG. In FIG. 19, “6H” indicates a tag peptide for purification consisting of six consecutive histidine (His; H) residues, “Cerulean” indicates a blue fluorescent protein Cerulean, and “L1H1” indicates a linker (Glu- Ala-Ala-Ala-Arg-Ser-Arg (EAAARSR); SEQ ID NO: 32), “IP ₃ R 2 (1-604) with R440Q” has affinity for the ligand binding part of rat type 2 IP ₃ receptor A peptide having an increased amino acid mutation R440Q, that is, a peptide consisting of amino acid sequence 1 to 604 from the N-terminal of rat type 2 IP ₃ receptor, and arginine (R) at position 440 of the sequence to glutamine (Q) The mutated peptide is shown.

Subsequently, Example 1 (4) [4-2] on the basis of the same technique as, cells resulting fluorescence IP ₃ sensor cyLIBRAvIISVd (cyLvIISVd) expressed the perforated saponin, Talon beads the supernatant ( Takara Bio Inc.) was added and incubated for 2 hours to prepare a bead-immobilized fluorescent IP ₃ sensor cyLIBRAvIISVd (cyLvIISVd). Further, this bead-immobilized fluorescent IP ₃ sensor LIBRAvIISVd (cyLvIISVd) was adhered to an experimental chamber coated with Celltac, and a fluorescent image was taken using a fluorescence inverted microscope TE2000 (Nikon Instech) and EM-CCD camera C9100-13. Then, the fluorescent competitor F-ADA, the fluorescent competitor F-LL, or FITC was allowed to act so that the final concentration was 10 nM, 30 nM, 100 nM, or 300 nM, and the imaging system of Example 1 (3) [3-2] The fluorescence change rate was calculated using (Hamamatsu Photonics Co., Ltd.). The results are shown in FIG.

As shown in FIG. 20, when F-LL or F-ADA was allowed to act, a concentration-dependent change in fluorescence ratio was observed, and the maximum fluorescence change rate by F-LL and F-ADA was 60% (1%, respectively). 6) and 75% (1.75) (FIGS. 20A and B). On the other hand, the fluorescence change rate was close to 0 even when FITC not bound to the IP ₃ receptor was added (FIG. 20D). From these facts, it becomes clear that the bead-immobilized fluorescent IP ₃ sensor cyLIBRAvIISVd (cyLvIISVd), which is a carrier-immobilized fluorescent IP ₃ sensor, changes its fluorescence ratio at 480 nm / 535 nm by specific binding with a fluorescent competitor. The Furthermore, this quantitative analysis revealed that a large reaction of about 10 times the change in the fluorescence ratio of 480 nm / 535 nm by the conventional fluorescent IP ₃ sensors LIBRAvIIS (LvIIS) and IP ₃ occurs.

(2) Examination of whether or not the fluorescent competitor competes with the test substance IP ₃ and binds to or dissociates from the ligand binding part of the IP ₃ receptor of the carrier-immobilized fluorescent IP ₃ sensor. A test for confirming whether or not it binds to or dissociates from the ligand binding part of the IP ₃ receptor of the carrier-immobilized fluorescent IP ₃ sensor in competition with ₃ was performed according to the following procedure.
After the fluorescent competitor F-LL is made to act on the carrier immobilized fluorescent IP ₃ sensor cyLIBRAvIISVd (cyLvIISVd) prepared in the present Example 5 (1) so that the final concentration is 100 nM, the final concentration is 0.1 μM, 1 μM The carrier immobilization using F-LL and IP ₃ was carried out based on the same procedure as in Example 1 (4) [4-3] except that IP ₃ was allowed to act stepwise to 10 μM. The fluorescence ratio between the fluorescence at 480 nm and the fluorescence at 535 nm and the rate of fluorescence change in the fluorescence IP ₃ sensor cyLIBRAvIISVd (cyLvIISVd) were calculated. The result is shown in FIG.

As shown in FIG. 21, when 100 nM F-LL is allowed to act on the bead-immobilized fluorescent IP ₃ sensor cyLIBRAvIISVd (cyLvIISVd), the 480 nm / 535 nm fluorescence ratio decreases, and when IP ₃ is added, F-LL and IP ₃ are competitive binding of the beads immobilized fluorescent IP ₃ sensor cyLIBRAvIISVd (cyLvIISVd) and F-LL is inhibited by, 480 nm / 535 nm fluorescence ratio is increased depending on the IP ₃ concentration (Figure 21A). That is, a sample containing F-LL and IP ₃ when allowed to act on the bead-immobilized fluorescent IP ₃ sensor cyLIBRAvIISVd (cyLvIISVd), was shown to be capable of measuring the IP ₃ concentration from the rate of increase in 480 nm / 535 nm fluorescence ratio. Since the binding of competing ligands is dependent on their respective concentrations, it is believed that lowering F-LL concentrations will allow lower concentrations of IP ₃ to be measured, and 30 nM F-LL and 100 nM F-LL each reacted further were compared by the action of IP _3, the street, that is by the action of low concentrations of F-LL, fluorescence change rate due to IP ₃ by further action of IP ₃ is increased ( Figure 21 B). From these results, it was shown that the carrier-immobilized fluorescent IP ₃ sensor and the carrier-immobilized IP ₃ measurement FRET molecular sensor having the fluorescent competitor enable highly accurate IP ₃ measurement.

<Example 6> IP ₃ receptors study of amino acid sequence variants of the ligand binding portions of and fluorescent IP ₃ Study IP ₃ receptor added 1 dozen amino acids in the amino acid sequence of the ligand binding portions of the fluorescent material in the sensor Whether or not a protein having an inserted amino acid sequence functions as a ligand binding part of an IP ₃ receptor, and whether a fluorescent IP ₃ sensor can use an organic fluorescent compound as a fluorescent substance instead of a fluorescent protein We examined at the same time whether or not.

(1) IP ₃ is cysteine binding fluorescent organic compound instead of creating a fluorescent protein variant proteins expressed genes of the ligand binding portion of the receptor N- [4- methyl-7- (dimethylamino) coumarin-3-yl] In order to use maleimide (DACM), LumioTag (LT, CCPGCC, SEQ ID NO: 33) is inserted between amino acids 82 and 83 located upstream of the ligand binding site of mouse type 1 IP ₃ receptor OP1-ma (EN) -E, OP1-ma (EN) -S with LumioTag inserted between amino acids 140 and 141 located upstream of the ligand binding site of rat type 1 IP ₃ receptor, and OP1-ma (EN) -ES from which the inserted LumioTag was removed was prepared according to the following procedure.

[1-1] Preparation of Lhb-ma gene First, a peptide chain consisting of 12 amino acids including a membrane-binding domain, a FLAG tag, a histidine tag and a LumioTag is added or inserted into the ligand binding site of the mouse type 1 IP ₃ receptor. Lhb-ma (SEQ ID NO: 34) was prepared. Specifically, a membrane-binding domain, a FLAG tag and a histidine tag are added, and LumioTag is located between amino acids 82 and 83 located upstream of the ligand binding site of mouse type 1 IP ₃ receptor, and amino acids 140 Lhb-1 plasmid (SEQ ID NO: 35) having a gene sequence inserted between No. 141 and No. 141 was synthesized by requesting GeneScript, and the obtained Lhb-1 plasmid and LIBRAvIII of Example 2 (1) ( LvIII) Plasmid (SEQ ID NO: 22) was cleaved with restriction enzymes NheI and NotI, and a DNA fragment containing the Lhb-1 gene was inserted into a LIBRAv vector to prepare the Lhb-ma gene.

[1-2] Preparation of OP1-ma (EN) gene The Lhb-ma gene obtained in Example 6 (1) [1-1] was cleaved with restriction enzymes EcoRV and Nru1, and then recombined. The LumioTag upstream of the ligand binding site of the mouse type 1 IP ₃ receptor was removed. This resulted in an OP1-ma (EN) gene containing LumioTag between amino acids 82 and 83 and between amino acids 140 and 141 of the ligand binding site of the mouse type 1 IP ₃ receptor. .

[1-3] Preparation of OP1-ma (EN) -S gene, OP1-ma (EN) -E gene, and OP1-ma (EN) -ES gene Example 6 (1) [1-2 The OP1-ma (EN) gene obtained in [1] was cleaved with the restriction enzyme ScaI, and then recombined, whereby the LumioTag between amino acids 82 and 83 of the ligand binding site of the mouse type 1 IP ₃ receptor. Was removed. As a result, an OP1-ma (EN) -S gene (SEQ ID NO: 36) containing LumioTag between amino acids 140 and 141 of the ligand binding site of the mouse type 1 IP ₃ receptor was obtained.

Next, the OP1-ma (EN) gene obtained in Example 6 (1) [1-2] was cleaved with the restriction enzyme Eco47III and then rebound to thereby bind the ligand of mouse type 1 IP ₃ receptor. The LumioTag between amino acids 140 and 141 of the junction was removed. As a result, an OP1-ma (EN) -E (SEQ ID NO: 37) gene containing LumioTag between amino acids 82 and 83 of the ligand binding site of the mouse type 1 IP ₃ receptor was obtained.

Subsequently, the obtained OP1-ma (EN) -E gene is cleaved with a restriction enzyme ScaI and then rejoined to thereby bind amino acids 82 and 83 of the ligand binding part of mouse type 1 IP ₃ receptor. Removed the LumioTag in between. As a result, an OP1-ma (EN) -ES gene (SEQ ID NO: 38) that did not contain LumioTag in the ligand binding site of the mouse type 1 IP ₃ receptor was obtained. FIG. 22 shows the procedure for preparing the OP1-ma (EN) -S gene, the OP1-ma (EN) -E gene, and the OP1-ma (EN) -ES gene. In FIG. 22, “→” indicates a part of a vector gene, “MT” indicates a membrane bound gene sequence, “FLAG” indicates a gene sequence of FLAG tag, and “LT” and “LT” indicate LumioTag gene “E1” indicates the ScaI cleavage site, “E2” indicates the Eco47III cleavage site, “1-82-E1-LT-E1-83-140-E2-LT-E2-141-604”, "1-82-E1-LT-E1-83-140-E2-141-604", "1-82-E1-83-140-E2-LT-E2-141-604" and "1-82-E1" -83-140-E2-141-604 "shows the gene sequence of the variant protein of the ligand binding part of IP ₃ receptor, and" Hx6 "consists of 6 consecutive histidine (His; H) residues Shows the gene sequence of the tag peptide.

(2) Fluorescent IP ₃ sensors OP1-ma (EN) -S, followed analysis by OP1-ma (EN) -E, and OP1-ma (EN) of -E-S expressing saponin perforated cell creation and fluorescence imaging method Based on the same method as in Example 1 (4) [4-1], each of OP1-ma (EN) -S, OP1-ma (EN) -E, and OP1-ma (EN) -ES After preparing cells expressing OP1-ma (EN) -S, OP1-ma (EN) -E, and OP1-ma (EN) -ES by introducing the expression plasmid of Based on the same procedure as in Example 1 (4) [4-2], OP1-ma (EN) -S, OP1-ma (EN) -E, and OP1-ma (EN) -ES are expressed, respectively. Saponin penetration from isolated cells The cells were obtained. Each saponin perforated cell obtained was treated with 2 μM of N- [4-methyl-7- (dimethylamino) coumarin-3-yl] maleimide (DACM), which is a cysteine-binding fluorescent organic compound, for 2 minutes. performed, fluorescence DACM bound IP ₃ sensors OP1-ma (EN) -S, OP1-ma (EN) -E, and OP1-ma a (EN) -E-S is expressed on the cell membrane. For these fluorescence IP ₃ sensors OP1-ma (EN) -S, OP1-ma (EN) -E, and OP1-ma (EN) -E- S expressing saponin drilling cells, fluorescence to a final concentration of 100nM Except that the competitor F-ADA was allowed to act, the fluorescent IP ₃ sensor OP1-ma (to which the fluorescence competitor F-ADA was caused to act, respectively, based on the same method as in Example 1 (4) [4-3] The fluorescence intensities of 480 nm fluorescence and 535 nm fluorescence in saponin-perforated cells expressing EN) -S, OP1-ma (EN) -E, and OP1-ma (EN) -E-S, and the 480 nm / 535 nm fluorescence ratio were calculated . The result is shown in FIG.

As shown in FIG. 23, FRET from DACM to the fluorescence competitor F-ADA increased the fluorescence at 480 nm 3 to 5 times, and greatly decreased the 480 nm / 535 nm fluorescence ratio. Therefore, IP ₃ receptors to several tens of amino acids are added to the amino acid sequence of a ligand binding portion of the protein having the amino acid sequence inserted such functions as a ligand-binding portion of the IP ₃ receptor and fluorescent in IP ₃ sensor has been shown that it is possible to use an organic fluorescent compound as a fluorescent substance instead of the fluorescent protein.

Since DACM is a compound that changes to a fluorescent substance by binding to cysteine, and LumioTag is a peptide containing 4 cysteine residues, DACM can bind to LumioTag. However, since a reaction was also observed in OP1-ma (EN) -E-S from which LumioTag was removed, DACM involved in FRET with F-ADA is ligand binding of IP ₃ receptor excluding inserted LumioTag. It was shown that it bound to cysteine in the part. In addition, OP1-ma (EN) -ES corresponds to the ligand binding part 253, 292, 326, 394, 530, 553 and 556 of the mouse type 1 IP ₃ receptor. It is known that there are a total of 7 cysteine residues (Bosanac et al., Nature, 420: 696-700, 2002). Therefore, even if an organic fluorescent compound is bound to these, a fluorescent IP ₃ sensor As a function was shown not to be lost.

Furthermore, in the IP ₃ receptor, a ligand binding part is constituted by about 700 amino acids at the N-terminal side. For example, in comparison with the N-terminal 734 amino acids, 1 to 31, 1 to 62, 1 to 146, 1 to 162, 1 to 183, 1 to 199, 1 in the N-terminal side IP ₃ binding capacity in the case of removing to 215 th amino acids greatly reduced but not eliminated. Furthermore, the binding ability is restored by removing the amino acids 1-220, 1-223 and 1-225, but the binding ability is restored, but 1-228, 1-238, 1-261, 1-283, 1-307 The binding ability disappears by removing the No. amino acid. Moreover, although the binding ability does not change even if the amino acids 579 to 649 are removed, the binding ability disappears when the amino acids 568 to 649 are removed. Furthermore, IP ₃ binding affinities of the peptide chains of number 224-579 is known to be about 10-fold compared to IP ₃ binding affinities of the peptide chains of number 1~734 (Yoshikawa et al., J. Biol. Chem. 271: 18277-18284, 1996), amino acids 224 to 579 are essential as the ligand binding part, and are called the core domain. Also, No. 241 Arginine (241R), No. 249 Lysine (249K), No. 265 Arginine (265R), No. 269 Arginine (269R), No. 504 Arginine (504R), No. 506 Arginine (506R), Ten amino acids of 508th lysine (508K), 511th arginine (511R), 568th arginine (568R), and 569th lysine (569K) are essential, and by mutating them, IP ₃ It is known that the binding ability is lost (Bosanac et al., Nature, 420: 696-700, 2002; and Yoshikawa et al., J. Biol. Chem. 271: 18277-18284, 1996).

On the other hand, other amino acids can be substituted, so far, lysine No. 100 has been substituted with glutamine (K 100 Q), lysine No. 101 has been substituted with glutamine (K 101 Q), lysine No. 235 has been substituted with glutamine (K K 235 Q), replace 257 arginine with glutamine, # 258 lysine with glutamine, and # 259 lysine with glutamine with R 257 Q / K 258 Q / K 259 Q, replace # arginine with glutamine (R 293 Q), # 304 Arginine to glutamine, and lysine 306 to glutamine (R304Q / K306Q), lysine 350 to glutamine (K350Q), 376 arginine to glutamine (R376Q), lysine 412 to lysine Substitution to glutamine (K412Q), Substitution of lysine No. 24 to glutamine (K 424 Q), substitution of lysine No. 427 to glutamine (K 427 Q), substitution of lysine No. 459 to glutamine (K 459 Q), substitution of arginine No. 470 and 471 to glutamine (R 470 Q / R 471 Q), substitution of lysine No. 501 with glutamine (K 501 Q), substitution of 537 arginine with glutamine (R 537 Q), substitution of 545 arginine with glutamine (R 545 Q), substitution of 554 arginine with glutamine (R 554 Q) , Substitution of arginine at position 561 to glutamine (R 561 Q), substitution of lysine at position 576 to glutamine (K 576 Q), substitution of arginine at position 603 to glutamine, and substitution of lysine at position 604 to glutamine (R 603 Q / K 604 Q), 623 The Argi The emissions to glutamine, and replace 624 No. lysine to glutamine (R623Q / K624Q), replacing the 626 th arginine to glutamine (R626Q), IP ₃ binding by the 629 th arginine substitution of glutamine (R629Q) Is known not to change significantly (Bosanac et al., Mol. Cell, 17: 193-203, 2005).

Furthermore, when examined at the ligand binding site of the IP ₃ receptor composed of 604 amino acids at the N-terminus, substitution of leucine 30 with lysine (L30K), substitution of leucine 32 with lysine (L32K), 33 No. valine is replaced with lysine (V33K), No. 34 aspartic acid is replaced with lysine (D34K), No. 36 arginine is substituted with glutamic acid (R36E), No. 54 arginine is substituted with glutamic acid (R54E), No. 127 Substituting lysine for glutamic acid (K127E), 54th arginine for glutamic acid, 127th lysine for glutamic acid (R54E / K127E), and removal of 67-108 increase IP ₃ binding affinity While the Kd value decreases by 30 to 90%, Substitution with sine (D35K), substitution of lysine 51 with glutamic acid (K51E), substitution of lysine 52 with glutamic acid (K52E), substitution of aspartic acid 97 with lysine (D97K), and substitution of 99th glutamic acid with lysine (E99K), No. 104 glutamic acid to lysine (E104K), No. 106 glutamic acid to lysine (E106K), No. 97 aspartic acid and No. 99 glutamic acid to lysine (D97A / E99A) ), by the 104 th and 106 th glutamic acid substitution to lysine (E104K / E106K) is, IP ₃ binding affinity is reduced to 50-80%.

As described above, arginine No. 241 (241 R), lysine No. 249 (249 K), arginine No. 265 (265 R), arginine No. 269 (269 R), arginine No. 504 (504 R), arginine No. 506 ( Removal and substitution of amino acids except for the 10 essential amino acids of 506R), lysine at position 508 (508K), arginine at position 511 (511R), arginine at position 568 (568R), and lysine at position 569 (569K) It can be said that the function of the ligand binding part of the IP ₃ receptor is not impaired even if the step is performed. Furthermore, replacing arginine at position 441 of the ligand binding part of mouse type 1 IP ₃ receptor with glutamine (R 441 Q) greatly enhances IP ₃ binding affinity, but the same substitution (R 441 Q) is carried out as mouse 2 type 2 IP ₃ The same increase in affinity occurs even if it is performed at position 441 of the ligand binding part of the receptor (Tanimura et al., J. Biol. Chem., 284: 8910-8917, 2009), IP ₃ receptor Regardless of the subtype and animal species, mutations are considered possible except for the 10 essential amino acids.

From <Example 7> IP ₃ results receptors fluorescent IP ₃ sensor study Example 6 inserting a circular permutation of the ECFP in the ligand binding part, the peptide chain within the ligand binding portion of the IP ₃ receptors be inserted, IP ₃ since the function of the ligand-binding portion of the receptor was not impaired, fluorescence IP ₃ inserting the circular permutation of the ECFP is inside fluorescent protein of the ligand binding portion of the IP ₃ receptors as well as consider the sensor was examined whether the fluorescent competitor in combination with a carrier immobilized IP ₃ measurements FRET molecular sensor of possible building.

(1) Preparation of fluorescent IP ₃ sensors OP1S-cpC157, OP1S-cpC173, and OP1S-cpC229 Example 6 (1) OP1-ma (EN) -E-S ligand binding site prepared in [1-3] Between amino acids 82 and 83, the fluorescent proteins cpSECFP157, cpSECFP173, and cpSECFP229, which are circular permutations of ECFP (Nagai et al., Proc. Natl. Acad. Sci. USA., 101: 10554-10559, 2004) was inserted. Specifically, vectors (SEQ ID NOS: 39 to 41) expressing cpSECFP157, cpSECFP173, and cpSECFP229 were digested with restriction enzymes EcoRV and Xca1 (SnaI) to purify each fluorescent protein gene. The purified fluorescent protein genes are inserted into the ScaI-cleaved OP1-ma (EN) -E-S expression plasmid, and the fluorescent IP ₃ sensor OP1S-cpC157, OP1S-cpC173 and OP1S-cpC229 expression plasmid (SEQ ID NO: 42) ~ 44) was created. FIG. 24 shows the procedure for preparing the OP1S-cpC157 gene, OP1S-cpC173 gene, and OP1S-cpC229 gene. In FIG. 24, “→” represents a part of the vector gene, “MT” represents the membrane-bound gene sequence, “FLAG” represents the gene sequence of the FLAG tag, “E1” represents the ScaI cleavage site, "E2" indicates the Eco47III cleavage site, "1-82-E 1-83-140-E 2-141-604" indicates the gene sequence of the variant protein of the ligand binding part of IP ₃ receptor, "Hx 6" indicates 6 Shows the gene sequence of a tag peptide consisting of consecutive histidine (His; H) residues, and “cpSECFP157”, “cpSECFP173” and “cpSECFP229” are circular permutations of ECFP, respectively, fluorescent proteins cpSECFP157, cpSECFP173, and cpSECFP229 The gene sequence of “1-82-cpSECF” P157-83-140-E2-141-604 "," 1-82-cpSECFP 173-83-140-E2-141-604 ", and" 1- 82-cpSECFP 229-83-140-E2-141-604 "are each shows a gene sequence inserted fluorescent protein cpSECFP157, cpSECFP173, and cpSECFP229 the IP ₃ into a modified protein of the ligand binding portion of the receptor is a circular permutation of ECFP.

(2) Preparation of fluorescent IP ₃ sensors OP1S-cpC157, OP1S-cpC173, and OP1S-cpC229-expressing saponin-perforated cells and analysis by fluorescence imaging method Subsequently, in the same manner as in Example 1 (4) [4-1] The fluorescent IP ₃ sensor OP 1 S-cpC 157, the fluorescent IP ₃ sensor OP 1 S-cp C 173, and the fluorescent IP ₃ sensor OP 1 S-cp C 229 are introduced into cells to express the fluorescent IP ₃ sensor OP 1 S-cp C 157, OP 1 S-cp C 173. And OP1S-cpC229 were respectively expressed, and then the fluorescent IP ₃ sensor OP1S-cpC157, OP1S-cpC173, and OP1S-cpC were prepared based on the same method as in Example 1 (4) [4-2]. Each saponin perforated cell was obtained from each cell in which 229 was expressed. Except that the fluorescent competitor F-ADA was allowed to act so that the final concentration would be 100 nM, the fluorescent competitor F-ADA was allowed to act on the same method as in Example 1 (4) [4-3]. The fluorescence intensities of 480 nm fluorescence and 535 nm fluorescence in the fluorescent IP ₃ sensors OP1S-cpC157, OP1S-cpC173, and OP1S-cpC229-expressing saponin-perforated cells were calculated, as well as the 480 nm / 535 nm fluorescence ratio. The result is shown in FIG.

As shown in FIG. 25, it was confirmed that these fluorescent IP ₃ sensors OP1S-cpC157, OP1S-cpC173, and OP1S-cpC229 function. This indicates that the fluorescent protein can be inserted into the ligand binding site of the IP ₃ receptor. In addition, in the case of the ligand binding part of rat type 1 IP ₃ receptor, 1 to 13, 23 to 24, 30 to 36, 40 to 51, 61 to 66 on the N terminal side forming a loop. No. 76 to 86, 109 to 119, 126 to 129, 140 to 146, 153 to 156, 160, 166 to 180, 187 to 192, 200 to 206, 212 to 216, 225, 229-238, 245-248, 257-259, 266-277, 281, 287-301, 308-312, 319-352, 359-363, 366, 373 ~ 386, 393-396, 406-414, 422-430, 435-436, 463-466, 485-503, 513-514, 524-551, 564-5 It is suggested that a fluorescent protein can be inserted at amino acids 67, 573-579 (Bosanac et al., Nature, 420: 696-700, 2002; Bosanac et al., Mol. Cell, 17: 193-203). 2005; and Tanimura et al., J. Biol. Chem. 271: 30904-30908, 1996).

<Example 8> A fluorescent test substance sensor having a fluorescent substance and a paratope-containing polypeptide part of an antibody, and specifically binding to or dissociating from a paratope-containing polypeptide part of the target antibody in competition with the test substance; Construction of analyte-measuring FRET molecular sensor with fluorescently modified substance and construction of competitive fluorescent antigen assay (CFAA) Fluorescent substance and paratope-containing polypeptide moiety of antibody Competing by constructing a sensor and a test substance measurement FRET molecular sensor comprising a substance that specifically binds or dissociates with a paratope-containing polypeptide portion of the target antibody in competition with the test substance and is fluorescently modified Fluorescent antigen assay (Compe An attempt was made to construct a Titive Fluorescence Antigen Assay (CFAA).

(1) C-terminus of human type 2 IP ₃ receptor CT2-18 paralogs of creating human IP ₃ receptor fluorescent peptide F-CT2-18 obtained by fluorescence-modified by FITC (human IP3R2), 2685~ A fluorescent peptide (F-CT2-18) obtained by fluorescently modifying CT2-18 (SEQ ID NO: 45), which is a peptide consisting of 18 amino acids CGFLGSNTPV NHHMPPH of No. 3001, with FITC, competes with CT2-18, and CT2 It is a fluorescent competitor specifically binding to the paratope-containing polypeptide portion of an antibody (ABsII) prepared using -18 as an antigen. The fluorescent peptide F-CT2-18 labeled with FITC at the N-terminus of the antigen peptide CT2-18 and CT2-18 used for preparation of the antibody was purchased from United Biosystems Inc. Dr. It was synthesized by asking CabinJohn, MD (USA).

(2) Preparation of antibody ABsII-binding agarose beads labeled with fluorescent substance CT2-18 described in a previous report (Tanimura et al., J. Biol. Chem., 275: 27488-27493, 2000) was prepared as an antigen. By acting 200 μM DACM for 30 minutes on antibody ABsII, a fluorescent ABsII-CT2-18 sensor having ABsII labeled with fluorescent substance DACM was produced. Specifically, 10 μM DACM was added to 1000 μL of a phosphate buffer (pH 7.4) containing about 10 μg of ABsII and 0.05% bovine serum albumin, and the mixture was allowed to stand at 4 ° C. for 2 hours, and then 10 μL of Protein. G agarose beads were added, and the fluorescent ABsII-CT2-18 sensor was bound to the agarose beads by further standing at 4 ° C. for 2 hours, followed by washing with phosphate buffer. The agarose beads coupled with the fluorescent ABsII-CT2-18 sensor were dispersed in 100 μL of phosphate buffer, and aliquoted into 20 μL aliquots of tubes A to D. A tube contains phosphate buffer, B tube contains 1 μM fluorescence competitor F-CT2-18, C tube contains 1 μM fluorescence competitor F-CT2-18 and 1 μM analyte CT2 100 μL each of 1 μM of the fluorescence competitor F-CT2-18 and 10 μM of the test substance CT2-18 were added to the tube of D-18, and allowed to stand for 2 hours.

(3) Analysis by Fluorescence Imaging Method Fluorescence due to the action of these agarose bead-bound fluorescence ABsII-CT2-18 sensor and fluorescence competitor F-CT2-18 was measured using the imaging system of Example 1 (3) [3-2] ( A fluorescence ratio of 480 nm / 535 nm was calculated using Hamamatsu Photonics Co., Ltd.). The result is shown in FIG. FIG. 26A shows the result of fluorescence measurement after washing the agarose bead-bound fluorescent ABsII-CT2-18 sensor with a phosphate buffer, and FIG. 26B shows the fluorescence measurement without washing the agarose bead-bound fluorescent ABsII-CT2-18 sensor. It is the result of having performed.

  As shown in FIG. 26A, the fluorescence ratio of 480 nm / 535 nm decreased by 85% by FRET of DACM and the fluorescent competitor F-CT2-18 in the fluorescent ABsII-CT2-18 sensor. This fluorescence change due to FRET was recovered depending on the concentration of CT2-18, and the fluorescence ratio increased 5 times by adding the test substance CT2-18 corresponding to 10 times the amount of the fluorescence competitor F-CT2-18. . In addition, as shown in FIG. 26B, when the same fluorescence measurement is performed without washing the agarose bead-bound fluorescence ABsII-CT2-18 sensor, the fluorescence ABsII-CT2-18 sensor is affected by the fluorescence in the solution. Although the change in fluorescence of DACM and the fluorescence competitor F-CT2-18 was slightly reduced, the recovery of the fluorescence ratio of 480 nm / 535 nm by addition of the test substance CT2-18 could be sufficiently confirmed.

  From the above results, a fluorescent test substance sensor having a fluorescent substance and a paratope-containing polypeptide part of the antibody, and a specific binding or dissociation with the paratope containing polypeptide part of the target antibody in competition with the test substance It has been shown that an analyte-measuring FRET molecular sensor with fluorescently modified material can be constructed to construct a competitive fluorescent antigen assay (CFAA).

<Example 9> Examination of FRET molecular sensor comprising a combination of nicotinic acetylcholine receptor labeled with a fluorescent substance and nicotinic acetylcholine labeled with FITC. A nicotinic acetylcholine receptor on the cell membrane surface is labeled with a fluorescent substance and labeled with FITC. By using a fluorescent competitor that is nicotinic acetylcholine, the in vivo acetylcholine concentration can be measured.

<Example 10> Solid-phased FRET molecular sensor using anti-c-Myc antibody (1) Preparation of fluorescent test substance sensor Anti-cMyc monoclonal antibody (1 mg) was added to 100 μL of protein G beads (protein G agarose) manufactured by SANTA CRUS. Antibody was added to the beads by adding 25 μL / mL) and incubating at 4 ° C. for 2 hours. After washing the antibody beads with a phosphate buffer (PBS), 8 μL of 10 mM DACM was added to 400 μL of the antibody bead solution and incubated at 4 ° C. for 30 minutes (final concentration 200 μM). Thereafter, 50 μM cysteine was added to stop the reaction, and a fluorescent test substance sensor in which an anti-c-Myc antibody labeled with DACM was immobilized on beads was prepared.

(2) Preparation of FITC-labeled c-Myc peptide 2 μm c-Myc peptide (amino acid sequence: EQKLISEEDL, SEQ ID NO: 46) solution in 100 μL (240 μg) FITC labeling reagent (Fluorescein Labeling Kit-NH manufactured by Dojindo) 5 μL The peptide was labeled with FITC by incubation at 4 ° C. for 10 minutes. Thereafter, 5 μL (500 μg) of 10% bovine serum albumin (BSA) was added, and the excess labeling reagent was reacted with BSA by incubation for 20 minutes. Thereafter, a fluorescent competitor was produced by recovering a fluorescent peptide (FITC-cMyc) having a molecular weight of <50 Kd by ultrafiltration.

(3) Preparation of non-immobilized fluorescent test substance sensor 8 μL of 10 mM DACM was added to 400 μL of PBS containing 25 μg of anti-cMyc monoclonal antibody, and incubated at 4 ° C. for 30 minutes. Thereafter, the reaction was stopped with 50 μM of cysteine, the fraction with a molecular weight of <50 Kd was removed by ultrafiltration, and a fluorescent test substance sensor which is an anti-c-Myc antibody labeled with DACM was prepared.

(4) Analysis by fluorescence imaging method The fluorescent test substance sensors produced in (1) and (3) were added to PBS containing 0.01% BSA, respectively, and 20 μM of the fluorescent competitor produced in (2) or fluorescence competition 20 μM of the substance and 400 μM of the test substance (c-Myc peptide not labeled with FITC) were added and reacted for 2 hours. Using a fluorescence imaging system (see Example 1, (4) and [4-3]) in which 5 μL of each reaction solution was dropped onto a cover glass and equipped with a × 20 objective lens, 480 nm by excitation at 425 nm And 535 nm fluorescence images were recorded, and the fluorescence ratio (480 nm / 535 nm) was measured. The result is shown in FIG. The graph on the left is the fluorescence ratio when using the fluorescent test substance sensor on which the solid phase is used, and the graph on the right is the fluorescence ratio when using the fluorescent test substance sensor on which the solid phase is not used.

  As shown in FIG. 27, the fluorescence ratio at 480 nm / 535 nm by FRET from DACM to FITC was increased due to the presence of the test substance. In particular, it was confirmed that the FRET molecular sensor provided with the immobilized fluorescent test substance sensor is clearly elevated, that is, the sensitivity is excellent.

<Example 11> FRET molecular sensor using anti-DYK antibody (1) Preparation of fluorescent test substance sensor In the same manner as in (1) of Example 10, an anti-DYK antibody labeled with DACM was immobilized on beads. A fluorescent test substance sensor was produced.

(2) Preparation of FITC-labeled DYK peptide: Requested to United Peptide, Inc., FITC-labeled DYK peptide (amino acid sequence: GDYKDDDDKCCPGCCGGGGGGGGHHHHH, represented as SEQ ID NO: 47, FT1) was prepared as a fluorescence competitor. In addition, a DYK peptide (FT2) that was not fluorescently labeled was prepared as a test substance.

(3) Analysis by fluorescence imaging method In the same manner as (4) in Example 10, a fluorescent test substance sensor in which DACM-labeled anti-DYK antibody was immobilized on beads and FT1 0.4 μM as a fluorescence competitor Alternatively, FT1 0.4 μM and test substance 4.0 μM were reacted, and the fluorescence ratio (480 nm / 535 nm) was measured using a fluorescence imaging system. The results are shown in FIG.

  As shown in FIG. 28, the fluorescence ratio at 480 nm / 535 nm by FRET from DACM to FITC was increased due to the presence of the test substance.

  From the results of Example 10 and Example 11, the solid-stated fluorescent test substance sensor using the specific antibody as the test substance binding site and the FRET molecular sensor equipped with the fluorescent competitor substance can detect the specific antigen of the antibody. It was confirmed that it could be used.

<Example 12> Preparation of FRET molecular sensor for protein detection with histidine tag (1) Preparation of fluorescent test substance sensor Anti-type 2 IP ₃ receptor antibody (ABsII) in 100 μL solution of protein G beads (protein G agarose from SANTA CRUS) Was added and incubated at 4 ° C. for 2 hours to prepare antibody beads. After washing the antibody beads with phosphate buffer (PBS), 8 μL of 10 mM DACM was added to 400 μL of antibody bead solution (final concentration 200 μM) and incubated at 4 ° C. for 30 minutes. The reaction was then stopped with 50 μM cysteine. Furthermore, 8 μL of 10 mM Isothiocyanobenzyl-NTA (manufactured by Dojindo) (final concentration 200 μM) was added and incubated at 4 ° C. for 30 minutes. Then, the reaction was stopped with 50 μM of cysteine, and 10 mM of NiCl ₂ was further added and incubated for 30 minutes to prepare a fluorescent test substance sensor labeled with DACM and having a Ni-NTA complex.

(2) Analysis by fluorescence imaging method In the same manner as in (4) of Example 10, the fluorescent test substance sensor prepared in (1) was added to PBS containing 0.01% BSA. The prepared FT1 100 μM, or FT1 100 μM and FT2 100 μM, which is a test substance (a peptide that competes with FT1), are reacted, and the fluorescence ratio (480 nm / 535 nm) using a fluorescence imaging system (the magnification of the objective lens is × 10). Was measured. The result is shown in FIG.

  As shown in FIG. 29, the fluorescence ratio at 480 nm / 535 nm by FRET from DACM to FITC was increased due to the presence of the test substance FT2. Both FT1 and FT2 are peptides having a histidine tag and bind to the Ni-NTA complex, but not to ABsII. From this, the above-mentioned increase in the fluorescence ratio is due to the binding of FT1 and FT2 to the Ni-NTA complex contained in the fluorescent test substance sensor, which means that such a fluorescent test substance sensor and the peptide having a histidine tag It means that FRET molecular sensor equipped with a certain fluorescent competitor can be used for detection, quantification and separation / purification of a peptide having a histidine tag.

<Example 13> FRET molecular sensor for antibody molecule detection (1) Preparation of DACM-labeled beads 8 μL of 10 mM DACM was added to 400 μL of Protein G beads (Stained Crus protein protein g agarose) solution (final concentration) (200 μM) Incubated at 4 ° C. for 30 minutes. Thereafter, 50 μM cysteine was added to stop the reaction, and Protein G beads (DACM-PG) labeled with DACM were produced as a fluorescent test substance sensor.

(2) FITC -labelled anti Type 2 IP ₃ receptor antibody Anti-Type 2 IP ₃ receptor antibody (ABsII) FITC labeled reagent PBS solution containing Preparation 100μg of (ABsII) (Dojindo Inc. Fluorescein Labeling Kit- NH) 5 μL was added, and ABsII was FITC labeled by incubation at 4 ° C. for 10 minutes. Thereafter, a fraction having a molecular weight of <50 Kd was removed by ultrafiltration, and FIs-labeled ABsII (FITC-ABsII) was collected to produce a fluorescence competitor.
(3) Analysis by fluorescence imaging method The fluorescent test substance sensor prepared in (1) is added to PBS containing 0.01% BSA, and the fluorescent competitor 0.1 μg / mL prepared in (2) or the fluorescence competitor 0.1 μg / mL and 0.1 μg / mL or 1.0 μg / mL of a test substance (ABsII not labeled with FITC) were added and allowed to react for 2 hours. Using a fluorescence imaging system (see Example 1, (4) and [4-3]) in which 5 μL of each reaction solution was dropped onto a cover glass and equipped with a × 10 objective lens, 480 nm by excitation at 425 nm And 535 nm fluorescence images were recorded, and the fluorescence ratio (480 nm / 535 nm) was measured. The result is shown in FIG.

  As shown in FIG. 30, the fluorescence ratio at 480 nm / 535 nm by FRET from DACM to FITC increased depending on the amount of the test substance. From this, the fluorescence test substance sensor that is a bead for antibody binding labeled with DACM and the FRET molecular sensor equipped with a fluorescence competitor that is any antibody labeled with fluorescence detect the detection of the antibody molecule in the test sample It was confirmed that it was available to

Claims (5)

A FRET molecular sensor for measuring a test substance using fluorescence resonance energy transfer (FRET),
A fluorescent test substance sensor having a fluorescent substance and a test substance binding part; and a fluorescent competitor that competes with the test substance and binds to or dissociates from the test substance binding part,
Here, the test substance binding portion is a ligand binding portion of inositol 1, 4, 5 triphosphate (IP ₃ ) receptor, and the fluorescence competitor is 9- [5-Deoxy-5- [N-[[1-]. [5-[[3 ′, 6′-dihydroxy-3-oxospiro (isobenfuran-1,9′-xanthen) -5-yl] amino] thioxoxyl] amino] propyl] -1H-1,2,3-triazol- 4-yl]-(3,4-di-O-phosphoryl-α-D-glucocopyrasyl) -2-O-phosphoryl-β-D-ribo-pentofuranosyl] adenine (F-ADA) or (2R, 3R, 4S) ) -4-O-phosphoryl-2- [N-[[1- [5-[[3 ', 6'] dihydroxy-3-oxospiro (isobenzofuran-1,9'-xanthen) -5-yl] amino] thioxomethyl] amino] propyl] -1H-1,2,3-triazol-4-yl] methyl-tetrahydrofuran-3-yl -3,4-di-O-phosphoryl-α-D-glucopyranoside (F-LL),
And the FRET molecular sensor utilizing FRET caused by the fluorescent substance and the fluorescent competitor.   The FRET molecular sensor according to claim 1, wherein the fluorescent substance is a fluorescent protein or a protein fluorescent labeling substance. The FRET molecular sensor according to claim 1 or 2, wherein the fluorescent test substance sensor is immobilized on a carrier. Contacting the subject with the FRET molecular sensor according to any one of claims 1 to 3 ,
A method of measuring a test substance in a subject by using the intensity of fluorescence as an index, comprising the steps of: irradiating fluorescence with excitation light; 9- [5-Deoxy-5- [N-[[1- [5-[[3 ', 6'-dihydroxy-3-oxospiro (isobenzofuran-1,9'-xanthen) -5-yl] amino] thiooxomethyl ] Amino] propyl] -1H-1,2,3-triazol-4-yl]-(3,4-di-O-phosphoryl-α-D-glucocopyrasyl) -2-O-phosphoryl-β-D-ribo -Pentofuranosyl] adenine (F-ADA) or (2R, 3R, 4S) -4-O-phosphoryl-2- [N-[[1- [5-[[3 ', 6'-dihydroxy-3-oxospiro ( isobenzofuran-1,9'-xanthen) -5-yl] amino] thioxomethyl] amino] propyl] -1H-1,2,3-triazol-4-yl] methyl-tetrahydrofuran-3-yl-3,4-di-O-phosphoryl-α-D-glucocopyanoside (F-LL Use as a fluorescence competitor in the FRET molecular sensor according to any one of claims 1 to 3 .

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