Novel fluorescent labeling method

Abstract

[Problem] To provide a method for fluorescent labeling of an intracellular protein using fluorescence ON / OFF control technology which can be applied to various cells. [Solution] A method for fluorescent labeling of an intracellular protein that includes fluorescent labeling of the target protein by obtaining, in a cell, a fusion protein of a protein to be labeled and an anti-DNP (dinitrophenyl compound) antibody, bringing a compound represented by formula (I) or a salt thereof into contact with the cell, and reacting the fusion protein with the compound represented by formula (I) or salt thereof. The anti-DNP antibody in the fusion protein is an anti-DNP antibody containing a light chain containing VL-CDR1 comprising an amino acid sequence represented by SEQ ID NO: 1, VL-CDR2 comprising an amino acid sequence represented by SEQ ID NO: 2, and VL-CDR3 comprising an amino acid sequence represented by SEQ ID NO: 3 and a heavy chain containing VH-CDR1 comprising an amino acid sequence represented by SEQ ID NO: 4, VH-CDR2 comprising an amino acid sequence represented by SEQ ID NO: 5, and VH-CDR3 comprising an amino acid sequence represented by SEQ ID NO: 6, or an antigen-binding fragment thereof. The anti-DNP antibody or an antigen-binding fragment thereof is a single-chain Fv (scFv).

Description

New fluorescent labeling method

[0001] The present invention relates to a novel anti-DNP antibody and a method for fluorescently labeling an intracellular protein using the antibody.

[0002] In order to understand the molecular mechanisms underlying cellular functions, fluorescent imaging techniques that can track the time evolution of intracellular distribution of functional molecules in real time are an effective tool. To date, visualization analysis using fusion proteins in which the fluorescent protein GFP has been genetically engineered into the target protein has been widely used in analyzing intracellular protein dynamics (Non-Patent Document 1).

[0003] Furthermore, in recent years, molecular tagging technology useful for fluorescence imaging has progressed through approaches incorporating chemical biology techniques. Halo-tagged proteins have been developed by genetically modifying a bacterial haloalkane dehalogenase (Non-Patent Document 2), and SNAP-tagged proteins have also been developed by modifying the DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (Non-Patent Document 3).

[0004] These tagging technologies enable specific fluorescent labeling of target molecules by covalently binding specific fluorescent ligands to Halo tag proteins and SNAP tag proteins, respectively. Here, because the fluorescent probes used in molecular tagging technologies such as Halo tag and SNAP tag are always fluorescent, fluorescence from fluorescent probes nonspecifically bound to the fluorescent probe specimen or from unlabeled fluorescent molecules present outside the cell is observed as background signals, hindering good-contrast microscopic observation of the target molecules. To reduce this background signal, the target molecules must be fluorescently labeled and then washed to remove unreacted fluorescent probes. However, washing biomolecules present inside cells or in living organisms is generally difficult, and in many cases, washing is not possible.

[0005] To solve the above problems, a promising technology is to develop a fluorescence ON / OFF control technique in which a fluorescent probe that is not bound to a target molecule is non-fluorescent (fluorescence OFF) and becomes fluorescent (fluorescence ON) only when the fluorescent probe binds to a target molecule. It has been shown that it is possible to detect rRNA with an RNA aptamer sequence by utilizing the property of some non-fluorescent dyes that become fluorescent ON when bound to a nucleic acid (RNA) aptamer (Non-Patent Documents 4 and 5), but a practically usable fluorescence ON / OFF control technology has not yet been developed.

[0006] The present inventors aimed to provide a novel method for fluorescently labeling intracellular proteins using a technology for controlling fluorescence ON / OFF, and considered utilizing the quenching phenomenon that occurs when a fluorescent dye comes into close proximity with an atomic group (quencher) that has fluorescence quenching ability to control fluorescence ON / OFF. They found that an excellent technology for controlling fluorescence ON / OFF can be provided by controlling the fluorescence quenching ability of a fluorescent substance using an anti-DNP (dinitrophenyl compound) antibody (Patent Document 1). This new technology for controlling fluorescence ON / OFF involves expressing a fusion protein between a protein to be labeled (protein to be labeled) and an anti-DNP antibody in a cell, introducing a fluorescent substance (DNP probe) into the cell, and allowing it to react with the fusion protein, causing the quenched (OFF state) fluorescent substance to emit fluorescence (ON state), thereby fluorescently labeling the protein to be labeled.

[0007] The inventors' development of this fluorescence ON / OFF control technology has led to the discovery of excellent anti-DNP antibodies (e.g., the anti-DNP antibody designated 5D4 clone in Patent Document 1) and various excellent DNP probes. The inventors have confirmed that, using the 5D4 clone, for example, fusion proteins with the fluorescent protein TagRFP, which is a protein to be labeled, can be expressed in HEK293T cells, and that fusion proteins with proteins to be labeled, such as ECFP, β-tubulin protein, or β-actin, can be expressed in HeLa cells, demonstrating that fluorophore-DNP becomes fluorescent only when 5D4 binds to DNP in these cells. However, further investigation by the inventors has revealed that prior art anti-DNP antibodies, such as the 5D4 clone, are unable to express fusion proteins in the desired cells at a sufficient rate, and that further improvement is necessary for practical use.

[0008] D. M. Chudakov, S. Lukyanov, K. A. Lukyanov, Fluorescent proteins as a toolkit for in vivo imaging. Trends in Biotechnology 23, 605-613 (2005). G. V. Los et al., HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS chemical biology 3, 373-382 (2008). T. Gronemeyer, C. Chidley, A. Juillerat, C. Heinis, K. Johnsson, Directed evolution of O6-alkylguanine-DNA alkyltransferase for applications in protein labeling. Protein engineering, design & selection : PEDS 19, 309-316 (2006). J. S. Paige, K. Y. Wu, S. R. Jaffrey, RNA mimics of green fluorescent protein. Science 333, 642-646 (2011). R. L. Strack, M. D. Disney, S. R. Jaffrey, A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA. Nat Methods 10, 1219-1224 (2013).

[0009] International Publication No. 2018-135598

[0010] An object of the present invention is to provide a method for fluorescently labeling an intracellular protein using a technology for controlling fluorescence ON / OFF, which can be applied to various (various) cells. Another object of the present invention is to provide an anti-DNP (dinitrophenyl compound) antibody that can be suitably used in the fluorescent labeling method and that is capable of stably expressing a fusion protein in various (various) cells.

[0011] As a result of intensive research to solve the above problems, the present inventors discovered that by using an anti-DNP antibody having a specific sequence, it is possible to provide a method for fluorescently labeling intracellular proteins using a technology for controlling fluorescence ON / OFF in various (various) cells, and thus completed the present invention.

[0012] That is, the present invention has the following configuration: [1] A method for fluorescently labeling an intracellular protein, comprising: obtaining a fusion protein of a protein to be labeled and an anti-DNP (dinitrophenyl compound) antibody in a cell; contacting the cell with a compound represented by the following formula (I) or a salt thereof; and reacting the fusion protein with the compound represented by the following formula (I) or a salt thereof, thereby fluorescently labeling the target protein: (In formula (I), S is a fluorescent group, L is a linker, and R ｂ and R ｃ is selected from the following combinations: ｂ , R ｃ ): (NO ２ , p-NO ２ ), (NO ２ , p-Br), (NO ２ , p-SO ２ Me), (NO ２ , p-Cl), (NO ２ , m-CN), (NO ２ , p-CN), (NO ２ , p-COOMe), (CF ３ , p-CF ３ ), (NO ２ , p-CONHMe), (NO ２ , m-COOMe), (NO ２ , H) (where p- and m- are Rｃ are located at the para and meta positions on the benzene ring relative to L. The fluorescent group is represented by any one of the following formulas (II) to (IV): (In formula (II), R １ represents a hydrogen atom or 1 to 4 identical or different monovalent substituents present on a benzene ring; R ２ represents a hydrogen atom, a monovalent substituent, or a bond; １ , R ２ is selected from the group consisting of an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 1 to 6 carbon atoms, an alkynyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, a hydroxyl group, a carboxy group, a sulfonyl group, an alkoxycarbonyl group, a halogen atom, and an amino group, and the alkyl group having 1 to 6 carbon atoms may have one or more substituents selected from the group consisting of a halogen atom, a carboxy group, a sulfonyl group, a hydroxyl group, an amino group, and an alkoxy group, and the bond is ２ means to be introduced at the position of R ３ and R ４ each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; R ５ and R ６ each independently represents an alkyl group having 1 to 6 carbon atoms or an aryl group, provided that when X is an oxygen atom, R does not exist; ７ and R ８ each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; X represents an oxygen atom or a silicon atom; * represents a bonding site to L in formula (I) at any position on the benzene ring. (In formula (III), R １ ~R ８ , X is as defined in formula (II); R ９ and R １０ each independently represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; R ９ and R １０ Let's get together and ９ and R １０may form a 4- to 7-membered heterocyclyl containing the nitrogen atom to which R is attached, ９ or R １０ Alternatively, R ９ and R １０ Both of these are R ３ or R ７ Together with ９ or R １０ may form a 5-7 membered heterocyclyl or heteroaryl containing a nitrogen atom to which R is bonded, and may contain 1 to 3 additional heteroatoms selected from the group consisting of oxygen atoms, nitrogen atoms and sulfur atoms as ring-constituting members, and the heterocyclyl or heteroaryl may be substituted with an alkyl having 1 to 6 carbon atoms, an alkenyl having 2 to 6 carbon atoms, an alkynyl having 2 to 6 carbon atoms, an aralkyl group having 6 to 10 carbon atoms, or an alkyl-substituted alkenyl group having 6 to 10 carbon atoms; R １１ and R １２ each independently represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; R １１ and R １２ Let's get together and １１ and R １２ may form a 4- to 7-membered heterocyclyl containing the nitrogen atom to which R is attached, １１ or R １２ Alternatively, R １１ and R １２ Both of these are R ４ or R ８ Together with １１ or R １２ is bonded to a nitrogen atom, and may contain 1 to 3 additional heteroatoms selected from the group consisting of oxygen atoms, nitrogen atoms, and sulfur atoms as ring-constituting members, and the heterocyclyl or heteroaryl may be substituted with an alkyl having 1 to 6 carbon atoms, an alkenyl having 2 to 6 carbon atoms, an alkynyl having 2 to 6 carbon atoms, an aralkyl group having 6 to 10 carbon atoms, or an alkyl-substituted alkenyl group having 6 to 10 carbon atoms; * represents a bonding point to L in formula (I) at any position on the benzene ring.) (In formula (IV), R １is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms which may have a substituent; R ２ is an alkylene group having 1 to 6 carbon atoms, which may be substituted; R ３ ~R ６ are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms which may have a substituent; R ７ ~R ８ are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms which may have a substituent, and R ７ and R ８ If there are two R ７ , R ８ may be the same or different; m1 and m2 are 1 or 2; R ９ ~R １０ are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms which may have a substituent, and R ９ and R １０ If there are two R ９ , R １０ may be the same or different; n1 and n2 are 1 or 2; R １１ ~R １４ are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms which may have a substituent; １ , R ３ ~R １４At least one of the groups is an alkyl group having 1 to 6 carbon atoms and having a water-soluble group; ----- indicates a single bond or a double bond; * indicates the site of bonding to the linker L. ) The anti-DNP antibody in the fusion protein is an anti-DNP antibody or an antigen-binding fragment thereof comprising: a light chain comprising a VL-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1, a VL-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and a VL-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3; and a heavy chain comprising a VH-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4, a VH-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and a VH-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6, wherein SEQ ID NO: 1: RSSQEISGYLGW SEQ ID NO: 2: AASTLD SEQ ID NO: 3: VQYASYPYT SEQ ID NO: 4: FSNYWMN SEQ ID NO: 5: EAELESNNYATHYAESVKG SEQ ID NO: 6: YSYDSRYGY A method for fluorescently labeling a protein, wherein the anti-DNP antibody or the antigen-binding fragment thereof is a single-chain Fv (scFv). [2] The method according to [1], wherein the anti-DNP antibody consists of an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 7, and includes the amino acid sequences shown in SEQ ID NOs: 1 to 6 as CDR sequences. SEQ ID NO: 7: DIVLTQSPSSLSASPGDRVTITCRSSQEISGYLGWLQQKPDGSIKRLIYAASTLDSGVPSRFSGSRSGTDYTLTISGLEPEDFGDYYCVQYASYPYTFGQGTKVEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGDSLRLSCAASGFTFSNYWMNWVRQAPGKGLEWVGEAELESNNYATHYAESVKGRFTISRDDSEGTVYLEMNGLRSEDTGVYYCTGYSYDSRYGYWGQGTLVTVSS [3] The method according to [2], wherein the amino acid sequence is SEQ ID NO: 7.[4] The method of [1], wherein the anti-DNP antibody has an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 7, and includes the amino acid sequences of SEQ ID NOs: 1 to 6 as CDR sequences, and has at least one of the following substitutions in the amino acid sequence of SEQ ID NO: 7: (a) any one of aspartic acid at position 41, arginine at position 66, tyrosine at position 71, glutamic acid at position 79, and aspartic acid at position 85, numbered from the N-terminus, substituted with another hydrophilic amino acid (provided that the total number of negative charges after the substitution remains unchanged); or (b) any one of aspartic acid at position 149, aspartic acid at position 209, glutamic acid at position 211, glutamic acid at position 217, and serine at position 223, numbered from the N-terminus, substituted with another amino acid having a hydroxyl group in its side chain. [5] The method according to any one of [1] to [4], wherein obtaining the fusion protein comprises obtaining a polynucleotide encoding the fusion protein, obtaining a plasmid or vector capable of expressing the fusion protein, expressing the fusion protein in a cell, or isolating the expressed fusion protein. [6] The method according to any one of [1] to [5], wherein the linker is represented by T-Y, where Y represents a linking group that bonds to the fluorescent group S, and T represents a crosslinking group. [7] The method according to [6], wherein the linking group is selected from an amide group, an alkylamide group, an ester group, an alkyl ester group, a carbonylamino group, or an alkyl ether group. [8] An anti-DNP antibody or an antigen-binding fragment thereof comprising: a light chain comprising a VL-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1, a VL-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and a VL-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3; and a heavy chain comprising a VH-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4, a VH-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and a VH-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6.SEQ ID NO: 1: RSSQEISGYLGW SEQ ID NO: 2: AASTLD SEQ ID NO: 3: VQYASYPYT SEQ ID NO: 4: FSNYWMN SEQ ID NO: 5: EAELESNNYATHYAESVKG SEQ ID NO: 6: YSYDSRYGY [9] The anti-DNP antibody or antigen-binding fragment thereof according to claim 9, wherein the anti-DNP antibody or antigen-binding fragment thereof is a single-chain Fv (scFv).

[10] The anti-DNP antibody or antigen-binding fragment thereof according to [8] or [9], consisting of an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 7 and comprising the amino acid sequences shown in SEQ ID NOs: 1 to 6 as CDR sequences. SEQ ID NO: 7: DIVLTQSPSSLSASPGDRVTITCRSSQEISGYLGWLQQKPDGSIKRLIYAASTLDSGVPSRFSGSRSGTDYTLTISGLEPEDFGDYYCVQYASYPYTFGQGTKVEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGDSLRLSCAASGFTFSNYWMNWVRQAPGKGLEWVGEAELESNNYATHYAESVKGRFTISRDDSEGTVYLEMNGLRSEDTGVYYCTGYSYDSRYGYWGQGTLVTVSS

[11] The anti-DNP antibody or antigen-binding fragment thereof according to

[10] , wherein the amino acid sequence is SEQ ID NO: 7.

[12] An anti-DNP antibody or an antigen-binding fragment thereof, consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 7, including the amino acid sequences shown in SEQ ID NOs: 1 to 6 as CDR sequences, and having at least one of the following substitutions in the amino acid sequence of SEQ ID NO: 7: (a) any one of aspartic acid at position 41, arginine at position 66, tyrosine at position 71, glutamic acid at position 79, and aspartic acid at position 85, numbered from the N-terminus, substituted with another hydrophilic amino acid (provided that the total number of negative charges after the substitution remains unchanged); or (b) any one of aspartic acid at position 149, aspartic acid at position 209, glutamic acid at position 211, glutamic acid at position 217, and serine at position 223, numbered from the N-terminus, substituted with another amino acid having a hydroxyl group in its side chain.

[13] An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of [8] to

[11] .

[14] The nucleic acid according to

[13] , consisting of the nucleotide sequence shown in SEQ ID NO: 8. SEQ ID NO: 8: GATATTGTGCTGACTCAGAGCCCATCCAGCTTGAGTGCCTCTCCCGGCGATCGAGTGACCATAACATGCAGAAGCTCCCAGGAAATCAGCGGATACCTGGGCTGGTTGCAGCAGAAACCGGATGGGTCTATCAAGCGCCTGATTTATGCGGCAAGCACCCTCGACAGTGGGGTTCCTTCTCGGTTCTCTGGATCTAGGTCTGGAACAGACTATACCCTGACAATCAGTGGCCTCGAACCCGAGGATTTTGGTGACTACTATTGCGTGCAGTATGCCAGCTATCCCTACACTTTTGGACAGGGGACCAAAGTGGAGATCAAGAGAGGTGGTGGAGGGTCAGGCGGTGGTGGGTCCGGCGGAGGCGGAAGCGGA

[15] An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof according to

[12] .

[16] A plasmid or vector comprising the nucleic acid according to any one of

[13] to

[15] .

[0013] The present invention provides a method for fluorescently labeling intracellular proteins using a technology for controlling fluorescence ON / OFF, which can be applied to a variety of cells. Furthermore, the anti-DNP antibody of the present invention is capable of stably expressing fusion proteins in a variety of cells and exhibits higher fluorescence intensity than when the inventors' prior art 5D4 clone is used, making it possible to provide highly accurate fluorescent imaging of a variety of proteins.

[0014] Furthermore, while conventional fluorescent imaging methods suffer from the problem of rapid signal decay during long-term observation, the novel fluorescent labeling technique based on the anti-DNP antibody of the present invention is capable of stably maintaining fluorescence intensity over long periods of time, providing a groundbreaking method that overcomes the essential challenges of conventional methods. Furthermore, fluorescence recovery assay (FRAP) experiments using the novel fluorescent labeling technique of the present invention confirmed that fluorescence rapidly recovers after photobleaching. Thus, the novel fluorescent labeling technique of the present invention demonstrates significant utility in long-term live cell imaging, particularly in precise analysis of protein dynamics.

[0015]

[0039] Figure 1 shows the amino acid sequence of the 5D4 clone (SEQ ID NO: 12).

[0040] Figure 1 shows the results of confirming the expression of a fusion protein between an intracellular protein and hum5D4NL1-NH2 in COS7 cells.

[0041] Figure 1 shows a comparison of the fluorescence intensity derived from 6SiR-DNP for ECFP-5D4 and ECFP-hum5D4NL1-NH2 in COS7 cells.

[0042] Figure 1 shows the results of confirming the expression of hum5D4NL1-NH2-β-Actin and Lifeact-hum5D4NL1-NH2 in cultured hippocampal neurons.

[0043] Figure 1 shows the results of confirming the expression of a fusion protein between hum5D4NL1-NH2 and a presynaptic molecule.

[0044] Figure 1 shows the results of confirming the expression of a fusion protein between hum5D4NL1-NH2 and a postsynaptic molecule. 1 shows the results of sequential imaging of HeLa cells expressing the novel labeling tag of the present invention (DQ-SQSTM1) fused with the conventional SNAP tag (SNAP-SQSTM1). 2 shows the results of fluorescent imaging of microtubules in COS-7 cells expressing the novel labeling tag of the present invention (DQ-SQSTM1), HaloTag, fused with β-tubulin.

[0016] One embodiment of the present invention is a method for fluorescently labeling an intracellular protein, which comprises obtaining a fusion protein of a protein to be labeled and an anti-DNP (dinitrophenyl compound) antibody in a cell, contacting the cell with a compound represented by the following formula (I) or a salt thereof, and reacting the fusion protein with the compound represented by the following formula (I) or a salt thereof, thereby fluorescently labeling the target protein (hereinafter also referred to as the "method of the present invention").

[0017]

[0018] Each component of the method of the present invention will now be described in detail.

[0019] 1. Anti-DNP (dinitrophenyl compound) antibody The anti-DNP antibody in the fusion protein obtained intracellularly by the method of the present invention (hereinafter also referred to as the "anti-DNP antibody of the present invention") is an antibody or fragment comprising the variable regions of the heavy and light chains of the antigen-binding antibody. The anti-DNP antibody of the present invention may be an antibody in which only the variable regions of the heavy and light chains of the antibody are linked by a short amino acid linker. Examples of the amino acid linker include those in which 1 to 5, preferably 1 to 4, amino acid sequence units consisting of GGGGS (Gly-Gly-Gly-Gly-Ser) are linked together, and those in which 1 to 5 flexible linkers consisting of G and S, such as GGSGG, are linked together.

[0020] The anti-DNP antibody of the present invention is a single-chain Fv (scFv). Furthermore, the anti-DNP antibody of the present invention is preferably an antibody with a molecular weight of approximately 30 kDa. A typical antibody is a molecule with a molecular weight of approximately 60 kDa, in which a heavy chain and a light chain are connected by a disulfide bond. Because full-length antibodies are in a reducing environment within cells, they are not suitable for forming the multiple disulfide bonds necessary for normal folding, making it difficult to express them intracellularly while maintaining a normal folding state. In contrast, the antibody of the present invention has a structure in which only the variable regions of the antibody's heavy and light chains are connected by a short amino acid linker, making it relatively easy to express them intracellularly.

[0021] The anti-DNP antibody of the present invention is an anti-DNP antibody or an antigen-binding fragment thereof comprising a light chain comprising a VL-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1, a VL-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and a VL-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3, and a heavy chain comprising a VH-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4, a VH-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and a VH-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6. SEQ ID NO: 1: RSSQEISGYLGW SEQ ID NO: 2: AASTLD SEQ ID NO: 3: VQYASYPYT SEQ ID NO: 4: FSNYWMN SEQ ID NO: 5: EAELESNNYATHYAESVKG SEQ ID NO: 6: YSYDSRYGY

[0022] Anti-DNP antibodies, such as the 5D4 clone, discovered by the present inventors in previous research have the excellent property that, for example, fusion proteins with the fluorescent protein TagRFP, which is the protein to be labeled, can be expressed in HEK293T cells, and fusion proteins with the protein to be labeled, such as ECFP, β-tubulin, or β-actin, can be expressed in HeLa cells, whereby the fluorophore-DNP becomes fluorescent only upon binding of 5D4 to DNP in these cells. However, further investigation by the present inventors revealed that prior art anti-DNP antibodies, such as the 5D4 clone, occasionally result in a localization different from the original intracellular localization when expressed in cells as a fusion protein with the protein to be labeled. This was thought to be due to the instability of the 5D4 clone within cells. Therefore, starting from the 5D4 clone (sequence shown in Figure 1), the present inventors aimed to stabilize the 5D4 clone by humanizing it, which utilizes extensive knowledge and know-how in antibody stabilization, in order to improve the expression of fusion proteins within cells. Further sequence optimization resulted in the humanized heavy chain sequence hum5D4VH, and for the light chain, a less humanized sequence (hum5D4VL-low) and a more humanized sequence (hum5D4VL-high) were obtained. Evaluation of scFvs containing light and heavy chains with these sequences revealed that a higher degree of humanization reduces antibody performance (expression level of fusion protein). Here, hum5D4VH had mutations added to the CDR sequence from the 5D4 clone, while hum5D4VL-low had the same CDR sequence as the 5D4 clone. Furthermore, comparison of the amino acid sequences of hum5D4VL-low and hum5D4VL-high revealed that hum5D4VL-low had more hydrophilic amino acid side chains and a negative net charge (a difference of 5). Therefore, based on an scFv (also called hum5D4-low) having a light chain of hum5D4VL-high and a heavy chain of hum5D4VH, combinations in which mutations were introduced into the heavy and light chains were examined.As a result, it was found that an anti-DNP antibody having light chain CDRs (VL-CDR1 to 3) consisting of the same amino acid sequence as the 5D4 clone, and heavy chain VH-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4, VH-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and VH-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6, has excellent performance.

[0023] As described above, when comparing the amino acid sequences of hum5D4VL-low and hum5D4VL-high, hum5D4VL-low had more hydrophilic amino acid side chains and a negative net charge (a difference of 5). Based on this finding, when introducing mutations into the heavy and light chains of hum5D4-low, sequence design was carried out with the goal of more aggressively increasing substitutions with hydrophilic amino acids, particularly targeting loops that were unlikely to alter the overall structure. While loops were found both within and outside the CDR regions, we selected sites and amino acids that were unlikely to be disrupted by introducing mutations, taking into account alanine scan data and the three-dimensional structure of the ROSE model. As a result, an anti-DNP antibody was discovered that had the amino acid sequence of SEQ ID NO: 7, and that had a light chain (also referred to as "hum5D4NL1") with the amino acid sequence of hum5D4VL-low and a heavy chain (also referred to as "hum5D4NH2") with mutations introduced into hum5D4VH5D.

[0024] That is, a preferred embodiment of the anti-DNP antibody of the present invention is an antibody or antigen-binding fragment thereof consisting of an amino acid sequence having 90% or more, preferably 95% or more, more preferably 98% or more identity with the amino acid sequence of SEQ ID NO: 7 below, and comprising the amino acid sequences shown in SEQ ID NOs: 1 to 6 as CDR sequences. SEQ ID NO: 7: DIVLTQSPSSLSASPGDRVTITCRSSQEISGYLGWLQQKPDGSIKRLIYAASTLDSGVPSRFSGSRSGTDYTLTISGLEPEDFGDYYCVQYASYPYTFGQGTKVEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGDSLRLSCAASGFTFSNYWMNWVRQAPGKGLEWVGEAELESNNYATHYAESVKGRFTISRDDSEGTVYLEMNGLRSEDTGVYYCTGYSYDSRYGYWGQGTLVTVSS

[0025] A preferred aspect of the anti-DNP antibody of the present invention is an antibody or an antigen-binding fragment thereof having the amino acid sequence of SEQ ID NO: 7.

[0026] Another preferred embodiment of the anti-DNP antibody of the present invention is an antibody or antigen-binding fragment thereof, which consists of an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 7, and which includes the amino acid sequences shown in SEQ ID NOs: 1 to 6 as CDR sequences, and which has at least one of the following substitutions in the amino acid sequence of SEQ ID NO: 7: (a) any one of aspartic acid at position 41, arginine at position 66, tyrosine at position 71, glutamic acid at position 79, and aspartic acid at position 85, numbered from the N-terminus, is substituted with another hydrophilic amino acid (provided that the total number of negative charges after the substitution remains unchanged); or (b) any one of aspartic acid at position 149, aspartic acid at position 209, glutamic acid at position 211, glutamic acid at position 217, and serine at position 223, numbered from the N-terminus, is substituted with another amino acid having a hydroxyl group in its side chain.

[0027] The anti-DNP antibody having the amino acid sequence of SEQ ID NO: 7 is characterized by the substitution of many hydrophilic amino acids in the light chain (hum5D4NL1) and the introduction of substitutions of amino acids having hydroxyl groups in the side chains in the heavy chain (hum5D4NH2) compared to hum5D4-low. Therefore, antibodies obtained by substituting hydrophilic amino acids substituted in regions other than the CDRs of the light chain in the amino acid sequence of SEQ ID NO: 7 with other hydrophilic amino acids, or by substituting amino acids having hydroxyl groups substituted in regions other than the CDRs of the heavy chain with other amino acids having hydroxyl groups, can also be expected to exhibit performance similar to that of the anti-DNP antibody of SEQ ID NO: 7. This is because amino acids outside the CDR regions of antibodies generally contribute little to antigen recognition, and substitutions between hydrophilic amino acids are unlikely to have a significant impact on antibody stability. However, it is necessary that the total number of negative charges in the light chain remain unchanged after the substitution.

[0028] 2. Protein to be Labeled In the fluorescent labeling method of the present invention, obtaining a fusion protein of a protein to be labeled and an anti-DNP antibody may include one or more steps selected from obtaining a polynucleotide encoding the fusion protein, obtaining a plasmid or vector capable of expressing the fusion protein, introducing the plasmid or vector into a cell, expressing the fusion protein in the cell, or isolating the expressed fusion protein. Furthermore, the step of obtaining a plasmid or vector capable of expressing the fusion protein of a protein to be labeled and an anti-DNP antibody described above may also include producing a viral vector (e.g., lentivirus, adeno-associated virus, adenovirus, etc., but are not limited to these) for introducing the fusion protein of the protein to be labeled and the anti-DNP antibody into target cells (cells to which the fluorescent labeling method of the present invention is applied). A polynucleotide encoding the fusion protein can be prepared by conventional methods using a polynucleotide encoding the protein to be labeled, a polynucleotide encoding the anti-DNP antibody, etc. to prepare a plasmid or vector capable of expressing the fusion protein. Fusion proteins can be generally prepared using standard techniques (including chemical conjugation). Briefly, DNA sequences encoding the polypeptide components can be assembled separately and ligated into an appropriate expression vector. The 3' end of the DNA sequence encoding one polypeptide component is linked, with or without a peptide linker, to the 5' end of the DNA sequence encoding the second polypeptide component, so that the reading frames of the sequences are in phase. This allows translation into a single fusion peptide that retains the biological activity of both component peptides. Alternatively, a linker sequence can be used to separate the first and second polypeptides by a sufficient distance so that each polypeptide can fold into its higher-order structure and not interfere with the function of the other. Such a linker can be a peptide, polypeptide, alkyl chain, or other conventional spacer molecule.

[0029] Any protein can be used as the protein to be labeled, including, for example, cytoskeletal proteins, ion channels, receptors, fluorescent proteins, adaptor proteins, cell adhesion proteins, enzymes, and the like.

[0030] In the fluorescent labeling method of the present invention, it is preferable to obtain the fusion protein by introducing a plasmid or vector capable of expressing the fusion protein into a cell or a living organism.

[0031] A plasmid or vector capable of expressing a fusion protein that can be used in the fluorescent labeling method of the present invention can be prepared, for example, using a vector that can be induced to express in mammalian cells. Examples of mammalian cell expression-inducible vectors that can be used include, but are not limited to, available, known mammalian cell expression-inducible vectors, such as pcDNA3.1(+), pCI (Promega), pCMV-Tag (Agilent / Stratagene), and pEF1α (Takara Bio). When a mammalian cell expression-inducible vector is used, for example, a cDNA fragment of an anti-DNP antibody and, optionally, a cDNA fragment of a linker are inserted into a mammalian cell expression-inducible vector, and a cDNA fragment of the protein to be labeled, amplified by PCR, is subcloned into the restriction enzyme recognition site of the resulting vector, thereby obtaining a plasmid or vector capable of expressing a fusion protein.

[0032] A plasmid or vector capable of expressing a fusion protein that can be used in the fluorescent labeling method of the present invention can also be prepared using, for example, a viral vector (e.g., a lentiviral vector, an adeno-associated virus, an adenovirus, etc.). Any commonly available vector can be used as the lentiviral vector, adeno-associated virus, or adenovirus. Examples of such lentiviral vectors include, but are not limited to, pLV-EF1a-IRES-Puro, the pLenti series, and the pLVX series. Such a plasmid or vector can be prepared preferably by the in-fusion cloning method or the ligation method.

[0033] Non-limiting examples of methods for preparing a plasmid or vector capable of expressing a fusion protein of a protein to be labeled and an anti-DNP antibody, which can be used in the fluorescent labeling method of the present invention, are shown in the Examples. That is, examples of preparing plasmids that express fusion proteins of various proteins to be labeled and hum5D4NL1-NH2, one of the anti-DNP antibodies of the present invention, are shown in Examples 2 and 5. By referring to the descriptions in these Examples, those skilled in the art will be able to prepare plasmids or vectors that express fusion proteins of various proteins to be labeled and the anti-DNP antibody of the present invention.

[0034] 3. Compound Represented by Formula (I) or Salt Thereof The method of the present invention comprises contacting a cell from which the above-mentioned fusion protein has been obtained with a compound represented by the following formula (I) or a salt thereof:

[0035] In formula (I), S is a fluorescent group, L is a linker, and R ｂ and R ｃ is selected from the following combinations: ｂ , R ｃ ): (NO ２ , p-NO ２ ), (NO ２ , p-Br), (NO ２ , p-SO ２ Me), (NO ２ , p-Cl), (NO ２ , m-CN), (NO ２ , p-CN), (NO ２ , p-COOMe), (CF ３ , p-CF ３ ), (NO ２ , p-CONHMe), (NO ２ , m-COOMe), (NO ２ , H) where p- and m- are R ｃ are located at the para and meta positions on the benzene ring relative to L.

[0036] In this specification, unless otherwise specified, an "alkyl group" or the alkyl portion of a substituent containing an alkyl portion (for example, an alkoxy group) means, for example, an alkyl group having 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, and more preferably about 1 to 3 carbon atoms, which is linear, branched, cyclic, or a combination thereof. More specific examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a cyclopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, a cyclopropylmethyl group, an n-pentyl group, an n-hexyl group, and the like.

[0037] As used herein, the term "halogen atom" may refer to any of a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, and is preferably a fluorine atom, a chlorine atom, or a bromine atom.

[0038] The fluorescent group S is represented by any one of the following formulas (II) to (IV).

[0039] A fluorescent group represented by formula (II):

[0040] In formula (II), R １ represents a hydrogen atom or 1 to 4 identical or different monovalent substituents present on a benzene ring. ２ represents a hydrogen atom, a monovalent substituent, or a bond.

[0041] R １ , R ２ is selected from the group consisting of an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 1 to 6 carbon atoms, an alkynyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, a hydroxyl group, a carboxy group, a sulfonyl group, an alkoxycarbonyl group, a halogen atom, and an amino group, and the alkyl group having 1 to 6 carbon atoms may have one or more substituents selected from the group consisting of a halogen atom, a carboxy group, a sulfonyl group, a hydroxyl group, an amino group, and an alkoxy group.

[0042] The bond is L to R ２ This means that the compound is introduced at the position

[0043] In one preferred embodiment of the present invention, R １ are all hydrogen atoms.

[0044] In one preferred embodiment of the present invention, R ２ is an alkyl group having 1 to 6 carbon atoms (preferably a methyl group), a carboxyl group, a methoxy group, a hydroxymethyl group, or a bond (i.e., L (i.e., a linker) is R ２ (introduced at position ).

[0045] In formula (II), R ３ and R ４ R each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom. ３ or R ４ When R represents an alkyl group, the alkyl group may contain one or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, etc., and for example, R ３ or R ４ The alkyl group represented by R may be a halogenated alkyl group, a hydroxyalkyl group, a carboxyalkyl group, etc. ３ and R ４ are preferably each independently a hydrogen atom or a halogen atom, and R ３ and R ４ are both hydrogen atoms, or R ３ and R ４ It is more preferable that both of are fluorine atoms or chlorine atoms.

[0046] In formula (II), R ５ and R ６ each independently represents an alkyl group having 1 to 6 carbon atoms or an aryl group, provided that when X is an oxygen atom, R ５ and R ６ does not exist. When X is a silicon atom, R ５ and R ６ are each preferably independently an alkyl group having 1 to 3 carbon atoms, and R ５ and R ６ It is more preferable that both R ５ and R ６The alkyl group represented by may contain one or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, etc., and for example, R ５ or R ６ The alkyl group represented by R may be a halogenated alkyl group, a hydroxyalkyl group, a carboxyalkyl group, etc. ５ or R ６ When represents an aryl group, the aryl group may be either a monocyclic aromatic group or a fused aromatic group, and the aryl ring may contain one or more ring-constituting heteroatoms (e.g., nitrogen atom, oxygen atom, sulfur atom, etc.). The aryl group is preferably a phenyl group. One or more substituents may be present on the aryl ring. The substituents may include, for example, one or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, etc.

[0047] In formula (II), R ７ and R ８ each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; R ３ and R ４ This is the same as that explained for R. ７ and R ８ are preferably both hydrogen atoms, both chlorine atoms, or both fluorine atoms.

[0048] X represents an oxygen atom or a silicon atom, and preferably represents an oxygen atom.

[0049] * represents a bonding point to L in formula (I) at any position on the benzene ring.

[0050] A fluorescent group represented by formula (III):

[0051] In formula (III), R １ ~R ８ , X is as defined in formula (II).

[0052] In formula (III), R ９ and R １０each independently represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. ９ and R １０ Let's get together and ９ and R １０ may form a 4- to 7-membered heterocyclyl containing a nitrogen atom to which R is bonded. ９ or R １０ Alternatively, R ９ and R １０ Both of these are R ３ or R ７ Together with ９ or R １０ may form a 5- to 7-membered heterocyclyl or heteroaryl containing a nitrogen atom to which is bonded. The ring may contain 1 to 3 additional heteroatoms selected from the group consisting of oxygen atoms, nitrogen atoms, and sulfur atoms, and the heterocyclyl or heteroaryl may be substituted with an alkyl having 1 to 6 carbon atoms, an alkenyl having 2 to 6 carbon atoms, an alkynyl having 2 to 6 carbon atoms, an aralkyl group having 6 to 10 carbon atoms, or an alkyl-substituted alkenyl group having 6 to 10 carbon atoms. In this case, the heterocyclyl or heteroaryl may have one or more substituents.

[0053] In formula (III), R １１ and R １２ R each independently represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. １１ and R １２ Let's get together and １１ and R １２ may form a 4- to 7-membered heterocyclyl containing a nitrogen atom to which R is bonded. １１ or R １２ Alternatively, R １１ and R １２ Both of these are R ４ or R ８ Together with １１ or R １２may form a 5- to 7-membered heterocyclyl or heteroaryl containing a nitrogen atom to which is bonded. The ring may contain 1 to 3 additional heteroatoms selected from the group consisting of oxygen atoms, nitrogen atoms, and sulfur atoms, and the heterocyclyl or heteroaryl may be substituted with an alkyl having 1 to 6 carbon atoms, an alkenyl having 2 to 6 carbon atoms, an alkynyl having 2 to 6 carbon atoms, an aralkyl group having 6 to 10 carbon atoms, or an alkyl-substituted alkenyl group having 6 to 10 carbon atoms. In this case, the heterocyclyl or heteroaryl may have one or more substituents.

[0054] * represents a bonding site to L in formula (I) at any position on the benzene ring. In formula (III), * represents a bonding site (bonding point, the same applies hereinafter) to L in formula (I) at any position on the benzene ring. L can be bonded to any position on the benzene ring bonded to the xanthene ring skeleton, but is preferably bonded to the 4-position of the benzene ring.

[0055] A fluorescent group represented by formula (IV):

[0056] In formula (IV), R １ is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms which may have a substituent. The alkyl group having 1 to 6 carbon atoms may have one or more substituents selected from the group consisting of a halogen atom, an alkoxy group, an amino group, and a water-soluble group. Here, examples of the water-soluble group include -SO ３ － , -OSO ３ － , -PO ３ ２－ , -OPO ３ ２－ , —OH or —COO － is selected from.

[0057] In formula (IV), R ２ is an alkylene group having 1 to 6 carbon atoms. The alkylene group may be substituted, and examples of the substituent include an alkyl group, a halogen atom, an alkoxy group, and an amino group.

[0058] In formula (IV), R ３ ~R ６ are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms which may have a substituent.

[0059] In formula (IV), R ７ ~R ８ are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms which may have a substituent. ７ and R ８ When there are two (when m1 and m2 are 2), each R ７ , R ８ may be the same or different.

[0060] m1 and m2 are 1 or 2. Here, R ７ , R ８ When a double bond is formed between the carbon atoms to which each of the carbon atoms is bonded, m1 and m2 are 1, and when a single bond is formed, m1 and m2 are 2.

[0061] In formula (IV), R ９ ~R １０ are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms which may have a substituent. ９ and R １０ When there are two (when n1 and n2 are 2), each R ９ , R １０ may be the same or different.

[0062] n1 and n2 are 1 or 2. Here, R ９ , R １０ When a double bond is formed between the carbon atoms to which n1 and n2 are bonded, n1 and n2 are 1, and when a single bond is formed, n1 and n2 are 2.

[0063] In formula (IV), R １１ ~R １４ are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms which may have a substituent.

[0064] R ３ ~R １４The alkyl group having 1 to 6 carbon atoms defined as: may each have one or more substituents selected from the group consisting of a halogen atom, an alkoxy group, an amino group, and a water-soluble group. ３ － , -OSO ３ － , -PO ３ ２－ , -OPO ３ ２－ , —OH or —COO － is selected from.

[0065] R １ , R ３ ~R １４ At least one of the groups is an alkyl group having 1 to 6 carbon atoms and having a water-soluble group. It is essential that the fluorescent group represented by formula (IV) has an alkyl group having 1 to 6 carbon atoms and having at least one water-soluble group in its molecule, which makes it possible to avoid non-specific staining by the fluorescent group. The alkyl group having 1 to 6 carbon atoms and having a water-soluble group is R １ , R ３ ~R １４ In particular, R ７ , R ９ It is preferable to introduce it into

[0066] In formula (IV), ----- represents a single bond or a double bond.

[0067] * indicates the site of binding to the linker L.

[0068] The linker of formula (I) can be represented by TY, where Y represents a linking group that bonds with the fluorescent group S, and T represents a bridging group.

[0069] The bonding group for Y is selected from an amide group (-CONH-, -CONR'-, -R-CONH-, -R-CONR'-), an alkylamide group (-CONH-R-, -CONR'-R-), an ester group (-COO-), an alkyl ester group (-R-COO-, -COO-R-), a carbonylamino group (-NHCO-, -NR'CO-), or an alkyl ether group (-RO-, -OR-). Here, R represents a divalent hydrocarbon group, preferably an alkylene group having 1 to 10 carbon atoms, more preferably an alkylene group having 1 to 5 carbon atoms. R' represents an alkyl group having 1 to 5 carbon atoms.

[0070] The bridging group of T can be any bridging group that functions as a spacer connecting the bonding group of Y and the benzene ring of the compound of formula (I). Examples include, but are not limited to, substituted or unsubstituted divalent hydrocarbon groups (alkanes, alkenes, alkynes, cycloalkanes, aromatic hydrocarbons, etc.), dialkyl ether groups (e.g., dimethyl ether, diethyl ether, methyl ethyl ether, etc.), ethylene glycol groups, diethylene glycol groups, triethylene glycol groups, polyethylene glycol groups, amide groups, carbonyl groups, and heterocyclic groups (e.g., divalent piperidine rings), and combinations of two or more thereof. In addition, the bridging group may have a functional group at one or both ends that can bond to Y and the benzene ring of the compound of formula (I). Examples of such functional groups include an amino group, an alkylamino group, an aminoalkyl group, a carbonyl group, a carboxyl group, an amide group, and an alkylamide group. In addition, the bridging group of T can be, for example, an amino group, an alkylamino group, an aminoalkyl group, a carbonyl group, a carboxyl group, an amide group, an alkylamide group, etc. １ -(W)-T ２ The formula: １ and T ２ When W is present, it is possible to use the bridging group exemplified above. １ and T ２Examples of such a cross-linking group include, but are not limited to, a group in which a triethylene glycol group and a diethylene glycol group are linked via an amide group, an alkylamide group, etc. Furthermore, T １ -(W)-T ２ The crosslinking group represented by the formula may have, at one or both ends thereof, a functional group (e.g., an amino group, an alkylamino group, an aminoalkyl group, a carbonyl group, a carboxyl group, an amide group, an alkylamide group, etc.) that can bond to Y, the benzene ring of the compound of formula (I).

[0071] Non-limiting examples of compounds represented by formula (I) or salts thereof are shown below.

[0072] The compound represented by formula (I) may have one or more asymmetric carbon atoms depending on the type of substituent, and may exist as stereoisomers such as optical isomers or diastereoisomers. Pure stereoisomers, any mixture of stereoisomers, racemates, etc. are all included in the scope of the present invention. In addition, the compound of the present invention represented by formula (I) or a salt thereof may exist as a hydrate or solvate, and all of these substances are included in the scope of the present invention. The type of solvent that forms the solvate is not particularly limited, and examples include solvents such as ethanol, acetone, and isopropanol.

[0073] The method for producing the compounds included in the compound represented by formula (I) is specifically described in the examples of Patent Document 1. A person skilled in the art can produce the compound of the present invention represented by formula (I) by appropriately selecting reaction raw materials, reaction conditions, reaction reagents, etc. based on these explanations and by modifying or altering these methods as necessary.

[0074] 4. Fluorescent Labeling Method of the Present Invention The method of the present invention comprises fluorescently labeling a target protein by reacting the above-described fusion protein with a compound represented by formula (I) or a salt thereof. In the fluorescent labeling method of the present invention, the step of reacting the fusion protein with a compound represented by formula (I) or a salt thereof may be carried out in a cell expressing the fusion protein, in a living body, or in a cell ex vivo, or may be carried out in vitro using an isolated fusion protein. When labeling is carried out in vitro, it may be carried out, for example, in a buffer solution (pH 7.4) at 25°C.

[0075] The compound represented by formula (I) or a salt thereof is quenched by DNP in the steady state and does not emit fluorescence. However, when DNP binds to an anti-DNP antibody expressed in a cell, the quenching ability of DNP disappears, and the fluorophore-dye becomes fluorescent, making it possible to fluorescently label a target protein in the cell.

[0076] Because the compound represented by formula (I) or its salt is sufficiently quenched when not bound to an anti-DNP antibody, the fluorescence from the compound represented by formula (I), even when present extracellularly but not bound to an anti-DNP antibody, is suppressed to a level where its effect on the spatial resolution in observing the target molecules or organelles is negligible. The method of the present invention is particularly useful in high-throughput screening (HTS) for drug discovery and other applications, as it does not require a step to remove unnecessary fluorescent dyes from the system during fluorescence observation. HTS is required to increase the overall efficiency of the screening system by reducing steps such as probe washing, thereby assaying a large number of samples with extremely high efficiency. Sequential reaction and measurement without washing and other processes is called the "mix and measure" or "homogeneous" method, and is considered particularly desirable for drug screening assays involving tens of thousands to hundreds of thousands of compounds. A screening system incorporating the DNP tag of the present invention and the compound represented by formula (I) enables the construction of an HTS system that does not require a washing process for excess fluorescent dyes.

[0077] The method of the present invention can be used to fluorescently label target proteins in various cells, including, for example, nerve cells, glial cells, immune cells, kidney cells, and fibroblasts.

[0078] 5. Anti-DNP Antibody or Antigen-Binding Fragment Thereof Another aspect of the present invention is an anti-DNP antibody or antigen-binding fragment thereof comprising a light chain comprising a VL-CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 1, a VL-CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 2, and a VL-CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and a heavy chain comprising a VH-CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a VH-CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a VH-CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 6 (hereinafter also referred to as "anti-DNP antibody 1 or antigen-binding fragment 1 thereof"). SEQ ID NO: 1: RSSQEISGYLGW SEQ ID NO: 2: AASTLD SEQ ID NO: 3: VQYASYPYT SEQ ID NO: 4: FSNYWMN SEQ ID NO: 5: EAELESNNYATHYAESVKG SEQ ID NO: 6: YSYDSRYGY The anti-DNP antibody of the present invention may be an antibody in which only the variable regions of the heavy and light chains of the antibody are linked by a short amino acid linker. Examples of amino acid linkers include those in which 1 to 5, preferably 1 to 4, amino acid sequence units consisting of GGGGS (Gly-Gly-Gly-Gly-Ser) are linked together, and those in which 1 to 5 flexible linkers consisting of G and S, such as GGSGG, are linked together.

[0079] The anti-DNP antibody or antigen-binding fragment thereof of the present invention is preferably a single-chain Fv (scFv).

[0080] A preferred embodiment of the anti-DNP antibody of the present invention is an antibody or antigen-binding fragment thereof consisting of an amino acid sequence having 90% or more, preferably 95% or more, more preferably 98% or more identity with the amino acid sequence of SEQ ID NO: 7 below, and comprising the amino acid sequences shown in SEQ ID NOs: 1 to 6 as CDR sequences (hereinafter also referred to as "anti-DNP antibody 2 or antigen-binding fragment 2"). SEQ ID NO: 7: DIVLTQSPSSLSASPGDRVTITCRSSQEISGYLGWLQQKPDGSIKRLIYAASTLDSGVPSRFSGSRSGTDYTLTISGLEPEDFGDYYCVQYASYPYTFGQGTKVEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGDSLRLSCAASGFTFSNYWMNWVRQAPGKGLEWVGEAELESNNYATHYAESVKGRFTISRDDSEGTVYLEMNGLRSEDTGVYYCTGYSYDSRYGYWGQGTLVTVSS

[0081] A preferred aspect of the anti-DNP antibody of the present invention is an antibody having the amino acid sequence of SEQ ID NO: 7 or an antigen-binding fragment thereof (hereinafter also referred to as "anti-DNP antibody 3 or antigen-binding fragment thereof 3").

[0082] The identity and similarity of anti-DNP antibodies can be readily calculated by known methods, including those described in "Computational Molecular Biology," Lesk, A. M. (ed.), Oxford University Press, New York (1988); "Biocomputing: Informatics and Genome Projects," Smith, D. W. (ed.), Academic Press, New York (1993); "Computer Analysis of Sequence Data," Part 1, Griffin, A. M. and Griffin, H. G. (ed.), "Computer Analysis of Sequence Data," Part 2, Griffin, A. M. and Griffin, H. G. (ed.), "Computer Analysis of Sequence Data," ... , Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., Eds., M. Stockton Press, New York (1991); and Carillo et al., SIAM J. Applied Math., 48:1073 (1988).

[0083] Another preferred aspect of the anti-DNP antibody of the present invention is an antibody consisting of an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 7, including the amino acid sequences shown in SEQ ID NOs: 1 to 6 as CDR sequences, and which has at least one of the following substitutions in the amino acid sequence of SEQ ID NO: 7: (a) any one of aspartic acid at position 41, arginine at position 66, tyrosine at position 71, glutamic acid at position 79, and aspartic acid at position 85, numbered from the N-terminus, is substituted with another hydrophilic amino acid (provided that the total number of negative charges after the substitution remains unchanged); or (b) any one of aspartic acid at position 149, aspartic acid at position 209, glutamic acid at position 211, glutamic acid at position 217, and serine at position 223, numbered from the N-terminus, is substituted with another amino acid having a hydroxyl group in its side chain; The antibody or antigen-binding fragment thereof consists of the amino acid sequence specified above (hereinafter also referred to as "anti-DNP antibody 4 or antigen-binding fragment thereof 4").

[0084] The method of the present invention can further include observing the fluorescent response using a fluorescent imaging means. The fluorescent response can be observed using a fluorometer with a wide measurement wavelength range, or the fluorescent response can be visualized using a fluorescent imaging means capable of displaying a two-dimensional image. The use of a fluorescent imaging means allows the fluorescent response to be visualized two-dimensionally, enabling instantaneous visualization of labeled proteins within cells. Any device known in the art can be used as the fluorescent imaging device. In some cases, the reaction between the sample to be measured and the fluorescent probe can be detected by changes in the ultraviolet-visible absorption spectrum (e.g., changes in absorbance at specific absorption wavelengths).

[0085] The method of the present invention may further include observing the fluorescence response using super-resolution imaging. The super-resolution imaging is preferably performed using stimulated emission depletion microscopy (STED microscopy) or single-molecule localization microscopy. Super-resolution imaging using single-molecule localization microscopy can be performed, for example, as described in M. J. Rust, M. Bates, X. Zhuang, "Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)." Nat Methods 3, 793-795 (2006)" or M. Heilemann, S. van de Linde, M. Schuettpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld, M. Sauer, "Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes." Angew Chem Int Ed Engl 47, 6172-6176 (2008)."Furthermore, super-resolution imaging using STED microscopy, for example, super-resolution imaging using stimulated emission depletion microscopy (STED microscopy), can be performed based on the description in, for example, the aforementioned Non-Patent Document 8 (SW Hell, J. Wichmann, Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt Lett. 19(11):780-782, 1994) or Non-Patent Document 9 (K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, SW Hell, STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature. 440(7086):935-939, 2006).

[0086] Another aspect of the present invention is an isolated nucleic acid encoding any of the above-mentioned anti-DNP antibodies or antigen-binding fragments thereof (i.e., anti-DNP antibodies 1 to 4, etc., and antigen-binding fragments thereof 1 to 4, etc.).

[0087] The preferred side of the nucleic acid of the present invention is the nucleic acid of the following sequence number 8: GATATTGTGCTGACTCAGAGCCCATCCAGCTTGAGTGCCTCTCCCGGCGATCGAGTGACCATAACATGCAGAAGCTCCCAGGAAATCAGCGGATACCTGGGCTGGTTGCAGCAGAAACCGGATGGGTCTATCAAGCGCCTGATTTATGCGGCAAGCACCCTCGACAGTGGGGTTCCTTTCCGTTC TCTGGATCTAGGTCTGGAACAGACTATACCCTGACAATCAGTGGCCTCGAACCCGAGGATTTTGGTGACTACTATTGCGTGCAGTATGCCAGCTATCCCTACACTTTTGGACAGGGGACCAAAGTGGAGATCAAGAGAGGTGGTGGAGGGTCAGGCGGTGGTGGGTCCGGCGGAGGCGGAAGCGGA GGAGGCGGATCAGAGGTGCAACTGGTCGAGAGCGGCGGTGGGCTCGTACAACCTGGCGACAGCCTGAGACTGTCCTGTGCTGCAAGTGGCTTCACCTTCTCCAACTACTGGATGAATTGGGTCAGGCAGGCTCCAGGTAAAGCCTGGAATGGGTTGGGGAGGCCGAGCTGGAGTCCAACAACTAC GCCACTCACTACGCTGAGTCAGTAAAGGGCCGGTTTACGATAAGTCGCGACGACAGCGAGGGGACAGTGTACCTCGATGAATGGGCTTCGTTCCGAAGATACTGGCGTGTACTACTGTACCGGGTATTCCTACGACTCAAGGTATGGGTATTGGGGCCAAGGCACACTTGTCACGGTTTCATCC

[0088] Furthermore, in the nucleic acid consisting of the nucleotide sequence of SEQ ID NO: 8, the initiation codon when encoding the anti-DNP antibody or antigen-binding fragment thereof of the present invention is ATG, and a nucleic acid consisting of a nucleotide sequence which has this as the first codon (a nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 9 below) is also within the scope of the present invention. SEQ ID NO: 9: ATGGATATTGTGCTGACTCAGAGCCCATCCAGCTTGAGTGCCTCTCCCGGCGATCGAGTGACCATAACATGCAGAAGCTCCCAGGAAATCAGCGGATACCTGGGCTGGTTGCAGCAGAAACCGGATGGGTCTATCAAGCGCCTGATTTATGCGGCAAGCACCCTCGACAGTGGGGTTCCTTCTCGGTTCTCTGGATCTAGGTCTGGAACAGACTATACCCTGACAATCAGTGGCCTCGAACCCGAGGATTTTGGTGACTACTATTGCGTGCAGTATGCCAGCTATCCCTACACTTTTGGACAGGGGACCAAAGTGGAGATCAAGAGAGGTGGTGGAGGGTCAGGCGGTGGTGGGTCCGGCGGAGGCGGAAGCG GAGGAGGCGGATCAGAGGTGCAACTGGTCGAGAGCGGCGGTGGGCTCGTACAACCTGGCGACAGCCTGAGACTGTCCTGTGCTGCAAGTGGCTTCACCTTCTCCAACTACTGGATGAATTGGGTCAGGCAGGCTCCAGGTAAAGGCCTGGAATGGGTTGGGGAGGCCGAGCTGGAGTCCAACAACTA CGCCACTCACTACGCTGAGTCAGTAAAGGGCCGGTTTACGATAAGTCGCGACGACAGCGAGGGGACAGTGTACCTCGAGATGAATGGGCTTCGTTCCGAAGATACTGGCGTGTACTACTGTACCGGGTATTCCTACGACTCAAGGTATGGGTATTGGGGCCAAGGCACACTTGTCACGGTTTCATCC

[0089] Another aspect of the invention is a plasmid or vector comprising a nucleic acid of the invention.

[0090] Another embodiment of the present invention is a kit for a method for fluorescently labeling a protein, the kit comprising a fluorescent probe used in the method for fluorescent labeling of the present invention, which comprises a compound of formula (I) or a salt thereof, and a plasmid or vector used in the method for fluorescent labeling of the present invention. The kit for the fluorescent labeling method of the present invention can be suitably used in a super-resolution imaging method.

[0091] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0092] [Example 1] Modification of the Amino Acid Sequence of the 5D4 Clone The amino acid sequence of the 5D4 clone was modified based on the following three principles. First, the framework of the 5D4 clone was changed to a framework derived from a human antibody, which has high folding efficiency and scFv thermodynamic stability and is highly homologous to the 5D4 clone in amino acid sequence. Specifically, a search using IGBLAST confirmed that the amino acid sequences of the light chain (VL) and heavy chain (VH) of the 5D4 clone are highly homologous to human IGKV1 and IGHV3, respectively. IGKV1 and IGHV3 were adopted as frameworks because they have been reported to contribute to improving the folding efficiency and thermodynamic stability of scFv (see Reference 1 below). However, for the amino acids predicted to be present at the contact surface between VL and VH (G34, L36, D41, R46, T53, D55, N163, E178, H189) (Reference 2 below), the amino acid residues of the 5D4 clone were maintained.

[0093] Second, to improve the folding efficiency and structural stability of the 5D4 clone, some amino acids in the framework were substituted with proline or glycine (L15P, S77G, S80P, A84G, E170G, A177G, S207G, N215G). Substitution with glycine contributes to protein flexibility, while substitution with proline contributes to controlling protein torsion. Both substitutions are thought to improve the folding efficiency and structural stability of scFv (References 2 and 3 below).

[0094] Third, to suppress intracellular aggregation of the 5D4 clone and improve its structural stability, hydrophobic amino acids, which are expected to improve intracellular solubility, were substituted with hydrophilic amino acids (see Reference 4 below), and basic amino acids, which pose a risk of intracellular aggregation, were substituted with acidic amino acids (see Reference 5 below) (I179A, R180E, K182E, Y230S).

[0095] References 1. S. Ewert, T. Huber, A. Honegger, A. Pluckthun, Biophysical properties of human antibody variable domains. J. Mol. Biol. 325, 531-553 (2003). 2. S. Ewert, A. Honegger, A. Pluckthun, Stability improvement of antibodies for extracellular and intracellular applications: CDR grafting to stable frameworks and structure-based framework engineering. Methods. 34, 184-199 (2004). 3. K. Watanabe, T. Masuda, H. Ohashi, H. Mihara, Y. Suzuki, Multiple proline substitutions cumulatively thermostabilize Bacillus cereus ATCC7064 oligo-1,6-glucosidase. Irrefragable proof supporting the proline rule. Eur. J. Biochem. 226, 277-283 (1994). 4. L. Nieba, A. Honegger, C. Krebber, A. Pluckthun, Disrupting the hydrophobic patches at the antibody variable / constant domain interface: improved in vivo folding and physical characterization of an engineered scFv fragment. Protein Eng. 10, 435-444 (1997). 5. E. Kvam, M. R. Sierks, C. B. Shoemaker, A.Messer, Physico-chemical determinants of soluble intrabody expression in mammalian cell cytoplasm. Protein Eng. Des. Sel. 23, 489-498 (2010).

[0096] Based on the first and second guidelines described above, starting with the 5D4 clone (sequence shown in Figure 1), the 5D4 clone was humanized and its sequence optimized to obtain a humanized heavy chain sequence: hum5D4VH (also referred to as "hum5D4NL1"), and a light chain sequence with a low degree of humanization: hum5D4VL-low (also referred to as "hum5D4NH1") and a light chain sequence with a high degree of humanization (hum5D4VL-high). Evaluation of scFvs containing the light and heavy chains with these sequences revealed that a high degree of humanization reduced the antibody performance (expression level of the fusion protein).

[0097] The sequences of hum5D4NL1 and hum5D4NH1 are shown below: The sequences were confirmed by DNA sequencing using the Sanger method.

[0098] <Sequence of hum5D4NL1> SEQ ID NO: 10: DIVLTQSPSSLSASPGDRVTITCRSSQEISGYLGWLQQKPDGSIKRLIYAASTLDSGVPSRFSGSRSGTDYTLTISGLEPEDFGDYYCVQYASYPYTFGQGTKVEIKR

[0099] <Sequence of hum5D4NH1> SEQ ID NO: 11: EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMNWVRQAPGKGLEWVGEIRLKSNNYATHYAESVKGRFTISRDNSKGTVYLQMNGLRAEDTGVYYCTGYYYDSRYGYWGQGTLVTVSS

[0100] Next, based on scFvs having the sequences of hum5D4NL1 and hum5D4NH1, combinations in which mutations were introduced into the heavy and light chains were examined, and the amino acid sequence was modified based on the third guideline above, resulting in hum5D4NL1-NH2. The sequence was confirmed by DNA sequencing using the Sanger method, and it was confirmed that hum5D4NL1-NH2 has CDRs (VL-CDR1-3) consisting of the amino acid sequences shown in SEQ ID NOs: 1 to 3, and CDRs (VH-CDR1-3) consisting of the amino acid sequences shown in SEQ ID NOs: 4 to 6, and that its amino acid sequence is shown in SEQ ID NO: 7.

[0101] Example 2: Preparation of Plasmids (1) Preparation of Plasmid for Expression of hum5D4NL1-NH2 Fusion Protein pECFP-hum5D4NL1-NH2 A plasmid (pECFP-hum5D4NL1-NH2) for introducing a fusion protein of ECFP and hum5D4NL1-NH2 into COS-7 cells was prepared according to the following procedure. hum5D4NL1-NH2 cDNA with added BglII and BamHI sites was synthesized as an artificial gene (Eurofins). This artificial gene was subcloned into the BglII / BamHI site of pECFP-C to obtain pECFP-hum5D4NL1-NH2. The sequence was confirmed by DNA sequencing using the Sanger method, and it was confirmed that the nucleic acid sequence of hum5D4NL1-NH2 cDNA is set forth in SEQ ID NO:8.

[0102] Expression plasmids for fusion proteins of hum5D4NL1-NH2 with β-tubulin, Zyxin, TOM20, Rab5a, Vinculin, or Stim1 The cDNA fragment of hum5D4NL1-NH2 and the cDNA fragment of the GGGSx4 linker were inserted into pcDNA3.1(+), which is inducible for expression in mammalian cells, to prepare pcDNA-hum5D4NL1-NH2-GGGSx4 and pcDNA-GGGSx4-hum5D4NL1-NH2. Using any of the plasmids, pcDNA-β-Tubulin-hum5D4NL1-NH2 was used, pcDNA-Histon2B-hum5D4NL1-NH2, pcDNA-Zyxin-GGGSx4-hum5D4NL1-NH2, pcDNA-TOM20-GGGSx4-hum5D4NL1-NH2, pcDNA-hum5D4NL1-NH2-GGGSx4-Rab5a, pcDNA-hum5D4NL1-NH2-GGGSx4-Vinculin, and pcDNA-hum5D4NL1-NH2-GGGSx4-Stim1 were prepared. The procedure for preparing the above plasmids is shown below.

[0103] The hum5D4NL1-NH2 cDNA fragment was amplified by PCR using pcDNA-hum5D4NL1-NH2-GGGSx4, pcDNA-GGGSx4-hum5D4NL1-NH2, and pECFP-hum5D4NL1-NH2 as templates (forward primer: 5'-CCCGAATTCACCATGGATATTGTGCTGACTCAGAGCCC-3', reverse primer: 5'-GGGTCTAGATTAGGATGAAACCGTGACAAGTGTGCC-3') and subcloned into the EcoRI / XbaI sites of pcDNA3.1(+) (pcDNA3.1-EcoRHum5D4NL1-NH2-XbaI). Furthermore, a cDNA fragment of hum5D4NL1-NH2 was amplified by PCR using pECFP-hum5D4NL1-NH2 as a template (forward primer: 5'-CCCGCTAGCACCATGGATATTGTGCTGACTCAGAGCCC-3', reverse primer: 5'-GGGAAGCTTGGATGAAACCGTGACAAGTGTGCC-3') and subcloned into the NheI / HindIII site of pcDNA3.1(+) (pcDNA3.1-NheI-hum5D4NL1-NH2-HindIII). To add a linker sequence (GGGSGGGSGGGSGGGS) to the N-terminus of hum5D4NL1-NH2, an oligo DNA encoding the linker sequence was inserted into the HindIII / EcoRI site of pcDNA3.1-EcoRI-hum5D4NL1-NH2-XbaI (pcDNA-GGGSx4-hum5D4NL1-NH2). To add a linker sequence (GGGSGGGSGGGSGGGS) to the C-terminus of hum5D4NL1-NH2, an oligo DNA encoding the linker sequence was inserted into the HindIII / EcoRI site of pcDNA3.1-NheHum5D4NL1-NH2-HindIII (pcDNA-hum5D4NL1-NH2-GGGSx4).

[0104] A β-Tubulin cDNA fragment was amplified by PCR using pcDNA-β-Tubulin-GGGSx4-hum5D4NL1-NH2 pTBB5-GGGSx4-5D4 as a template (forward primer: 5'-GTTTAAAACTTAAGCTTACCATGAGGGAAATCGTGC-3', reverse primer: 5'-AACCTCCACCAAGCTTGGCCTCCTCTTCTGCCTC-3') and subcloned into the HindIII site of pcDNA-GGGSx4-hum5D4NL1-NH2 using In-Fusion Snap Assembly Master Mix (TAKARA, hereafter referred to as In-fusion).

[0105] pcDNA-Histon2B-hum5D4NL1-NH2. The cDNA fragment of GGGSx4-hum5D4NL1-NH2 was amplified by PCR using pcDNA-GGGSx4-hum5D4NL1-NH2 as a template (forward primer: 5'-GGGACCGGTCGCCACCATGGATATTGTGCTGACTCAGAGCCC-3', reverse primer: 5'-GTCGCGGCCGCTTAGGATGAAACCGTGACAAGTGTGC-3'). This fragment was then subcloned into the AgeI / NotI sites of pH2B-emiRFP703 (Addgene #136567) to obtain pcDNA-Histon2B-hum5D4NL1-NH2.

[0106] pcDNA-Zyxin-GGGSx4-hum5D4NL1-NH2: A Zyxin cDNA fragment was amplified by PCR using tdTomato-Zyxin (Addgene #58148) as a template (forward primer: 5'-GTTTAAAACTTAAGCTTACCATGGCGGCCCCCCCGC-3, reverse primer: 5'-AACCTCCACCAAGCTTCGTCTGGGCTCTAGCAGT-3'). This was then subcloned into the HindIII site of pcDNA-GGGSx4-hum5D4NL1-NH2 by in-fusion to obtain pcDNA-Zyxin-GGGSx4-hum5D4NL1-NH2.

[0107] pcDNA-TOM20-GGGSx4-hum5D4NL1-NH2 A TOM20 cDNA fragment was amplified by PCR using pTOM20-ddGFP-B (Addgene #40291) as a template (forward primer: 5'-GTTTAAAACTTAAGCTTACCATGGTGGGTCGGAAC-3', reverse primer: 5'-AACCTCCACCAAGCTTTTCCACATCATCTTCAGCC-3') and subcloned into the HindIII site of pcDNA-GGGSx4-hum5D4NL1-NH2 by in-fusion to obtain pcDNA-TOM20-GGGSx4-hum5D4NL1-NH2.

[0108] A Rab5a cDNA fragment was amplified by PCR using pcDNA-hum5D4NL1-NH2-GGGSx4-Rab5a mTagBFP2-Rab5a (Addgene #55322) as a template (forward primer: 5'-AGGTGGCTCAGAATTCATGGCTAGTCGAGGCGCA-3', reverse primer: 5'-GATATCTGCAGAATTCTTAGTTACTACAACACTGATTC-3'). This was then subcloned into the EcoRI site of pcDNA-hum5D4NL1-NH2-GGGSx4 by in-fusion to obtain pcDNA-hum5D4NL1-NH2-GGGSx4-Rab5a.

[0109] A Vinculin cDNA fragment was amplified by PCR using pcDNA-hum5D4NL1-NH2-GGGSx4-Vinculin mTagBFP2-Vinculin (Addgene #55334) as a template (forward primer: 5'-AGGTGGCTCAGAATTCATGCCAGTGTTTCATACGC-3', reverse primer: 5'-GATATCTGCAGAATTCCTACTGGTACCAGGGAGT-3'). This was then subcloned into the EcoRI site of pcDNA-hum5D4NL1-NH2-GGGSx4 by in-fusion to obtain pcDNA-hum5D4NL1-NH2-GGGSx4-Vinculin.

[0110] pcDNA-hum5D4NL1-NH2-GGGSx4-Stim1 Using pcDNA-hum5D4NL1-NH2-GGGSx4 as a template, a cDNA fragment of hum5D4NL1-NH2-GGGSx4 with NotI and XhoI sites added was amplified by PCR (forward primer: 5'-CAAGCGGCCGCATGGATATTGTGCTGACTCAGAGCCC-3', reverse primer: 5'-GGGCTCGAGAGACCGCGGGGATGAAACCGTGACAAGTGTGCC-3') and subcloned into the NotI / XhoI sites of pcDNA3.1(+)-5D4-Stim1 to obtain pcDNA-hum5D4NL1-NH2-GGGSx4-Stim1.

[0111] (2) Lentivirus production plasmid pLenti-CaMKII-HC20-EGFP HC20 pEYFPN1 anti-Homer1 A HC20 cDNA fragment was amplified by PCR using nanobody (Addgene #135220) as a template (forward primer: 5'-CGGGCTGCAGGAATTCATGGCCGAGGTGCAGCTG-3', reverse primer: 5'-GCCCTTGCTCACCATAGCGTAGTCCGGCAGCTG-3'). A EGFP cDNA fragment was amplified using pLenti-CaMKII-EGFP as a template (forward primer: 5'-ATGGTGAGCAAGGGCGAGG, reverse primer: 5'-GCTTGATATCGAATTCTTACTTGTACAGCTCGTCCATGCCG-3'). This cDNA fragment was subcloned into the EcoRI site of pLenti-CaMKII by in-fusion to obtain pLenti-CaMKII-HC20-EGFP.

[0112] pLenti-CaMKII-GPHN.FingR-eGFP pCAG_GPHN.FingR-eGFP-CCR5TC (Addgene #46296) was digested with SmaI and XhoI to obtain the GPHN.FingR-eGFP-CCR5TC fragment, which was then subcloned into the SmaI / XhoI site of pLenti-CaMKII to obtain pLenti-CaMKII-GPHN.FingR-EGFP.

[0113] pLenti-CaMKII-hum5D4NL1-NH2-Actin pLenti-CaMKII-EGFP-WPRE, pLenti-CaMKII-hum5D4NL1-NH2-Rab3a, and pLenti-CaMKII-mEGFP-Actin were constructed by the following procedure. To generate pLenti-CaMKII-EGFP-WPRE, WPRE was amplified by PCR from pAAV-HC20-5D4i(Y96F)-Fvclasp-2A-EGFP (forward primer: 5′-TCGAGGGGGGGGCCCGAATCAAACCTCTGGATTACAAAATTTGTGAAAGATTGAC-3′, reverse primer: 5′-TCATTGGTCTTTAAAGGCGGGGGAGGCGGCC-3′) and subcloned into the KpnI site of pLenti-CaMKII-EGFP by In-Fusion to obtain pLenti-CaMKII-EGFP-WPRE.

[0114] To generate pLenti-CaMKII-hum5D4NL1-NH2-Rab3a, the hum5D4NL1-NH2 fragment was amplified by PCR using pECFP-hum5D4NL1-NH2 as a template (forward primer: 5′-CCCGAATTCGCCACCATGGATATTGTGCTGACTCAGAGCCC- 3′, reverse primer: 5′CCCACTAGTACCGGTGTCGACGGATGAAAACCGTGACAAGTGTGCC-3′), which was subcloned into the EcoRI / SalI site of pLenti-CaMKII-5D4-GGGSx4-Rab3a to obtain pLenti-CaMKII-hum5D4NL1-NH2-Rab3a.

[0115] To prepare pLenti-CaMKII-mEGFP-Actin, mEGFP-Actin was amplified by PCR using pCAG-mEGFP-Actin as a template (forward primer: 5'-TGGGGGGCAGCGGATCCATGGTGAGCAAGGGCGAG-3', reverse primer: 5'-CGGGCCCCCCCCTCGAGCTAGAAGCATTTGCGGTGGACGAT-3'). This was then introduced by infusion into pLenti-CaMKII-EGFP-WPRE cleaved with BamHI and XhoI, and cloned to obtain pLenti-CaMKII-mEGFP-Actin.

[0116] To construct pLenti-CaMKII-hum5D4NL1-NH2-Actin, the hum5D4NL1-NH2 sequence was amplified by PCR using pLenti-CaMKII-hum5D4NL1-NH2-Rab3a as a template (forward primer: 5'-TGGGGGGCAGCGGATCCGCCACCATGGATATTGTGCTGACTC-3', reverse primer: 5'- The Actin sequence was amplified by PCR using pLenti-CaMKII-mEGFP-Actin as a template (forward primer: 5'-TCCGGACTCAGATCTCGAGCTATGG-3', reverse primer: 5'-CCCCCCTCGAGGTCGACCTAGAAGCATTTGCGGTGGACGATG-3'). This was introduced by infusion into pLenti-CaMKII-EGFP-WPRE cleaved with BamHI and SalI, and then cloned to obtain pLenti-CaMKII-hum5D4NL1-NH2-Actin.

[0117] pLenti-CaMKII-Lifeact-hum5D4NL1-NH2 pLenti-CaMKII-Lifeact-hum5D4NL1-NH2 was prepared via the construction of pLenti-CaMKII-hum5D4NL1-NH2-CAST, pLenti-CaMKIHum5D4NL1-NH2, pLenti-CaMKII-Munc13-1-hum5D4NL1-NH2, and pLenti-CaMKII-Munc13-1-GGGGGSx4-hum5D4NL1-NH2. The construction procedures for each plasmid are shown below.

[0118] ・pLenti-CaMKII-hum5D4NL1-NH2-CAST The hum5D4NL1-NH2 cDNA fragment was amplified by PCR using pECFP-hum5D4NL1-NH2 as a template (forward primer: 5'-TGGGGGGCAGCGGATCCATGGATATTGTGCTGACTCAGAGCCC-3', reverse primer: 5'-CGCTCCCGTAGGATCCGATATCGGCGGAGGGCCCGGATGAAACCGTGACAAGTGTGCC-3') and subcloned into the BamHI site of pLenti-CaMKII-EGFP-CAST by in-fusion to obtain pLenti-CaMKII-hum5D4NL1-NH2-CAST.

[0119] pLenti-CaMKII-hum5D4NL1-NH2: The cDNA fragment of hum5D4NL1-NH2 was amplified by PCR using pLenti-CaMKII-hum5D4NL1-NH2-CAST as a template (forward primer: 5'-CCCCTCGAGACCGGTATGGATATTGTGCTGACTCAGAGCCC-3', reverse primer: 5'-CCCGGTACCCTAGGATGAAACCGTGACAAGTGTGCC-3') and subcloned into the XhoI / KpnI sites of pLenti-CaMKII to obtain pLenti-CaMKII-hum5D4NL1-NH2.

[0120] ・pLenti-CaMKII-Munc13-1-hum5D4NL1-NH2 The xDNA fragment of Munc13-1 was amplified by PCR using pLenti-CaMKII-Munc13-1-EGFP as a template (forward primer: 5′-CCCGAATTCGCCACCATGTCTCTGCTGTGCGTGGGAGTCAAAAAAAG-3′, reverse primer: 5′-CCCGTCGACACTAGTCGGCGCAGGCGCGGC-3′) and subcloned into the EcoRI / SalI site of pLenti-CaMKII-hum5D4NL1-NH2 to obtain pLenti-CaMKII-Munc13-1-hum5D4NL1-NH2.

[0121] pLenti-CaMKII-Munc13-1-GGGGGSx4-hum5D4NL1-NH2 This was prepared by annealing oligo DNA encoding the GGGGSx4 linker fragment (5'-CTAGTGGAGGTGGCGGTAGCGGCGGAGGGGGTTCTGGTGGGCGGAGGTAGTGGGGGAGGCGGTTCTA-3' and 5'-CCGGTAGAACCGCCTCCCCCCACTACCTCCGCCACCAGAACCCCCTCCGCCGCTACCGCCACCTCCA-3'). The obtained fragment was subcloned into the SpeI / AgeI site of pLenti-CaMKII-Munc13-1-hum5D4NL1-NH2 to obtain pLenti-CaMKII-Munc13-1-GGGGSx4-hum5D4NL1-NH2.

[0122] pLenti-CaMKII-Lifeact-hum5D4NL1-NH2 To obtain the Lifeact sequence with EcoRI at the N-terminus and SpeI at the C-terminus, oligo DNA (5'-AATTCACCATGGGCGTGGCCGACTTGATCAAGAAGTTCGAGTCCATCTCCCAAGGAGGAGGGGGATCCACCGGTCGCCA-3' and 5'-CTAGTGGCGACCGGTGGATCCCCCTCCTCCTTGGAGATGGACTCGAACTTCTTGATCAAGTCGGCCACGCCCATGGTG-3') were annealed. The obtained EcoRI-Lifeact-SpeI was introduced by ligation into pLenti-CaMKII-Munc13-1-GGGGSx4-hum5D4NL1-NH2 which had been cleaved with EcoRI and SpeI, and the resulting product was cloned.

[0123] pLenti-CaMKII-hum5D4NL1-NH2-RimBP2 To prepare a lentiviral vector (pLenti-CaMKII-hum5D4NL1-NH2-RimBP2) for introducing the fusion protein of hum5D4NL1-NH2 and RimBP2 into neurons, pECFP-hum5D4NL1-NH2 was used as a template, and PCR was performed to amplify the hum5D4NL1-NH2 sequence, which had EcoRI at the N-terminus and SpeI at the C-terminus (forward primer: 5'-CCC GAATTCGCCACCATGGATATTGTGCTGACTCAGAGCCC-3′, reverse primer: 5′-CCCACTAGTACCGGTGTCGACGGATGAAACCGTGACAAGTGTGCC-3′), and subcloned into the EcoRI / SpeI sites of pLenti-CaMKII-EGFP-RimBP2 to obtain pLenti-CaMKII-hum5D4NL1-NH2-RimBP2.

[0124] pLenti-CaMKII-hum5D4NL1-NH2-Rim1α To create a lentiviral vector (pLenti-CaMKII-hum5D4NL1-NH2-Rim1α) for introducing the fusion protein of hum5D4NL1-NH2 and Rim1α into neurons, pLenti-CaMKII-hum5D4NL1-NH2-Rim1α was prepared by introducing the 1st to 1192nd amino acids of Rim1α into pLenti-CaMKII-Rim1α (1-1192) and the full-length Rim1α plasmid pLenti-CaMKII-Rim1α. Then, pLenti-CaMKII-hum5D4NL1-NH2-Rim1α was obtained by introducing the cDNA fragment of hum5D4NL1-NH2 into the N-terminus of Rim1α. The method for preparing the above plasmids is shown below.

[0125] pLenti-CaMKII-Rim1α(1-1192) pLenti-CaMKII-EGFP-Rim1α was cleaved with EcoRI and KpnI to obtain a cDNA fragment containing amino acid residues 1 to 1192 of Rim1α. This fragment was then subcloned into the EcoRI / KpnI site of pLenti-CaMKII to obtain pLenti-CaMKII-Rim1α(1-1192).

[0126] pLenti-CaMKII-Rim1α Amino acid residues 1193 to 1462 of Rim1α [Rim1α(1193-1462)] were obtained from pLenti-CaMKII-EGFP-Rim1α by PCR (forward primer: 5'-CCGACACGGCCGTGGG-3', reverse primer: 5'-TCATTGGTCTTTAAAGGTACCCTATGACCGGATGCAGGGAGG-3'), and Rim1α(1193-1462) was subcloned into the KpnI site of pLenti-CaMKII-Rim1α(1-1192) by in-fusion to obtain pLenti-CaMKII-Rim1α.

[0127] pLenti-CaMKII-hum5D4NL1-NH2-Rim1α The hum5D4NL1-NH2 cDNA fragment was amplified by PCR using pLenti-CaMKII-hum5D4NL1-NH2-Rab3a as a template (forward primer: 5'-CGGGCTGCAGGAATTCGCC-3', reverse primer: 5'-ATCCACCTCCGAATTCTGAGCCACCGCCCGATCC-3') and subcloned into the EcoRI site of pLenti-CaMKII-Rim1α to obtain pLenti-CaMKII-hum5D4NL1-NH2-Rim1α.

[0128] pLenti-CaMKII-hum5D4NL1-NH2-NR1 A lentiviral vector for introducing the fusion protein of hum5D4NL1-NH2 and NR1 into neurons was constructed as follows. To obtain the nucleotide sequence encoding the signal peptide, oligo DNA (5'-AATTCACCATGAGCACCATGCACCTGCTGACATTCGCCCTGCTTTTTTCCTGCTCCTTCGCCCGCGCCGCCG-3' and 5'-TCGACGGCGGCGCGGGGCGAAGGAGCAGGAAAAAAAGCAGGGCGAATGTCAGCAGGTGCATGGTGCTCATGGTG-3') was annealed and subcloned into the EcoRI / SalI site of pLenti-CaMKII to obtain pLenti-CaMKII-signal peptide (NR1). To generate pLenti-CaMKII-hum5D4NL1-NH2-NR1, the cDNA fragment of hum5D4NL1-NH2 was amplified from pLenti-CaMKII-hum5D4NL1-NH2-Rab3a by PCR (forward primer: 5′-CCGCGCCGCCGTCGACATGGATATTGTGCTGACTCAGAGCCC-3′, reverse primer: 5′-GATCTTGGGGGTCGCATGAGCCACCGCCCGATCC-3′). NR1 cDNA was amplified using pCI-EGFP-NR1 wt (Addgene 45446) as a template (forward primer: 5'-TGCGACCCCAAGATCGTCAAC-3', reverse primer: 5'-CCCCCTCGAGGTCGACTCAGCTCTCCCCTATGACGGGAAC-3') and subcloned into the SalI site of pLenti-CaMKII-signal peptide by in-fusion to obtain pLenti-CaMKII-hum5D4NL1-NH2-NR1.

[0129] pLenti-CaMKII-hum5D4NL1-NH2-NR2A A lentiviral vector for introducing a fusion protein of hum5D4NL1-NH2 and NR2A into neurons was constructed as follows. To obtain the nucleotide sequence encoding the signal peptide, oligo DNA (5'-AATTCACCATGGGCAGATTGGGCTACTGGACCTTGCTGGTATTGCCGGCCCTTCTGGTCTGGCGCGATCCGGCGCAGAACGCGG-3' and 5'-TCGACCGCGTTCTGCGCCGGATCGCGCCAGACCAGAAGGGCCGGCAATACCAGCAAGGTCCAGTAGCCCAATCTGCCCATGGTG-3') was annealed and subcloned into the EcoRI / SalI site of pLenti-CaMKII to obtain pLenti-CaMKII-signal peptide (NR2A). To generate pLenti-CaMKII-hum5D4NL1-NH2-NR2A, hum5D4NL1-NH2 was synthesized by PCR from pLenti-CaMKII-hum5D4NL1-NH2-Rab3a (forward primer: 5′-GCAGAACGCGGTCGACATGGATATTGTGCTGACTCAGAGCC-3′, reverse primer: 5′-ACCCTTCTCCGCCGCTGAGCCACCGCCCGATCC-3′) and pCI-EGFP-NR2a wt (Addgene). NR2A was amplified from the hum5D4NL1-NH2 gene (forward primer: 5'-GCGGCGGAGAAGGGTCC-3, reverse primer: 5'-CCCCCTCGAGGTCGACTTAAACATCAGATTCGATACTAGGCATTTTCTTGTAC-3) and subcloned into the SalI site of pLenti-CaMKII-signal peptide (NR2A) by infusion to obtain pLenti-CaMKII-hum5D4NL1-NH2-NR2A.

[0130] pLenti-CaMKII-GABAA α1-hum5D4NL1-NH2 A lentiviral vector for introducing a fusion protein of GABAA α1 and hum5D4NL1-NH2 into neurons was constructed as follows. A GABAA α1 cDNA fragment was amplified by PCR using alpha1-EGFP (Addgene #118952) as a template (forward primer: 5'-CGGGCTGCAGGAATTCATGAAGAAAGTCGGGGTCTCTCTCTGAC-3', reverse primer: 5'-CGCCACCTCCACTAGTCTGATGGGGTGTGGGGGC-3') and subcloned into the EcoRI / SpeI sites of pLenti-CaMKII-Munc13-1-GGGGGSx4-hum5D4NL1-NH2 to obtain pLenti-CaMKII-GABAA α1-hum5D4NL1-NH2.

[0131] pLenti-CaMKII-PSD95-hum5D4NL1-NH2 pLenti-CaMKII-GABAA Using α1-hum5D4NL1-NH2 as a template, the hum5D4NL1-NH2 cDNA fragment was amplified by PCR (forward primer: 5'-AGAGAGACTCACTAGTATGGATATTGTGCTGACTCAGAGCCC-3, reverse primer: 5'-TAAAGGTACGACGCGTTTAGGATGAAAC-3') and subcloned into the SpeI / MluI sites of pLenti-CaMKII-PSD95-GGGGSx4-hum5D4NL1-NH2 by in-fusion to obtain pLenti-CaMKII-PSD95-hum5D4NL1-NH2. The sequences of all plasmids were confirmed to be correct by Sanger sequencing.

[0132] Example 3: Culturing of 293T and COS-7 cells, and fluorescent imaging of COS-7 cells 293T and COS-7 cells were cultured in Dulbecco's modified Eagle's medium (WAKO) containing 10% fetal bovine serum (FBS, Sigma) at 5% carbon dioxide and 37°C. COS-7 cells were transfected with a COS-7 cell expression plasmid using XtremeGENE HP DNA (Roche), and observed 36 to 48 hours after transfection. Observations were performed in Hepes-buffered saline (25 mM HEPES, 125 mM sodium chloride, 2.5 mM potassium chloride, 2 mM calcium chloride, 1 mM magnesium chloride, 30% glucose, hereafter referred to as HBS) using an inverted microscope (IX-71, Olympus) equipped with a xenon arc lamp and a 10x objective lens (Olympus UPlanFL, numerical aperture 0.3). Fluorescence images were acquired using an EM-CCD camera (iXon, Andor). A U-MCFPHQ filter set (Olympus) was used to capture fluorescent images of ECFP, and a Cy5-4040C-OMF filter set (Semrock) was used to capture fluorescent images of 6SiR-DNP, represented by the following formula. All images were saved in 16-bit format.

[0133] Figure 2 shows the results of confirming the expression of a fusion protein between an intracellular protein and hum5D4NL1-NH2 in COS7 cells. Figure 2 shows fluorescent images obtained by expressing a fusion protein of hum5D4NL1-NH2 with β-tubulin, histone 2B, TOM20, vinculin, zyxin, Rab5a, or STIM1 in COS7 cells and loading them with 6SiR-DNP. The obtained images were analyzed using ImageJ (NIH).

[0134] To quantify the SiR fluorescence intensity in COS-7 cells transfected with ECFP-5D4 or ECFP-hum5D4NL1-NH2, cells were surrounded as a region of interest (ROI) in the ECFP-derived fluorescence image, and the ROI was reflected in the SiR-derived fluorescence image to obtain the SiR fluorescence intensity for each cell. The results are shown in Figure 3. Figure 3A shows a fluorescent image of ECFP and a stained image of 6SiR-DNP in a specimen transfected with ECFP-hum5D4NL1-NH2, ECFP-5D4, or ECFP. The scale bar represents 100 μm. Figure 3B shows a comparison of the fluorescence intensity when 6SiR-DNP was loaded onto a specimen transfected with ECFP-hum5D4NL1-NH2 or ECFP-5D4. The vertical axis represents the relative fluorescence intensity, with the mean fluorescence intensity from ECFP-5D4-transfected cells set to 1. p = 2 × 10 －１６ , Mann-Whitney U test, ECFP-hum5D4NL1-NH2: n = 83 cells (derived from one specimen), ECFP-5D4: n = 62 cells (derived from one specimen). Here, the fluorescence intensity in the area where no cells were present was taken as the background fluorescence intensity. The background fluorescence intensity was subtracted from the fluorescence intensity of SiR, and the resulting value was taken as a value indicating the expression levels of ECFP-5D4 and ECFP-hum5D4NL1-NH2 in the cells. It has been shown that the relative fluorescence intensity of ECFP-hum5D4NL1-NH2 is significantly improved compared to ECFP-5D4.

[0135] [Example 4] Culture of dissociated hippocampal neurons Fetuses were removed from Sprague-Dawley rats on day 21 of pregnancy, and the hippocampi were quickly removed from the fetal brains. The removed hippocampi were digested with trypsin (Nacalai Tesque) and DNase I (Sigma) to dissociate neurons. Neurons were cultured with rat hippocampal glial cells in a monolayer on 0.12-0.17 mm thick coverslips (Matsunami Glass, 25 mm x 25 mm) coated with poly-L-lysine (Sigma) and laminin (Invitrogen) in Neurobasal medium (Gibco) containing 2% B-27 supplement (Gibco), 1% Glutamax (Gibco), 1% penicillin-streptomycin mixed solution (Nacalai Tesque), and 1 mM sodium pyruvate (WAKO) at 37°C under 5% CO2 for 19 to 30 days. Cytosine arabinoside (final concentration 2.5 mM, Sigma) was added 2 and 5 days after the start of culture, and half of the medium was replaced every 5 to 7 days thereafter. All animal experiments were conducted in accordance with the University of Tokyo Animal Experiment Manual and with the approval of the Graduate School of Medicine Animal Experimentation Committee.

[0136] Lentivirus Production and Infection into Cultured Hippocampal Neurons. To generate lentiviruses that express fusion proteins of β-Actin, Lifeact, and synaptic molecules with hum5D4NL1-NH2 in neurons, 293T cells were transfected with the lentivirus production plasmids psPAX2 and pMD2.G using XtremeGENE HP DNA (Roche). 24 hours after transfection, the medium was replaced with neuronal culture medium. 48 hours after transfection, the entire medium was collected and centrifuged (4°C, 6 minutes, 800xg) to obtain a lentivirus-containing supernatant. Using a similar procedure, a virus solution was also prepared that expresses tetracycline-regulated transactivator (tTA) under the CaMKII promoter using pLenti 6PW STB. The resulting virus solution was stored at -80°C until use. For lentivirus infection of neurons, 10 to 100 μl of lentivirus solution was added to hippocampal neurons on days 12 to 17 of culture, and half of the medium was replaced the next day. After that, the neurons were cultured for 4 to 9 days and then used for fluorescence imaging.

[0137] The results of confirming the expression of hum5D4NL1-NH2-β-Actin and Lifeact-hum5D4NL1-NH2 in cultured hippocampal neurons are shown in Figure 4. Figure 4A shows fluorescent images of EGFP and 6SiR-DNP obtained by fluorescent staining with 6SiR-DNP in neurons into which EGFP and hum5D4NL1-NH2-β-Actin were introduced via lentivirus. The fluorescent image on the right is an enlarged version of the fluorescent image on the left. Figure 4B shows fluorescent images of EGFP and 6SiR-DNP obtained by fluorescent staining with 6SiR-DNP in neurons into which EGFP and Lifeact-hum5D4NL1-NH2 were introduced via lentivirus. The image on the right is an enlarged version.

[0138] Live cell fluorescence imaging of neurons using a confocal microscope. For fluorescence imaging using a confocal microscope, the hum5D4NL1-NH2 fusion protein with a synaptic molecule and the hum5D4NL1-NH2 fusion protein expressed in cultured hippocampal neurons expressing a synaptic marker were fluorescently labeled with 10 nM 6SiR-DNP, 100 nM 6SiR-5-pCNoNP (chemical formula shown below), or 100 nM ATTO655-2-DNP in HBS at room temperature.

[0139]

[0140] A confocal microscope (Leica TCS SP8) equipped with a pulsed white light laser, a HyD-SMD detector, and a 100x oil immersion objective (HC PL APO CS2, NA = 1.4) was used. The excitation wavelengths were set to 488 nm for EGFP and 633 nm for SiR. Other imaging conditions are listed in Table 1 below. All images were acquired using LAS-X software and saved in 8-bit or 16-bit format. Image analysis was performed using ImageJ (NIH).

[0141]

[0142] The results of fluorescent imaging of presynaptic molecules using hum5D4NL1-NH2 are shown in Figure 5. The results of fluorescent imaging of postsynaptic molecules using hum5D4NL1-NH2 are shown in Figure 6.

[0143] Figure 5 shows the results of confirming the expression of the fusion protein between hum5D4NL1-NH2 and a presynaptic molecule, and shows fluorescent images of a specimen transfected with EGFP-CAST (A), a specimen transfected with EGFP-CAST and hum5D4NL1-NH2-RIM1a (B), and a specimen transfected with EGFP-CAST and hum5D4NL1-NH2-RBP2 (C) stained with 6SiR-DNP. The lower figures are enlarged views.

[0144] Figure 6 shows the results of confirming the expression of fusion proteins between hum5D4NL1-NH2 and postsynaptic molecules, and shows fluorescent images of specimens transfected with EGFP-HC20 and hum5D4NL1-NH2-GABAA1α (A), EGFP-HC20 and PSD95-hum5D4NL1-NH2 (B), EGFP-HC20 and hum5D4NL1-NH2-GluA1 (C), EGFP-HC20 and hum5D4NL1-NH2-NR1 (D), and EGFP-HC20 and hum5D4NL1-NH2-NR2A (E) stained with 6SiR-DNP. The figures below each show enlarged views.

[0145] Example 5: Evaluation of the usefulness of a new fluorescent labeling method using anti-DNP antibodies in long-term imaging. Tracking protein dynamics within living cells over long periods of time is essential for elucidating the molecular mechanisms of cellular function, but long-term observation is difficult with conventional labeling methods due to photobleaching. The novel fluorescent labeling technology of the present invention, based on anti-DNP antibodies, is a groundbreaking method that overcomes the essential challenges of long-term observation that conventional fluorescent imaging methods face. The usefulness of the new fluorescent labeling method in long-term imaging is examined below.

[0146] (1) Preparation of N / C-Terminal Tag-Fusion Lentiviral Cloning Plasmids. PCR-amplified DNA fragments were inserted into the BamHI / XbaI site of pLV-EF1a-IRES-Puro (Addgene #85132) using the in-fusion cloning method to prepare plasmids. The primers for the DNA fragments were designed as follows: (1) the BamHI site was inactivated and a new EcoRI site was introduced; (2) the XbaI site was maintained; and (3) a BamHI site was placed between the tag sequence and the target gene insertion site. Details of the primers and templates used to amplify the DNA fragments are shown in Tables 2-1 and 2-2. All tag-fusion plasmids and all plasmids prepared based on these plasmids by inserting a target gene were sequenced by Sanger sequencing to confirm that the coding sequences in the plasmids were as designed. As the tag, in addition to the hum5D4NL1-NH2 of the present invention (hereinafter also abbreviated as "DQ"), HaloTag protein (HT), SNAP protein, or monomeric green fluorescent protein (mEGFP) was used.

[0147] Table 2-1 Primers and templates used to amplify DNA fragments for plasmid construction

[0148] Table 2-2. Primer sequences

[0149] (2) Preparation of pLef2 Series Plasmids Plasmids for expressing tag-fused target proteins were prepared by in-fusion cloning or ligation. Details of the prepared plasmids, methods, vectors and their insertion sites, and inserted DNA fragments are shown in Table 3-1, and the sequences of the primers used are shown in Table 3-2.

[0150] Table 3-1 Plasmid construction methods

[0151] Table 3-2 Primer sequences

[0152] (3) Preparation of lentivirus HEK293T cells were seeded at 1,250,000 cells per 35 mm dish and incubated in 5% CO ２ The cells were cultured in medium (DMEM containing 10% FBS) at 37°C. The next day, 800 ng of pLef2 plasmid, 800 ng of psPAX2 (Addgene #12260), 400 ng of pMD2.G (Addgene #12259), and 200 μL of OptiMEM were mixed, followed by the addition of 6 μL of X-tremeGENE and mixing thoroughly. After allowing to stand at room temperature for 15 minutes, the prepared solution was added to the HEK293T cell medium and gently mixed. After culturing the cells for one day, the medium was replaced with fresh medium. After culturing for another day, the medium was collected and filtered through a 0.45 μm filter. A PEG solution (32% w / v PEG 6000, 400 mM NaCl, 40 mM HEPES (pH 7.4)) was added to the resulting virus solution in a volume of 1 / 4, mixed, and then allowed to stand at 4°C for at least one day. The mixture was centrifuged at 1,500g for 30 minutes, the supernatant was removed, and the precipitate was thoroughly suspended in 100 μL of medium to obtain a virus concentrate, which was then stored at -80°C.

[0153] (4) Establishment of a stable expression cell line HeLa cells (15,000 cells / well) were seeded in a 96-well plate and incubated under 5% CO ２ , 37 ｏ The cells were cultured for one day in medium (DMEM containing 10% FBS) under C conditions. A series of virus solutions was prepared by diluting the virus concentrate with DMEM containing 6 μg / mL polybrene, and the medium was removed before adding the virus to the wells. The 96-well plate was centrifuged at 32°C at 1,200 g for 60 minutes, and then incubated in 5% CO ２ , 37 ｏThe cells were cultured for one day under C conditions. After removing the virus solution, fresh medium was added and cultured for another day. If necessary, the cells were stained with a ligand for the tag, and then expression of the target protein in the cells was evaluated using a fluorescence microscope, and wells with a multiplicity of infection of less than approximately 0.3 were selected. Using the cells from the selected wells, single clone cell lines were established by limiting dilution. Using the above method, a HeLa cell line stably expressing mEGFP-PRKN (HeLa / mEGFP-PRKN) was obtained. Then, by infecting HeLa / mEGFP-PRKN with an additional lentivirus using the same method, a HeLa cell line also stably expressing TOMM20-HT (HeLa / mEGFP-PRKN_TOMM20-HT) was obtained. Furthermore, HeLa cell lines stably expressing DQ-SQSTM1 or SNAP-SQSTM1 (HeLa / mEGFP-PRKN_TOMM20-HT_DQ-SQSTM1 and HeLa / mEGFP-PRKN_TOMM20-HT_SNAP-SQSTM1, respectively) were obtained by infecting HeLa / mEGFP-PRKN_TOMM20-HT with additional lentiviruses using a similar method. HeLa / mEGFP-PRKN_TOMM20-HT_DQ-SQSTM1 and HeLa / mEGFP-PRKN_TOMM20-HT_SNAP-SQSTM1 were used in the following Experiment A (a comparative experiment between the novel labeling tag of the present invention and the SNAP tag system by continuous observation).

[0154] (5) Transient gene transfer to cultured cells COS-7 cells were cultured under 5% CO ２ The cells were cultured in DMEM containing 10% FBS at 37°C. pLef2-Tubb5-DQ_Pur or pLef2-Tubb5-HT_Pur was introduced into COS-7 cells using X-tremeGENE HP DNA Transfection Reagent (Sigma-Aldrich). After one day of culture, the cells were detached and re-plated on glass-bottom dishes (Matsunami Glass). After an additional day of culture, they were subjected to the following experiment B (a comparison experiment of the developed chemical tag and HaloTag using fluorescence recovery photoacceleration (FRAP)).

[0155] (6) Fluorescence Imaging Fluorescence imaging was performed using established stably expressing HeLa cell lines or transiently transfected COS-7 cells. In Experiment A, stably expressing HeLa cell lines were stained with 6SiR-5-pCNoNP for DQ-SQSTM1, TMR-CA for TOMM20-HT, and SiR-BG for SNAP-SQSTM1. Cells were loaded with CCCP (carbonyl cyanide-m-chlorophenylhydrazone) as a mitophagy induction condition and cultured for 2 hours before fluorescence imaging. In Experiment B, COS-7 cells expressing Tubb5-DQ were stained with 6SiR-5-pCNoNP, and cells expressing Tubb5-HT were stained with SiR-CA before fluorescence imaging. A Leica TCS SP8 confocal microscope system (Leica Microsystems) was used for imaging. A pulsed white light laser was used as the excitation light source, and the excitation light wavelengths were set to 488 nm for mEGFP, 561 nm for TMR-CA, and 633 nm for SiR (6SiR-5-pCNoNP, SiR-BG, SiR-CA). The fluorescence detection wavelengths were set to 495-550 nm for mEGFP, 565-590 nm for TMR-CA, and 640-700 nm for SiR (6SiR-5-pCNoNP, SiR-BG, SiR-CA). A 100x oil immersion objective (HC PL APO CS2, NA = 1.4) was used as the objective lens. Image analysis was performed using ImageJ (NIH).

[0156] (7) Experiment A: Comparative experiment of the novel labeling tag of the present invention and the SNAP tag system by continuous observation The results of continuous imaging of HeLa cells fused and expressed with the novel labeling tag of the present invention (DQ-SQSTM1) or the conventional SNAP tag (SNAP-SQSTM1) are shown in Figure 7. Fluorescent staining was performed using 6SiR-5-pCNoNP as a probe in the novel labeling method using an anti-DNP antibody, and SiR-BG in the SNAP tag method, and the HeLa cells were treated with the mitochondrial uncoupling drug CCCP at 20 μM for 2 hours.

[0157] The upper panel of each panel shows a fluorescent image of the target protein, with #1 and #100 (DQ-SQSTM1) or #50 (SNAP-SQSTM1) representing the frame number, respectively. The lower panel shows a fluorescent image of a mitochondrial marker (TOMM20-HT) in the same cell. The scale bar represents 1 μm. Panel C quantitatively compares the time course of fluorescence intensity between the new chemical tag (DQ-SQSTM1) and the conventional method (SNAP-SQSTM1). The vertical axis represents normalized fluorescence intensity, the horizontal axis represents frame number, and the scale bar represents 25% intensity and 10 frames.

[0158] The novel chemical tagging system (DQ-SQSTM1) of the present invention, shown in Figure 7A, maintains a nearly constant fluorescence signal intensity throughout the entire observation period, from frame #1 to frame #100. Comparison with a mitochondrial marker (TOMM20-HT) in the same cell demonstrates the ability to stably visualize target proteins over long periods of time. In contrast, the conventional labeling method (SNAP-SQSTM1) shown in Figure 7B exhibits a significant decay in fluorescence intensity after only about 50 frames of observation. As shown in the quantitative analysis graph in Figure 7C, the novel labeling method of the present invention maintains stable fluorescence intensity over long periods of time, whereas the conventional method experiences rapid signal decay. This excellent photostability provides a crucial advantage in continuous live cell imaging, enabling the long-term tracking of minute changes in the dynamics of biomolecules.

[0159] (8) Experiment B: Comparison of the Developed Chemical Tag and HaloTag Using Fluorescence Recovery and Photobleaching (FRAP). Figure 8 shows the results of fluorescent imaging of microtubules in COS-7 cells expressing the novel labeling tag of the present invention (DQ-SQSTM1; Figure 8A) or HaloTag (Figure 8B) fused to β-tubulin. Fluorescent staining was performed using 6SiR-5-pCNoNP as the probe for the novel labeling method using anti-DNP antibodies, and SiR-CA for the HaloTag method. Furthermore, the novel labeling method was performed under conditions where 50% of the tag was bound to the probe and in equilibrium. The left image in the upper left panel and the right image are images after FRAP, respectively. The dotted circle indicates the area that was photobleached by irradiating it with strong excitation light. The graph in the lower left panel shows the time course of fluorescence intensity in the region of interest. The vertical axis indicates normalized fluorescence intensity, the horizontal axis indicates time (seconds), and the scale bar indicates 50% intensity and 10 seconds.

[0160] The results of the fluorescence recovery assay (FRAP) experiment shown in Figure 8 show that the new labeling method rapidly recovers fluorescence after photobleaching, whereas the conventional HaloTag system fails to do so. This demonstrates the reversible molecular binding properties of the new tagging system, demonstrating its advantage in enabling continuous observation of dynamic molecular behavior. This technology is particularly useful for long-term live cell imaging, particularly for precise analysis of protein dynamics. Continuous observation of spatiotemporal expression patterns and intermolecular interactions of intracellular proteins is expected to contribute to elucidating the dynamic control mechanisms of cellular function and to the advancement of high-throughput screening systems.

Claims

1. A method for fluorescently labeling an intracellular protein, comprising: obtaining a fusion protein of a protein to be labeled and an anti-DNP (dinitrophenyl compound) antibody within a cell; contacting the cell with a compound represented by the following formula (I) or a salt thereof; and reacting the fusion protein with the compound represented by the following formula (I) or a salt thereof, thereby fluorescently labeling the protein of interest: (In formula (I), S is a fluorescent group, L is a linker, and R ｂ and R ｃ is selected from the following combinations: ｂ , R ｃ ): (NO ２ , p-NO ２ ), (NO ２ , p-Br), (NO ２ , p-SO ２ Me), (NO ２ , p-Cl), (NO ２ , m-CN), (NO ２ , p-CN), (NO ２ , p-COOMe), (CF ３ , p-CF ３ ), (NO ２ , p-CONHMe), (NO ２ , m-COOMe), (NO ２ , H) (where p- and m- are R ｃ are located at the para and meta positions on the benzene ring relative to L. The fluorescent group is represented by any one of the following formulas (II) to (IV): (In formula (II), R １ represents a hydrogen atom or 1 to 4 identical or different monovalent substituents present on a benzene ring; R ２ represents a hydrogen atom, a monovalent substituent, or a bond; １ , R ２ is selected from the group consisting of an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 1 to 6 carbon atoms, an alkynyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, a hydroxyl group, a carboxy group, a sulfonyl group, an alkoxycarbonyl group, a halogen atom, and an amino group, and the alkyl group having 1 to 6 carbon atoms may have one or more substituents selected from the group consisting of a halogen atom, a carboxy group, a sulfonyl group, a hydroxyl group, an amino group, and an alkoxy group, and the bond is ２ means to be introduced at the position of R ３ and R ４ each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; R ５ and R ６ each independently represents an alkyl group having 1 to 6 carbon atoms or an aryl group, provided that when X is an oxygen atom, R does not exist; ７ and R ８ each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; X represents an oxygen atom or a silicon atom; * represents a bonding site to L in formula (I) at any position on the benzene ring. (In formula (III), R １ ~R ８ , X is as defined in formula (II); R ９ and R １０ each independently represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; R ９ and R １０ Let's get together and ９ and R １０ may form a 4- to 7-membered heterocyclyl containing the nitrogen atom to which R is attached, ９ or R １０ Alternatively, R ９ and R １０ Both of these are R ３ or R ７ Together with ９ or R １０ may form a 5-7 membered heterocyclyl or heteroaryl containing a nitrogen atom to which R is bonded, and may contain 1 to 3 additional heteroatoms selected from the group consisting of oxygen atoms, nitrogen atoms and sulfur atoms as ring-constituting members, and the heterocyclyl or heteroaryl may be substituted with an alkyl having 1 to 6 carbon atoms, an alkenyl having 2 to 6 carbon atoms, an alkynyl having 2 to 6 carbon atoms, an aralkyl group having 6 to 10 carbon atoms, or an alkyl-substituted alkenyl group having 6 to 10 carbon atoms; R １１ and R １２ each independently represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; R １１ and R １２ Let's get together and １１ and R １２ may form a 4- to 7-membered heterocyclyl containing the nitrogen atom to which R is attached, １１ or R １２ Alternatively, R １１ and R １２ Both of these are R ４ or R ８ Together with １１ or R １２ is bonded to a nitrogen atom, and may contain 1 to 3 additional heteroatoms selected from the group consisting of oxygen atoms, nitrogen atoms, and sulfur atoms as ring-constituting members, and the heterocyclyl or heteroaryl may be substituted with an alkyl having 1 to 6 carbon atoms, an alkenyl having 2 to 6 carbon atoms, an alkynyl having 2 to 6 carbon atoms, an aralkyl group having 6 to 10 carbon atoms, or an alkyl-substituted alkenyl group having 6 to 10 carbon atoms; * represents a bonding point to L in formula (I) at any position on the benzene ring.) (In formula (IV), R １ is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms which may have a substituent; R ２ is an alkylene group having 1 to 6 carbon atoms, which may be substituted; R ３ ~R ６ are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms which may have a substituent; R ７ ~R ８ are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms which may have a substituent, and R ７ and R ８ If there are two, each R ７ , R ８ may be the same or different; m1 and m2 are 1 or 2; R ９ ~R １０ are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms which may have a substituent, and R ９ and R １０ If there are two, each R ９ , R １０ may be the same or different; n1 and n2 are 1 or 2; R １１ ~R １４ are each independently a hydrogen atom or an alkyl group having 1 to 6 carbon atoms which may have a substituent; １ , R ３ ~R １４ At least one of the groups is an alkyl group having 1 to 6 carbon atoms and having a water-soluble group; ----- indicates a single bond or a double bond; * indicates the site of bonding to the linker L. ) The anti-DNP antibody in the fusion protein is an anti-DNP antibody or an antigen-binding fragment thereof comprising: a light chain comprising a VL-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1, a VL-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and a VL-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3; and a heavy chain comprising a VH-CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4, a VH-CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and a VH-CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6, wherein SEQ ID NO: 1: RSSQEISGYLGW SEQ ID NO: 2: AASTLD SEQ ID NO: 3: VQYASYPYT SEQ ID NO: 4: FSNYWMN SEQ ID NO: 5: EAELESNNYATHYAESVKG SEQ ID NO: 6: YSYDSRYGY A method for fluorescently labeling a protein, wherein the anti-DNP antibody or the antigen-binding fragment thereof is a single-chain Fv (scFv).

2. The method according to claim 1, wherein the anti-DNP antibody consists of an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 7 and includes the amino acid sequences shown in SEQ ID NOs: 1 to 6 as CDR sequences. SEQ ID NO: 7: DIVLTQSPSSLSASPGDRVTITCRSSQEISGYLGWLQQKPDGSIKRLIYAASTLDSGVPSRFSGSRSGTDYTLTISGLEPEDFGDYYCVQYASYPYTFGQGTKVEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGDSLRLSCAASGFTFSNYWMNWVRQAPGKGLEWVGEAELESNNYATHYAESVKGRFTISRDDSEGTVYLEMNGLRSEDTGVYYCTGYSYDSRYGYWGQGTLVTVSS 3. The method of claim 2, wherein the amino acid sequence is SEQ ID NO:

7.

4. The method of claim 1, wherein the anti-DNP antibody consists of an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 7, and includes the amino acid sequences shown in SEQ ID NOs: 1 to 6 as CDR sequences, and wherein at least one of the following substitutions is made in the amino acid sequence of SEQ ID NO: 7: (a) any one of aspartic acid at position 41, arginine at position 66, tyrosine at position 71, glutamic acid at position 79, and aspartic acid at position 85, numbered from the N-terminus, is substituted with another hydrophilic amino acid (provided that the total number of negative charges remains unchanged after the substitution); or (b) any one of aspartic acid at position 149, aspartic acid at position 209, glutamic acid at position 211, glutamic acid at position 217, and serine at position 223, numbered from the N-terminus, is substituted with another amino acid having a hydroxyl group in its side chain.

5. The method of any one of claims 1 to 4, wherein obtaining the fusion protein comprises obtaining a polynucleotide encoding the fusion protein, obtaining a plasmid or vector capable of expressing the fusion protein, expressing the fusion protein in a cell, or isolating the expressed fusion protein.

6. The method of claim 1, wherein the linker is represented by TY, where Y represents a linking group that bonds with the fluorescent group S, and T represents a bridging group.

7. The method of claim 6, wherein the linking group is selected from an amide group, an alkylamide group, an ester group, an alkylester group, a carbonylamino group, or an alkylether group.

8. An anti-DNP antibody or antigen-binding fragment thereof comprising a light chain comprising a VL-CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 1, a VL-CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 2, and a VL-CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and a heavy chain comprising a VH-CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 4, a VH-CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a VH-CDR3 consisting of the amino acid sequence set forth in SEQ ID NO:

6. SEQ ID NO: 1: RSSQEISGYLGW SEQ ID NO: 2: AASTLD SEQ ID NO: 3: VQYASYPYT SEQ ID NO: 4: FSNYWMN SEQ ID NO: 5: EAELESNNYATHYAESVKG SEQ ID NO: 6: YSYDSRYGY 9. The anti-DNP antibody or antigen-binding fragment thereof according to claim 8, wherein the anti-DNP antibody or antigen-binding fragment thereof is a single-chain Fv (scFv).

10. The anti-DNP antibody or antigen-binding fragment thereof according to claim 8, which consists of an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 7 and contains the amino acid sequences shown in SEQ ID NOs: 1 to 6 as CDR sequences. SEQ ID NO: 7: DIVLTQSPSSLSASPGDRVTITCRSSQEISGYLGWLQQKPDGSIKRLIYAASTLDSGVPSRFSGSRSGTDYTLTISGLEPEDFGDYYCVQYASYPYTFGQGTKVEIKRGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGDSLRLSCAASGFTFSNYWMNWVRQAPGKGLEWVGEAELESNNYATHYAESVKGRFTISRDDSEGTVYLEMNGLRSEDTGVYYCTGYSYDSRYGYWGQGTLVTVSS 11. The anti-DNP antibody or antigen-binding fragment thereof according to claim 10, wherein the amino acid sequence is SEQ ID NO:

7.

12. An anti-DNP antibody or antigen-binding fragment thereof, consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 7, including the amino acid sequences shown in SEQ ID NOs: 1 to 6 as CDR sequences, and having at least one of the following substitutions in the amino acid sequence of SEQ ID NO: 7: (a) any one of the following amino acids from the N-terminus: aspartic acid at position 41, arginine at position 66, tyrosine at position 71, glutamic acid at position 79, and aspartic acid at position 85, is substituted with another hydrophilic amino acid (provided that the total number of negative charges remains unchanged after the substitution); or (b) any one of the following amino acids from the N-terminus: aspartic acid at position 149, aspartic acid at position 209, glutamic acid at position 211, glutamic acid at position 217, and serine at position 223, is substituted with another amino acid having a hydroxyl group in its side chain.

13. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 8 to 11.

14. The nucleic acid according to claim 13, consisting of the base sequence shown in SEQ ID NO:

8. SEQ ID NO: 8: GATATTGTGCTGACTCAGAGCCCATCCAGCTTGAGTGCCTCTCCCGGCGATCGAGTGACCATAACATGCAGAAGCTCCCAGGAAATCAGCGGATACCTGGGCTGGTTGCAGCAGAAACCGGATGGGTCTATCAAGCGCCTGATTTATGCGGCAAGCACCCTCGACAGTGGGGTTCCTTCTCGGTTCTCTGGATCTAGGTCTGGAACAGACTATACCCTGACAATCAGTGGCCTCGAACCCGAGGATTTTGGTGACTACTATTGCGTGCAGTATGCCAGCTATCCCTACACTTTTGGACAGGGGACCAAAGTGGAGATCAAGAGAGGTGGTGGAGGGTCAGGCGGTGGTGGGTCCGGCGGAGGCGGAAGCGGA GGAGGCGGATCAGAGGTGCAACTGGTCGAGAGCGGCGGTGGGCTCGTACAACCTGGCGACAGCCTGAGACTGTCCTGTGCTGCAAGTGGCTTCACCTTCTCCAACTACTGGATGAATTGGGTCAGGCAGGCTCCAGGTAAAGGCCTGGAATGGGTTGGGGAGGCCGAGCTGGAGTCCAACAACTAC GCCACTCACTACGCTGAGTCAGTAAAGGGCCGGTTTACGATAAGTCGCGACGACAGCGAGGGGACAGTGTACCTCGAGATGAATGGGCTTCGTTCCGAAGATACTGGCGTGTACTACTGTACCGGGTATTCCTACGACTCAAGGTATGGGTATTGGGGCCAAGGCACACTTGTCACGGTTTCATCC 15. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof of claim 12.

16. A plasmid or vector containing a nucleic acid according to any one of claims 13 to 15.

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